THE MICROTOMIST'S VADE-MECUM A HANDBOOK OF THE METHODS OF MICROSCOPIC ANATOMY ARTHUR BOLLES LEE FOURTH EDITION LONDON J. & A. CHURCHILL 7, G15KAT MARLBOROUGH STREET 1896 * , -* * ' a 5 ? ' * * O e * I/:X-V-::JA...- V s - \ 7/2- / 3 PREFACE THE short period of three years that has elapsed since the publication of the last edition of this work has not brought with it any radical change in the methods of histological research. Such progress as has been realised has consisted rather in improvements in the detail of already-established methods than in the introduction of new methods or new reagents. Nevertheless, the present edition has undergone a most thorough revision a revision indeed so thorough as to amount to extensive re-writing in many parts. It has seemed to me advisable in the interest of the be- ginner, and indeed in the interest of readers who are not beginners at all, to enter more fully than was hitherto done into the detail of the more important processes, to explain more fully the principles on which they are founded, and to add in many cases a critical estimate of their rationality and practical value. In consequence of this re-writing, and in spite of strenuous efforts to keep down the bulk of the work, it has turned out to be considerably increased. I regret, however, this increase the less, in so far as it is due rather to the ampler treatment that has been accorded to the more valuable methods than to increase in the number of processes described. The number of new processes described is in fact a smaller one than I have had to deal with in the preparation of any edition since the first. The classification of the various methods has received most careful attention, and has been in many cases greatly simpli- fied, whilst at the same time a large number of superfluous vi PREFACE processes have been rejected. Advice to the beginner con- cerning the choice of methods has been given wherever practicable, and I think that notwithstanding the abundance and complexity of the matters treated of, there can hardly be aoy risk that the student may be unable to see the wood for the trees. The chapters treating of Staining and of the Carmine and Haematein stains have had the great advantage of revision by Dr. Paul Mayer, who, it is superfluous to remind the reader, has made af speciality of this subject, with results brilliant alike in theory and in practice. Not indeed that the present English text has been directly revised by him, but that it has been prepared from a recent text so revised. Dr. Mayer was good enough to revise most carefully the three corresponding chapters prepared by me for the recent new edition of the Traite des Methodes Techniques de VAna- tomie Microscopique (LEE et HENNEGUY), and in the prepara- tion of the present English text I have closely followed the chapters so revised. No less obligation have I to express to Professor van Gehuchten, who with great kindness has thoroughly revised for me the three chapters entitled Neurological Methods. It occurred to me that my treatment of this complicated subject could not but gain greatly by the advice of an observer who is not only one of the foremost of the new school of neuro" legists but at the same time an instructed and capable cytologist, and therefore likely to sympathise with my feeling that it would be much to be deplored that the study of nervous anatomy should degenerate into a mere study of topographical relations, to the neglect of the inner mechanism of nervous elements. By Professor van Gehuchten's advice I have entirely re-arranged the contents of these three chapters according to a scheme worked out by him, thereby effecting a great gain in clearness of exposition. I cannot but acknowledge that the arrangement adopted in previous editions resulted in something like a chaos ; whilst the new I'UKI-'AC!-: vii arrangement may, I think, fairly claim to be natural, logical, and easily comprehensible. By his advice too I have entirely re-written the account of the bichromate of silver impregna- tions of Golgi ; the account as it now stands is, I believe, the only complete one that has appeared in the English language. I am under the greatest obligation to Professor van Gehuchten, as well as to Dr. Paul Mayer, for the generous assistance which enables me to affirm that the important subjects in question have been treated with all the requisite accuracy and thoroughness. The essential feature of the first edition was that it was an altogether exhaustive collection of all the methods of pre- paration that had up to that time been recommended as useful for the purposes of microscopic anatomy, and its primary intention that of being a work of reference for the instructed anatomist. Its character of a guide to the be- ginner was secondary only. It contained indeed a general introduction and much explanatory matter in the different chapters, but, on the whole, the didactic matter bore but an insufficient proportion to the historical matter. This has now been rectified. It has come to pass that during the repeated operations of revision to which the book has been subjected, the explanatory and didactic element has been continually increasing, whilst at the same time the historical element has been continually diminishing diminishing, that is, in all parts of the book relatively to the former element, and in some parts absolutely (as may be seen, for instance, by com- paring the number of formulae given in the chapters on carmine and haematoxylin with the number given in former editions). On the one hand the book has been lightened by the jettison of much useless matter, and on the other hand there has been accorded to the matter that has been retained a far ampler share than before of explanation and detail. To such an extent, indeed, have the instructions to students and other explanatory matter been amplified that I am not Viii PREFACE acquainted with any modern work on the subject that con- tains anything like so complete an account of the various fundamental operations of histological technique fixing, im- bedding, staining, and the like. I only felt justified in claiming for the first edition that it " went far to make up a formal treatise on the art/' Through the changes above- mentioned the book has come to assume altogether the character of a formal treatise, and now contains in due pro- portions both the grammar and the dictionary of the art. The rejection of superfluous matter above referred to re- lates chiefly to old methods that have been before the public for so long a time that there can be no doubt that they have no good claim to further survival, whilst recent methods, which may be considered to be still on their probation, have been treated with the accustomed -fulness. , SWITZEBLAND ; September, 1896. CONTENTS PART I CHAPTER I. PAGE INTBODUCTOBY. The General Method . . . . .1 CHAPTER II. KILLING . . . . . . . .11 CHAPTER III. FIXING AND HARDENING . . . . . .18 CHAPTER IV. FIXING AGENTS : MINERAL ACIDS AND THEIB SALTS . . .24 CHAPTER V. FIXING AGENTS : CHLOBIDES, OBGANIC ACIDS, AND OTHEBS . . 38 CHAPTER VI. HABDENING AGENTS . . . . . . .56 CHAPTER VII. CLEABING AGENTS . . . . 64 CHAPTER VIII. IMBEDDING METHODS. INTBODUCTION . . . .71 CHAPTER IX. ^ IMBHI Dora MKTHODS : PARAFFIN AND OTHEB FUSION MA*-I> . 79 Paraffin, 79 ; Soap, 94; Gelatin, V7. b CONTENTS. CHAPTER X. PAGE COLLODION (CELLOIDIN) AND OTHER IMBEDDING METHODS . . 99 Collodion or Celloidin, 99 ; other Evaporation Masses, 113 ; Freezing Masses, 115. CHAPTER XI. SERIAL SECTION MOUNTING . . . . .119 Methods for Paraffin Sections, 119 ; Methods for Watery Sections, 128; Methods for Celloidin Sections, 128. CHAPTER XII. STAINING ........ 135 CHAPTER XIII. CARMINE AND COCHINEAL STAINS ..... 145 Theory of Carmine Staining, 145 ; Aqueous Carmines, Acid, 149 ; Neutral and Alkaline, 153 ; Alcoholic Carmines and Cochineals, 156. CHAPTER XIV. HJEMATEIN (H^EMATOXYLIN) STAINS ..... 161 Theory of Staining with Hsematoxylin, 161 ; Haematein (HaBma- toxylin), 163; Alumina-ha?matein Lakes, 167; other Ha3ma- tein Lakes, 173. CHAPTER XV. ON STAINING WITH COAL-TAE COLOURS . . . .179 Basic, Acid and Neutral Coal-tar Colours, 179 ; Progressive and Regressive Stains, 181; General Directions for the Regressive Method, 182. CHAPTER XVI. THE COAL-TAR CHROMATIN STAINS ..... 189 Regressive Stains, 189 ; Progressive Stains, 197. CHAPTER XVII. METHYLEN BLUE ....... 202 CHAPTER XVIII. THE COAL-TAR PLASMA STAINS . . . 215- CONTENTS. xi CHAPTER XIX. PAGE OTHER ORGANIC STAINS AM. COMBINATIONS . . . 233 Other Organic Stains, 233 ; Carmine combinations, 235 : Haema- tei'n Combinations, 237. CHAPTER XX. METALLIC STAINS (IMPREGNATION METHODS) .... 241 Silver. 243; Gold, 250; other Metallic Stains, 258. CHAPTER XXI. EXAMINATION AND PRESERVATION MEDIA .... 262 Aqueous Liquids, 262 ; Mercurial Liquids, 266 ; other Fluids, 267 ; Glycerin Media, 270 ; Resinous Media, 277. CHAPTER XXII. CEMENTS AND VARNISHES . 284 PART II SPECIAL METHODS AND EXAMPLES. CHAPTER XXIII. INJECTIONS : GELATIN MASSES . . . . . 293 Carmine, 296 ; Blue, 300 ; other Colours, 304. CHAPTER XXIV. INJECTIONS : OTHEE MASSES ... . 307 White of Egg, Gum, 307 ; Glycerin, 308 ; Aqueous, 309 ; Cel- loidin, 310 ; other Masses, 310. CHAPTER XXV. MACERATION AND DIGESTION . . . . . .312 Maceration, 312 ; Digestion, 320. CHAPTER XXVI. CORROSION, DECALCIFICATION, AND BLEACHING . . . :]'2'2 Corrosion, 322 ; Decalcification and Desilicification, 323; Bleach- ing, 328. xii CONTENTS. CHAPTER XXVII/ PAGE EMBRYOLOGICAL METHODS . . 331 CHAPTER XXVIII. -CYTOLOGICAL METHODS . . 354 CHAPTER XXIX. TEGUMENTARY ORGANS ... . 370 CHAPTER XXX. MUSCLE AND TENDON (NERVE-ENDINGS) .... 378 Striated Muscle, 378 ; Tendon, 381 ; Smooth Muscle, 382. CHAPTER XXXI. NEUROLOGICAL METHODS. INTRODUCTION AND SECTION METHODS . 385 CHAPTER XXXII. NEUROLOGICAL METHODS. NERVE-FIBRE STAINS (Weigert and others) 405 CHAPTER XXXIII. NEUROLOGICAL METHODS. AXIS-CYLINDER AND PROTOPLASM STAINS (Golgi and others) ...... 415 Retina, 439 ; Inner Ear, 442. CHAPTER XXXIV. SOME OTHER HlSTOLOGICAL METHODS ... . 444 Connective Tissues, 444; Blood, 456 ; Glands, 461. CHAPTER XXXV. SOME ZOOLOGICAL METHODS ...... 466 Tunicata, 467 ; Mollusca, 468 ; Arthropoda, 472 ; Veraies, 476 ; Echinodermata, 485 ; Coelenterata, 488 ; Porifera, 493 ; Pro- tozoa, 495. APPENDIX ... . . 501 INDEX . . 507 THE MICROTOMIST'S VADE-MECUM. CHAPTER I. INTRODUCTORY. 1. The General Method. The methods of modern microscopic anatomy may be roughly classed as General and Special. There is a General or Normal method, known as the method of sections, which consists in carefully fixing the structures to be examined, staining them with a nuclear stain, dehydrating with alcohol, and mounting series of sections of the structures in balsam. It is by this method that the work is blocked-out and very often finished. Special points are then studied, if necessary, by Special Methods, such as examination of the living tissue elements, in situ, or in " indifferent " media ; fixation with special fixing agents ; staining with special stains; dissociation by teasing or maceration; injection; impregnation ; and the like. There is a further distinction which may be made, and which may help to simplify matters. The processes of the preparation of tissues may be divided into two stages, Pre- liminary Preparation and Ulterior Preparation. Now the pro- cesses of preliminary preparation are essentially identical in all the methods ; essential divergencies being only found in the details of ulterior preparation. By preliminary prepara- tion is meant that group of processes called by German anatomists ConservirungsmetJtoden, those namely whose object it is to get the tissues into a fit state for passing unharmed through all the ulterior processes to which it may be desired to submit them. Preliminary preparation comprehends the operations of (1) killing; (2) fixing; (3) the washing and other manipulations necessary for removing the fixing agent 1 CHAPTER I. from the tissues, and substituting for it the preservative liquid or other reagents which it is desired to employ. 2. Preliminary Preparation. The first thing to be done with any structure is to fix its histological elements. (This state- ment applies equally to all classes of objects, whether it be desired to cut them into sections or to treat them in any other special way.) Two things are implied by the word " fixing ; " first, the rapid killing of the element, so that it may not have time to change the form it had during life, but is fixed in death in the attitude it normally had during life ; and second, the hardening of it to such a degree as may enable it to resist without further change of form the action of the reagents with which it may subsequently be treated. Too much stress can hardly be laid on this point, which is the most distinctive feature of modern histological practice ; without good fixation it is impossible to get good stains, or good sections, or prepa- rations good in any way. The structure having been duly fixed by one of the pro- cesses described in the chapter on FIXING AGENTS, is washed in order to remove from the tissues as far as possible all traces of the fixing reagent. The kind of liquid with which washing out is done is not a matter of in- difference. If corrosive sublimate (for instance) or osmic acid, or a solution into which chromic acid or a chromate enters, have been used for fixing, the washing may be done with water. But if picric acid in any form has been used, the washing must be done with alcohol. The reason of this difference is that the first-named reagents (and, indeed, all the compounds of the heavy metals used for fixing) appear to enter into a state of chemical combination with the elements of tissues, rendering them insoluble in water ; so that the hardening induced by these agents is not removed by subsequent treatment with water. Picric acid, on the other hand, produces only a very slight hardening of the tissues, and does not "appear to enter into any combination whatever with their elements, as it is entirely removeable by treating the tissues with water or alcohol. If the removal be effected by means of water, the tissue elements are left in a soft state in which they are obnoxious to all the hurtful effects of water. Alcohol must therefore be taken to remove the picric acid and to effect the necessary hardening at the same time. Instruc- tions for washing out are given, when necessary, in the discussion of the different fixing agents in the following parts of this work. These operations having been duly performed, two roads become open. The object may be further prepared by what may be termed the ivet method, in which all subsequent INTRODUCTORY. 3 operations are performed by means of aqueous media. Or it may be further prepared by what may be termed the Dehydra- tion method, which consists in treatment with successive alcohols of gradually increasing strength, final Dehydration with absolute alcohol, imbibition with an essential oil or other elf arintj agent, and lastly either mounting at once in balsam or other resinous medium or imbedding in paraffin for the purpose of making sections. The dehydration method is the course which is generally preferred. The chief reason for this lies in the great superiority of the dehydration methods, as regards the preservation of tissues. The presence of water is the most important factor in the conditions that bring about the decomposition of organic matter, and its complete removal is the chief condition of permanent preservation. It is of course not intended here to suggest that wet methods of pre- paration should be altogether discarded. They have great value, they are even indispensable, for special ends ; and all that is intended to be suggested is, that they should be re- garded not as general but as special methods. The further course of preliminary preparation by the de- hydration method is as follows : 3. Dehydration and Preservation. At the same time that the superfluous fixing agent is being removed from the tissues, or as soon as that is done, the water of the tissues must be removed. This is necessary for two reasons ; firstly, in the interest of preservation, the presence of water being the condition of all others that most favours post-mortem decomposition ; and secondly, because all water must be removed in order to allow the tissues to be impregnated with the imbedding material necessary for section- cutting, or with the balsam with which they are to be finally preserved. (The cases in which aqueous imbedding and preserving media are employed are exceptional, and will be treated of in the proper places.) The dehydration is performed as follows : The objects are brought into weak alcohol, and are then passed through successive alcohols of gradually increased strength, remaining in each the time necessary for complete saturation, and the last bath consist- ing of absolute or at least very strong alcohol. In dealing with extremely delicate objects, it may be necessary to take special precautions in order to avoid injury to them through the violent 4 CHAPTER I. diffusion-currents that are set up in the passage from water to alcohol, or from one bath of alcohol to another of considerably different density. Some kind of diffusion-apparatus should be used in these cases. The objects may be placed in a tube plugged at one end and closed at the other by a diaphragm of chamois skin or other suitable membrane, the tube being then immersed in a vessel containing the grade of alcohol that it is desired to add to the liquid in the tube, and the whole allowed to remain until by diffusion through the diaphragm the two liquids have become of equal density. Or COBB'S differentiator (Proc. Linn. Soc., N.S.W., v, 1890, p. 157 ; Journ. Boy. Mic. Soc., 1890, p. 821) may be employed. Or, more conveniently in most cases, the apparatus described and figured by HASWELL (Proc. Linn. Soc., N.S.W., vi, 1891, p. 433 ; Journ. Roy. Mic. Soc., 1892, p. 696). This consists of two wash-bottles connected in the usual way by tubing, and furnished, the one with an overflow-tube, and the other with a feeding-tube leading from a reservoir connected with it by means of a regulating tap or drop arrangement. The objects are placed in the first bottle ; some of the same liquid as that containing the objects is placed in the second bottle ; and alcohol of the grade that it is desired to add is led into it from the reservoir. The mixture of liquids therefore takes place in the bottle that does not contain the objects, and the mixture itself is gradually led over to the objects through the siphon-tube connecting the two bottles. Another apparatus for rapid dehydration, devised by CHEATLE, will be found described in Journ. Pathol. and Bacteriol., i, 1892, p. 253, or Journ. Roy. Mic. Soc., 1892, p. 892. I would here call attention to the varied usefulness of the " Siebdosen " or sieve-dishes of STEINACH, ZIMMEEMANN, and SUCHANNEK (vide Zeit.f. wiss. Mik., iv, 4, 1887, p. 433, and vii, 2, 1890, p. 159). They consist of a covered glass capsule into which is fitted a "sieve " made of a watch-glass pierced with holes and supported on legs. It is evident that the arrange- ment is very handy, not only for staining, washing out, treatment with vapours, &c., but for any operation in which it is desirable to have speci- mens supported in the upper layers of a quantity of reagent. They are sent out in a very neat form by Dr. Griibler. FAIECHILD'S perforated porcelain cylinders for washing seem to be a very neat idea. These are made small enough to be floated by the cork that closes them. See the descriptive paper in Zeit.f. wiss. Mik., xii, 3, 1896, p. 301. It is sometimes stated that it is necessary that the last alcohol bath should consist of absolute alcohol. This however is incorrect, a strength of 90 per cent., or at all events 95 per cent., being sufficient in almost all cases. For the small amount of water that remains in the tissues after treatment with these grades of alcohol is efficiently removed in the bath of clearing agent, if a good clearing agent be employed. Oil of cedar will remove the remaining water from tissues saturated with 95 per cent, alcohol ; oil of bergamot will " clear " from 90 per cent, alcohol, and anilin oil will clear from 70 per cent, alcohol. It is not generally necessary that pure ethylic alcohol be employed for dehydration ; methylated spirit will suffice in most cases. I am not aware of any substance that can entirely take the place of alcohol for dehydration and preservation. Acetone, and inethylal, have been INTRODUCTORY. 5 lately i PARKER, Zool Anz., 403, 1892, p. 376) substituted for alcohol in the dehyhvation of methvlen-blue preparations ; but the great boon of an efficient substitute for alcohol in general work remains yet to be discovered. For- maldehyde (see under Fixing and Hardening Agents) is now known to be a most admirable medium for the preservation of museum specimens, being for that purpose in many cases greatly superior to alcohol ; but experience is wanting as to how far it is available for the preservation of histological material, whilst of course, occurring as it does in the form of an aqueous solution, it can have no dehydrating effect. Considered as a mere dehydrating agent, alcohol fulfils its functions fairly well. But considered as a histological preservative agent, it is far less satisfactory. If tissues be left in alcohol for only a few days before sub- jecting them to the further stages of preparation, the injurious effects of a sojourn in alcohol will perhaps not be very disagreeably evident. But it is otherwise if, as must often be the case, they are put away for many weeks or months before the final preparation can be carried out. The dehydrating action of the alcohol being continuously prolonged, the minute structure of tissues is sometimes considerably altered by it ; they become overhard and shrink, and become brittle, and their capacity for taking stains well becomes seriously diminished. KULTSCHITZKY (Zeit. f. wiss. Mik., iv, 3, 1887, p. 349) has proposed to remedy this by putting up objects, after fixation and washing out with alcohol, in ether, xylol, or toluol. FLEMMING has lately (Arcli.f. mik. Anat., xxxvii, 1891, p. 685) advised putting up objects after fixation in a mixture of alcohol, glycerin, and water, in about equal parts, pointing out that objects thus preserved may be at any moment either pre- pared for sectioning by treatment with pure alcohol, or softened for dissec- tion or teasing by a little soaking in water, and that they do not become so hard and brittle as alcohol specimens, and retain their staining power much better. After extensive experience of this plan, I can highly recommend it, and would only further suggest that the action of the liquid seems to me to be in many cases much improved by addition of a little acetic acid (say 0'5 to 0'75 per cent.). For material that is intended only for section-cutting, I find that by far the best plan is to clear and imbed at once in paraffin. This affords, as far as I can see, an absolutely perfect preservation. I have been working lately on some material that has been preserved in this way for over seven years. The preservation of the tissues, down to the finest details of cell-structure, appears to be perfect, and the stain- ing, though perhaps somewhat slowed, is as precise as when the specimens were first put up, or more so. The only notice- able defect is that the tissues are rather brittle, and do not cut well ; but it is not certain that that is not owing to their having been over-hardened in the first instance. Cedar- wood oil is, I find, nearly, if not quite, as good as paraffin. 6 CHAPTER I. 4. Ulterior Preparation. Clearing. The water having been thus sufficiently removed, the dehydration method proceeds as follows : The alcohol is in its turn removed from the tissues, and its place taken by some anhydrous substance, generally an essential oil, which is miscible with the material used for imbedding. This operation is known as Clearing. It is very important that the passage from the last alcohol to the clearing agent be made gradual. This is effected by placing the clearing medium under the alcohol. A sufficient quantity of alcohol is placed in a tube (a watch-glass will do, but tubes are generally better), and then with a pipette a sufficient quantity of clearing medium is introduced at the bottom of the alcohol. Or you may first put the clearing medium into the tube, and then carefully pour the alcohol on to the top of it. The two fluids mingle but slowly. The objects to be cleared being now quietly put into the super- natant alcohol, float at the surface of separation of the two fluids, the exchange of fluids takes place gradually, and the objects slowly sink down into the lower layer. When they have sunk to the bottom, the alcohol may be drawn off with a pipette, and the objects will be found to be completely penetrated by the clearing medium. (It may be rioted here that this method of making the passage from one fluid to another applies to all cases in which objects have to be trans- ferred from a lighter to a denser fluid for instance, from alcohol, or from water to glycerine. It is a more exact method than that of successive baths of mixture of alcohol and clear- ing agent.) It should also be noted here that this is the proper stage for carrying out minute dissections, if any such have to be done, a drop of clearing medium agent being a most helpful medium for carrying out such dissections in, as will be further explained later on ( 8). 5. Imbedding, and Treatment of Sections. The objects are now imbedded. They are removed from the clearing medium, and soaked until thoroughly saturated in the imbedding medium. This is, for small objects, generally paraffin, liquefied by heat, and for large objects generally a solution of collodion or "cello'idin" (in this latter case the clearing is omitted, the tissues beins- imbedded direct from the INTRODUCTORY. 7 alcohol). The imbedding medium containing the object is then made to' solidify, as described in the chapter on im- bedding processes, and sections are made with a microtome through the imbedding mass and the included objects. The sections are then mounted on a slide by one of the methods described in the chapter on Serial Section Methods, the imbedding material is removed from them (in the case of paraffin), they are stained in situ 011 the slide, dehydrated \vith alcohol, cleared and mounted in balsam or damar. Or they may be stained, washed, dehydrated, and cleared in watch-glasses, and afterwards mounted as desired the im- bedding medium being first removed if desirable. It is not always desirable to remove the imbedding mass ; celloidin sec- tions stain well without being freed from it, and are usually even dehydrated, cleared, and mounted without removal of the mass, which becomes quite transparent in balsam. This plan has the advantage, which is a very im- portant one for large sections, of allowing the sections to remain during the whole of the manipulations protected by a supporting mass that holds all their parts together. The plan of staining sections on the side is of somewhat recent introduc- tion ; before it had been worked out the practice was to stain structures in toto, before cutting sections. In this case the object, after having been fixed, and washed out, is taken from the water, or while still on its way through the lower alcohols (it should not be allowed to proceed to the higher grades of alcohol before staining, if that can be avoided), and passed through a bath of stain (generally alcoholic borax-carmine or other alcoholic stain) of sufficient duration, then dehydrated with successive alcohols, passed through a clearing medium into paraffin, cut, and treated as above described, the sections m this case being mounted direct from the turpentine, naphtha, or other solvent with which the paraffin is removed. If aqueous staining media be applied (and it is sometimes very desirable for particular pur- poses to prepare specimens with some aqueous stain) the structures should either be stained in toto immediately after fixing and washing out, or sections may be stained on the slide, the objects being passed through suc- cessive baths of alcohol of gradually decreasing strength before being put into the aqueous stain (a precaution which will not be necessary for chromic objects (see below, 9) ). But it is generally advisable not to stain in bulk material that is intended to be sectioned ; much time is saved by staining it as sections, with the further advantage that the staining can be much better controlled, and that many excellent stains can in this way be employed that are not available for staining in bulk. 6. Resume of the Section-Method. It was stated in the first edition of this work that " the great majority of preparations are made by fixing either with sublimate or a picric acid com- 8 CHAPTER I. bination, washing out with alcohol, staining with alcoholic borax-carmine, imbedding in chloroform paraffin, cutting with a sliding microtome, and mounting the sections in series in Canada balsam/' But histological practice has greatly changed since then, and I would now suggest the following : Fix either in sublimate, or preferably in many cases in Flemming's chromo-aceto-osmic mixture, or one of the other fixing agents recommended in later chapters ; wash out ; dehydrate; clear with oil of cedar-wood; imbed in paraffin ; mount sections on the slide with Mayer's albumen medium ; stain as desired ; and mount in balsam or damar. That, or something like that, is now the practice of many of the most advanced workers ; and I know of no method that seems to have equal claims to be considered a classical method of general morphological investigation. As regards the method to be used for imbedding, I take it that the paraffin method is the method par excellence for small objects (objects up to 5 or 7 millimetres diameter); whilst the collodion or " cello'idin " method is the method par excellence for large objects. As above said, I consider the practice of staining section material in bulk to be superannuated. 7. Preparation of Entire Objects, or of Material that is not to be Sectioned. The treatment of objects which can be studied without being cut into sections is identical with that above described, with the omission of those passages that relate to imbedding processes. Its normal course may be described as fixation, washing out, staining, treatment with successive alcohols of gradually increasing strength, final dehydration with absolute alcohol, clearing, and mounting in balsam. This method is usually preferred, as a general method, to the wet methods, for the reasons that have been given above ( 2), and for some others, amongst which may be noted the greater transparency given to tissues by mounting them in media of high refractive index, such as balsam. In the preparation of entire objects or structures that are intact and covered by an integument not easily permeable by liquids, special care must be taken to avoid swelling from endosmosis on the passage of the objects from any of the liquids employed to a liquid of less density, or shrinkage from exosmosis on the passage to a liquid of greater density. This applies most specially to the passage from the last alcohol INTRODUCTORY. 9 into the clearing medium. A slit should be made in the integument, if possible, so that the two fluids may mingle without hindrance. And in all cases the passage is made gradual by placing the clearing medium under the alcohol, as above described. Fluids of high diffusibility should be employed as far as possible in all the processes. Fixing agents of great penetrating power (such as picrosulphuric acid or alcoholic sublimate solution) should be employed where the objects present a not easily permeable integument. Washing out is done with successive alcohols, water being used only in the case of fixation by osmic acid, or the chromic mixtures or other fixing solutions that render washing by water imperative. Staining is done by preference with alcoholic staining media. The stains most to be recommended are Grenadier's borax-carmine, or one of Mayer's new carminic acid or haematein stains (for all of which see STAINING AGENTS). Anilin stains are rarely applicable to this class of preparations. Aqueous stains are more seldom used, though there are many cases in which they are admissible, and some in which they are preferable. 8. Minute Dissections. These are best done, if in a drop of clearing agent. I recommend cedar-wood oHjor.. this purpose, as it gives to the tissues a consistency favorable for dissection, whilst its viscosity serves to lend support to delicate structures. Clove oil has a tendency to make tissues that have lain in it for some time very brittle. The brittleness is, however, sometimes very helpful in minute dissections. Another property of clove oil is that it does not easily spread itself over the surface of a slide, but has a tendency to form very convex drops. This property also makes it frequently a very convenient medium for making minute dissections in. 9. General Principles. Following Paul Mayer, I gave in the previous editions the following reasons for employing for entire objects alcoholic rather than aqueous staining media. Since, in most cases, treatment with alcohol forms part of the fixing process, alcoholic solutions are logically indicated for staining. For by means of them it is possible to avoid the bad effects that follow on passing delicate tissues from alcohol into water, violent diffusive currents being thereby set up which sometimes carry away whole groups of cells; swellings being caused in the elements of the tissues; and, if the immersion in the aqueous medium be prolonged, as is generally 10 CHAPTER I. necessary in order to obtain a thorough stain, maceration of the tissues supervening. But alcoholic staining fluids have still other advantages ; they are in general more penetrating ; with them alone is it possible to stain through chitinous integuments ; and, if it be desired to stain slowly, tissues may be left in them for days without hurt. Applied to the case now under consideration, the preparation in toto of objects protected by not easily permeable investments, this doctrine is evi- dently a wise one. For such objects must necessarily be fixed by some highly- penetrating but not permanently hardening agent such as picric acid, and must necessarily be washed out with alcohol ; and it is a good maxim for tissues so fixed that an object that has once been in alcohol should not be allowed to go back into water, if that can possibly be avoided. But in the case of structures that have been well fixed in a strongly and permanently coagulating medium such as chromic acid, this precaution is much less necessary. Sections of tissues that have been fixed for twenty- four hours in Fleniming's solution may be passed with relative impunity from absolute alcohol into an aqueous stain, and from that back again direct into absolute alcohol. It is this property of tissues fixed in chromic solution that determines me to recommend the practice of staining sections, instead of staining objects in toto. As regards the quality of the stain, aqueous solutions are in general the best. No alcoholic carmine or hsematoxylin, for instance, will give a stain equal in precision and delicacy to those given by alum-carmine or Bohmer's hematoxylin. In a recent publication Mayer himself states that he has somewhat cooled in his preference for alcoholic staining media. For an excellent exposition of the principles underlying the practice above recommended, the reader may consult with advantage the paper of Paul Mayer, in Mitth. Zool. Stat. Neapel, ii (1881), p. 1, et seq. See also the abstract in Journ. Roy. Mic. Soc. (N.S), ii (1882), pp. 866881, and that in Amer. Natural., xvi (1882), pp. 697706, in which two last some im- provements are mentioned which have been worked out since the publication of Mayer's paper. CHAPTER II. KILLING. 10. In the majority of cases, the first step in the prepara- tion of an organ or organism consists in exposing it as rapidly and as completely as possible to the action of one of the Fixing Agents that are discussed in the next chapter. The organ or organism is thus taken in the normal living state ; the fixing agent serves to bring about at the same time, and with sufficient 'rapidity, both the death of the organism and that of its histologies! elements. But this method is by no means applicable to all cases. There are many animals, especially such as are of a soft con- sistence, and deprived of any rigid skeleton, but possessing a considerable faculty of contractility such as many Coelen- terata, Bryozoa, and Serpulida, for instance which if thus treated contract violently, draw in their tentacles or branchiae, and die in a state of contraction that renders the pre?erved object a mere caricature of the living animal. In these cases, special methods of killing must be resorted to. Sudden Killing. 11. Heat. Speaking generally, there are two ways of dealing with these difficult cases. You may kill the animal so suddenly that it has not time to contract; or you may paralyse it by narcotics before killing it. The application of Heat is a good means of killing suddenly. It has the great advantage of allowing of good staining sub- sequently, and of hindering less than any other method the application of chemical tests to the tissues. By it the tissues are fixed at the same time that somatic death is brought about. The difficulty consists in hitting off the right temperature, 12 CHAPTER II. which is of course different for different objects. I think that a temperature of 80 to 90 C. will generally be amply suffi- cient, and that very frequently it will not be necessary to go beyond 60 C. An exposure to heat for a few seconds will generally suffice. Small objects (Protozoa, Hydroids, Bryozoa) may be brought into a drop of water in a watch-glass or on a slide and heated over the flame of a spirit lamp. For large objects, the water or other liquid employed as the vehicle of the heat may be heated beforehand and the animals thrown into it. As soon as it is supposed that the protoplasm of the tissues is coagulated throughout, the animals should be brought into alcohol (30 to 70 per cent. alcohol) (if water be employed as the heating agent). An excellent plan for preparing many marine animals is to kill them in hot fresh water. Some of the larger Nemertians are better preserved by this method than by any other with which I am acquainted. 12. Slowly Contracting Animals. Animals that contract but slowly, such as Alcyonium and Veretillum, and some Tunicates, such as Pyrosoma, are very well killed by throwing them into some very quickly acting fixing liquid, either used hot or cold. Glacial or very strong acetic acid (VAN BENEDEN'S method) is an excellent reagent for this purpose; it may be used, for example, with some Medusas. After an immersion of a few seconds or a few minutes, according to the size of the animals, they should be brought into alcohol of at least 50 per cent, strength. See " Acetic acid "and " Tunicata." Lemon juice employed in this way has given me very good results with small Annelids and Hirudinea. Corrosive sublimate is another excellent reagent for this purpose. Narcotisation. 13. The secret of narcotisation consists in adding some anesthetic substance very gradually, in very small doses, to the water containing the animals, and waiting patiently for it to take effect slowly. THE TOBACCO-SMOKE METHOD for Actiniae (HEKTWIG, Die Actinien, 1879) used to be practised as follows : A dish containing the animals in water is covered with a bell-glass, under which passes a curved glass or rubber tube, which dips into the water. Tobacco-smoke is blown into the water for some time, through the tube, and the animals are then left for some hours in order that narcotisation may become fully established. The animals are irritated from time to time by touching a tentacle with a needle. KILLING. 13 A- .mn as it is observed that an animal begins to react slowly, that is to say as soon as it is found that the contraction of the tentacle does not begin until a considerable time after it has been irritated by the needle, the nar- cotisation may be considered sufficient. A quantity of some fixing liquid sufficient to kill the animals before they have time to contract is then added to the water. A space of several hours is necessary in order to thoroughly narcotise an Actinia by this method. 14. Nicotin in solution may be used instead of tobacco smoke (ANDRES, Atti R. Accad. dei Lincei, v, 1880, p. 9; see Jonrn. //"//. Mic. Soc., N.S., ii, 1882, p. 881). Andres employs a solution of 1 gramme of nicotin in a litre of sea-water. The animal to be anaesthetised is placed in a jar containing half a litre of sea- water, and the solution of nicotin is gradually con- ducted into the jar by means of a thread acting as a syphon. The thread ought to be of such a thickness as to be capable of carrying over the whole of the solution of nicotin in twenty- four hours. 15. Chloroform may be employed either in the liquid state or in the state of vapour. KOROTNEFF (Mitth. Zool. Stat. X.'apel, v, Hft. 2, 1884, p. 229; Zeit. f. iciss. Mik., 2, 1885, p. 230) operates in the following manner with Siphonophora. The animals being extended, a watch-glass containing chloro- form is floated on the surface of the water in which they are contained, and the whole is covered with a bell-glass. As soon as the animals have become insensible they are killed by means of hot sublimate or chromic-acid solution plentifully poured on to them. Liquid chloroform is employed by squirting it in small quantities on to the surface of the water containing the animals. A syringe or pipette having a very small orifice, so as to thoroughly pulverise the chloroform, should be employed. Small quantities only should be projected at a time, and the dose should be repeated every five minutes, until the animals are anaesthetised. I have seen large Medusae very completely anaesthetised in the state of extension in an hour or two by this method. ANDRES finds that this plan does not succeed with Actiniae, as with them maceration of the tissues supervenes before anaes- thesia is established. 14 CHAPTER II. 16. Ether and Alcohol may be administered in the same way. ANDRES has obtained good results with Actiniae by the use of a mixture (invented by SALVATORE LO BLANCO) containing* 20 parts of glycerin, 40 parts of 70 per cent, alcohol, and 40 parts of sea- water. This mixture should be carefully poured on to the surface of the water containing the animals, and allowed to diffuse quietly through it. Several hours are sometimes necessary for this. EISIG employs alcohol in the same way. 17. Methyl-alcohol. CORI (Zeit. f. iviss. Mik., vi, 4, 1890, p. 438) prefers methyl-alcohol to all other reagents. It has, amongst other advantages, that of having but a slight action on albumins. CORI recommends a mixture composed of 10 c.c. methyl-alcohol (of 96 per cent, strength), 90 c.c. water (fresh or sea-water), and 0'6 g. of sodium chloride (to be added only when fresh water is taken, the addition of the salt having for its object to prevent maceration). It may be well to add to this mixture a very few drops of chloroform (for Cristatella ; Zeit.f. wiss. ZooL, Iv, 1893, p. 626; Zeit.f. wise. Mik., x, 4, 1893, p. 475). 18. Hydrate of Chloral, which was first recommended, I believe, by Foettinger (Arch, de BioL, vi, 1885, p. 115), gives very good results with some subjects. Foettinger operates by dropping crystals of chloral into the water containing the animals. For Alcyonella he takes 25 to 80 centigrammes of chloral for each hundred grammes of water. It takes about three quarters of an hour to render a colony sufficiently insensible to allow of fixing. Foettinger has obtained satis- factory results with marine and fresh-water Bryozoa, with Annelida, Mollusca, Nemertians, Actinia^ and with Aster acan- thion. He did not succeed with Hydroids. I am bound to state that I have never had the slightest success with Ne- mertians. VEEWOEN (Zeit.f. wiss. Zool., xlvi, 9, 1887, p. 99 ; see also Journ. Roy. Mic. Soc., 1888, p. 148) operates differently for fresh-water Bryozoa. He puts Cristatella for a few minutes into 10 per cent, solution of chloral, in which the animals sooner or later become extended. KiizENTHAL (Zeit. f. wiss. Mik., iv, 3, 1887, p. 378; Journ. Roy. Mic. Soc., 1888, p. 509) has obtained good results with some Annelids, by means of a solution of one part of chloral in 1000 parts of sea-water. 15 The chloral method gives rise to maceration with some sub- jects, and has been found to distort nuclear figures. (This, I suspect, will probably be found to be the case with most of these methods.) 19. Cocaine (RICHARD; Zool. Anz., 196, 1885, p. 332) has been found to give good results. Richards puts a colony of Bryozoa into a watch glass with 5 c.c. of water, and adds gradually 1 per cent, solution of hydrochlorate of cocaine in water. After five minutes, the animals are somewhat numbed, and half a cubic centimetre of the solution is added, and the tentacles are caused to contract by irritating them with a needle. Ten minutes later the animal should be found to be dead in a state of extension. This method is stated to succeed with Bryozoa, Hydra, and certain worms. It is the best method for Rotifers (ROUSSELET). It has been pointed out (bj GOBI, in the paper quoted above) that unfor- tunately when fixing agents, such as sublimate solution, are added to the animals, the cocaine is thrown down on them as a white precipitate. 20. Hydroxylamin. HOFER (Zeit. f. wiss. Nik., vii, 3, 1890, p. 318) has employed hydroxylamin as a paralysing agent with success with the most varied animal forms. Either the sulphate or the hydrochlorate of the base may be used. He recommends that the hydrochlorate be taken. This, as found in commerce, is usually contaminated with HC1. It should be dissolved in water (spring or sea-water, according to the habitat of the organisms in no case distilled water) and the solution exactly neutralised by addition of carbonate of soda. A 1 per cent, solution should be made up, and further diluted for use. The organisms are placed in the diluted solution, which may be taken of a strength vary ing from Ol per cent., used for thirty minutes or less (as for Infusoria), to 0'25 per cent., used for from fifteen minutes to one hour (Hydra), 1 per cent., one half to two hours (Hirudo), or as much as ten to twenty hours (Helix and Anodonta) . It should be remembered that hydroxylamin is an extremely powerful reducing agent. Care must therefore be taken not to treat the paralysed animals with easily reducible fixing agents, such as osmic acid, chromic acid, sublimate, chlorides 16 CHAPTER IF. of gold or platinum, &c., unless it have been possible first to sufficiently wash out the hydroxylamin with water. 21. Chloride of Magnesium. TULLBEEG (Arch. Zool. Exper. et Gen., x, 1892, p. 11 ; Journ. Roy. Mic. Soc., 1892, p. 435) has obtained some results with this salt. For Actiniae, a 33 per cent, solution of the salt is to be very slowly added to the water containing the expanded animal, until the vessel contains 1 per cent, of the salt (thus for one litre of sea-water 33 c.c. of the solution must be added). The addition must be made gradually ; but it must be effected within half an hour. Thirty minutes later the animal will be found to be anaesthetised, and may be fixed. For terrestrial and fresh-water Invertebrates, rather stronger solutions should be used. REDENBAUGH (Amer. Natural., xxix, 1895, p. 399; Journ. Roy. Mic. Soc., 1895, p. 385) has obtained good results by means of Sulphate of Magnesium, either added in crystals to the sea-water containing the animals, until a saturated solu- tion is obtained ; or in the shape of a saturated solution into which they are thrown (Annelids). 22. Poisoning by small doses of some fixing agent is sometimes a good method. SALVATOEE LO BLANCO employs the following method for pre- serving Ascidiae in an extended state. A 1 per cent, solution of chromic acid acidulated with acetic acid is poured on to the surface of the water contain- ing the animals, and allowed to diffuse slowly through it. The operation takes four or five days (v. GAKBINI, Manuale per la Technica Mod. del Microscopio, p. 168). Osmic acid, or Kleinenberg's solution, is sometimes employed in the same way. I have seen Medusae killed in a satisfactory manner by means of crystals of corrosive sublimate added to the water containing them. Morphia, Curare, Strychnin, Prussic Acid, and other paralysing drug have also been employed. 23. Asphyxiation may be sometimes successfully practised. Terrestrial Gastropods may be killed for dissection by putting them into a jar quite full of water that has been deprived of its air by boiling, and hermetically closed. After from twelve to twenty-four hours the animals are generally found dead and extended. The effect is obtained somewhat quicker if a little tobacco be added to the water. Good results are sometimes obtained with aquatic animals by simply leaving them to exhaust the oxygen of the water in which they are contained. I have sometimes succeeded with KILLING. 17 Holothuriae and other Echinoderms in this way ; and WARD (see Amer. Nat., xxv, 1891, p. 398) has succeeded with Hydroids, Actinias, and similar forms. If the animals be found to be imperfectly expanded when narcosis has set in, they may be got to expand by putting them back for a short time into pure sea-water ; and as soon as they are expanded should be quickly thrown into some rapidly-killing reagent. 24. Carbonic Acid Gas has been recommended (by FOL, ZooL Anz., 128, 1885, p t 698). The water containing the animals should be saturated with the gas. The method is stated to succeed with most Coelenterata and Echinodermata, but not with Molluscs or Fishes. It has not been found successful at the Naples Zoological Station. I have had most excellent results with small Annelids and Hirudinea. It is not neces- sary to employ a generator for obtaining the gas. It suffices to take an ordinary " soda-water " siphon, and squirt its con- tents into the water containing the animals. Narcotised animals recover very quickly on being put back into pure water. 25. Marine Animals are sometimes successfully killed by simply putting them into fresh water. Warm Water will sometimes serve to immobilise and even kill both marine and fresh-water organisms. CHAPTEE III. FIXING AND HARDENING. 26. The Necessity of Fixing. The meaning of the term " fixing" has been explained above (2). It only remains here to insist on the absolute necessity of the employment of fixing agents, and to briefly illustrate this necessity by a single example. If a portion of living retina be placed in aqueous humour, serum, or other so-called "indifferent" medium, or in any of the media used for permanent preserva- tion, it will be found that the rods and cones will not preserve the appearance they have during life for more than a very short time ; after a few minutes a series of changes begins to take place, by which the outer segments oi: both rods and cones become split into discs, and finally disintegrate so as to be altogether unrecognisable, even if not totally destroyed. Further, in an equally short time the nerve -fibres become varicose, and appear to be thickly studded with spindle- shaped knots ; and other post-mortem changes rapidly occur. If, however, a fresh piece of retina be treated with a strong solution of osmic acid, the whole of the rods and cones will be found perfectly preserved after twenty-four hours' time, and the nerve-fibres will be found not to be varicose. After this preliminary hardening, portions of the retina may be treated with water (which would be ruinous to the structures of a fresh retina), they may even remain in water for days without harm ; they may be stained, acidified, hardened, imbedded, cut into sections, and mounted in either aqueous or resinous media without suffering. 27. The Action of Fixing Agents consists in the coagulation of certain of the constituents of tissues, of their albuminoids, their gelatin, their mucin. Some fixing agents seem to have further the property of combining chemically with the tissues, FIXING AND HARDENING. 19 so that they cannot be easily removed from them by washing. This is a consideration of great importance in view of ulterior operations, and most particularly in view of staining. Chromic acid and its salts, osmic acid, the chlorides of palladium, of gold, and of iron, are reagents that seem to combine chemi- cally with the tissues, and render necessary a special after- nvatment and special modes of staining, whilst picric acid, nitric acid, and corrosive sublimate do not appear to enter into that kind of combination, and can be entirely removed from the tissues by washing, and leave the tissues in a state in which they are susceptible of any kind of staining. Practically it amounts to this, that if you fix with a chromic or osmic mixture, you cannot stain with carmine, but can only stain with haBmatoxylin, if you wish to stain your objects in toto ; or you may make sections, and stain them with safranin or some other coal-tar colour. Whilst if you fix with subli- mate or a picric acid mixture, you may do as you like in the matter of staining. The after-treatment appropriate to each fixing agent is indicated in the special paragraphs. 28. Choice of a Fixing Agent. Indications concerning the proper fixing agent to employ for the different tissues and organs of the animal kingdom will be found in Part II. The following remarks are intended as hints for beginners only. The chief fixing agents for general work are FLEMMING'S Hii.t'ture, HERMANN'S platinum-chloride mixture, corrosive sub- limate, and PERENYI'S mixture. I recommend that Flemmiiig's mixture should be used wherever it is possible, as 1 believe it to be in general by far the best fixing agent yet invented, with the exception of Hermann's mixture, which is unfortunately too expensive to be employed in large quantities. But it will not always be found possible to use it. Its low power of penetration, for instance, puts it out of court in the case of very impermeable objects, such as are frequently offered by the Arthropoda. For these, picro-sulphuric acid may be recommended. For very small objects, such as may be mounted whole, osmic acid is nearly as good as Flemming's mixture, and is frequently much more convenient to use. 20 CHAPTER TIT. For objects a little larger; -and for much embryological work on objects that it is convenient to have stained in toto, corrosive sublimate may be recommended. I think it is in general a much better preservative of the forms of anatomical elements than either Altmann's nitric acid or Kleinenberg's fluid. 29. The Practice of Fixation. Hints and Cautions. See that the structures are perfectly living at the instant of fixation,, otherwise you will only fix pathological states or post-mortem states. Do all you can to facilitate the rapid penetration of the fixing agent. To this end, let the structures be divided into the smallest portions that can conveniently be employed, and if entire organs or organisms are to be fixed whole, let openings, as large as possible, be first made in them. The penetration of reagents is greatly facilitated by heat. You may warm the reagent and put it with the objects to be fixed in the paraffin stove, or you may even employ a fixing agent heated to boiling point (as boiling sublimate solution for certain corals and Hydroids, or boiling absolute alcohol for certain Arthropods with very resistant integuments) . Let the quantity of fixing agent employed be at least many times the volume of the objects to be fixed. If this precaution be not observed the composition of the fixing liquid may be seriously altered by admixture of the liquids or of the soluble substances of the tissues thrown into it. For a weak and slowly acting fixing agent, such as picric acid, the quantity of liquid employed should be in volume about one hundred times that of the object to be fixed. Reagents that act very ener- getically, such as Flemming's solution, may be employed in smaller proportions. Be careful to use the appropriate liquid for washing out the fixing agent after fixation. It is frequently by no means a matter of indifference whether water or alcohol be employed for washing out. Sometimes water will undo the whole work of fixation (as with picric acid). Sometimes alcohol causes precipitates that may ruin the preparations. Instructions on this head are given in the paragraphs devoted to the different fixing agents. Use liberal quantities of liquid for washing. FIXING AND HAKI'KNIXG. 21 Change the liquid as often as it becomes turbid, if that should happen. The process of washing out is often greatly facilitated by Ji'-at. Picric acid, for instance, is nearly twice as soluble in alcohol warmed to 40 C. as in alcohol at the normal tempe- rature (Fol). 3t. Fixation of Marine Animals. In the case of marine organisms it may be stated as a general rule that their tissues are more refractory to the action of reagents than are the tissues of corresponding fresh-water or terrestrial forms, and fixing solutions should in consequence be stronger (about two to three times stronger, according to Laugerhans). Marine animals ought to be freed from the sea-water adhe- rent to their surface before treating them either with alcohol or any fixing reagent that precipitates the salts of sea-water. If this be not done, the precipitated salts will form on the surfaces of the organisms a crust that prevents the penetra- tion of reagents to the interior, thus allowing maceration to be set up, and hindering the penetration of staining fluids. Fixing solutions for marine organisms should therefore be such as serve to keep in a state of solution, and finally remove, the salts in question. They should never be made with sea- water as a menstruum, as some workers have inconsiderately proposed. If alcohol be employed it should be acidified with hydrochloric or some other appropriate acid. Picro-sulphuric acid is a reagent that fulfils the conditions here spoken of. (On this subject see Paul Mayer, in Mitth. Zool. Stat. Xeapel, ii (1881), p. 1 et seq. See also the abstract in Journ. Roy. Mic. Soc. (N.S.), ii (1882), pp. 866881, and that in Amer. Natural., xvi (1882), pp. 697706.) 31. The Obligation of Hardening. Methods of imbedding have now been brought to such a degree of perfection that the thorough hardening of soft tissues that was formerly neces- sary in order to cut thin sections from them is now, in the majority of cases, no longer necessary; by careful infiltration with paraffin or some other good infiltration mass, most soft objects can be satisfactorily cut with no greater an amount of previous hardening than is furnished by the usual passing of the tissues after fixing through successive alcohols in order to 22 CHAPTER III. prepare them for the paraffin bath. But there are some exceptions. Such are, for instance, the cases in which it is desired to cut very large sections, such as sections of the entire human brain. Such an organ as this cannot be duly infiltrated with alcohol in a few hours, and it is doubtful whether it can be duly infiltrated with paraffin or any other imbedding mass in any reasonable time. And certain organs that are either extremely delicate or inaccessible, such as retina or cochlea, will require to be specially hardened in order to give the best results. The processes employed for hardening such specimens as these will be described when treating of the organs in question. In this chapter I confine myself to such general statements concerning the employment of the usual hardening agents as appear likely to be generally useful. 32. The Practice of Hardening Hints and Cautions. Employ in general a relatively large volume of hardening liquid, and change it very frequently. The exact proportions may be made out by experiment for each reagent and each class of objects. If the volume of liquid be insufficient its composition will soon become seriously altered by the diffusion into it of the soluble substances of the tissues ; and the result may be a macerating instead of a hardening liquid. Further, as soon as, in consequence of this diffusion, the liquid has acquired a composition similar in respect of the proportions of colloids and crystalloids contained in it to that of the liquids of the tissues, osmotic equilibrium will become established, and dif- fusion will cease. That is to say, the hardening liquid will cease to penetrate. This means, of course, maceration of in- ternal parts. On the other hand, it appears that a certain slight proportion of colloids in the hardening liquid is favor- able to the desired reaction, as it gives a better consistency to the tissues by preventing them from becoming brittle. Hence the utility of employing a certain proportion of harden- ing agent. Hardening had better be done in tall cylindrical vessels, the objects being suspended by a thread at the top of the liquid. This has the advantage of allowing diffusion to take place as freely as possible, whilst any precipitates that may form fall harmlessly to the bottom. FIXING AND HARDENING. 23 Always begin hardening with a weak reagent, increasing the strength gradually, as fast as the tissues acquire a consist- ence that enables them to support a more energetic action of the reagent. Let the objects be removed from the hardening fluid as soon as they have acquired the desired consistency. As to the choice of a hardening reagent, if you wish, above all, for a rapid and energetic action, take chromic acid. If you wish for a more moderate and more equable action, take a chromic salt, or one of the compounds of which the chromic salts are the chief ingredients, or one of the platinum chloride mixtures. CHAPTER IV. FIXING AGENTS. MINERAL ACIDS AND THEIR SALTS. 33. Osmic Acid. The tetroxide of osmium (Os0 4 ) is the substance commonly known as osmic acid, though it does not possess acid properties. It is a substance that is exceedingly difficult to keep in use for any length of time. It is extremely volatile, and in the form of an aqueous solution becomes par- tially reduced with great readiness in presence of the slightest contaminating particle of organic matter. (It is generally believed that the aqueous solutions are reduced by light alone, but this is not the case : they may be exposed to the light with impunity if dust be absolutely denied access to them. It would even seem that the solutions are improved for some purposes by exposure to sunlight, vide infra, the remarks on gold chloride solutions. (Some observations communicated to me by Dr. Lindsay-Johnson go to prove that, if dust be avoided, solutions keep better in the light, with occasional "sunning," than in the dark.) Great stress is laid by authors on the fact that the vapour of osmium is very irritating to mucous tissues. It is said that the slightest exposure to it is sufficient to give rise to serious catarrh, irritation of the bronchial tubes, laryngeal catarrh, conjunctivitis, &c. I have never myself suffered in this way, but there is no doubt that many persons do, and such susceptible sub- jects should be very careful in handling osmium in any form. 34. How to keep the Solutions. After having carefully tried several of the plans that have been recommended for keeping the working solutions free from dust, I have come to the con- clusion that the following is the most practical plan for preventing them from " going bad " : The solution of osmic acid in chromic acid solution is not, like the solution in pure water, easily reducible, but may be kept without any special precautions. I therefore keep the bulk of my osmium in the shape of a 2 per cent, solution of osmic acid in 1 per cent. FIXING AGKNTS. MJNEKAL ACIDS AND THEIR SALTS. 25 aqueous chromic acid solution. This solution serves for fixation by osmium vapours, and for making up solution of Flemming (or solution of Hermann) which is the form in which osmium is most generally employed. A small quantity of osmic acid may also be made up in 1 per cent, solution in distilled water, and kept carefully protected from dust for use in special cases. Those who have to do a great deal of fixing by means of the vapours may also keep a supply of the solid oxide for this purpose. GRUBLER AND Co. now send out osmic acid in tubes con- taining one tenth of a gramme. CORI (Zeit. f. wiss. Mik., vi, 4, 1890, p. 442) finds that solutions in distilled water keep perfectly if there be added to them enough permanganate of potassium to give a very slight rosy tint to the liquid. From time to time, as the solution becomes colourless, further small quantities of the salt should be added, so as to keep up the rosy tint. 35. Regeneration of reduced Solutions. BRISTOL (Amer. Nat., xxvii, 1893, p. 175; Journ. Roy. Mic. Soc., 1893, p. 564; Ref. Handbook Ned. Sci., Supp., p. 442) says that reduced solutions may be regenerated by oxidising them by means of peroxide of hydrogen. The reaction is stated to be identical with that which takes place in the bleaching of osmium-blackened tissues by peroxide. It is admitted that the tetroxide of osmium, Os0 4 , is reduced by contact with organic matter into the deutoxide, Os0 2 . Then, OsO 3 + 2H 2 2 = Os0 4 + 2H 2 0. According to Bristol, for regenerating 100 c.c. of 1 per cent, solution of osmic acid (erratim 10 per cent in Journ. Roy. Mic. Soc.), ten to twenty drops of fresh peroxide solution should be added. KOLOSSOW (Zed. f. iciss. Mik., ix, 1, 1892, p. 40) says that half-reduced solutions, so long as they have not lost their characteristic odour, may be regenerated by the addition of a little powdered potash-alum. 36. Fixation by the Vapours. Osmic acid is frequently em- played in the form of vapour, and its employment in this form is indicated in most of the cases in which it is possible to expose the tissues directly to the action of the vapour. The tissues are pinned out on a cork which must fit well into a 26 CHAPTER IV. wide-mouthed bottle in which is contained a little solid osmic acid (or a small quantity of 1 per cent, solution will do). Very small objects, such as isolated cells, are simply placed on a slide, which is inverted over the mouth of the bottle. They remain there until they begin to turn brown (isolated cells will generally be found to be sufficiently fixed in thirty seconds, whilst in order to fix the deeper layers of relatively thick objects, such as retina, an exposure of several hours may be desirable). It is well to wash the objects with water before staining, but a very slight washing will suffice. For staining, methyl-green may be recommended for objects destined for study in an aqueous medium, and, for permanent preparations, alum-carmine, picro-carmine, or heematoxylin. In researches on nuclei, it is possible and. may be useful to employ the vapours of a freshly-prepared mixture of osmic and formic or acetic acid (Gilson, La Cellule, i, 1885, p. 96). The reasons for preferring the process of fixation by vapour of osmium, where practicable, are that osmium is more highly penetrating when em- ployed in this shape than when employed in solution, and produces a more equal fixation, and that the arduous washing out required by the solutions is here done away with. In many cases delicate structures are better pre- served, all possibility of deformation through osmosis being here eliminated. 37. Fixation by Solutions. When employed in aqueous solu- tions osmic acid is used in strengths varying from -fo per cent, to 2 per cent. Solutions of ^ per cent, to 1 per cent, have been very largely used, but the tendency of modern practice seems to be towards weaker solutions and longer immersion. For Infusoria | per cent, for a few seconds ; for Porifera -^ to yL per cent, for some hours; for Mollusca 1 to 2 per cent, for twenty-four hours ; for epithelia -J- to | per cent, for an hour or two ; for meroblastic ova y 1 ^- per cent, for twenty-four hours ; for medullated nerve-fibre -jL to 1 per cent, for from twenty minutes to two hours; for tactile corpuscles -J- to 1 per cent, for twenty-four hours; for retina i to 2 per cent, for from ten minutes to twenty-four hours ; for nuclei T V to 2 per cent, for two or three hours. Such figures as these will serve to give a general idea of the practice, whilst more precise in- structions will be given when dealing with the tissues in detail. (The durations here quoted appear to me exaggerated, except lor very voluminous specimens.) A little acetic or formic acid (0'5 to 1 per cent.) may fre- FIXIX'J AiiKXTS. MINERAL ACIDS AXI) TIIKIK SALTS. 27 (jiientlv with advantage be added to the solutions just before using. If solutions made with pure water be used, they must be kept protected from the liglit during the immersion of tissues. This precaution is not necessary if Flemining's or Hermann's solution be used. If the immersion is to be a long one the tissues must be placed with the solution in well-closed vessels, Miiium is ver volatile. The principle of combining osmium solutions with alcohol is due to HAN VIKIJ KT VIGNAL (RANYIER, Leg. d'Anat. Gen., " App. term, des muscles de la vie org.," p. 76 ; VIGNAL, Arch, de Physiol, 1884, p. 181). They take equal volumes of 1 per cent, osmic acid and 90 per cent, alcohol (freshly mixed). They wash out in 80 per cent, alcohol, then wash with water and stain for forty-eight hours in picro-carmine or haematoxylin. Viallanes has applied this method to the histology of insects. KOLOSSOW (Zeit.f. wiss. Mik., v, 1, 1888, p. 51) has recommended a 0'5 per cent, solution of osmium in 2 or 3 per cent, solution of nitrate or acetate of uranium, as having a greatly enhanced penetrating power. He has more lately (op. cit., ix, 1, 1892, p. 39) recommended for the same reason a mixture of 50 c.c. absolute alcohol, 50 c.c. distilled water, 2 c.c. concentrated nitric acid, and 1 to 2 g. osmium. This mixture is said to keep indefinitely in a cool place. MANX (Zeit.f. wiss. Mile., xi, 4, p. 481) recommends a freshly-prepared mixture of equal volumes of 1 per cent, osmic acid solution and saturated solution of corrosive sublimate in normal salt solution (for nerve centres). 38. After-treatment. The excess of osmic acid must be well washed out before proceeding to any further steps in prepara- tion ; water should be used for washing. Notwithstanding the greatest care in soaking, it frequently happens that some of the acid remains in the tissues, and causes them to over- blacken in time. To obviate this it is necessary to wash them out in ammonia-carmine or picro-carmine, or to soak them for twenty-four hours in a solution of bichromate of potash (Miiller's solution or Krlicki's will do), or in O5 per cent. solution of chromic acid, or in Merkel's solution, or in a weak solution of ferrocyanide of potassium or cyanide of potassium. The treatment with bichromate solutions has the great advan- tage of highly facilitating staining with carmine or haema- toxylin. Max Schultze recommended washing, and mounting permanently in acetate of potash ; but I believe the virtues attributed to this method are illusory. Fol has recommended treatment with a weak solution of carbonate of ammonia. 28 CHAFTEK IV. But the best plan of all is to properly bleach the preparations (see "Bleaching"). This is perhaps most conveniently done (as recommended by FOL, BRASS, and OVERTON) by means of peroxide of hydrogen, which regenerates the osmium to osinic acid. OVERTON (Zeit. /. wiss. Mik., vii, 1, 1890) finds that bleaching is completed in a few minutes in a mixture of 1 part commercial peroxide of hydrogen with 10 to 25 parts 70 per cent, alcohol. (The commercial peroxide, slightly acidulated with HC1, will keep well in the dark; but the mixture with alcohol must be made fresh for use.) CARAZZI'S peroxide of sodium may be found convenient for this purpose. BINET (Jour n. de V Anat. et de la PhysioL, xxx, 1894, p. 449) has successfully used permanganate of potash. The same stains recommended for objects fixed by vapour will be found useful here, with the addition of ammonia-carmine, which is really very use- ful for strongly fixed specimens. For sections, of course in both cases safranin and the other nuclear anilin stains may be employed with ad- vantage. 39. Characters of the Fixation with Osmic Acid. In general osmic acid, especially when used in the form of vapour, preserves the forms of cells more faithfully than any other reagent. But there are some drawbacks over and above those before mentioned ( 27). The penetrating power of the solu- tions is very low, so that if any but very small pieces of tissue be taken the outer layers become over-fixed before the action of the reagent has penetrated to the deeper layers. Over-fixed cells have a certain homogeneous, glassy or colloid look, owing to all their constituents having been raised by coagulation to so high an index of refraction that little or no detail is visible in them. They stain very badly, or not at all. Such cells are known as " osmicated cells, osmirte Zellen." There is no remedy for this state of things if once it has occurred. For this reason it is important to avoid using stronger solutions than is necessary. The danger of osmi- cation is lessened by using the osrnic acid in conjunction with certain other reagents, such as chromic acid. But it is not thereby entirely removed; FLEMMING'S mixture, especi- ally the strong formula, will readily osmicate superficial cells if care be not taken. For ordinary histological work osmica- tionof superficial layers is not of much consequence. But for cytological work care should be taken not to draw conclusions FIXING AiiKXTS. MINERAL ACIDS AND THEIK SALTS. 29* as to the structure of cells from osmicated specimens, and attention should be confined to cells four or five layers deeper down, which will generally be found to present the desired intensity of fixation. Osmic acid stains all fatty structures black ; it must therefore be avoided for tissues in which much fat is present ; or if not, the preparations must be subsequently very thoroughly bleached. 40. Chromic Acid. Chromic anhydride, CrO 3 , is found in commerce in the form of red crystals that dissolve readily in water, forming chromic acid, H 2 Cr0 4 . These crystals are very deliquescent, and it is therefore well to keep the acid in stock in the shape of a 1 per cent, solution. Care must be taken not to allow the crystals to be contaminated by organic matter, in the presence of which the anhydride is readily reduced into sesquioxide. Chromic acid is employed in solution either in water or in alcohol. The mosf useful strengths in which it is employed in aqueous solution are from Ol to TO per cent, for a period of immersion of a few hours (structure of cells and ova). For nerve-tissues weaker solutions are taken, -g^th to ^th per cent, for a few hours. Stronger solutions, such as 5 per cent., should only be allowed to act for a few seconds. The object should be washed out with water before passing into alcohol or staining fluids. Long washing in water is necessary to prepare them for staining, except an anilin stain be used. It is possible to wash out in alcohol, and this may be useful in special cases, but in general I think the practice is not to be recommended. It is well to wash for many hours in running water. Tissues that have been fixed in chromic acid are best stained in aqueous solutions, as water does not appear to have an injurious effect on them ; the acid appears to enter into some chemical combination with the elements of the tissues, forming with them a compound that is not affected either physically or chemically by water. The best stain to follow chromic acid is hasmatoxylin, or, for sections, some anilin stain. But the previous washing out with water must be very thorough if good results are to be insured ; it may take days. 30 CHAPTER IV. Chromic acid is not a very penetrating reagent, and for this reason, as well as for others, is seldom used pure, but plays an important part in the mixtures described below, of which the chief is certainly the mixture of Flemming. A chief objection to the use of chromic acid is that it precipitates certain of the liquid albuminoids of tissues in the form of filament* or net- ^vorks, which are often of great regularity, and simulate struc- tural elements of the tissues. This objection applies to all mixtures into which chromic acid enters. 41. Action of light on alcohol containing chromic objects. When objects that have been treated by chromic acid or a chromate are put into alcohol for hardening or preservation, it is found that after a short time a fine pre- cipitate is thrown down on the surface of the preparations, thus forming a certain obstacle to the further penetration of the alcohol. Previous washing by water does not prevent the formation of this precipitate, and changing the ajcohol does not prevent it from forming again and again. It has been found by Hans Virchow (Arch.f. mik. Anat., Bd. xxiv, 1885, p. 117) that the formation of this precipitate may be entirely prevented bv simply keeping the preparations in the dark. The alcohol becomes yellow as usual (and should be changed as often as this takes place), but no precipitate is formed. If this precaution be taken, previous washing with water may be omitted, or at all events greatly abridged. The brownish-green colour of chromic objects may be removed by treat- ing them with peroxide of hydrogen (Unna, in Arch. f. mik. Anat., Bd. xxx, 1887, p. 47 ; cf. Journ. Roy. Mic. Soc., 1887, p. 1060 ; and see the int structions for bleaching osmic acid preparations at the end of 39). 42. Chromic Acid and Spirit. A mixture of 2 parts of -i- per cent, chromic acid solution with one part of methylated spirit was much used by Klein in his investigations into the structure of cells and nuclei, and found to give better results than the ordinary reagents (including even osmic acid). HaBmatoxylin was used for staining. The addition of alcohol to augment the penetrating power of chromic acid seems to be a step in the right direction, and it is matter for surprise that such mixtures are not more used. The alcohol should be added to the acid in aqueous solution, as if strong alcohol be added to crystals of chromic anhydride, a very violent reaction is set up. The mixture should be kept in the dark. 43. Chromo-acetic Acid (FLEMMING, Zellsbz. Kern u. Zelltli., p. 382). Chromic acid . 0'2 to 0*25 per cent. Acetic acid . . O'l per cent., in water. FIXING AGENTS. MINERAL ACIDS AND THEIlt SALTS. 31 FJemmiog finds this the best reagent for the study of the achromatic elements of karyokinesis. (Flemming wrote this in 1882, and 1 doubt whether it would now hold good.) Stain with htematoxylin (the preparations are not favorable for staining with safranin or other coal-tar colours). I can recommend as a good fixing and hardening mixture for Annelids in general, and probably for other forms, the following fluid due to EHLERS (I do not know whether it has been published elsewhere) : To 100 c.c. of chromic acid of 0'5 to 1 per cent, add from 1 to 5 drops of glacial acetic acid. The proportion of acetic acid indicated is sufficient to counter- act any tendency to shrinkage due to the chromic acid. 44. Chromo-formic Acid (RABL, Morph. Jahrb., x, 1884, pp. 215, 216). Four or five drops of concentrated formic acid are added to 200 c.c. of 0'38 per cent, chromic acid solution. The mixture must be freshly prepared at the instant of using. Fix for twelve to twenty-four hours, wash out with water, harden in alcohol, stain with haematoxylin or safranin. For the study of karyokinesis. This is acknowledged to be one of the very best reagents for the purpose. 45. Chromo-osmic Acid (MAX FLESCH, Arch. f. mik. Anat., xvi, 1879, p. 300). This mixture (osmic acid O'lO, chromic acid 0'25, water lOO'O) origi- nally introduced for the preparation of the auditory organ of vertebrates, is of general application. It does not require to be kept in the dark. Objects may remain in it for twenty-four or thirty-six hours without risk of the osmic acid over-blackening them. Flemming found it to preserve nuclear figures well, but the preparations are pale, and difficult to stain well. He finds that the action of the mixture is improved (for nuclear figures) by the addition of acetic, formic, or other acid. This addition brings out the figures more sharply, and has the further advantage of allowing of a sharper stain with haematoxylin, picro-carmine, or gentian violet. He recommends the following formula, which may be considered to have superseded Max Flesch's. 46. Chromo-aceto- osmic Acid (FLEMMING, Zellsulstanz, Kern und Zelltheilung , 1882, p. 381) FIRST or WEAK formula. Chromic acid . . 0*25 per cent. A Osmic acid . . O'l per cent. [ In water. Glacial acetic acid . 0*1 per cent, j The best results (as regards faithfulness of fixation) are ob- tained with this mixture when it is allowed to act for only a short time (about half an hour). 32 CHAPTER IV. But it ma}', without inconvenience, be allowed to act for many hours or days, or according to some workers, even weeks or months. Wash out, very thoroughly, in water. Stain with haematoxylin, if you wish to stain in toto (staining in this way with other reagents is possible, but very difficult, and not be recommended). Stain sections with safranin, or other anilin, or with haematoxylin or Kernschwarz. To make up this mixture with the usual stock solutions, you take : Chromic acid of 1 per cent. . . 25 volumes. Osmic acid of 1 per cent. . . 10 Acetic acid of 1 per cent. . 10 ,, Water 55 If you keep your osmium in 2 per cent, solution in chromic acid of 1 per cent., as I have recommended, you will have to take only 20vols. of chromic acid, 5 of your osmium solution, and 65 of water. See also the remarks on the deterioration of these solutions by keeping, in the next . It has been already stated more than once that Flemming's solution is, with the exception of Hermann's solution, probably the very best fixing re- agent in general yet discovered. It has, however, been criticised. Faussek (Zeitschr.f. wiss. Zool, Bd. xlv, 1887, pp. 694, et seq.) found it totally in- applicable to the histology of the intestine of insects. He states that it caused the intima to disappear, and the cells to run together into a compact mass. Arnold (Arch. /. mik. Anat., Bd. xxx, 1887, p. 205) states that it does not preserve cell-bodies faithfully. And A. Kotlarewsky (Mitth. d. naturf. Ges. Bern., 1887 ; cf. Zeit.f. wiss. Mik., iv, 3, 1887, p. 387) found that it preserved the forms of nerve-cells (spinal ganglia) less faithfully than any of the reagents tried. I have not, myself, been struck by any defect in the preservation of cytoplasmic structures in my preparations made by this reagent. It is not necessary in all cases to observe the exact proportions of the in- gredients in this mixture. FOL (Lchrb. d. vergl. mik. Anat., 1884, p. 100) recommends the following : 1 per cent, chromic acid 25 vols. 1 per cent, osmic acid 2 2 per cent, acetic acid 5 ,, Water 68 That is to say, a mixture much weaker in osmium than Flemming's. In the Traite des Methodes Techniques, &c., Lee et Henneguy, 1887, I recom- mended this mixture, as giving better results in general, but am now inclined to think that, at all events as regards fidelity of fixation, it is a step in the wrong direction. Fol's formula has the advantage of allowing better staining with carmine, that is all. FIXING AGENTS. MINERAL ACIDS AND THEIR SALTS. 33 A mixture still weaker than this in osmium, viz. with 1 vol. osmium solu- tion instead of 2, has been recommended by GOBI (Zeit.f. wiss. Mik., vi, 1, 1890, p. 441), but I still adhere to the opinion above expressed. 47. Chromo-aceto-osmic Acid (FLEMMING, Zeit.f. wiss. Mik., 1, 1884, p. 349), SECOND or STRONG formula. 1 per cent, chromic acid . . . 15 parts. 2 per cent, osmic acid . 4 Glacial acetic acid . . . 1 If 2 per cent, osmium solution should not be at hand, you may conve- niently make the mixture by taking 10 per cent, chromic acid . . . .15 parts. atciot / 1 per cent, osmic acid 80 {&>,. Glacial acetic acid 10 \ Water 95 X^~ - _^_ If this mixture be kept in stock in large quantities, it may go bad, probably on account of the large proportion of organic acid contained in it. I therefore recommend that it be made up from time to time from stock solutions, in which the osmium is kept separate from the acetic acid. The propor- tions being as follows : CrO 3 0-15 Os 0-08 Acid, acet 1-00 Aq 19-00 You may make up and keep separately (A) 1 per cent, chromic acid . .11 parts. Distilled water . . . 4 Glacial acetic acid . . . 1 and (Bj a 2 per cent, solution of osmium in 1 per cent, chromic acid solution, and when required, mix four parts of A with one of B ; or, of course, if you prefer it, you may keep the osmic and chromic acid ready mixed in the propor- tions given, and add 5 per cent, of acetic acid at the moment of using. In any case, it is better not to make up very large quan- tities of the mixture at once, as osmium being very volatile it will be found that solutions that have been long in use no longer contain the proper proportion of that ingredient, and the hardening action being thus weakened the swelling action of the acetic acid will be insufficiently controlled. 3 34 CHAPTER IV. I am convinced that for some purposes there is advantage in diminishing notably the proportion of acetic acid. Merk (DenJcsch. d. Math. Naturw. Cl d. K. Acad. d. Wiss. Wien, 1887 ; cf. Zeit.f. wiss. Mik., v, 2, 1888, p. 237) proposes to make up separately (A) 2 per cent, chromic acid 7 '5 parts. Water . 3'5 Acetic acid 1 and (B), some 1 per cent, osmium solution, and to mix for use 12 parts of A with 8 of B. But this plan leaves you in the old difficulty of keeping your osmium in aqueous solution. It does not appear necessary to observe the exact proportions of the ingre- dients of these mixtures, a certain latitude is allowable. Thus CAENOY (La Cellule, 1, 2, 1885, p. 211) has employed a mixture one third stronger in osmium and twice as strong in chromic acid, viz. Chromic acid of 2 per cent, (or even stronger) 45 parts. Osmic acid of 2 per cent . . . . 16 Glacial acetic acid . . . . . 3 PODWYSSOZZI recommends (for glands especially) the following modifi- cation : 1 per cent. Cro. 3 dissolved in 0'5 per cent, solution of corrosive sublimate ....... 15 c.c. 2 per cent, osmium solution ..... 4 c.c. Glacial acetic acid 6 to 8 drops. The sublimate is said to augment the penetration of the osmium, but is unfavorable to staining. The proportion of acetic acid is reduced in order to avoid swelling of the tissue-elements (ZIEGLEE'S Beitrdge x.path.Anat., i, 1886 ; cf. Zeit.f. wiss. Mik., iii, 3, 1886, p. 405). The strong formula was recommended by FLEMMING in the first instance merely for a very special purpose, the hunting for karyokinetic figures, and not for general purposes. Further experience has shown that it is applicable 'to general purposes, and is in many cases considerably superior to the weak formula. But it is not suited to all objects, and FLEMMING has lately pointed out that some workers have used it for pur- poses for which it is not fitted. Arnold, in the place quoted in the last paragraph, says that it is to be avoided if you wish to demonstrate the structure of certain nuclei (of wandering cells) ; and the other objections there quoted as applying to the weak formula are intended to apply more or less to the present formula. It will be well not to attach too much importance to them. Let delicate struc- tures be fixed for twenty-four hours or more, washed in running water for an hour, and in successive alcohols for twenty-four hours, sectioned, and stained with safranin or gentian violet, and there will be little complaint of defective preservation. FIXING AGENTS. MINERAL ACIDS AND THEIR SALTS. 35 The strong mixture does not brown tissues more than the weak mixture, but rather less. Fat is blackened by these mixtures ; but the blackened fat can be entirely dissolved out of the tissues by treating them for a few hours with turpentine that has been exposed to sunlight for an hour or two (see Flemming in Zeit.f. iviss. Mik., vi, 1, 1889, p. 39 ; and vi, 2, 1889, p. 178). The remarks on over-fixation made in 39 apply to both these mixtures, and more especially to the stronger one. For staining see last . 48. Platino-aceto-osmic Acid (HERMANN'S solution). This extremely important reagent is historically a modification of Flemming's solution, platinum chloride being taken instead of chromic acid. See 65. 49. Nitric Acid (ALTMAXN, Arch. Anat. u. Phys., 1881, p. 219). Altmann employs dilute nitric acid, containing from 3 to 3^ per cent, pure acid. Such a solution has a sp. gr. of about 1'02 ; an araeometer may con- veniently be used to determine the concentration of the solution. Stronger solutions have been used, but do not give such good final results. After extensive trial I am convinced that ALTMANN'S solution is much too weak a reagent for general work, and should be discarded. His (ibid., 1877, p. 115) recommended a 10 per cent, solution. Flemming at one time employed solutions of 40 to 50 per cent, for the ova of inverte- brates. This of course has the advantage of a very rapid fixing action. Nitric acid has the valuable property of hardening yolk without making it brittle. But, for general purposes at all events, the pure nitric acid solu- tions may be considered to be superseded by Perenyi's chromo-nitric acid mixture (below, next ). 50. Chromo-nitric Acid (PERENYI'S formula, Zool. Anzeig., v, 1882, p. 459). 4 parts 10 per cent, nitric acid. 3 parts alcohol. 3 parts 0'5 per cent, chromic acid. These are mixed, and after a short time give a fine violet- coloured solution. The objects are immersed for four to five hours, and then passed through 70 per cent, alcohol (twenty-four hours), strong alcohol (some days), absolute alcohol (four to five days). They are then fit for cutting. The advantage of the process is, amongst others, that segmentation spheres and nuclei are 36 CHAPTER IV. perfectly fixed, the ova do not become porous, and cut like cartilage. Chromo-nitric acid is not only an embryological reagent, and a very important one, but also an admirable one for general work. I have found it altogether excellent for pre- serving marine organisms, especially large forms. Strong alcohol need only be used if the objects are destined to be sectioned. For a special formula for embryological purposes, see Part II, " Embryological Methods." 51. Picro-chromic Acid (Fol. Lehrb., p. 100). Picric acid, sol. sat. in water . . . .10 vols. 1 per cent, chromic acid solution . . . 25 Water 65 At the instant of using, you may add O'OOo of osmic acid, which makes the action more energetic. Wash with water (hot, nearly boiling water is best), and then with alcohol. Fol says, "This reagent hardens tissues admirably, without hindering staining in any way ; but it is not very pene- trating and fixes slowly." I find it gives very good results with Annelids. I have seen Fol's formula, with the addition of a trace of acetic acid, quoted as " liquid of Haensel " I know not with what justification. 52. Chromic Acid and Platinic Chloride (Merlcel's solution from Mitth. Zool. Stat. Neapel, 1881, p. 11). Equal volumes of 1'400 solution of chromic acid and T400 solution of platinic chloride (PtCl 4 ). Objects should remain in it for several hours or even days, as it does not harden very rapidly. After washing out with alcohol of 50 per cent, to 70 per cent., objects stain excellently, notwithstanding the admixture of chromic acid. This is a very delicate and admirable fixative. If objects that have been fixed by osmium be put into it for some hours, blackening is effectually prevented. Salts. 53. Bichromate and Cupric Sulphate Mixture (KULTSCHITZKY, Zeit.f. wiss. Mik., iv, 3, 1887, p. 348). A saturated solution of bichromate of potash and sulphate of copper in 50 per cent, alcohol, to which is added at the instant of using a little acetic acid, five or six drops per 100 c.c. To make the solution, add the finely powdered salts to the alcohol in- excess, and leave them together in total darkness, for twenty-four hours. Fix for twelve to twenty-four hours in the dark, otherwise the salts will be precipitated. Then treat with strong alcohol for twelve to twenty-four hours, and make sections. The rationale of this mixture is stated to be that it fixes tissues faithfully, I-'IXIXU ACJKNTS. MIXUKAL A<'II>S AXD TIIKIU SALTS. :!/ without causing the production of tlie delusive reticuhir precipitates of albuminoids which we have mentioned as beiriij produced by chromic acid that is the part played by the bichromate and sulphate ; and that it also tixes faithfully the chromatiu of nuclei that is the part played by the organic acid. 53a. For LINDSAY- JOHNSON'S Bichromo-osmic Mixture, \vliich may be used with excellent results as a gentle fixative, see 5 97. 54. The Chromates are useful as hardening rather than fixing agents. They have a very mild and even action on tissues, but are not at all pene- trating and act very slowly. They may still be found useful for fixing certain tissues, some of those of Mollusca for example. For mixtures that may be used for such a purpose, see the chapter on HARDENING AGENTS. 55. Cupric Sulphate. Not of general utility. See " Siphonophora." 56. Alum. Alum has been used for fixing purposes. After an extended experience of it, I only quote it in order to recommend that it be avoided at all costs. CHAPTER V. FIXING AGENTS. CHLORIDES, ORGANIC ACIDS, AND OTHEKS. Chlorides. 57. Bichloride of Mercury (Corrosive Sublimate). Corrosive sublimate is stated in the books to be soluble in about sixteen parts of cold and three of boiling water. It will probably be found that the aqueous solution contains about 5 per cent, of the sublimate at the temperature of the laboratory. It is more soluble in alcohol than in water, and still more so in ether. Its solubility in all these menstrua is augmented by the addition of hydrochloric acid, ammonious chloride, or camphor. With sodium chloride it forms a more easily soluble double salt; hence sea water may dissolve as much as 15 per cent., and hence the composition of the liquid of Lang. The simple aqueous solutions frequently deteriorate in even a short time through the formation of a pulverulent precipitate. The nature of this precipitate is unknown to me, and I have been unable to find any certain means of preventing its formation Thinking that it may be due in part to ammonia derived from the air, I have lately been in the habit of adding a little nitric acid to my solutions, and certainly have found that they thus keep much better. The addition of one drop of nitric acid for each cubic centimetre of sublimate solution lias already, I have since found, been re- commended by FRENZEL (see MANN, in Zeit. f. wiss. Milt., xi, 4, 180 1, p. 480). In any case, for work in which it is desired to obtain as energetic a fixing action as possible, it is well to use only freshly made up solutions. For fixing, corrosive sublimate may be, and very frequently is, used pure; but in most cases a finer fixation will be ob- tained if it be acidified with acetic acid, say about I percent, of the acid. I find that a saturated solution in 5 per cent. acetic acid is a very good formula for marine animals. VAN BKNEDEN has recommended a saturated solution in 25 per cent, acetic acid. It is sometimes advisable to take the most concentrated FIXING AGENTS. CHLORIDES, ORGANIC ACIDS, ETC. 39 solution obtainable. The cold saturated aqueous solution will suffice in most cases; but for some very contractile forms (coral polypes, Planaria), a concentrated solution in warm or even boiling water should be employed. For Arthropoda the alcoholic solution is frequently indicated. Delicate objects, however, may require treatment with weak solutions. HARTING found solutions of 0*2 to 0'5 per cent, suitable for blood-corpuscles, and PACINI'S fluids are much of the same strength. For these see the chapter on Examination Media. Objects should in all cases be removed from the fixing bath as soon as fixed, that is, in other words, as soon as they are seen to have become opaque throughout, which is practically as soon as they are penetrated by the liquid. Small objects are fixed in a few minutes. I have found that a " salivary " gland of the larva of Chironomus is thoroughly fixed in three seconds. \Vash out with water or with alcohol. I consider alcohol always almost preferable. Alcohol of about 70 per cent, may be taken. The extraction of the sublimate is hastened by the addition of a little camphor to the alcohol. Or, better, a little tincture of iodine may be added to the liquid, either alcohol or water, used for washing, and the liquid changed until it no longer becomes discoloured by the objects. It is important that the sublimate be thoroughly removed from the tissues, otherwise they become brittle. They will also become brittle if they are kept long in alcohol. It has been advised that solution of iodine in potassium iodide be taken instead of tincture of iodine for the washing out. But that is quite wrong, iodine in potassium iodide precipitates corrosive sublimate. I am much obliged to my friend Prof. GILSON for calling my attention to this point. It may happen that if the extraction of the excess of subli- mate from the tissues in bulk has been insufficient, crystals of sublimate may form in the sections after they have been mounted in balsam. This may easily be prevented by treating the sections themselves with tincture of iodine for a quarter of an hour before mounting. This will do away with the necessity of treating the tissues in bulk with iodine, which is frequeutly a very long process (unless it is desired to keep the material for a long time in alcohol before making the sections). 40 CHAPTER V. You may stain in any way you like. Carmine stains are peculiarly brilliant after sublimate (owing, it has been said, to the formation of mercuric carminate). It is not necessary that the objects be thoroughly washed out before staining; the staining processes themselves may be made to constitute a part of the washing-out process. It must be remembered that the solutions must not be touched with iron or steel, as these produce precipitates that may hurt the preparations. To manipulate the objects, wood, glass, or platinum may be used; for dissecting them, hedge- hog spines, or quill pens. When properly employed, sublimate is for general work un- doubtedly a fixing agent of the very highest order. It is applicable to most classes of objects. It is perhaps less applicable, in the pure form, to Arthropods, as it possesses no great power of penetrating chitin. For cytological work it is, according to my experience, not to be trusted, and not to be recommended. 58. Corrosive Sublimate (LANG'S formula, ' Zool Anzeiger,' 1878, i, p. 14). For Planaria. Take Distilled water . . . 100 parts by weight. Chloride of sodium . . 6 to 10 parts. Acetic acid . . . . 6 to 8 Bichloride of mercury . . 3 to 12 (Alum, in some cases . . ). Second formula (ibid., 1879, ii, p. 46). Make a concentrated solution of corrosive sublimate in picro-sulphuric acid, to which has been added 5 per cent, of acetic acid. 59. Other Simple Solutions. A solution containing 5 g. sublimate, 0'5 g. sodium chloride, and 100 c.c. water, has been quoted as " solution of GrAULE." REISER'S solution consists of 10 g. sublimate, 3 g. glacial acetic acid, and 300 g. distilled water (from Zeit. f. wiss. Mik. t xi, 3, p. 378). M. HEIDENHAIN has recommended a 0*5 per cent, solution of sodium chloride saturated while hot with sublimate. 60. GILSON'S Mercuro-nitric Mixture. I am indebted to Prof. GILSON for kindly sending the latest formula (1895), which is as follows : FIXING AGENTS. CHLORIDES, ORGANIC ACIDS, ETC. 41 Nitric Acid of 46 strength (this would be sp. gr. 1*456, or 80 per cent., nearly) .... 78 c.c. Glacial acetic acid . . . 22 Corrosive sublimate . . . 95 to 100 grms. GO per cent, alcohol . . . 500 c.c. Distilled water .... 4400 When required for 'marine animals add a few crystals of iodine, which will prevent the formation of precipitates of sea salts. If in any case the preparations should show a granular precipitate, due probably to an abundance of phosphates in the tissues, the precipitate may be removed by washing with water containing a little tincture of iodine (not iodide of potassium, which would precipitate the sublimate). I have tried this mixture and find that it affords in general a faithful and delicate fixation, and gives to tissues an excellent consistency. Objects may remain in it for a con- siderable time without hurt. Tissues are left in a state very favorable for staining. The liquid has a high degree of penetration. A treatment for a few days with it will serve to remove the albumen from the ova of Batrachians. This liquid may be recommended to beginners, as it is very easy to work with. For some objects, as I have found, the pro- portion of sublimate may be increased with advantage. 61. KABL'S Pier o- sublimate (Zeit. f. wiss. Mik., xi, 2, 1894, p. 165). Sublimate, saturated solution in water, 1 vol.; a similar solution of picric acid, 1 vol. ; distilled water, 2 vols. Embryos may be left in it for twelve hours, washed for two hours in water, and brought into weak alcohol. 62. MANN'S Picro- sublimate (op. dt., xi, 4, 1895, p. 480). ! per cent, of picric acid with or without 1 per cent, of tannin in a saturated solution of sublimate in normal salt solution. The same author's Alcoholic Picro-sublimate (Anat. Anz., 8, 1893, pp. 441443) consists of absolute alcohol 100 c.c., picric acid 4 grms., sublimate 15 grriis., tannin 6 to 8 grms. The tannin is added in order to prevent excessive hardening. The same author's Mercurio-osmic Mixture has been given ( 37). Vom RATH'S Picro-sublimate (Anat. Anz., xi, 9, 1895, p. 286). 42 CHAPTER V. Cold saturated solution of picric acid, 1 part; hot saturated solution of sublimate, 1 part ; glacial acetic acid, J to 1 per cent. Fix for several hours and bring direct into alcohol. The same author's Picro-sublimate-osmic Mixture (loc. cit.) consists of the above with the addition of 10 per cent, of 2 per cent, osmic acid solution. 63. ZENKER'S Mixture (Milncliener med. Wochenschr., 24, 1894, p. 534 ; quoted from MEECIEE, Zeit. f. wiss. Mik., xi, 4, 1894, p. 471, where will be found minute instructions for using it). Five per cent, of sublimate and 5 per cent, of glacial acetic acid, dissolved in solution of MULLER. Fix for several hours, wash out with water, treat the tissues in bulk, or the sections, with alcohol containing tincture of iodine. 63a. FOA'S Mixture (Quart. Journ. Mic. Sci., 1895, p. 287 ; Journ* Roy. Mic. Soc., 1895, p. 486). Equal parts of saturated solution of sublimate in normal salt solution, and of liquid of Miiller, or 5 per cent, solution of bichromate. The rationality of these mixtures is not apparent. The addition of the chrome salts is unfavorable to staining with carmine or hsematin. 64. Chloride of Platinum (Platinic Chloride, PtClJ. An extremely valuable reagent, originally introduced for the study of karyokinesis, but of general application. RABL, to whom we owe the introduction of this agent, employed an aqueous solution of 1'300. The objects remain in it for twenty-four hours, and are then washed with water, hardened in alcohol, and sectioned. Babl stained with Delafield's hasmatoxylin, or with safranin. The action of platinum chloride is similar to that of gold chloride, with the advantage that there is no blackening of the preparations. Rabl finds it give better results (for the study of karyokinesis) than any other reagent except chro- moformic acid ( 44). It causes a slight shrinkage of the chromatin elements, a condition that renders the granules of Pfitzner and the longitudinal division of the elements very distinctly visible (see Rabl's well-known paper in Morph. Jahrl., Bd. x, 1884, p. 216). Platinum chloride is an extremely deliquescent salt, and for this reason had better be procured in solution. Ten per cent, solutions are found in commerce. For MERKEL'S solution (chromo-platinic mixture) see ante, 52. KIXINC; .UJKNTS. Ml LORI 1>KS, OliCANK' ACIDS, KTC. 43 For RABL'S Platino-sublimate Mixture, see $mbryoloyieal Methods, 65. Platino-aceto-osmic Mixture (HERMANN, Arch. /. mik. Annt., xxxiv, 1889, p. 58). The author obtained excellent results bv substituting 1 per cent, platinic chloride for the chromic acid in Flemming's strong formula for chromo-aceto- osmic acid ( 47), the other ingredients either remaining as before, or the osmium being diminished one half. Thus, 1 per cent, platinic chloride 15 parts, glacial acetic acid 1 part, and 2 per cent, osmic acid either 4 parts or only 2 parts. Hermann found that protoplasmic structures are thus better preserved than with the chromic mixture. It was noted above ( 40) that a chief objection to the use of chromic acid is that it precipitates certain of the liquid albuminoids of tissues in the form of filaments or networks, which are often <>!' great regularity, and simulate structural elements of the tissues. This platinum chloride does not do. The after-treatment and staining should be the same as for objects treated with Flemming's solution. The remarks in 47, as to the deterioration of Flemming's solution by evaporation of osmium, apply with equal force to Hermann's mixture. After considerable experience of this reagent I find that it has the advantage of giving more colourless preparations, and, it may be in some cases, a more delicate fixation. But the fixation is certainly not in all cases superior, the hardening is less energetic, and in some cases not so energetic as may be desired. As this is the most expensive of all reagents, I am glad to iind that for prder to obtain the best results you should not employ portions of tissue of more than an inch cube. For a human spinal cord you should take two litres of solution, and change it for fresh after a few days. Six weeks or two months are necessary to complete the hardening. The solution should be taken weak at first, and the strength increased after a time. The objects should be removed from the solution as soon as they have acquired the desired con- sistency, as if left too long they will become brittle. (These HARDENING AGENTS. 57 precautions are peculiarly necessary in the case of chromic acid.) They may be preserved till wanted in alcohol (95 per cent.). It is well to wash them out in water for twenty -four or forty-eight hours before putting them into the alcohol. I think it is frequently useful to add a little glycerin to the hardening solution ; there is less brittleness and, I think, less shrinkage. The reader's attention is called to the statements made in 41 concerning the action of light on the alcohol containing chromic objects. Further directions for the employment of chromic acid will be given in the special paragraphs. Chromic acid is a most powerful and rapid hardening agent (by it, you may obtain in a few days a degree of hardening that you would hardly obtain it in as many weeks with bichromate, for instance.) It has the defect of a great tendency to cause brittleness. 89. Chromic Acid and Spirit (UBBAN PEITCHABD, Quart. Journ. Mic. Sci., 1873, p. 427). Chromic acid, 1 part ; water, 20 parts ; rectified spirit, 180 pails. Dissolve the chromic acid in the water first, and then add the spirit (violent action will ensue if the dry chromic acid be added directly to the spirit). The colour of the solution soon becomes brown. If, after a few days, it turns semi-gelatinous, it should be changed for fresh. From a week to ten days is required to harden such tissues as retina, cochlea, &c., for which this fluid is particularly well adapted. 90. Chromo-osmic Acid (M^x FLESCH.) Chromo-aceto-osmic Acid (FLEMMING.) Either of these mixtures may be used for prolonged hardening, and are admirable. The weak form of Flemming's solution is the one that should generally be taken for hardening purposes. (See 45 and 46.) For delicate objects perhaps even better results may be obtained by means of Chromic Acid and Platinic Chloride (MEEKEI/S Solution). See 52 ante. 91. Picro-chromic Acid. This fixative may be found useful for hardening objects that are only penetrable with difficulty, some Tunicata, for instance. See 51, ante. 92. Osmic Acid. Hardly used for anything but nervous tissue. See Neurological Methods in Part II. 93. Nitric Acid. Hardly used for anything but brain. 'See also Part II. 58 CHAPTER VI. Salts. 94. Bichromate of Potash. Perhaps the most important of all known hardening agents, sensu stricto. It hardens slowly, much more so than chromic acid, but it gives an incompar- ably better consistency to the tissues, and it has not the same tendency to make them brittle if the reaction be prolonged. They may remain almost indefinitely exposed to its action without much hurt. The strength of the solutions employed is from 2 to 5 per cent. As with chromic acid, it is extremely important to begin with weak solutions and proceed gradually to stronger ones. About three weeks will be necessary for hardening a sheep's eye in solutions gradually raised from 2 to 4 per cent. Spinal cord requires from three to six weeks ; a brain, at least as many months. After hardening, the objects should be well soaked out in water before being put into alcohol. They had better be kept in the dark when in alcohol (see above, 41 ). If you wish to have a good stain with carmine, especially ammonia-carmine, which is admirable for portions of nervous system so hardened, you should not put the objects into alcohol at all, even for a second, until they have been stained. You may stain either with carmine or hgematoxylin. Bichromate objects have an ugly yellow colour which cannot be removed by soaking in water. It is said that it can be removed by washing for a few minutes in a 1 per cent, solution of chloral hydrate. Gierke, however, says that this treatment is prejudicial to the preservation of the tissues. Prof. GILSON writes me that alcoholic solution of sulphurous anhydride (S0 2 ) is very convenient for the rapid decoloration of bichromate objects. A few drops suffice. See also 41, and " Bleaching." 95. MULLER'S Solution. Bichromate of potash . . . 2 2.J parts. Sulphate of soda ... 1 part. Water 100 parts. The duration of the reaction is about the same as with the simple solution of chromic salts. This fluid was very highly in vogue for many years, but seems -lately to be much less used. I fancy that the supe- riority of this mixture over the simple bichromate solution HARDENING AGENTS. 59 is not illusory, and is due to the formation in it of a trace of free chromic acid. Fol says that for mammalian embryos, for which it has been recommended, it is worthless. 96. ERLICKI'S Solution ( Warschauer med. Zeit.,xx.i\, Kos. 15 and 18). Bichromate of potash . . . 2'5 parts. Sulphate of copper . . . I'O part. Water lOO'O parts. Here the addition of the cupric sulphate is intelligible. This salt is itself a hardening agent of some energy, and may well serve to reinforce the somewhat slow action of the bi- chromate. As a matter of fact, " Erlicki " hardens very much more rapidly than either simple bichromate or Mutter's solu- tion. A spinal cord may be hardened in it in four days at the temperature of an incubator, and in ten days at the normal temperature (FoL, Lehrb. d. vergl. mik.Anat., p. 106). I believe it to be one of the best hardening agents known for voluminous objects. Human embryos of several months may be conveniently hardened in it. Nerve-centres that have been hardened in Eiiicki's fluid frequently con- tain dark spots with irregular prolongations, simulating ganglion-cells. These were at one time taken to be pathological formations, but they are now known to consist of precipitates formed by the action of the* hardening fluid. They may be removed by washing with hot water, or with water slightly acidified with hydrochloric acid, or by treating the specimens with 0*5 per cent, chromic acid before putting them into alcohol (TscnisCH, Virchow*8 Arch., Bd. xcvii, p. 173 ; EDINGEB, Zeit.f. wiss. Mik., ii, 2, p. 245 ; LOEWENTHAL, Rev. med. de la Suisse romande, 6me annee, i, p. 20). 97. Bichromate and Platinic Mixture (LINDSAY JOHNSON'S Mixture). Latest Formula, 1895, communicated by Dr. Lindsay Johnson. Bichromate of potash (2*5 per cent.) . 70 parts. Osmic acid (2 per cent.) . . . 10 Platinic chloride (1 per cent.) . . 15 Acetic or formic acid . . . . 5 It is not well to take the platinum chloride stronger than here given, as too strong solutions have a tendency to crys- tallize out on the tissues. HENNEGUY, who has worked a great deal with this reagent, and recommends it highly, says (Lemons sur la Cellule, Paris, Carre, 1896, p. 61) that it is well 60 CHAPTER VI. only to add the acetic or formic acid just before using, as it frequently provokes a spontaneous reduction of the osmium and platinum to such an extent that the mixture becomes quite black. This mixture was imagined for the preliminary hardening of retina, being allowed to act for two hours only, and then being followed by final hardening in pure bichromate solution. But it has proved applicable to other structures, and gives excellent results. The function of the osmic acid in the present formula is to enhance the hardening energy of the mixture. Dr. Lindsay Johnson writes me that " it greatly reduces the length of time necessary for hardening, three days being the time .from removal of the organ to its being in celloidin under dilute spirit." " If the osmium has a tendency to blacken, this may be entirely prevented and a beautiful delicate chestnut-brown deepening towards Bartholozzi-red tint obtained by adding ten parts of 5 per cent, solution of nitrate of uranium, which forms a layer of uranium on the top of the reduced platinum and osmium (one or both)." It has already been pointed out, No. 53A, that this mixture may be used for fixing, in some cases with the best results. HENNEGUY, I. c., says it contracts the more spongy sorts of protoplasm less than mixture of FLEMMING, and that it does not produce the delusive precipitates that that mixture fre- quently does produce. I am convinced that for some purposes (e. g. delicate researches on protoplasmic structures) it will be found well to diminish the proportion of organic acid. 98. Bichromates and Alcohol. Mixtures of either bichro- mate with alcohol may be employed, and have a more rapid action than the aqueous solution. Thus HAMILTON takes for hardening brain a mixture of 1 part methylated spirits with 3 parts of solution of Miiller (see the chapter on the Central Nervous System in Part II ; see also KULTSCHIZKY'S Mixture, ante, 53). Preparations should be kept in the dark during the process of hardening in these mixtures. 99. Bichromate of Ammonia. A review of the literature of the sub- ject shows that this salt is in considerable favour, for what precise motive is not apparent. Its action is very similar to that of the potassium salt. Fol says that it penetrates somewhat more rapidly, and hardens somewhat more HARDENING AGENTS. 61 slowly. It should be employed in somewhat stronger solutions, up to 5 per cent. 100. Neutral Chromate of Ammonia is preferred by some anatomists. It is used in the same strength as the bichromate. Klein has recommended it for intestine, which it hardens, in 5 per cent, solution, in twenty-four h GUI'S. Chlorides and others. 101. Platinic Chloride Mixture (MERKEI/S Solution). The formula of this admirable reagent has been given above, 52. It is an admirable hardening medium for delicate objects. Merkel states that he allowed from three to four days for the action of the fluid for the retina ; for Annelids Eisig employs an immersion of three to five hours, and transfers to 70 per cent, alcohol ; for small leeches Whitman finds " one hour sufficient, and transfers to 50 per cent, alcohol." Whitman recommends, for the hardening of pelagic fish ova, a stronger mixture (due, I believe, to Eisig), viz. : 0'25 per cent, solution of platinum chloride . 1 vol. 1 per cent, solution of chromic acid . . 1 The ova to remain in it one or two days (WHITMAN, Methods in Micro. Anat., p. 153). 102. Palladium Chloride (F. E. SCHULTZE, Arch. mik. Anat, iii, 1867, p. 477). This reagent was recommended by Schultze partly as giving to tissues a better consistency than chromic acid or Miiller's solution, and partly on account of a special faculty for penetrating organs rich in connective tissue that he attributes to it. It is an impregnation reagent, staining certain elements of tissues in various tones of brown. For the somewhat lengthy details of the manner of employing it, the reader is referred to the paper quoted. 103. Chloride of Zinc is only employed for brain, see post, Part II. 104. Picric Acid taken alone is a weak hardening agent, little used. It should be employed in saturated solution. But it is a useful ingredient in mixtures, serving to enhance the penetrating power. See Picro-chromic Acid, ante, 51 ; also Gage's Picric Alcohol, 81. 105. Acetate of Lead. Both the neutral acetate (sugar of lead) and the basic acetate have been used for hardening nerve tissues. ANNA KOTLA- EEWSKY found that nerve-cells hardened in 10 per cent, solution of sugar of lead were admirably preserved. See her " Inaug.-Diss." in Mitth. d. naturf. Ges. Bern., 1887, and Zeit.f. wise. Mik., iv, 3, 1887, p. 387. 62 CHAPTER VI. 106. Iodine may be used in combination with alcohol, and render service through its great penetrating power. See the method of BETZ, post, Part II. 107. Pyridin. Pyridin has been recommended as a hardening agent (by A. DE SOUZA). It is said to harden, dehydrate, and clear tissues at the same time. They may be stained after hardening by anilin dyes dissolved in the pyridin, or passed through water and stained by the usual processes. It is said to harden quickly, and to give particularly good results with brain. See Comptes Rendus hebd. de la Soc. de Biologic, 8 ser., t. iv, No. 35, p. 622 ; Zeit.f. wiss. Mik., v, i, 1888, p. 65 ; Journ. Roy. Hie. Soc., 1888, p. 1054. 108. Alcohol. When used alone, alcohol is inferior as a hardening agent to most of the reagents discussed above ; but when judicially employed to complete the action of a good fixing agent, it renders most valuable services. 90 to 95 per cent, is the most generally useful strength. Weaker alcohol, down to 70 per cent., is often indicated. Absolute alcohol is seldom advisable. You ought to begin with weak, and proceed gradually to stronger, alcohol. Large quanti- ties of alcohol should be taken. The alcohol should be fre- quently changed, or the tissue should be suspended near the top of the alcohol, in order to have the tissue constantly sur- rounded with pure spirit (the water and colloid matters extracted from the tissue falling to the bottom of the vessel) . Many weeks may be necessary for hardening large specimens. Small pieces of permeable tissue, such as mucous membrane, may be sufficiently hardened in twenty-four hours. 109. Formaldehyde (Formol, Formalin, Formalose). This important reagent has been in part described, ante No. 86. Considered as a hardening agent, sensu stricto its most im- portant use is for hardening nervous tissue. This will be considered in Part II. BLUM (L c., No. 86), found solutions containing 4 per cent, of formaldehyde hardened voluminous pieces of liver, kidney, stomachal mucosa, brain, &c., considerably quicker than alcohol, the preservation being excellent. HERMANN (I. c., No. 86) found that such a large organ as a calf's heart was hardened by a 0*5 to 1 per cent, solution in twelve to twenty-four hours. Entire eyes are so hardened in the 1 per cent, solution in twenty-four hours that they may be cut in two with a sharp knife like an apple. Hermann found HARDENING AGENTS. 63 this disadvantage, that tissues hardened in formaldehyde solution suffer when they are put into alcohol for the purpose of dehydration. The paper in question contains interesting observations on the property formaldehyde has of preserving the natural colours and transparent and life-like aspect of tissues. BLUM (Anat. Anz., ix, 1894, p. 229), recapitulating, says that very voluminous pieces of material are hardened quickly and without shrinkage. The tissues stain well. Cells and nuclei preserve their forms; karyokinetic figures are fixed. Mucin is not precipitated, but remains transparent; fat is not dissolved. Micro-organisms retain their specific staining reactions. As to the degree and kind of hardening obtained by for- maldehyde the authors are not so explicit as could be wished. As far as I can see myself, the hardening obtained is gentle and tough, giving an elastic and not a brittle consistency. It probably varies greatly with different tissues. For prolonged hardening, considerable volumes of liquid should be taken and the liquid should be renewed from time to time. For the formaldehyde fixes itself on the tissues with which it comes in contact, deserting the solution, which thus becomes progressively weaker. For the employment of formaldehyde in hardening nervous tissue, see Part II. CHAPTER VII. CLEARING AGENTS. 110. Introductory Remarks. Clearing agents are liquids one ot whose functions it is to make microscopic preparations transparent by penetrating amongst the highly refracting elements of which the tissues are composed, the clearing liquids themselves having an index of refraction not greatly inferior to that of the tissues to be cleared. Hence all clearing agents are liquids of high index of refraction. The same substances have also a second function, which consists in getting rid of the alcohol in which preparations are generally preserved, and facilitating the penetration of the paraffin used for imbedding, or the balsam or other resinous medium in which preparations are, in most cases, finally mounted. Hence all of the group of bodies here called " clearing agents " must be capable of expelling alcohol from tissues, and must be at the same time solvents of Canada balsam and the other resinous mounting media. The majority of clearing ag*eiits are essential oils. 111. The Practice of Clearing. It is important to note again, notwithstanding some repetition, the manner of employing these agents. The old plan was to take the object out of the alcohol and float it on the surface of the clearing medium in a watch-glass. This plan was faulty, because the alcohol escapes from the surface of the object, into the air quicker (in most instances) than the clearing agent can get into it ; hence the object must shrink. To avoid or lessen this cause of shrinkage, clearing is now generally done by the method suggested by Giesbrecht, which consists in putting the clearing medium under the alcohol containing the object. This is done in the following manner. Take a short glass tube, and put into it enough alcohol to contain the objects (a watch-glass will often do well, but a tube is safer) . With a pipette carefully put CLEARING AGENTS. 65 under the alcohol a sufficient quantity of clearing medium (or carefully pour the alcohol on to the clearing medium). Then put the objects into the alcohol. They will sink down to the level of separation of the two liquids at once ; and after some time they will be found to have sunk to the bottom of the clearing medium. They may then be removed by means of a pipette, or the supernatant alcohol drawn off and the prepara- tions allowed to remain until wanted. They should not be considered to be perfectly penetrated by the clearing medium until the wavy refraction-lines caused by the mixture of the two liquids at their surface have ceased to form. The penetration of all clearing media may be hastened by using them warm. It frequently happens that the essential oil with which ob- jects are being treated in a watch-glass or on a slide becomes cloudy after a short time, and fails to clear the tissues. This is owing to a combination between the essential oil and mois- ture, derived, I think, rather from the air than from the ob- jects themselves. The cloudiness can usually be removed by warming (as pointed out by HATCHETT JACKSON, Zool. Anzeig., 1889, p. 630), but this remedy is not always successful, for in certain states of the atmosphere the cloudiness will persist, notwithstanding continued warming. It is for this reason that I advise that clearing be done, whenever possible, in shallow well-corked tubes, under which conditions the pheno- menon rarely occurs. 112. Classification of Clearing Agents. For an account of STIEDA'S experiments with essential oils, see previous editions. XEELSEN and SCHIEFFERDECKER (Arch.f. Anat. u. Phys., 1882, p. 206) examined a large series of ethereal oils (prepared by -Schimmel and Co., Leipzig), with the object of finding a not too expensive substance that should combine the properties of clearing quickly alcohol preparations, not dissolving out anilin colours, clearing celloidin without dissolving it, not evaporating too quickly, and not having a too disagreeable smell. Of these, the following three fulfil the conditions and can be recommended : Cedar-wood, Origanum, Sandal-wood. To these should be added the others recommended in the following paragraphs. 5 66 CHAPTER VII. It would be important to possess a list of the exact indices of refraction of the substances used for clearing. I have, unfortunately, not been able to obtain sufficient information of a trustworthy nature for the compilation of such a list. Cedar oil has nearly the index of crown glass (this is true of the oil in the thick state to which it is brought by exposure to the air, not of the new, thin oil, which is less highly refractive), it therefore clears to the same extent as Canada balsam. Clove oil has a much higher index, and therefore clears more than balsam. Turpentine, bergamot oil, and creasote have much lower indices, and therefore clear less. 113. Choice of a Clearing Agent. Special directions for clearing are given when necessary under the heads of the different organs and tissues. It will suffice here to advise the beginner to keep on his table the following : Oil of cedar, for general use and for preparing objects for imbedding in paraffin, see " Imbedding Methods Paraffin " ; clove oil, for making minute dissections in cases in which it is desirable to take advantage of the property of that essence of forming very convex drops on the slide and of imparting a remarkable brittleness to soft tissues, and for much work with safranin, &c. ;. oil of bergamot which will clear from 90 per cent, alcohol, and which does not extract coal-tar colours ; carbolic acid, for rapidly clearing very imperfectly dehydrated objects. For special clearers for celloidln sections see Cf Collodion (Celloidin) Imbedding Methods." 114. Cedar Oil (NEELSEN and SCHIEFPEEDECKEK, op. ciL, 112). For finest cedar- wood oil, price per kilo varies from fifteen to twenty shillings, say about sevenpence halfpenny per ounce for small quantities, or about the price of clove oil. Very thin, colour light yellow or greenish, odour slight (of cedar- wood), evaporates slowly, is not changed by light, is miscible with chloroform balsam, and with castor oil. Clears readily tissues in 95 per cent, alcohol, without shrinkage ; does not extract anilin colours, Celloidin sections are cleared in five to six hours. Cheap, but requires an inconvenient length of time for the clearing of celloidin sections. The observer should be careful as to the quality of the cedar oil he obtains. I have examined the clearing properties of a sample obtained from the celebrated firm of Rousseau, Paris. This sample was absolutely colourless. It totally CLEARING AGENTS. 67 failed to clear absolute alcohol objects after many days. I always use the thickened oil as supplied for use with immersion objectives. The authors think that a laboratory supplied with cedar oil and origanum oil is fully equipped for all possible cases (the origanum oil being used merely to take the place of cedar- wood oil for the special case of celloidin sections) . Cedar oil is very penetrating, and for this and other reasons is, in my experience, the very best of all media for preparing objects for paraffin imbedding. I find it to be less hurtful to cells and delicate tissue-structures than any other medium known to me. 115. Clove Oil. Samples of clove oil of very different shades of colour are met with in commerce. It is frequently recom- mended that only the paler sorts should be employed in histology. A word of explanation is here necessary. Doubt- less it is, in general, best to use a pale oil, provided it be pure, but it is not always easy to obtain a light-coloured oil that is pure. Clove oil passes very readily from yellow to brown with age, so that in choosing a colourless sample you run great risk of obtaining an adulterated sample, for clove oil is one of the most adulterated substances in commerce. Two important properties of clove oil should be noticed here. It does not easily spread itself over the surface of a slide, but has a tendency to form very convex drops. This property makes it a very convenient medium for making minute dis- sections in. The second property I wish to call attention to is that of making tissues that have lain in it for some time very brittle. This brittleness is also sometimes very helpful in minute dissections. These qualities may be counteracted if desired by mixing the clove oil with bergamot oil. Clove oil has, I fancy, the highest index of refraction of all the usual clearing agents ; it clears objects more than balsam. It dissolves celloidin (or collodion), and therefore should not be used for clearing sections cut in that medium, without special precautions. Notwithstanding the opinion of Schieffer- decker, I consider this to be one of the best of clearing agents, and very valuable on account of the properties to which attention has been called above. New clove oil washes out 68 CHAPTER VI [. anilin colours more quickly than old. It is well to possess trustworthy samples of both new and old oil. 116. Cannel Oil. Greatly resembles clove oil, but is in general thinner. An excellent medium, which I particularly recommend. 117. Oil of Bergamot. SCHIEFFERDECKER (Arch. Anat. vt; Phys., 1882 [Anat. Abth.], p. 206) finds that this oil has many good qualities ; it clears 95 per cent, alcohol prepara- tions and celloidin preparations quickly, does not attack anilin colours, but the strong odour is disagreeable ; it is as dear as -oil of cloves, twice as dear as oil of origanum, and three times as dear as oil of cedar. He considers its action preferable to that of oil of cloves, but all things considered, gives the palm to cedar and origanum. I think that this is a very valuable medium, and though I do not agree with Schiefferdecker in thinking its action superior to oil of cloves, I think it should always be kept at hand. Bergamot oil is, I believe, the least refractive of these essences, having a lower index than even oil of turpentine. SUCHANNEK (Zeit. f. wiss. Mik., vii, 2, 1890, p. 158) says that bleached, colourless bergamot oil will not take up much water, whereas a green oil will take up as much as 10 per cent. VAN DER STRICHT (Arch, de Biol., xii, 1892, p. 741) says that bergamot oil will, with time, dissolve out the fatty granules of certain ova. 118. Oil of Origanum (NEELSEN and SCHIEFFERDECKER, Arch. Anat.u.Phi/s.,1882,p. 204). Price per kilo 15 marks ( = los.). Thin, light brown colour, odour not too strong, agreeable, does not evaporate too quickly, is not changed by light, is miscible with chloroform balsam and with castor oil. Ninety- five per cent, alcohol preparations are cleared quickly, and so are celloidin sections, without solution of the celloidin. Anilin colours are somewhat extracted. For work with celloidin sections care should be taken to obtain 01. Origani Cretici (" Spanisches Hopfenol "), not 01. Orig. Gallici (v. GIBSON; see Zeit. f. iviss. Mik., iv, 4, 1887, p. 482). Specimens of origanum oil vary greatly in their action on celloidin sections, and care should be taken to obtain a good sample. CLEARING AGENTS. 69 SQUIRE, in his Methods and Formulse, &c., p. 81, says that origanum oil (meaning the commercial product) is nothing but oil of white thyme more or less adulterated, and that the product sold as 01. Origani Cretici is probably oil of marjoram. 119. Oil of Thyme. FISH (Proc. Amer. Mic. Soc., 1893, Zeit. f. iciss. Mik., xi, 4, p. 503), following BUMPUS, says that for most of the purposes for which origanum oil has been re- commended, oil of thyme will do just as well if not better. After one distillation of the crude oil of thyme it is of a red- dish-brown colour, and is called the red oil of thyme ; when again distilled it becomes colourless, and is distinguished as the white oil. The red oil is just as efficient as the white for clearing. 120. Sandal-wood Oil (NEELSEN and SCHIEFFEEDECKEE, 1. c., 118). Very useful ; but its high price is prohibitive. 121. Turpentine. Generally used for treating sections that have been cut in paraffin, as it has the property of dissolving out the paraffin and clearing the sections at the same time ; but many other reagents (see No. 126) are preferable for this purpose. If used for alcohol objects it causes considerable shrinkage, and alters the structure of cells more than any other clearing agent known to me, unless used in the thickened state, a method which is much liked for some purposes in Germany. Thickened turpentine (" Verbalizes Terpentinol " of German writers) is prepared by exposing rectified turpentine in thin layers for some days to the air. All that is necessary is to pour some turpentine into a plate, cover it lightly so as to protect it from dust without excluding the air, and leave it until it has attained a syrupy consistency. Turpentine has, I believe, the lowest index of ref raction of all the usual clearing agents except bergamot oil ; it clears objects less than balsam. 122. Carbolic Acid. Best used in concentrated solution in alcohol. Clears instantaneously, even very watery prepara- tions. This is a very good medium, but it is better avoided for preparations of soft parts which it is intended to mount in balsam, as they generally shrink by exosinosis when placed in the latter medium. It is, however, a good medium for celloidin sections. 123. GAGE'S Mixture (Proc. Amer. Soc. Micr., 1890, p. 120; Journ. Roy. Mic. Soc., 1891, p. 418). Carbolic acid crystals melted, 40 c.c. ; oil of turpentine, 60 c.c. 70 CHAPTER VII. 124. Creasote. Much the same properties as carbolic acid. Beech-wood creasote is the sort that should be preferred for many purposes, amongst others, for clearing celloidin sections, for which it is a very good medium. 125. Anilin Oil. This is a rather important reagent on account of its ability to clear excessively watery objects. Common anilin oil will readily clear sections from 70 per cent, alcohol, and with certain precautions (for which, see tl^ paper of SUCHANNEK quoted below) objects may be cleared from watery media without the intervention of alcohol at all. This pro- perty renders anilin valuable in certain cases as a penetrating medium for preparing for paraffin imbedding. For ordinary work the usual commercial anilin will suffice ; and it is immaterial whether it be colourless or have become brown through oxidation. For difficult work, it is well to use a perfectly anhydrous oil. For directions for preparing this see SUCHANNEK, Zeit.f. wiss. Mik., vii, 2, 1890, p. 156, or the last edition of this work. Anilin is chiefly used for clearing celloidin sections, and is sometimes found very valuable for this purpose. 126. Xylol, Benzol, Toluol, Naphtha, Chloroform. Too volatile to be recommendable as general clearing agents, but very useful for celloidin sections or for paraffin sections. I find naphtha is quite good enough for removing paraffin from sections destined to be afterwards passed through alcohol and stained ; but that it should not be used for objects destined to be passed direct into balsam, as it frequently precipitates resinous media. Of the three first mentioned liquids, benzol is the most volatile, then toluol, and xylol is the least volatile, in the proportion of 4 : 5 : 9 (SQUIRE, Methods and Formulae, p. 20). Chloroform is injurious to some delicate stains. . CHAPTER VIII. IMBEDDING METHODS INTRODUCTION. 127. A Word on Microtomes. It is no part of the purpose of this work to discuss instruments, yet a word on this subject may be helpful to the student. The freezing microtome so generally employed in England is less than any other form adapted to the wants of the zoologist. Very thin sections can be obtained by it more readily than with any other microtome, but they are of little use when obtained. The relations of the parts of the organs are deranged by the freezing and by the thawing, and the aqueous nature of the process prevents it from being readily applicable to the mount- ing of series of sections. The microtome of the zoologist, therefore, must be an imbedding microtome. Now there are two methods of imbedding in general use the paraffin method and the celloidin method. In the paraffin method the object is cut dry, with the knife set square ; whilst in the celloidin method the object is usually cut wet, and in a softer and more elastic state than paraffin objects, and always with an obliquely-set knife. It so happens that the most precise and beautiful microtomes that have been constructed are designed in view of the paraffin method, and cannot be applied, or at all events are much less adapted, to work with celloidin objects. A thoroughly equipped laboratory should therefore possess two microtomes, one for paraffin work, and one for celloidin material, or other material that has to be cut in the wet way. If the anatomist cannot afford two instruments, he will perhaps do well not to choose one of those that are adapted only for paraffin, but to choose an all- round instrument, one that without being absolutely of the highest attain- able precision in paraffin work will yet give sufficiently good results in that way, and will also cut in the wet way. Amongst microtomes fulfilling these conditions various forms will be found almost equally convenient. Zeiss makes a good one ; Schanze, of Leipzig, makes a good one ; Reichert, of Vienna, makes a good one. All these are relatively cheap, and, being at the same time perfectly efficient for easy work, may be recommended. Amongst more precise instruments of this class the first place in order of date belongs to the THOMA sliding micro- tome. This is made in several sizes by R. Jung, Mechaniker in Heidelberg. For zoological and general histological work I recommend the medium size (No. 2a or 4), with the newest Naples object-holder and newest form of knife and knife-holder. This instrument is described in Journ. Hoy. Hie. Soc. (N.S.), vol. iii, 72 CHAPTER VIII. p. 298 ; the new Naples object-holder (which I consider essential for the zoologist) is described and figured p. 915. The BECKER microtome is in many respects an improvement on the Thoma model. It is essentially on the same principle, but possesses a mechanical arrangement for moving the knife-carrier ; that is, the knife- carrier is not only guided by a mechanical arrangement, as in the Thoma model, but is put in motion by mechanism. This, I think, is certainly an advantage. Another improvement is that the slides are made of glass in- stead of metal ; this allows one to dispense with the use of oil to the slides, which in the Thoma model gives rise to inequality in the thickness of sections. A minor point is that the instrument is somewhat cheaper than the Thoma form. It is made by Aug. Becker, Gottingen. Descriptions of two forms (Spengel and Schiefferdecker) will be found in Journ. Roy. Mic. Soc., 1886, pp. 884 and 1084. The Naples object-holder can be fitted to the Becker microtome. The instruments above described are " all-round " microtomes ; by which is meant that they may be used either with a square-set knife or an obliquely- set knife, and will cut either celloidin sections or frozen preparations (if a freezing apparatus be added to them) just as well as paraffin sections. They will not, according to my experience, cut series of paraffin sections with any- thing like the same infallible regularity, certainly not with the same rapidity as the instruments next to be mentioned. But they give excellent results, and in view of their adaptability to celloidin or other semi-soft preparations, I think that one of them, the Becker by preference, should be the instru- ment chosen by the worker who desires not to be entirely confined to the paraffin method, and who cannot conveniently possess more than one microtome. All the instruments mentioned hitherto are sliding microtomes, that is instruments in which the object to be cut is a fixture during cutting, and tjie knife is moved on a slide and is only attached to its holder at one end. This arrangement will not allow the highest possible accuracy to be obtained with paraffin objects or any other hard objects. For with hard objects the , knife is free to tilt slightly on meeting the object, instead of cutting its way through it. This defect is fatal to the attainment of perfectly cut series of sections of equal thickness throughout. For the highest class of work it is necessary to employ a microtome constructed on the opposite principle, namely one in which the knife is a fixture, and fixed at both ends as near as possible to the cutting point ; the object being moved against it. The following instruments are constructed on this principle, and for accurate cutting of paraffin sections are undoubtedly superior to any sliding micro- tome ; they also work incomparably quicker. The Cambridge rocking microtome (furnished by the Cambridge Scientific Instrument Company, Carlyle Road, Cambridge, price 4 4s., or by Messrs. Swift and Son, or by Jung) is only adapted for cutting paraffin sections (Mr. Swift has shown me an arrangement for inclining the knife so as to give it the position required for cutting celloidin ; but I feel pretty sure that this will prove a failure in practice). This instrument is extremely simple and extremely rapid, and, what is more important, cuts more level series of sections than any other microtome I am personally acquainted with. It IMBEDDING METHODS INTRODUCTION. 73 should be fitted with an adjustable object-holder, allowing of precise orienta- tion of the object. This, I believe, has been done in the newest instruments. Or the object-holder of Henneguy and Vignal (Compt. Rend. Soc. BioL, 1885, p. 647), may be added to it. (This, as well as the entire instrument, is manufactured in France by Dumaige, 24, Rue Saint-Merri, Paris, or Messrs. Swift on request will furnish such an arrangement, or it may be obtained, with or without the entire instrument, from Jung, of Heidelberg). See also in Zeit. f. wiss. Mik., iv, 4, 1887, p. 465, the description of an object-holder adapted to the rocking microtome by HASSELAEE ; further, the price list of JUXG; also a paper in Zeit.f. wiss. Mik., vii, 2, 1890, p. 165. It has been objected to this instrument by Schiefferdecker (see Zeit. f. wiss. Mik., ix, 2, 1892, p. 171, a description and criticism of the instrument as made by Jung) that it does not cut plane sections, but sections ha the form of segments of a cylinder. This is true ; but it does not follow, as Schiefferdecker concludes, that the instrument is inapplicb many morphological purposes, and especially to embryological researc practice, the slight deviation of the sections from a plane figure is be quite inappreciable, and therefore unimportant. And if I rightly under- stand, this slight defect has been overcome in the new model (1895). Rather more costly (8 15s.) is the MINOT microtome made by E. Zimmer- niann, Mechaniker, 21, Emilien Strasse, Leipzig. A description and figures of this instrument will be found in Zeit.f. wiss. Mik., ix, 2, 1892, p. 176, or in Journ. Roy. Mic. Soc., 1889, p. 143. It is worked on the sewing- machine principle : the knife is fixed as in the Cambridge instrument, and the object is made to impinge on it by means of a rotary motion given to a wheel by the hand, and converted by a crank and slide iever into a vertical one given to a slide carrying the object. This microtome cuts with very great rapidity, and those who have worked with it speak very highly of it. It is said that owing to the construction of the slide, which is subject to un- compensated wear and tear, its work is liable to fail in accuracy. The object-holder does not appear to be so scientifically constructed as the Naples one. Like the Cambridge instrument, this microtome is only adapted for paraffin work, and for this reason and the others stated above I do not feel satisfied that it should be preferred to the Becker or Thoma by those who have to be content with a single instrument. The most beautiful of all these instruments is the RKINHOLD-GILTAY. It is constructed on essentially the same principle as the Minot, but the detail has been further elaborated, with the result of obtaining an instru- ment that is at the same time more precise in operation and more resistant to wear and tear, all working parts being compensated throughout. I have not had the advantage of working with this machine myself, but I have seen it in operation, and can testify that with a common razor I have seen it cut perfectly continuous ribbons of sections of one micron in thickness, from a block of paraffin half an inch square. The sections were all of them entire and without any defect, further than that some of them were some- what compressed, a fault which is, of course, the expression of insufficient keenness in the knife, not of any want of accuracy in the machine. The Reinhold-Giltay has an arrangement for allowing the cutting of collodion material. I am unable to say whether this is a success. It is made 74 CHAPTER VIII. by J. W. GILTAY, Delft, and costs about twenty pounds. A description will be found in Zeit. f. wiss. Mik., ix, 4, 1893, p. 445, and in Journ. Roy. Mic. Soc., 1893, p. 70(3. 128. Imbedding Methods. The processes known as Im- bedding Methods are employed for a twofold end. Firstly, they enable us to surround an object, too small or too delicate to be firmly held by the fingers or by any instrument, with some plastic substance that will support it on all sides with firmness but without injurious pressure, so that by cutting sections through the composite body thus formed, the included object may be cut into sufficiently thin slices without dis- tortion. Secondly, they enable us to fill out with the im- bedding mass the natural cavities of the object, so that their lining membranes or other structures contained in them may be duly cut in situ; and, further, they enable us to surround with the supporting mass not only each individual organ or part of any organ that may be present in the interior of the object, but each separate cell or other anatomical element, thus giving to the tissues a consistency they could not other- wise possess, and ensuring that in the thin slices cut from the mass all the details of structure will precisely retain their natural relations of position. Such a process of im- bedding is at the same time practically a process of hardening in so far as it gives to tissues a degree of firmness that could otherwise only be obtained by the employment of chemical processes such as prolonged treatment with chromic acid and the like. These ends are usually attained in one of two ways. Either the object to be imbedded is saturated by soaking with some material that is liquid while warm and solid when cold, which is the principle of the processes here called Fusion Imbedding Methods; or the object is saturated with some substance which whilst in solution is sufficiently fiuid to penetrate the object to be imbedded, whilst at the same time, after the evaporation or removal by other means of its solvent, it acquires and irnparjts to the imbedded object sufficient firm- ness for the purpose of cutting. The collodion process suffi- ciently exemplifies this principle. If a piece of soft tissue be dehydrated, and soaked first in ether and then in collodion, and if the ether contained in the collodion be allowed slowly to evaporate, the tissue and surrounding mass of collodion will IMBEDDING. METHODS INTRODUCTION. 75 acquire a consistency such as to admit of thin sections being cut from them. The methods founded on this principle are here called Evaporation Imbedding Methods. In any of these cases the material used for imbedding is technically termed an " imbedding mass " Einbettungsmasse masse d' inclusion. Imbedding methods are spoken of by French writers as methodes d' inclusion, or methodes d'enrob- age. As before stated, the method most generally employed, and the one which may be considered the normal anatomical method, is the paraffin method. 129. Imbedding Manipulations. Before proceeding to de- scribe in detail the more important imbedding methods, it is necessary to give an account of the manipulations of the pro- cess of imbedding in general. Imbedding in a melted mass such as paraffin is performed in one of the following ways. A little tray or box or thimble is made out of paper, some melted mass is poured into it ; at the moment when the mass has cooled so far as to have a consistency that will not allow the object to sink to the bottom, the object is placed on its surface, and more melted mass poured on until the object is enclosed. Or the paper tray being placed on cork, the object may be fixed in position in it whilst empty by means of pins and the tray filled with melted mass at one pour. The pins are removed when the mass is cold. In either case, when the mass is cold the paper is removed from it before cutting. To make paper trays, proceed as follows. Take a piece of stout paper or thin cardboard, of the shape of the annexed figure (Fig. 1) ; thin (foreign) post-cards do very well indeed. Fold it along the lines a a and b b' ', then along c c and d d', taking care to fold always the same way. Then make the folds A A', B B r , C C', D D', still folding the same way. To do this you apply A c against A a, and pinch out the line A A', and so on for the remaining angles. This done, you have an imperfect tray with dogs' ears at the angles. To finish it, turn the dogs' ears round against the ends of the box, turn down outside the projecting flaps that remain, and pinch them down. A well-made post-card tray will last through 76 CHAPTER VIII. several imbeddings, and will generally work better after having been used than when new. A f ^N y S / X / - - , ~A~ ~B C D 7 s^ ,' \ / / >S e' FIG. 2. D FIG. 1. To make paper thimbles, take a good cork, twist a strip of paper several times round it so as to make a projecting collar, and stick a pin through the bottom of the paper into the cork. For work with fluid masses, such as celloidin, the cork may be leaded at the bottom to prevent it from floating when the whole is thrown into spirit or other liquor for hardening (Fig. 2). LEUCKHART'S Imbedding Boxes are made of two pieces of type-metal (Fig. 3). Each of these pieces has the form of a carpenter's " square " with the end of the shorter arm triangularly enlarged outwards. The box is constructed by placing the two pieces together on a plate of glass which has been wetted with glycerin and gently warmed. The area of the box will evidently vary according to the position given to the pieces, but the height can be varied only by using different sets of pieces. In FIG. 3. IMBEDDING METHODS INTRODUCTION. 77 such a box the paraffin may be kept in a liquid state by warming now and then over a spirit lamp, and small objects be placed in any desired position under the microscope (Journ. Roy. Mic. Soc. [N.S.], ii, p. 880). SELENKA has described and figured a simple but perhaps more efficacious apparatus having the same object. It con- sists of a glass tube, through which a stream of warm water may be passed and changed for cold as desired, the object being placed in a depression in the middle of the tube (see Zool. Anz. y 1885, p. 419). A modification of this method is described by ANDREWS in Amer. Natural., 1887, p. 101 ; cf. Zeit.f. wi88. Mik., iv, 3, 1887, p. 375. For small paraffin objects the following procedure is very useful. The object is removed from the paraffin solution, the superfluous fluid is removed by means of blotting-paper, and the object placed on a cylinder of paraffin. A piece of stout iron wire is now heated in the flame of a spirit lamp, and with it a hole is melted in the end of the cylinder ; the specimen is pushed into the melted paraffin, and placed in any desired position. The advantages of the method lie in the quickness and certainty with which it can be performed. I strongly recommend the reader not to neglect this simple method, which is capable of sometimes rendering services which no other method can. Those who have to do work with objects so small that their position can only be made out with the aid of a powerful lens ought to know how to arrange an object with a heated needle under a dissecting microscope, or on the object-carrier of the microtome. [In the first edition this procedure was attributed to KINGSLEY. It appears to have been first published by BOKN, see " Die Plattenmodellirmethode," in Arch. f. mik. Anat., 1883, p. 591.] There remains the watch glass method. Melt paraffin in a watch-glass, and throw the object into it ; or place the object in the watch-glass, add solid paraffin, and heat. After the mass has hardened, cut out a block containing the object (this is of course applicable to other masses, such as celloidin). If paraffin be used you may, instead of cutting out a block, turn out the whole mass of paraffin by simply warming rapidly the bottom of the glass, but I find it is far safer to cut out a block. To facilitate the removal of the mass some persons 78 CHAPTER VIII. lubricate the watch-glass before pouring in the mass. To do this a drop of glycerin should be smeared over it and wiped off with a cloth until hardly a trace of it remains. But this is not necessary. As regards the merits of the watch-glass process, I wish to say that, as regards small objects at all events, I consider it the very best process of any. 130. Choice of a Method. Amongst the very various methods of imbedding that have been proposed two are pre-eminently important the paraffin method for small objects, and the celloidin or collodion method for large objects. The subject of the respective merits of paraffin and celloidin still affords matter for discussion to some persons. The case, however, seems to be a very simple one. Celloidin does not afford by a long way the thinnest sections that are obtainable with small objects. For such objects it is, therefore, not equal to the demands made by modern minute anatomy, and paraffin must be taken. On the other hand, paraffin (as at present employed) will only cut very thin sections with fairly small objects; with objects of much over half an inch in diameter you cannot get with paraffin thinner sections than you can with celloidin; and if you try to cut in paraffin objects of still greater size, say an inch and upwards, it will frequently happen that you will not get perfect sections at all, blocks of paraffin of this size having a tendency to split under the impact of the knife ; so that for very large objects celloidm gives better results. T have not been able to satisfy myself that the preservation of the tissues is better in celloidm sections than in paraffin sections; so that convenience apart the case remains as above stated, paraffin for small sections, celloidm for large ones. To this must be added aqueous masses, such as gum or gelatin, for very special cases. It will be the purpose of the next chapter to describe the paraffin method, and to mention some other masses that can be employed in a similar manner, the celloidin method and the other methods that remain to be described being treated of in the following Chapter, CHAPTER IX. IMBEDDING METHODS PARA KFIN AND OTHER FUSION MASSES. 131. Penetration or Clearing. The first stage of the paraffin method consists in the penetration or infiltration of the object by some substance which is a solvent of paraffin. The process may be called a clearing process, since the chief substances used for infiltration are also " clearing" agents. The process of penetration or clearing should be carefully performed with well-dehydrated objects in the manner de- scribed in 111. Penetration liquids being merely liquids that are, on the one hand, miscible with alcohol, and on the other hand good solvents of paraffin, are as numerous as could be wished. Amongst them may be mentioned essence of turpentine, clove oil, bergamot oil, creasote, benzol, xylol, toluol, naphtha, oil of cedar wood, chloroform, and anilin oil. Turpentine penetrates well, and mixes readily with paraffin. I do not, however, recommend it, because in ray experience it is of all others the clearing agent that is the most hurtful to delicate structures. Clove oil penetrates well, and preserves delicate structures well; but it mixes very slowly with paraffin, and quickly renders tissues brittle. Benzinhas been recommended by BRASS (Zeit.f. wiss. Mik., ii, 1885, p. 301). Toluol (or toluen) has been recommended by HOLL (Zool. Anz,, 1885, p. 223). Naphtha has been recommended by WEBSTER (Journ. Anat. and PhyswL, xxv, 1891, p. 278). For large specimens it has the advantage of being very cheap. Dr. Webster writes me that a quality known as " Persian naphtha " is best for fine 80 CHAPTER IX. work, but the common pure naphtha is sufficient for ordinary work. FIELD and MARTIN (Zeit. f. Wiss. Mik., xi, 1, 1894, p. 10) recommend a light petroleum known as " petroleum-aether.' 5 Xylol is said by M. HEIDENHAIN (Kern und Protoplasma, p. 1 14) to be a cause of shrinkage in cells ; he employs oil of bergamot. Chloroform mixes well with paraffin, and after evaporation in a paraffin bath (in the manner described in the next para- graph) leaves behind a pure and very homogeneous paraffin, having but little tendency to crystallise. But it is deficient in penetrating power, so that it requires an excessive length of time for clearing objects of any size ; and it must be very thoroughly got rid of by evaporation in the paraffin bath, or by successive baths of paraffin, as if the least trace of it remains in the paraffin used for cutting it will make it soft. The process of removal requires a very long time, in some cases days. Chloroform ought therefore to be reserved for small and easily penetrable objects. Cedar-wood oil is, according to my continued experience, for the reasons stated by me in Zool. Anz., 1885, p. 563, in general the very best clearing agent for paraffin imbedding. It penetrates rapidly, preserves delicate structure better than any clearing agent known to me, does not make tissues brittle, even though they may be kept for weeks or months in it, and has the great advantage that if it be not entirely removed from the tissues in the paraffin bath it will not seriously impair the cutting consistency of the mass; indeed, I fancy it sometimes improves it by rendering it less brittle. I always use the thickened oil as supplied for use with immersion objectives. In some difficult cases anilin oil is indicated (see 125). 132. The Paraffin Bath. The objects having been duly " penetrated " or " cleared," the next step is to substitute melted paraffin for the penetrating or clearing medium. Some authors lay great stress on the necessity of making the passage from the clearing agent to the paraffin as gradual as possible, by means of successive baths of mixtures of clearing agent and paraffin kept melted at a low temperature, say 35 C. With oil of cedar or toluol, at all events, this is not necessary. All that is necessary is to bring the objects into melted paraf- IMBEDDING METHODS PARAFFIN, ETC. 81 fin kept just at its melting-point, and keep them there till they are thoroughly saturated ; the paraffin being changed once or twice for fresh only if the objects are sufficiently voluminous to have brought over with them a notable quantity of clearing agent. The practice of giving successive baths first of soft and then of hard paraffin appears to me entirely illusory. It is important to keep the paraffin dry that is, protected from vapour of water during the bath. It is still more important to keep it as nearly as possible at melting-point. If it be heated for some time to a point much over its normal melting-point, the melting-point will rise, and you will end by having a harder paraffin than you set out with. And as regards the preservation of tissues, of course the less they are heated the better. The duration of the bath must, of course, vary according to the size and nature of the object. An embryo of the size of a pea ought to be thoroughly saturated after an hour's bath, or often less, if cedar oil has been used for clearing. In any case, the preparations should be cooled (see below, 135) as soon as saturated. If left for many hours in a warm bath, as is sometimes done, delicate structures may be seriously injured. Indeed, the great point to be attended to in paraffin work of the finer order is to minimise the action of heat. It is therefore important both to employ a paraffin of the lowest melting-point that will give good sections (see below, 142), and to abbreviate the warm bath as much as possible. If chloroform or other volatile agent be taken, choice may be made of two methods : either, as in Giesbrecht's method, the chloroform containing the object is heated to the melting- point of the paraffin, and the paraffin gradually added, and the mass kept at the melting-point of the pure paraffin until all the chloroform is driven off ; or, as in Biitschli's method, the objects are simply passed direct from chloroform into a solution of paraffin in chloroform, in which they remain until thoroughly impregnated (half to one hour), and which is then evaporated at the melting-point of the paraffin. Biitschli recommends a paraffin solution melting at 35. (Such a solu- tion is made of about equal parts of chloroform and paraffin of 50 melting-point.) Or, in the case of larger objects, instead of evaporating the chloroform (which is often a very 6 82 CHAPTER IX. long process, as the chloroform must be completely driven off, or the mass will remain too soft for cutting), Biitschli simply transfers them from the bath of paraffin solution to a bath of pure paraffin. Giesbrecht's method (Zool. Anz., 1881, p. 484), more fully stated, is as follows : Objects to be imbedded are saturated with absolute alcohol and then brought into chloroform (to which a little sulphuric ether has been added if necessary, in order to prevent the objects from floating). As soon as the objects are saturated with the chloroform, the chloroform and objects are gradually warmed up to the melting-point of the paraffin employed, and during the warming small pieces of paraffin are by degrees added to the chloroform. So soon as it is seen that no more bubbles are given off from the objects, the addition of paraf- fin may cease, for that is a sign that the paraffin has entirely displaced the chloroform in the objects. This displacement having been a gradual one, the risk of shrinkage of the tissues is reduced to a minimum. 133. Stoves and Water-baths. It is important that the paraffin . *J< H>). Balsam heated till brittle when cold, then dissolved in chloroform. Heat the objects in the mass on a water-bath. For further details see Journ. Roy. Mic. Soc., 1888, p. 1042. Congelation Masses. 177. The Freezing Method. Fresh tissues may be, and are, frequently frozen without being included in any mass, and in 116 CHAPTER X. certain cases very satisfactory sections can be obtained in this manner. But the formation of ice crystals frequently causes tearing of delicate elements, and it is better to infiltrate the tissues with a mass that does not crystallise in the freezing mixture, but becomes hard and tough. Gum arabic affords such a mass. Some workers used common gum water, which is either poured into the well of the microtome or round the object on the object plate, according to the form of microtome used. 178. Syrup and Gum Congelation Mass (HAMILTON, Journ. of Anat. and Phys., xii, 1878, p. 254). The hardening reagent having been soaked out by water, the tissues are prepared for freezing in the following manner, which it is important to observe, otherwise it will be found that the crystals of ice so break up delicate tissue as' to render it totally useless for minute examination. The tissues are to be well soaked in syrup. The sugar somewhat retards the freezing*, and besides, seems to alter the manner of crystallisation, so that instead of the ice being spicular in form it becomes granular, and does no injury to the parts. The syrup requires to be of a particular strength, viz. double refined sugar, 2 ounces ; water, 1 fluid ounce. Wash the superfluous syrup from the surface, and put into the ordinary mucilage for an hour or so before cutting. Imbed in the freezing microtome with mucilage in the usual way. Float the sections into water. 179. Gum and Syrup Congelation Mass (CoLE, Methods of Microscopical Research, 1884, p. xxxix ; Journ. Roy. Mic. Soc. [N.S.], iv, 1884, p. 318). Gum mucilage (B.P.), 5 parts; syrup, 3 parts. (For brain and spinal cord, retinas, and all tissues liable to come in pieces put 4 parts of syrup to five of gum.) Add 5 grains of pure carbolic acid to each ounce of the medium. (Gum mucilage [B.P.] is made by dissolving 4 ounces of picked gum acacia in 6 ounces of water.) The syrup is made by dissolving 1 pound of loaf sugar in 1 pint of water and boiling. This medium is employed for soaking tissues previous to COLLODION AND OTHER IMBEDDING METHODS. 117 freezing. They may remain in it for " any length of time, all the year round " if desired. The freezing is conducted as follows : the gum and syrup is removed from the outside of the object by means of a cloth ; the spray is set going and a little gum mucilage painted on the freezing plate ; the object is placed on this and surrounded with gum mucilage ; it is thus saturated with gum and syrup, but surrounded when being frozen with mucilage only. This combination prevents the sections from curling up on the one hand, or splintering from being too hard frozen on the other. The mass ought to cut like cheese. Should freezing have been carried too far, wait for a few seconds. 180. Dextrin Congelation Mass (WEBB, The Microscope, ix, 1890, p. 344; Journ. Roy. Mic. Soc., 1890, p. 113). Thick solution of dextrin in solution of carbolic acid in water (1 in 40). Use heat for making the solution if desired. This medium is much cheaper than the gum and syrup mass, and, according to Webb, possesses superior cutting qualities. 181. Gelatin Congelation Mass (SOLLAS, Quart. Journ. Mic. oe.,'xxiv, 1884, pp. 163, 164; Journ. Roy. Mic. Soc. [N.S.], iv, 1884, p. 316). "Instead of gum one uses gelatin jelly. This is prepared and clarified in the usual manner. It should set into a stiff mass when cold The tissue to be cut is transferred from water to the melted jelly, and should remain in it till well permeated." The sections are transferred to a slide as soon as cut. On touching the glass they adhere to it. When enough sections have been thus arranged they are covered with a drop of glycerin ; a cover is put on, and the mount closed with any suitable cement. In process of time the glycerin will per- meate the gelatin and convert it into glycerin jelly; this may be hastened by placing the slide in an oven kept at about 20 to 30 C. 182. Gum-Gelatin Congelation Mass (JACOBS, Amer. Natural., 1885, p. 734; Journ. Roy. Mic. Soc., 1885, p. 900). Gum arabic, 5 parts; gum tragacanth, 1 part; gelatin, 1 part. Dissolve in enough warm water (containing one sixth of glycerin) to give a mass of the consistency of thin jelly when cold. 118 CHAPTER, X. 183. White of Egg Congelation Mass (ROLLETT, Denksclir. math, naturw. Kl. It. Acad. Wiss. Wien, 1885 ; Zeit.f. wiss. Mile., 1886, p. 92). Small portions of tissue brought in the white of a freshly laid egg on to the freezing stage, frozen, and cut. The knife must be well cooled. 184. Oil of Aniseed Congelation Mass (KUHNE, Centralb. f. Bakteriol.,xn, 1892, p. 28 ; Journ. Roy. Mic. Soc., 1892. p. 706). Soak in oil of aniseed for twelve to twenty-four hours, freeze and cut, and remove the oil from the sections by means of alcohol. V. A. MOOEE (Amer. Hon. Mic. Journ., 1894, p. 373 ; Journ. Roy. Mic. Soc., 1895, p. 247) says that sections may be transferred direct into Canada balsam, which is miscible with anise oil. CHAPTER XI. SERIAL SECTION MOUNTING. 185. Choice of a Method. All the following methods are excellent if properly carried out. I recommend for general work the following : For paraffin sections that have been already stained, Schallibaum's collodion. For paraffin sections that are to be stained on the slide, Mayer's albumen, unless the stain to be employed be one that will stain the albumen or if the sections be badly folded, in which case take one of the water or alcohol methods given in the next section. For collodion sections, Mayer's albumen. For very large collodion sections, Weigert's process. Methods for Paraffin Sections. 186. The Water or Alcohol Method. The principle of this method is'due tolGAULE (Arch.f. Anat. u. Phys. [Phys. Abth.], 1881, p. 156), who practised it as follows : A slide is moistened with alcohol, the sections are arranged on it by means of a camel-hair brash, also moistened with alcohol ; the slide is slightly warmed so as to cause the sections to stick to the slide ; a cover is put on ; the excess of paraffin is removed by means of a drop of pure xylol, and the mount is completed by means of xylol balsam. Both the moistening with alcohol and the heating are necessary for the attachment of the sections to the slide ; the effect is not obtainable by means of one of these mano3uvres alone. In the primitive form given above, Gaule's method is only applicable to the purpose of mounting a small series of sections that do not require to be stained or otherwise further manipulated on the slide. Later workers have by improvements in the details of the process brought it to a state of considerable perfection, so that it may now be said to be a fairly safe process for extensive series of sections, and will allow of staining on the slide. SUCHANNEK (Zeit. f. wiss. Mik., vii, 4, 1891, p. 464) pointed out that the slides must be absolutely free from grease in order that the alcohol (50 per cent.) or distilled water, which may be used instead, may spread out in a thin and uniform layer. Secondly, that the slides should not be warmed to 120 CHAPTER XI. more than 40 C., it being important both thattfie alcohol should evaporate slowly and that the paraffin should only be softened, not melted, until the evaporation is complete. HOYEE had already for this reason advised slow evaporation at the temperature of the laboratory. GULLAND (Journ. of Anat. and Physiol., xxvi, 1891, p, 56; Journ. Boy. Mic. Soc., 1892, p. 161) floats sections on to the surface of warm water (not warm enough to melt the paraffin), or alcohol if preferred, in a dish, and thence floats them into position on the slide. The slide is drained, and the water evaporated from it at a low temperature as described above. When the water of the sections has evaporated completely they become more transparent, and look dry. The fixation is then complete, the paraffin may be melted and removed by means of any desired solvent, and the sections may be mounted, or be stained in any medium, or otherwise manipulated as desired. Thin sections will generally be fixed in about an hour ; thick ones will require six hours or more. Here it may be pointed out that the degree of adhesion seems to depend very much on the nature of the sections. SCHIEFFERDECKER (Zeit.f. wiss. Mik., ix, 2, 1892, p. 202) finds that the larger and thinner sections are, the better do they stick, and vice versa. And Mr. ANDREW PRINGLE writes me that he finds that tissues thoroughly fixed in chrome solutions, so that their albuminoid substances have become quite insoluble, do not adhere sufficiently without the aid of some substance to fix them to the slide. He prefers arranging sections on the slide with cold water, and then warming until the sections flatten out. M. HEIDENHAIN (Kern ^lnd Protoplasma, p. 114, in " Festschr. f. Kolliker," 1892) also proceeds in this way, and advises that the heating be not carried to above 35 C., at which temperature several hours at least are necessary to ensure fixation. He also finds that water is preferable to alcohol, which is too mobile on the slide and evaporates too quickly. DURHAM'S method (Quart. Journ. Mic. Sci., xxxiii, 1891, p. 116 ; Journ. Roy. Mic. Soc., 1892, p. 293) is the same as the last described, with the exception that he uses 70 per cent, alcohol instead of water. WLASSAX (MERCIER'S Coupes du Systeme Nerveux Central, 1894, p. 118) places the sections first of all on a drop of warm water on the slide, then exposes them to vapour of boiling distilled water, and then puts them in the stove. The advantage of the water or alcohol method is that as there is nothing on the slide that can stain in plasma stains, it gives very clean preparations. The disadvantages are that, besides being excessively lengthy, it is certainly not safe for chrome-osmium preparations. 187. Schallibaum's Collodion Method (Arch. f. mik. Anat., 1883, p. 565). One part of collodion is shaken up with three to four volumes (according to the consistency of the collodion) of clove oil or lavender oil. This should give a clear solution. A little is spread thinly on a slide with a small brush. After arranging the sections on the prepared surface, warm over a SKKIAL SECTION MOUNTING. 121 water-bath, gently, until the clove oil has evaporated (five to ten minutes). The sections are then found to be fixed, and can be treated for days with turpentine, chloroform, alcohol, and watery fluids, without becoming detached. The advan- tage of this method is that it allows of staining on the slide. If after staining any cloudiness should appear between the sections, dehydrate the slide and treat it several times with absolute alcohol and turpentine, warming it gently the while ; or brush the space between the sections repeatedly with a brush moistened with clove oil. This cloudiness only arises from the collodion solution having been taken too concentrated, or having been laid too thick on the slide. I find it is not necessary to evaporate over a water-bath. It is sufficient to hold the slide over a spirit lamp until the paraffin has melted and the clove oil has collected in drops between the sections. Schallibaum has stated elsewhere that long evaporation of the slide is necessary if the sections are to be secured firmly enough to allow of staining on the slide. That is not so. What is necessary is that the paraffin and clove oil be thoroughly removed from contact with the sec- tions ; and that can be done in a second by melting and blowing the paraffin and oil away as described in 140. EABL (Zeit. f. iviss. Mik., xi, 2, p. 179) says that new solution of Schallibaum, not more than four or five days old, will stick fast enough to resist absolute alcohol. He gives the proportions of three parts of clove oil to two of col- lodion. Personally I do not consider Schallibaum's method so safe as Mayer's albumen (and some other methods) for objects that are to be stained on the slide. I recommend it for already stained objects, because it is found to work very pleasantly. Its great defect is that it does not readily lend itself to any device for the flattening out of folded sections. I recommend xylol or naphtha for clearing, in preference to turpentine. FIELD and MARTIN (Bull. Soc. Zool. de France, 1894, p. 48) say that treatment with xylol, toluol, or benzin makes col- lodion more readily soluble in alcohol, and recommend for clearing after Schallibaum's fixative either petroleum ether (a light petroleum, density 0'650) or chloroform. GAU.EMAERTS (Bull. Soc. Beige de Micro., xv, 1889, p. 56; 122 CHAPTER XI. Zeit.f. wiss. Mik., vi, p. 4, 1889, p. 493), following DRASH, em- ploys a saturated solution of gun-cotton in acetone, diluted to the requisite thinness with absolute alcohol. GAGE prefers preparing slides with a layer of pure collodion, which is allowed to dry, and is rendered adhesive at the instant of using by brushing with clove oil. SUMMERS (Amer. Mon.Mic. Journ., 1887, p. 73 ; Zeit.f. wiss. Mik., iv, 4, 1887, p. 482) also employs a dry layer of collodion, which he renders adhesive after the sections are arranged on it, by wetting with a mixture of equal parts of alcohol and ether. As soon as the mixture has evaporated, the sections are found to be fixed. Good collodion is essential in this process. STBASSEE (Zeit.f. wiss. Mik., iv, 1, 1887, p. 45) recommends a mixture of 2 parts collodion, 2 parts ether, and 3 parts castor oil ; or (ibid., vi, 2, 1889, p. 153) 2 parts of collodion with one of castor oil, the sections being painted over with a thicker solution, viz. collodium concentratum duplex 2 to 3 parts, castor oil 2 parts, and the slide being plunged at once, without warming, into a bath of turpentine, in which it remains till the paraffin is dissolved (two to ten hours, somewhat less if the whole be put in a stove). The turpentine suffices to harden the collodion (benzin, benzol, and chloro- form have the same effect). 188. Strasser's Collodion-Paper Method (Zeit.f. wiss. Mik., iii, 3, 1886, p. 346). This is an extremely complicated modification of Weigert's method for celloidin sections, and is only adapted for use with STEASSEE'S automatic ribbon-microtome. See the original papers in Zeit.f. wiss. Mile., iii, 3.. 1886, p. 346 ; vi, 2, 1889, p. 154 ; vii, 3, 1890, p. 290; ib., p. 304; and ix, 1, 1892, p. 8 ; see also a very short abstract of the last paper in Journ. Roy. Mic. Soc., 1892, p. 703, in which is a figure of the " Schnitt- Aufklebe-Mikrotom ;" and Zeit. f. wiss. Mik., xii, 2, 1895, p. 154 (Journ. Roy. Mic. Soc., 1895, p. 702). 189. The Shellac Method (GIESBEECHT, Zool. Anz., 1881, p. 484). Prepare a stock of slides covered with a thin and even film of shellac. This is done as follows : Make a not too strong solution of brown shellac in abso- lute alcohol, filter it thoroughly ; warm the slides, and spread over them a layer of shellac by means of a glass rod dipped in the solution and drawn once over each slide. Let the slides dry. Just before beginning to cut your sections take a prepared slide and brush it over very thinly with kreasote applied by means of a brush ; this forms a sticky surface on which the sections are now arranged one by one as cut, care being taken to bring them on to the slide with as little surrounding paraffin as possible. When all the sections are arranged the slide is heated on a water-bath for SKRIAL SECTION MOUNTING. 123 about a quarter of an hour at the melting-point of the paraffin. The slide is allowed to cool, and the sections are now found to be firmly fixed in the shellac. The paraffin is dissolved away by dropping turpentine on to the sections, which are then mounted in Canada balsam. There is no danger of the sections being floated away by the turpentine, because turpentine does not dissolve shellac. In the note in the Zool. Anz. above quoted, the shellac solution is stated to be prepared with common brown shellac (choosing, of course, by prefer- ence the paler sorts), on account of the insolubility of white shellac in alcohol. In the Mitth. d. Zool. Stat. of Naples, of the same year, " bleached white shellac" is recommended to be dissolved as before in absolute alcohol. In the Journ. Roy. Mic. Soc. (N.S.), vol. ii, 1882, p. 888, it is stated (on whose authority is not clear) that the solution is made by mixing 1 part of bleached shellac with 10 parts absolute alcohol, and filtering. In the same place it is added that " Dr. Mark uses the bleached shellac in the form in which it is prepared for artists as a * fixative ' for charcoal pictures." The account given in the Mitth. d. Zool. Stat. further varies in one other detail from that given in the Zool. Anz. It directs that the shellac slides be brushed before cutting with oil of cloves instead of kreasote, the slide being slightly warmed before brushing. The white shellac of commerce is sometimes not easily soluble in alcohol. KINGSLET (see WHITMAN'S Methods in Microscopical Anat., p. 117) recom- mends that brown shellac be taken, and bleached by exposure to the sun. CALDWELL (Quart. Jourti. Mic. Soc. [N.S.], Ixxxvii, 1882, p. 336) simplifies the method by merely brushing over the side (thinly) at the moment of using with a strong solution of shellac in anhydrous kreasote. (To make the solution, warm the kreasote.) In both the foregoing methods it often happens that the shellac becomes granular or cloudy on the slide. P. MAYER attributes this to the kreasote or clove oil, and proposes to remedy it by employing carbolic acid instead (Amer. Natural, 1882, p. 733 ; Zeit. f. wiss. Mik., iv, 1, 1887, p. 77 ; Journ. Roy. Mic. Soc., 1885, p. 910). Powdered white shellac is heated with crystallised carbolic acid till it dissolves, and the solution filtered warm. But more recently (Intern. Monatschr.f. Anat., &c., 1887, Heft 2; Zeit. f. wiss. Mik., iv, 1, 1887, p. 77) the same author, on the ground that hot car- bolic acid attacks some tissues, recommends another method. Slides are pre- pared with alcoholic shellac according to Giesbrecht's plan. The sections are arranged on the dry film and gently pressed down on to it, then exposed for half a minute to vapour of ether. Chloroform 'softens shellac ; therefore chloroform balsam is n< mounting medium for sections fixed by these methods. These methods do not allow of staining on the slide. I feel bound to say that I am at a loss to understand by what virtue it is that the shellac method continues to survive, as it certainly seems to do, in the face of far more convenient and efficient processes. 190. Mayer's Albumen (Mitth. Zool. Stat. Neapel, iv, 1883; Journ. lio !f . Mic. Soc. [N.S.], iv, 1884, p. 317; Internat. 124 CHAPTER XI. Monatschr.f. Anat., 1887, Heft 2 ; Journ. Roy. Hie. Soc., 1888, pi 160). White of egg, 50 c.c. ; glycerin, 50 c.c. ; salicylate of soda, 1 grm. Shake them well together, and filter into a clean bottle. The filtering may take days or a week, but the preparation does not spoil meanwhile. FOL (Lehrb., p. 134) takes whipped white of egg, filters it through a Bunsen filter, and adds the glycerin and a little camphor or carbolic acid. I find it convenient to beat up the egg with a little water before adding the glycerin and filtering, the salicylate being dissolved in the water in the first instance. But in view of the good preservation of the mixture it is perhaps better not to add water ; the salicylate will dissolve without it with sufficient shaking. According to my experience carbolic acid is perfectly effi- cient as a preservative, but is not to be recommended because it precipitates a great deal of the albumen. A thin layer of the mixture is spread on a cold slide with a fine brush, and the sections laid on it and warmed for some minutes on a water-bath. (Press the sections ivell down into the albumen with a brush.) As the paraffin in the sections melts it carries the albumen away from them, and this is one of the advantages of the method. The sections may be treated with turpentine, alcohol, and aqueous or other stains without any danger of their moving. It is not necessary to use a water-bath for warming the slide. I prefer to warm for an instant over a flame until the paraffin melts, and then blow away the melted paraffin as described, 140. The remaining paraffin is instantly removed by means of xylol, toluol, or the like. It is not necessary to warm the slide at all ; the paraffin can be removed in the cold if desired by putting the slide into toluol, xylol, or the like. But the slide should be very thoroughly treated with alcohol after removal of the paraffin, in order to get rid of the glycerin, which will cause cloudiness if not perfectly removed. The function of the glycerin is merely to keep the layer of albumen moist. Miss A. M. CLAYPOLE has written a paper complaining that the method is uncertain, because too much heat may injure the tissues, and if too little be SERIAL SECTION MOUNTING 125 applied the albumen will not coagulate. This is a misapprehension. No heat whatever is required to coagulate the albumen ; the alcohol will do that sufficiently. The complications proposed by Miss Ciaypole are not only unnecessary but undesirable. The only object in using heat is to enable one to get rid of the paraffin quicker; and it cannot be supposed that tissues that have been for perhaps several hours in a bath of paraffin can suffer through being exposed for a single instant more to the temperature required to melt away the same paraffin from the sections. This method allows of the staining of sections on the slide with perfect safety, both with alcoholic and aqueous stains. This method can be combined with the water process for flattening out sections ( 186), as described by HENNEGUY (Jour n. de I'Anat. ct cle la Physiol., 1891, p. 398). A drop of water is spread by means of a glass rod on a slide prepared with white-of-egg mixture, the sections are arranged on it, the whole is warmed (not to the melting-point of the paraffin) until the sections flatten out; the water is then evaporated off at a temperature of about 40 C., and as soon as it has entirely disappeared the paraffin is melted, and the slide further treated as above described. See also the careful and detailed description of this method given by OHLMACHEK (Journ. Amer. Med. Ass., April, 1893), who has independently worked out the same process. According to my experience the albumen method is abso- lutely safe, and is the one that should in general be preferred for staining on the slide. It has the defect that certain plasma-stains (not chromatin stains) colour the albumen very strongly and cannot be removed from it. This produces very ugly mounts. It sometimes happens that the mixture after it has stood for some time becomes turbid, a change which is attributed to the development of a microbe. I know of no means of preventing the mixture from going bad in this way, though I have found that it keeps better when freely exposed to the sun. It has been stated (VOSSELEE, Zeit. /. wiss. Hik., vii, 4, 1891, p. 457) that as soon as the mixture has become turbid it loses its adhesive properties and should be thrown away. That is not my experience. I find the liquid first becomes milky, then altogether turbid, and at last coagulates, passing into a caseous state. But up to the very last it does not in the least degree lose its adhesive properties. As long as 126 CHAPTER XI. there is enough moisture in it to moisten the brush, it will stick as well as the first day. 191. Mann's Albumen Method (Zeit. f. wiss. Mik., xi, 4, 1894, p. 486). Shake up white of egg with ten volumes of distilled water and filter twice through the same paper. Spread this on a stock of slides with a glass rod, let them drain and dry. Float the sections on to water warmed to 40 C., pass a slide beneath them, arrange them in order, lift them out, put the slide for five minutes on a stove heated to 35 C., then treat with xylol and alcohol. 192. Flogel's Gum Method (Zool. Anz., 1883, p. 565). Make a solution of one part gum arabic in twenty parts water ; filter, and add a little alcohol to prevent the formation of mould. Slides are prepared by pouring the solution over them, and draining. (It is important that the slides be so perfectly clean as to be evenly wetted all over by the gum solution.) Sections may now be cut and laid on the gum surface before it has become dry, and floated into the proper position ; this is the best plan for sections of T ^ mm. thickness, and for large sections. For thinner and small sections it is best to take slides that have completely dried, arrange the sections dry on the gum film, and then breathe on it until the gum has become sticky. A very neat method for cases in which it is not required to treat the slide with watery fluids. WADDINGTON (Journ. Queh. M. Club, vi, 1881, p. 199 ; Journ. Roy. Mic. Soc. [N.S.], i, 1881, p. 704) gives the following process for preparing " arabin," a purified gum arabic which has the advantage of not presenting a granular appearance under the microscope as ordinary gum arabic does. Dissolve clear and white gum arabic in distilled water to the consistency of thin mucilage. Filter. Pour the filtrate into rectified alcohol, and shake well ; the arabin separates as a white pasty mass. Place it on filter-paper, and wash with pure alcohol until the washings are free from water. Dry. The white powder thus obtained should be dissolved in distilled water and filtered twice. It may then be placed on slides, which are drained, dried, and put away till wanted. In this condition it may be preserved indefinitely. 193. Frenzel's Gum Method (Arch. f. mik. Anat., Bd. xxv, 1885, p. 51). Gum arabic is dissolved in water to the con- sistency of a thin mucilage, and to this is added aqueous SERIAL SECTION MoUXTIM,. 127 solution of chrome-alum. An ^excess of t*he latter does no harm. Finally add a little glycerin and a trace of alcohol (1. c., p. 142). The slide is prepared with this in the usual way ; the sections (either cut dry or in the wet way) are gently pressed on to it with a brush and slightly melted on, and heated for at most a quarter of an hour at a temperature of 30 to 45 C., which suffices to render the gum insoluble. This layer has the advantage of not staining with the majority of staining fluids; fuchsin and safranin are the only ones that stain it to a harmful degree. In the other anilius, and in carmine or haematoxylin, it does not stain. Watery stains (it is stated) may be used with it. 194. Born and Wieger's Quince-Mucilage (Zeit. f. wiss. 1885, p. 346). To two volumes of the ordinary pharma- ceutical quince-mucilage (Mucilago cydonii) add one volume of glycerin and a trace of carbolic acid. Spread in a thin layer on a carefully cleaned slide, and arrange the sections on the moist surface. Heat for twenty minutes at a tempe- rature of 30 to 40 C. After removal of the paraffin by turpentine the slide is brought for half an hour into absolute alcohol. You may then mount, or pass through successive alcohols, and stain. Alkaline staining fluids must be avoided, as they soften the mucilage and cause the sections to become detache'd. 195. GRAVIS'S Agar-agar (Bull. Soc. Beige de Micr., xv, 1889, p. 72 ; Zeit. f. wi88. Mik., vi. 4, 1889, p. 494). Solution of agar-agar in 1000 parts of water, to be used as in last section. 196. GRAY'S Gelatin Process (The Microscope, ix, 1889, p. 325 ; Journ. Roy. Mic. Soc., 1890, p. 117). Solution of gelatin in 100 parts of water. Use as gum arabic solution, taking care not to melt the paraffin ; let the slide dry spontaneously overnight, and remove the paraffin with a suitable solvent ; remove the solvent with alcohol, and then treat for five minutes with 2 per cent, solution of potassium bichromate to render the gelatin insoluble. Stain as desired, or mount. HEXNEGUY (Lemons sur la Cellule, p. 62) takes a solution of gektin of about 1 : 5000, to which a trace of bichromate of potash is added at the instant of using. The sections are flattened out by warming, the slide is drained, and dried for some hours exposed to the light. ALLEGEE (Proc. Amer. Mic. Soc., xv, 104 and 192 ; quoted from FISH, ibid., xvii, 1895, p. 321) uses formalin in much the same way. " A few drops of formalin are added to each gramme of a O'o to 1 per cent, gelatin 128 CHAPTER XI. solution. A gentle heat is applied to the slide until the paraffin is softened, and the superfluous gelatin allowed to drain from the edge of the slide." EISEN (Proc. Gal. Acad. Sci., v, 1895, p. 4 ; Journ. Roy. Mic. Soc., 1895, p. 486) practises the same process, explaining that " the fixing should be .allowed to harden in the air for at least four hours, or better during the night," after the sections have been arranged on it. 197. VAN WALSEM'S Gelatin Process. Extremely complicated. See the six pages of description in the original, Zeit.f. wiss. Mik., xi, 2, 1894, pp. 229 to 235, or the abstract in Journ. Roy. Mic. Soc., 1895, p. 122. 198. OBREGIA'S process given below, for celloidin sections, is also applicable to paraffin sections. Methods for Watery Sections. 199. Fol's Gelatin (FoL, Lehrb., p. 132). Four grammes of gelatin are dissolved in 20 c.c. of glacial acetic acid by heating on a water-bath and agitation. To 5 c.c. of the solution add 70 c.c. of 70 per cent, alcohol and 1 to 2 c.c. of 5 per cent, aqueous solution of chrome-alum. Pour the mixture on to the slide and allow it to dry. In a few hours the gelatin passes into the insoluble state. It retains, however, the property of swelling and becoming somewhat sticky in pre- sence of water. The slide may then be immersed in water containing the sections ; these can be slid into their places, and the whole lifted out : the sections will be found to be fixed in their places. This method is especially useful for sections made under water, large celloidin sections amongst others. 200. POLI (Malpighia, ii, 1888, 2, 3 ; Zeit. f. wiss. Mik., v, 3, 1888, p. 361) arranges sections on a layer of melted Kaiser's gelatin, adds glycerin, and covers. See also supra, 196. 201. Frenzel and ThrelfalFs Gutta-percha (or Caoutchouc) Method (Zool. Anz., 1883, pp. 51, 301, and 423). This extremely elegant method is not perfectly safe, the gutta-percha film being liable to tear ; and is now, I believe, very generally abandoned. Methods for Celloidin Sections. 202. The Albumen Method. I find that celloidin sections may be mounted on Mayer's albumen, and have the celloidin SERIAL SECTION MOUNTING. 129 removed if desired by putting them into ether-alcohol. Care must be taken to press them down very thoroughly on to the albumen. 203. SUMMER'S Ether Method (Amer. Hon. Mic.Journ., 1887, p. 7:3; Zeit. f. wiss. Mik., iv, 4, 1887, p. 482 ; Journ. Roy. Mic. Soc., 1887, p. 523). Besides the method given above ( 187), which is applicable to celloidin sections, but is needlessly complicated, Summers recommends the following simpler method : Place the sections in 95 per cent, alcohol for a minute or two, arrange on the slide, and then pour over the sections sulphuric ether vapour, from a bottle partly full of liquid ether. The celloidin will immediately soften and become perfectly transparent. Place the slide in 80 percent, alcohol, or even directly in 95 per cent, if desired. The sections, it is said, will be found to be firmly fixed, and may be stained if desired. I have myself not found this method safe. SCHIEFFERDECKER (Zeit. f. iviss. Mik., v, 4, 1888, p. 507) re- commends that the slide be one that has been previously pre- pared with a layer of collodion if it is desired to stain on the slide ; but if not a clean slide is perfectly sufficient. The slide may of course be treated with ether vapour in a prepara- tion glass or similar arrangement. GAGE (Proc. Amer. Soc. Mic., 1892, p. 82) advises that the slide be one that has been previously coated with a 0'5 per -cent, solution of gelatin and dried ; the collodion adheres much more strongly to a gelatinised surface. 204. EYCLESHEIMER'S Method (Amer. Natural., xxvi, 1892, p. 354 ; Journ. Roy. Mic. Soc., 1892, p. 565). The sections are arranged on a slide with enough alcohol to keep them moist, but not enough to float them. They are covered with a strip of toilet paper, which is kept down in place by winding thread round the slide. Care should be taken to have the turns of the thread passing between the sections, not over them. You may now stain and clear in any way that may be desired. After clearing, you cub the thread, remove the paper by rolling it up from one end, and mount. 205. APATHY'S Oil of Bergamot Method (Mitth. Zool Stat. Neapel, 1887, p. 742; Zeit. f. wiss. Mik., v, 1, 1888, p. 46, 9 130 CHAPTER XI. and v, 3, 1888, p. 360; Journ. Roy. Mic. Soc., 1888, p. 670). Cut with a knife smeared with vaselin ( 162) and wetted with 95 per cent, alcohol. Float the sections, as cut, on bergamot oil (must be green, must mix perfectly with 90 per cent, alcohol, and must not smell of turpentine) . The sections spread themselves out on the surface of the oil ; before they sink, each one is pushed by means of a needle into its place on a slip of tracing-paper dipped into the oil. (A good size for the paper is about as broad as the slide, and three times as long as the cover.) When the requisite number of sections have been arranged on the paper, you drain the paper, dry the- under side of it with blotting-paper, turn it over, and gently press it down with blotting-paper on to a carefully dried slide. Remove the paper by rolling it up from one end. The sections remain adhering to the slide, and may have the remaining bergamot oil removed from them by means of a cigarette paper. If they are already stained, nothing remains but to add balsam and a cover. In the case of unstained or very small objects, it is well to add a little alcoholic solution of safranin to the bergamot oil. The celloidin of the sec- tions becomes coloured in it in a few seconds, and makes them readily visible. The colour disappears after mounting in a few days. If the sections are to be stained, the slide after removal of the bergamot oil is exposed for a few minutes to the vapour of a mixture of ether and alcohol, then brought into 90 per cent, alcohol, and after a quarter of an hour therein may be stained in any fluid that contains 70 per cent, alcohol or more. If it be desired to stain in a watery fluid, care must have- been taken when arranging the sections to let the celloidin of each section overlap that of its neighbours at the edges, so that the ether vapour may fuse them all into one continuous plate. This will become detached from the slide in watery fluids, and may then be treated as a single section. 206. APATHY'S Series-on-the-Knife Method (Zeit.f. wiss. Mik.,, vi, 2, 1888, p. 168). The following is in some respects more convenient than the oil of bergamot method. The knife is well smeared with yellow vaselin rubbed evenly on with the finger, and is wetted with alcohol of 70 to 90 per cent. As fast as the sections are cut they are drawn with a needle or SERIAL SECTION MOUXTIXC. 131 small brush to a dry part of thel^lade, and there arranged in rows, the celloidin of each section overlapping or at least touching that of its neighbours. The rows are the length of the cover-glass, and are arranged one under the other so as to form a square of the size of the cover-glass. When a series (or several series, if you like) has been thus completed, the sections are dried by laying blotting-paper on them (there is no risk of their becoming attached to it, as they are held down by the vaselin). The series is then painted over with some of the thickest celloidin solution used for imbed- ding, is allowed to evaporate for five minutes in the air, and is then either wetted with 70 per cent, alcohol, and allowed to remain whilst cutting is proceeded with, or (if no more sections are to be cut, or if the knife is now full) the knife is removed and brought for half an hour into 70 per cent, alcohol. This hardens the celloidin around the sections into a continu- ous lamella, which can be easily detached by means of a scalpel, and stained, or further treated as desired. It is well to bring it at once on to a slide, moisten the edges of the celloidin plate with ether and alcohol mixture, so that it may not become detached, and bring the whole into the staining solution. 207. WEIGERT'S Collodion Method (Zeit. f. iviss. Mik., 1885, p. 490) . Sections are cut wet with alcohol. Care should be taken not to have so much alcohol on the knife as to cause the sections to float. Prepare a slip of porous but tough paper (Weigert recommends " closet paper"), of about twice the width of the sections. Soak it in alcohol, take it by both ends, stretch it slightly, and lower it on to the section that is on the knife. The section will adhere to the paper, and is taken up by moving the slip horizontally or slightly upwards, away from the edge of the knife. Take up the first section towards the end of the paper that you hold in your left hand, and let the remaining sections follow in order from left to right. After each section has been taken up, the slip is placed, whilst the next section is being cut, with the sections upwards on a moist surface prepared by arranging several layers of blotting-paper, covered with one layer of closet paper, in a plate, and saturating the whole with alcohol. When all the sections have been arranged on the slip, you 132 CHAPTER XI. pass to the next stage of the process, the collodionisation of the series. This is done in two steps. The first of these consists in transporting the series on to a plate of glass prepared with collodion. The plate is prepared beforehand by pouring on to it collodion and causing it to spread out into a thin layer, as photographers do, and allowing it to dry. (A number of the plates maybe prepared and kept indefinitely in stock; microscope slides will do for series of small sections.) Take one of these plates; lay the slip of paper with the sections on the plate, the sections downwards ; press it down gently and evenly, and the sections will adhere to the collodion, then carefully remove the paper. (Do not place more than one or at most two lines of sections on the same plate, for those first placed run the risk of becoming dry whilst you are placing the others.) This finishes the first stage of the collodionising process. Now remove with blotting-paper any excess of alcohol that may remain on or around the sections, pour collodion over them, and get it to spread in an even layer. As soon as this layer is dry at the surface you may write any necessary indi- cations on it with a small brush charged with methylen blue (the colour will remain fast throughout all subsequent manipu- lations) . The plate may now either be put away till wanted in 80 per cent, alcohol, or may be brought into a staining fluid. Wei- gert recommends his hsematoxylin process, but other watery stains may be used. The watery fluid causes the double sheet of collodion to become detached from the glass, holding the sections fast between its folds. It is then easy to stain, wash, dehydrate, and mount in the usual way, merely taking care not to use alcohol of more than 90 to 96 per cent, for dehy- dration. Weigert recommends for clearing the above- described mixture of xylol and carbolic acid ( 164). Both the dehydration and the clearing take rather longer with the collodionised series than with free sections. The series should be cut into the desired lengths for mounting whilst in the alcohol. It is perhaps safer to lay them out for cutting on a strip of closet paper saturated with alcohol. SERIAL SECTION MOUNTING. 133 It is hardly necessary to comment on the great value of this beautiful method. It is suggested by SiBASSERthat gummed paper might be an improvement on the glass plates used in this process especially for very large sections. See ante, 187. The modification of Weigert's method proposed by WINTERSTEINEE (Zeit. f.wiss. Mik., x, 3, 1893, p. 316) consists in suppressing the alignment of the sections on the strip of paper, and slipping them direct from the knife onto the prepared glass. 208. OBREGIA'S Method. This method was originally de- scribed in the Neurolog isclies Centralb. for 1890, and is given in the third edition of WOODHEAD'S Practical Pathology. It has been recommended for class purposes as being very safe and convenient by GULLAND (JouiTi. of Path., February, 1893). Slides, or glass plates of any size, are coated with a solution made of Syrupy solution of powdered candy- sugar made with boiling distilled water . . . . . .30 c.c. Absolute alcohol . . . . 20 Transparent syrupy solution of pure dextrin made with distilled water . 10 ,, They are dried slowly for two or three days until the surface is just sticky to the moist finger. Sections are arranged and heated for a few minutes to a temperature slightly above the melting-point of the paraffin. The paraffin is removed by some solvent, such as xylol or naphtha, and this is in turn removed by alcohol. The alcohol is poured off, and the sections are covered with solution of celloidin or with a solution of 3 per cent, of photoxylin in a mixture of equal parts of ether and absolute alcohol. The plates are left to evaporate in a horizontal position, and when the sec- tions are required the sheet of collodion is cut into ribbons, which are floated off in water, and further treated as desired, e.g. as in Weigert's process, 207. (It is well to divide the sheet of collodion into ribbons by running the point of a knife down it as soon as evaporation has produced a very slight solidification, and the evaporation must not be artificially hastened.) This is the process for paraffin sections ; for celloidin 134 CHAPTER XI. sections the sections are taken up in order on a strip of paper as in WEIGERT'S method, and laid down on the glass in the same way, and then covered with the photoxylin solution and evaporated as described. The advantage of Obregia's process is that it is equally applicable to paraffin sections, to celloidin sections, and to sections of material that has not been imbedded at all. 209. GIACOMINI'S collodion-gelatin process for large sections, see Gazzeita delle Cliniche, November, 1885,Zeit.f. iviss. Mik.,1885, p. 531, or the first edition of the Traite of LEE et HENNEGUT, p. 392. CHAPTER XII. STAINING. 210. The Kinds of Stains. Stains are either General or Special (otherwise called Specific, or Selective, or Elective). A general stain is one that takes effect on all the elements of a preparation. A special, specific, selective, or elective stain is one that takes effect only on some of them, certain elements being made prominent by being coloured, the rest either remaining colourless or being coloured with a different intensity or in a different tone. To obtain this differentiation is the chief object for which colouring reagents are employed in microscopic anatomy. Two chief kinds of this selection may be distinguished, histological selection and cytological selection. In the former an entire tissue or group of tissue-elements is promi- nently stained, the elements of other sorts present in the preparation remaining colourless or being at all events differently stained, as in a successful impregnation of nerve- endings by means of gold chloride. This is the kind of stain that is generally meant by a specific stain. In the latter the stain seizes on one of the constituent elements of cells in general, namely, either the nucleus or the extra- nuclear parts. Stains that thus exhibit a selective affinity for the sub- stance of nuclei nuclear or chromatin stains form at present by far the most important class of stains in zootomy at any rate. What the zootomist wants, and the histologist too, in the great majority of cases, is either to differentiate the intimate structures of cells by means of a colour reaction, in order to study them for their own sakes, or to have the nuclei of tissues marked out by staining in the midst of the unstained material in such a way that they may form land- marks to catch the eye, which is then able to follow out with 136 CHAPTER XII. ease the contours aud relations of the elements to which the nuclei belong ; the extra-nuclear parts of these elements being expressly left unstained in order that as little light as possible may be absorbed in passing through the preparation. Pos- sibly this may be an irrational procedure, but it has hitherto been found in practice to be the most efficient for general work. To these two kinds of selective stains must be added a third group, the plasmatic stains, consisting of those few stains that take effect in a special way on cytoplasm, or formed tissue or ground-substance, leaving the chromatic substance of nuclei as far as possible unstained. In this book, therefore, stains are looked upon as being (1) General stains; (2) Selective stains ; the latter group being subdivided into (a) Nuclear, (6) Plasmatic, (c) Histologically Selective, or Specific. This classification, however, is not followed in the arrange- ment of the special paragraphs, it being more practical to follow an order based on the chemical nature of the staining agents, and on convenience of exposition. Some writers have divided stains into nuclear, general, and selective. This arrangement appears to me faulty because every nuclear stain is eo ipso selective, and because it ignores the subdivisions of selective stains. 211. The Methods of Staining. Colouring matters possessing so great an affinity for certain elements of tissues that they may be left to produce the desired electivity of stain without any special manipulation on the part of the operator, are un- fortunately rare. In practice, selective staining is arrived at in two ways- In the one, which may be called the progressive or direct method, you make use of a colouring reagent that stains the element desired to be selected more quickly than the elements you wish to have unstained ; and you stop the process and fix the colour at the moment when the former are just sufficiently stained, and the latter not affected to an injurious extent, or not affected at all, by the colour. This is what happens, for instance, when you stain the nuclei of a preparation by treatment with very dilute hasmatoxylin : you get, at a certain moment, a fairly pure nuclear stain ; but if you were to prolong the treatment, the extra-nuclear elements would take up the colour, and the selectivity of the stain would be lost. It may be noted of this method that it STAIXTNG. 137 is in general the method of fast stains (" echte Farbung "), and that it renders great services in the colouring of specimens in toto, a procedure which is not possible with the chief stains of the other class (the anilins). It is the old method of carmine and hjematoxylin staining. The second, the regressive or indirect method, is the method of overstaining followed by partial discoloration. You begin by staining all the elements of your preparation indis- criminately, and you then wash out the colour from all the elements except those which you desire to have stained, these retaining the colour more obstinately than the others in virtue of a certain not yet satisfactorily explained affinity. This is what happens, for instance, when you stain a section of one deep red in all its elements with safranin, and then treating it for a few seconds with alcohol, extract the colour from all but the chroinatin and nucleoli of the nuclei. It is in this method that the coal-tar colours find their chief employment. It is in general applicable only to sections, and not to staining objects in toto (the case of borax-carmine is probably only a seeming exception to this statement). It is a method, however, of very wide applicability, and gives, perhaps, the most brilliant results that have hitherto been attained. In previous editions the expressions ''direct" and "indirect" staining methods were alone used. The expressions " progressive " and " regressive " are due to M. HEIDENHAIX, and appear to me to be preferable. 212. The State of the Tissues to be Stained. It is generally found that precise stains can only be obtained with carefully fixed (i. e. hardened) tissues. Dead, but not artificially hard- ened tissues stain indeed, but not generally in a precise manner. Living tissue elements in general do not stain at all, but resist the action of colouring reagents till they are killed by them (see, however, next section) . It appears probable, as was first pointed out, I believe, by PAUL MAYER, that the usual histological stains obtained with fixed tissues are brought about in two ways. Either they result from the combination of the colouring agent with certain organic or inorganic salts, phosphates, for instance, that existed in the tissue elements during life and were thrown down in *Itn by the fixing or hardening agent employed, as j 138 CHAPTER XII. seems to happen when such a fixing agent as alcohol is employed. Or they result from the combination of the colouring agent with certain compounds that did not pre-exist in the tissues, but were formed by the combination of the con- stituents of the tissues with the chemical elements brought to them by the fixing agent, as seems to happen when such a fixing agent as chromic acid is employed the compounds in question being probably chiefly metal albuminates. These considerations will serve to show to how great an extent the quality of a stain is dependent on the nature of the previous treatment the tissues have undergone. 213. Staining "intra vitam." Some few substances possess the property of staining or rather, tingeing living cells without greatly impairing their vitality. Such are in very dilute solutions cyanin (or quinoleiu), inethylen blue, Bismarck brown, anilin black, and, under certain conditions, dahlia and eosin, gentian violet, with perhaps methyl violet, and some others whose action is not yet sufficiently esta- blished by experiment. Cpngo, even in strong solution, is not toxic to some organisms, and stains some structures (see SCHOLTZ, Centralb.f. d. med. Wiss., 1886, p. 449; also Journ. Roy. Mic. Soc., 1886, p. 1092). Living Eotifera are in part successfully stained by it during life. (The paper of Martinotti, Zeit. f. wiss. Mik., v, 3, 1888, p. 305, may be consulted on this subject.) More recently, neutral red (Neutralroth) has been greatly recommended by Ehrlich (see Chap. XVI). As to the employment of these reagents, it may be noted that they must be taken in a state of extreme dilution, and in neutral or feebly alkaline solution acids being of course toxic to cells. Thus employed, they will be found to tinge with colour the cytoplasm of certain cells during life (never, so far as I know, nuclear chromatin during life ; if this stain, it is a sign that death has set in). The stain is sometimes diffused throughout the general substance of the cytoplasm, sometimes limited to certain granules in it, which have been taken, perhaps without sufficient reason, to be identical with the granules of Altmann (Altmann's Studienilber die Zelle, 1886). Since the publication of the last edition I have made a considerable number of observations on this subject, and have STAINING. 139 come to the same conclusion as GALLEOTTI (Zeit.f. iviss. Mik., xi, '2, 1894, p. 172), namely, that the so-called "intravitam" stains are not true stains at all. The diffused coloration above mentioned appears always, if the cell that shows it have remained in a state of unimpaired vitality, to be due to simple absorption or imbibition of the colouring matter by the cell, not to a chemical combination of the colouring matter with any of the constituents of the cell. If a cell thus coloured be transported into a medium free from the colouring matter it will give up unchanged the colour it had imbibed, which seems to be a sufficient proof that the colouring matter had not entered into any chemical combination with the elements of the cell, but was simply held in a mechanical way in the interstices of its substance. If, on the other hand, there has been produced the above-mentioned coloration of certain granules or other cell-contents, it is possible that this may be a true stain in the sense of being a chemical combination. It may be so, but it certainly is not always so, as may sometimes be proved with the greatest ease by putting the cell into a colourless medium and observing the supposed stain disappear. And in cases in which this does not happen, in which therefore a more or less fast stain has been obtained, it is invariably found that the stain is limited to cell- contents that do not form an integral part of the living texture of the cell ; the cell itself may be living, but they are not. These granules or other cell-contents may be granules formed of substances that have been absorbed by the cell from without food-granules ; or they may be katabolic products, consist- ing of matter that is no longer alive and is destined to be shortly expelled from the cell ; or they may be elements that form indeed an integral part of the living texture of the cell but have been injuriously affected by the colouring matter, and for that or some other reason are in a state of diminished vitality, they are parts of the cell that are being killed by the colouring reagent or that have been totally killed by it whilst the rest survives ; in no case do they consist of matter that is fully and perfectly alive. I am inclined to think that the chief scientific value of the so-called vital or intra-vitam stains will be found to lie in the fact that they may furnish us with the means of distinguishing the living constituents of a cell from the non-living ones, and even of A 140 CHAPTER XII. recognising amongst the living ones those that possess only a relatively low or impaired degree of vitality. Apart, however, from the question whether the elements stained by the so-called " vital" stains are truly living or not, it must be conceded that this mode of treating living cells has frequently a measure of practical utility. It often enables us to map out physiological or morphological tracts that would otherwise be unrecognisable or less readily recog- nisable in the living state. I have frequently found gentian, dahlia, and methylen blue, added to indifferent liquids, extremely useful in the exa- mination of tissue-cells. Quinolein and Bismarck brown are well-known aids to the study of Infusoria. Methylen blue has a specific affinity for sensory nerves, and is an extremely important reagent (see post, Chap. XYI). According to my experience, methylen blue is the most generally useful of these stains. It has the valuable point that it is perfectly soluble in saline solutions, and may therefore be employed with marine organisms by simply adding it to sea water. The others are not thus soluble to a practical extent, but I find that gentian and dahlia become so if a trace of chloral hydrate 0'25 per cent, is ample enough be added to the saline solution. Any of these reagents may be rubbed up with serum, or other ' ' indifferent >} liquid. Methylen blue may be fixed in the tissues, and permanent preparations made, by one or other of the methods described in Chap. XVI. Bismarck brown stains may be fixed with 0*2 per cent, chromic acid or with sublimate solution (MAYEE), and the preparations may be stained with safranin, care being taken not to expose them too long to the action of alcohol. 214. Substantive and Adjective Staining; Mordants. In the industry of dyeing, colouring matters are divided into two classes, according to their behaviour with respect to the / material to be dyed. Certain dyes are absorbed directly from their solution by the material immersed therein, and combine with it directly. In this case the material is said to be sub- xtantively dyed, and the colouring matter is called a substantive colouring matter. Other dyes do not combine directly with the material to be acted on, but this material must first be charged with some STAINING. 141 substance known as a mordant^ (generally a metallic salt or hydrate) before it will combine with the colouring matter. These are known as adjective colouring matters.* Animal tissues have in general a considerable affinity for colouring matters, taking them up directly from their solutions. In consequence, the great majority of histological stains are obtained by substantive staining of the tissues. Still, as has been already pointed out, it seems probable that many of the histological stains that are obtained without intentional mordanting of the tissues, should yet in strictness be attributed to the class of adjective stains. This would be the case whenever there is reason to suppose that the stain obtained results from a combination of the colouring matter with some metallic salt or hydrate that is not a constituent of the living tissue, but has been brought into it by the fixing or hardening reagents, these reagents playing the part of mordants though only intentionally employed for another purpose. This would appear to be the case with the stains, or some of them, ob- tained after fixation with corrosive sublimate, alum, salts of iron, of platinum, of palladium, of uranium, and, for certain tissue elements and certain colours, chromium. And further, the mordanting substance may not only be present unin- tentionally in the fixing or hardening agents, it may be present unintentionally, or with imperfect realisation of its import, in the staining solutions themselves. Such is pre- sumably the part played by alum in many of the stains in which it figures as an ingredient. Iodine also plays in some staining processes a part which seems only explicable on the supposition that it acts as a mordant. In some staining processes, however, mordants are inten- tionally resorted to in order to fix the stain. Mordanting has long been employed in some ha3matein staining processes, such as that of M. HEIDENHAIX. More lately it has been resorted to for staining with tar colours, as in the curious " inversion " process of RAWITZ. These processes will be explained in their respective paragraphs. Here it remains only to note of what sort are the advantages secured by this mode of staining. It must be admitted that mordants are in some cases of use by * For an excellent popular exposition of this subject see BENEDIKT and KNECHT'S ' Chemistry of the Coal-tar Colours ' (George Bell and Sons). 142 CHAPTER XI r. enabling us to fix colouring matter in tissue elements that would otherwise be rebellious to staining. And they have in some cases the advantage of affording a very convenient means of regressive staining. For it happens that the colour- compounds thrown down in mordanted tissues are in many cases specially soluble in an excess of the mordant ; so that the solution of the mordant itself forms a very appropriate decolourising agent. Recognising these advantages, it must still, I think, be said that there seems to be some danger at the present moment that the practice of employing mordants may degenerate into an abuse. For surely the primary use and intention of an histological stain (not of an industrial dye) is, that it should select and reveal those elements of tissues that have a natural affinity for its colouring matter. That end is attained in the manner least open to objection by the use of substantive stains, the natural affinities of the tissues and the colouring matter here coming spontaneously and unconstrained into play. Not so in the case of adjective staining. Here the colour is as it were forcibly compelled into an unnatural union with all or many of the elements of the tissue, including many which have no natural affinity whatever for the colour. In such preparations the distinction between chromatic and achromatic elements is obliterated; and the interpretation of the images afforded by them is open to more serious causes of error than in the case of substantive stains. Attention may be called here to the theoretically interesting " invert " stain obtained by RAWITZ by the use of mordants (see post, Chap. XVI). In this curious process, a colour safranin, for instance which when used substantively is a pure chromatin stain, becomes by the use of mordants a pure plasma stairi, staining cytoplasm deeply, and leaving the chromatin of nuclei absolutely unaffected. This strange effect can hardly be satisfactorily explained at present. Are we to suppose that the affinities of the nucleic acid of the chromatin are so fully satisfied by combination with the mordants, that it is unable further to combine with the colouring matter ? It may be noted in a general way that the following sub- stances may be found useful as mordants, for enhancing the resistance of many tar colours to the alcohol employed for de- colourising, and for producing a stronger stain. STAINING. 143 Iodine : sections may be treated for a few minutes before staining with tincture of iodine. Permanganate of potash : see HENNEGUY'S process. Formaldehyde : see OHLMACHER'S process. Aniline : see SAFRAXIX. Chromic acid : see BIZZOZERO'S process. 215. Choice of a Stain. The following may be recommended with confidence for general work : For sections, MAYER'S hfpmalum ; or, for chromosmium objects more especially, safranin or tliionin. If a plasma stain be required at the same time as a chromatin stain, I strongly recommend Kern- scJtwarz, followed by safranin (see Kernschwarz) . For staining in toto, Grenadier's alcoholic borax-carmine or Mayer's carmalum, unless the object be so impermeable as to require a more highly alcoholised stain, in which case take Mayer's paracarmine, or for chromic acid objects Mayer's h&macalcium. For fresh tissues or small entire objects, methyl green, if it is not important to have permanent preparations ; if it is, take carmalum or alum-carmine. Picric acid may be used for double-staining in bulk after carmine or bsematoxylin. Many others of the numerous stains discussed in the follow- ing chapters render most valuable services, and will be found recommended in the special paragraphs as occasion dictates. The beginner will probably do well not to use a double stain where a single one will do. To do so is too often to go farther and fare worse. 216. Staining Reagents and Chemicals. You are not likely to succeed in staining, especially in the beautiful processes of staining with coal-tar colours, unless you see to it that you are working with chemicals of the proper quality. You cannot ensure this by going to a generally trustworthy house for chemical products at all events, not in the case of coal-tar colours. It is not sufficient that these should be what they arc commercially described to be ; they may be pure, and yet not give good stains. They must (in the case of anilins, at all events) be the identical products used in their work by the 144 CHAPTER Xll. authors who have described and recommended them (see the note on the numerous safranins in the market, sub voce Safranin) . I therefore feel constrained to advise everybody to get his reagents at all events his anilins from the well- known chemists Griibler or Miinder. Griibler has all the tried reagents in stock, and supplies only such as have been found by experiment with tissues to furnish the desired stain. He also makes up fixing and staining solutions, injection and imbedding masses, &c., according to the classical formulas, and sends them out neatly packed and ready for use. From experience I can most highly recommend these preparations, which are in nine cases out of ten better than those the observer is likely to make for himself. They may be ordered from the price list, or by quoting the numbers of the formulas in this work. The address is : Herrn Dr. G. GRUBLER & Co., Ohemiker, Baiersche Strasse 63, Leipzig. Griibler can cor- respond in English. His preparations can be obtained in London from Mr. R. KANTHACK, 21, Golden Square, Kegent Street, W., who is also the authorised agent for the microscopes and apparatus of Zeiss, also for the microtomes of Becker, and the bacterio- logical apparatus of F. & M. Lauteiischlaeger, &c. Miinder's address is : Herrn Dr. G. MUNDER, Mikroskopisch- chemisches Institut, Gottingen. Messrs. SQUIRE & SONS, chemists, 413, Oxford Street, London, W., also make a speciality of microscopical reagents, and I am told furnish excellent products. CHAPTER XIII. CARMINE AND COCHINEAL STAINS. 217. The Theory of Carmine Staining. I take the following from the important paper of MAYEE, " Ueber das Farben mit Carmin, Cochenille, und Hamatein-Thonerde," in JJitth a. d. Zunl. Station zu Neapel, Bd. x, Heft 3, 1892, p. 480. The rationale of staining with carmine has hitherto been obscured by the erroneous notion that carmine is nothing but carminic acid with at most certain impurities. This is not the case. According to the analysis of LIEBEKMAXN (Ber. d. Chem. Ges., Jahrg. 18, 1886, pp. 1969 1975) carmine is a very peculiar aliimina-lime-protein compound of carminic acid, a true chemi- cal compound from which at all events aluminium and calcium can no more be absent than sodium from salt. Analysis gives about 17 per cent, of water, 20 per cent, nitrogenous matters, 56 per cent, carminic acid, at least 3 per cent, alumina, and 3 per cent, lime, together with a small proportion of magnesia, potash, soda, phosphoric acid, and a trace of wax. Mayer has come to the conclusion that in the processes of histological staining (not of industrial dyeing) the active factors of the compound are, besides the carminic acid, only the alumina, and in some cases, the lime. The other bases are inactive; the nitrogenous matters, so far as they have any influence at all, are an obstacle, as it is they that give rise to the well-known putrefaction of the solutions. Having arrived at these conclusions, it seemed logical to admit that carminic acid, instead of carmine, should be taken . a. Acid. 223. Mayer's Carmalum (Mitth. Zool. Stat. zuNeapel, x, 3, 1892, p. 489). Carminic acid, 1 grin. ; alum, 10 grins.; distilled water, 200 c.c. Dissolve with heat (if necessary : I have been able to make my solutions in the cold). Decant or filter. Add some antiseptic, either a few crystals of thymol, or 0*1 percent, salicylic acid, or 0'5per cent, salicylafce of soda. The solution will then keep. A clearish red fluid with a violet tinge. It stains well in bulk even osmium objects. If washed out with distilled water only, the plasma will remain somewhat stained. If this be not desired, wash out carefully with alum solution, or, in difficult cases, with weak acid. The general effect is that of an alum-carmine stain. A notable difference between the two is that carmalum stains well in bulk, which alum-carmine is not very suitable for when used in the ordinary way ; but see 227. . A weaker solution may be made by taking from three to five times as much alum and five times as much water, and dissolving in the cold, which may be convenient. This is a very close equivalent of alum-carmine, giving, however, a somewhat redder stain. I find this solution very weak for ordinary work. It should be noted that with either solution the objects to be stained should not have an alkaline reaction. The other properties of these solutions are very similar to those of alum- carmine. 150 CHAPTER XIII. 224. MAYER'S Aqueous Aluminium-Chloride-Solution (Mitth. Zool. Stat. zu Neapel, x, 3, 1892, p. 490). Carminic acid 1 grm., chloride of aluminium, 3 grms. ; water, 200 c.c. Add an antiseptic, as for carmalum. Use as carmalum. The stain is of a blue-violet colour, very powerful, and elective. But it is not so pure a stain as that of carmalum, plasma being more strongly coloured. It is recommended only as a substitute for carmalum in cases in which the latter is counter-indicated on account of the presence of alum or the like. 225. Alum- carmine (GTEENACHEE'S formula, Arch. mik. Anat. y xvi, 1879, p. 465). An aqueous solution (of 1 to 5 per cent, strength, or any other strength that may be preferred) of com- mon or ammonia alum is boiled for ten or twenty minutes with J to 1 per cent, of powdered carmine. (It is perhaps the safer plan to take the alum solution highly concentrated in the first instance, and after boiling the carmine in it dilute to the desired strength.) When cool filter. This stain must be avoided in the case of calcareous struc- tures that it is wished to preserve. TIZZONI (Bull Sc. Med. Bologna, 1884, p. 259), PISENTI (Gazz. degli Ospetali,'No.24 l ', Zeit.f. wiss. Mik., ii, 1885, p. 378), and GEIEB (Mem. Soc. Hal. Sci., t, vi, No. 9, 1887 ; Zeit. f. wiss. Mik., vii, 1, 1890, p. 47) have given modifications of Grenadier's formula which do not appear to me rational. Alum-carmine is one of the best stains to be found outside the coal-tar colours. It is particularly to be recommended to the beginner, as it is easy to work with ; it is hardly possible to overstain with it (except muscle). Its chief defect is that it is not very penetrating, and therefore quite unsuitable for staining objects of considerable size in the mass. This defect may, however, be to some extent overcome by employing the acid formula of Henneguy ( 227), if it be not convenient to use Mayer's carmalum. The stain is permanent in balsam, and in aqueous media if not acid. 226. Cochineal Alum-Carmine (PAETSCH, Arch. f. mik. Anat., xiv, 1877, p. 180). Powdered cochineal is boiled for some time in a 5 per cent, solution of alum, the decoction filtered, and a little salicylic acid added to preserve it from mould. Another method of preparation has been given by CZOKOR (Arch. /. mik. Anat., xviii, 1880, p. 413). Mayer has care- CARMINE AND COCHINEAL STAINS. 151 fully examined both, and finds that Partsch's is the more rational, the proportion of alum in it being exactly right, whilst in Czokor's it is insufficient. Partsch's fluid has also the ad vantage of keeping better. The formula known as Klein's cochineal fluid (which appears to have been first published in the Ann. and Mag. Nat. Hist., viii, 1881, p. 232) is identical with that of Czokor. The formula recommended by RABL in Zeit. f. wise. Mik., xi, 2, 1894, p. 168, is only an insignificant modification of that of Czokor. Both these solutions are to all intents and purposes "alum- carmines." They give a stain that is practically identical with that of alum-carmine made from carmine, with perhaps even more delicate differentiations (but that depends so much on the quality of the carmine, the quality of the cochineal, and the nature of the objects to be stained that no absolute rule can be stated). On the whole, it seems to be a mere matter of convenience whether the one or the other should be preferred. The cochineal fluids should be used in exactly the same way as the carmine fluid. It has been lately recommended by HERRICK (Journ. Comp. Neur. t Cincinnati, vol. i, 1891, p. 134) as a " vast improve- ment " to make the solution with sulphate of aluminium instead of alum (quoted from Mayer's paper, Mitth. Zool. Stat. Neapel, x, 3, 1892, p. 496). 227. Acetic Acid Alum- Carmine (HENNEGUY, Traite des Meth. techn., LEE et HENNEGUY, 1887, p. 88). Excess of carmine is boiled in saturated solution of potash alum. After cooling add 10 per cent, of glacial acetic acid, and leave to settle for some days. The deposit of carmine and alum that forms during that time is removed by filtration. For staining, enough of the solution is added to distilled water to give it a deep rose tint. In order to ensure rapid diffusion, it is well to bring the tissues into the stain direct from alcohol. ' Stain for twenty-four to forty-eight hours, and wash for an hour or two in distilled water. (It is important that the water should be distilled in order to avoid the formation of crystals.) Dehydrate with alcohol and mount in balsam. You can mount in glycerin, but the pre- parations do not keep so well as in balsam. J52 CHAPTER XIII. The advantage of this carmine is that it has greater power of penetration than the non-acidified alum- carmine, and stains deep-seated layers of tissue just as well as the superficial ones. The colour of the stain is a somewhat inelegant violet, but this can be changed to a warmer tone by treating the objects with dilute HC1, as for borax-carmine objects. 228. Alum- Carmine and Picric Acid. Alum-carmine objects may be double-stained with picric acid. LEGAL (Morph. Jahrb.j viii, p. 353) combines the two stains by mixing ten vols. of alum-carmine with one of saturated picric acid solution. I consider this to be a very recommendable practice. 229. Aceto-Carmine (Acetic Acid Carmine) (SCHNEIDER'S formula, Zool. Anzeig., No. 56, 1880, p. 254). To boiling acetic acid of 45 per cent, strength add carmine until no more will dissolve, and filter. (Forty-five per cent, acetic acid is, according to Schneider, the strength that dissolves the largest proportion of carmine). To use the solution you may either dilute it to 1 per cent, strength, and use the dilute solution for slow staining ; or a drop of the concentrated solution may be added to a fresh preparation under the cover-glass. If you use the concentrated solution it fixes and stains at the same time, and hence may render service for the study of fresh objects. It is very pene- trating, a quality that enables it to be used where ordinary reagents would totally fail. The stain is a pure nuclear one. Unfortunately the pre- parations cannot be preserved, and for this and other reasons the stain is of very restricted applicability. ZACHARIAS (seeZeit.f. wiss. Mik., v, 3, 1888, p. 371) adds to this solution wood vinegar (acetum pyrolignosum] in the proportion of 1 drop to 10 c.c. A similar stain has been prepared with formic acid by PIANESE (see Zeit.f. wiss. Mik., x, 4, 1894, p. 502). Probably for almost all the purposes for which aceto-carmine is useful, methyl-green will give better results. 230. Iron Carmine (ZACHARIAS, Zool. Anz., No. 440, 1894, p. 62). Stain thoroughly for several hours (Zacharias stains in an aceto-carmine,, of which I suppress the formula, for, as pointed out to me by Dr. MAYER, and as I have verified, carmalum does just as well). Kinse the objects with dilute acetic acid, and bring them (taking care not to touch them with metallic instruments, if the aceto-carmine have been taken) into a 1 per cent, solution of ammoniated citrate of iron (the pharmaceutical Ferri et Ammonite Citras). Leave them till thoroughly penetrated, for as much as two or three hours if need be. In this solution they take on a black tint CARMINE AND COCHINEAL STAINS. 153 (with sections this happens in a few minutes). They should be removed as soon as the reaction has taken place throughout, otherwise there is risk of over-blackening. Wash for several hours in distilled water, dehydrate and mount in balsam. This is at the same time a chromatin stain and a plasma stain. In my preparations chromatin is blue and plasmatic elements brown. I consider the method may render service in some cases. Amongst other things, spindles and spindle- relics are fairly well brought out by it. It can be applied to the coloration of entire objects (provided that they be small, MAYER). /3. So-called "Neutral," and Alkaline. 231. As to Picro-carmine. The term " picro-carmine " is commonly used to denote a whole tribe of solutions in which carmine, ammonia, and picric acid exist unconibined in haphazard proportions. BAXVIEB, to whom we owe the invention of picro-carmine, claims that when prepared by his process it results as a definite chemical substance, a double salt of picric and carminic acid and ammonia, or picro-carminate of ammonia. But this has never been scientifically demonstrated. Picro-carmine gives good differen- tiations, and has the great merit of being less hurtful to most tissues than other aqueous alkaline carmines. It should be understood that the chief value of picrocarmine does not lie in its capacity of affording a double stain. The double stain, if that is all that is wanted, can be just as well or better obtained by staining first with borax-carmine, or the like, and after-staining with picric acid. The essential point about picro-carmine is that it is a fairly neutral fluid. Banvier was, in fact, led to add picric acid to ammo- niacal solution of carmine by the desire of neutralising the ammonia, that is all. For slow staining, dilute solutions may advantageously have 1 or 2 per cent, of chloral hydrate added to them. Overstains may be washed out with hydrochloric acid, say O'o per cent., in water, alcohol, or glycerin. Preparations should be mounted in balsam, or if in glycerin this should be acidulated with 1 per cent, of acetic or, better, formic acid. 232. BANVIEE'S Picro-carmine or " Picro-carminate of Ammonia.'* The method of preparation employed in the Laboratory of Histology of the College de France, kindly communicated to myself and Henneguy for our Traite des Meth. techn. (q. v. p. 451) by M. VIGNAL, one of the assist- ants there, is as follows : Take- Water .... . 1000 parts. Picric acid 20 Carmine ........ 10 Ammonia . 50 154 CHAPTER XIII. Put them into a stoppered bottle and leave them for two or three months in a warm place. Then put them into a large crystallising dish and let them putrefy. When the liquid has become reduced by evaporation to four fifths of its original volume, remove the crystals that have formed at the bottom, dry them, and dissolve them in a little warm water. Filter the solu- tion, and examine it with the microscope to see whether the carmine is really dissolved. If not, add water and ammonia, and let the solution putrefy again ; evaporate and examine as before. When you have got your carmine combined, evaporate the solution to dryness in a stove, and reduce the picro-carminate to powder. For staining, dissolve 1 grm. of the powder in 100 grins, of water, and add a crystal of thymol to prevent the development of mould. 233. KANVIEE'S Original Formula (Traite, p. 100) was as follows : To a saturated solution of picric acid add carmine (dissolved in ammonia) to saturation. Evaporate down to one fifth the original volume in a drying oven ; and separate by filtration the precipitate, pour in carmine, that forms in the liquid when cool. Evaporate the mother-liquid to dryness, and you will obtain the picro-carminate in the form of a crystalline powder of the colour of red ochre. It ought to dissolve completely in distilled water ; a 1 per cent, solution is best for use. 234. Other Formulae for Picro-carmine. I have tried most of the following, and found no real advantage in any of them: GAGE, Am. M. Mic. Journ., i, 1880, p. 22 ; Journ. Roy. Mic. Soc., vol. iii, p. 501 (very elaborate and has not afforded me a soluble carmine.). FOL, Lehrb. d. vergl. mile. Anat., p. 195. KTJTHEBFOED, Pract. Hist., p. 173. PAUL MATEE, Mitth. Zool. Stat. Neapel, ii, p. 20. BABEE, Mon. Micro. Journ., xii, p. 48. PEEGENS, Carney's Biologie cellulaire, p. 92. HOYEE, Biol. Centralb., ii, 1882, p. 17. BIZZOZEEO, Zeit.f. wiss. Mik., 1885, p. 539. KLEHENSIEWICS, Sitzb. Akad. Wiss. Wien, Ixxviii, 1878, iii, Juni ; Zeit.f. wiss. Mik., i, 1884, p. 501. CUCCATI, Zeit. f. wiss. Mik., vi, 1, 1889, p. 42. For/SocZa-Picro-CarmmeseeLowENTHAL, Anat. Anzeig., ii, 1887, No. 1, p. 22, and Zeit. f. wiss. Mik., x, 3, 1893, p. 313 ; Anon., Journ. Roy. Mic. Soc., 1888, p. 518 ; SQUIEE'S Methods and Formulse, &c, 1892, p. 35. WEIGEET corrects unsatisfactory samples of picro-carmine in the following way (Virchow's Archiv, Bd. Ixxxiv, pp. 275, 315 ; Zool. Jahrb., 1881, p. 40) : Small quantities of acetic acid are added " until the first slight precipitate appears even after stirring." The whole is again put away for twenty -four hours more, when it will be found that there has formed a pre- cipitate that can only partially be removed by filtration ; ammonia is then added drop by drop at intervals of twenty-four hours, until the solution becomes clear. If the solution stains too yellow, acetic acid is added ; if it overstains red, a little ammonia is again added. All badly staining samples of picro-carmine may, according to Weigert, be improved in the same way by addition of acetic acid or ammonia. I consider it to be a very happy-go- lucky process. CARMINE AND COCHINEAL STAINS. 155 235. Lithium-Carmine (0TH, Berlin, klin. Wochenschr., xxviii, 1883, p. 421). Superfluous, and macerates considerably (MAYER). 236. Ammonia-Carmine. In my opinion there is no valid excuse for using ammonia-carmine at all at the present day. If, however, such a stain be used, care should be taken to get rid of the free ammonia as completely as possible. This may be done by boiling until the excess of ammonia has evaporated. (So long as free ammonia is present large bubbles are formed in the fluid, and the latter shows a dark purple colour. When the free ammonia has evaporated small bubbles appear, and the solution takes a brighter red tint.) One per cent, each of carmine and ammonia in distilled water is a good proportion. But a safer mode of preparation is that of RAXVIER, as follows (kindly communicated by Dr. MALASSEZ, see Traite des Methodes techniques, &c., of Lee and Henneguy, 1st. edit., p. 82). Make a simple solution of carmine in water with a slight excess of ammonia, and expose it to the air in a deep crystallising dish until it is entirely dried up. It should be allowed to putrefy if possible. Dissolve the dry deposit in pure water, and filter. 7. Other Aqueous Carmines (Acid and Alkaline). 237. SCHWEIGGER-SEIDEL'S Acid Carmine (EAXVIER, Traite, p. 99). HAMAXN'S Acid Carmine (Intern. Mon. f. Anat. u. Hist., i, 5, 1884; Zeit. f. wiss. Hilt., ii, 1885, p. 87). Neutral Borax-Carmine (NiKi- FOROW, Zeit.f. wiss. Mik., v, 3, 1888, p. 337). Neutral Borax-Carmine (GRENACHER, Arch.f. mik. Anat., xvi, 1879, p. 466). HAUG'S modifications of this, Zeit. f. wiss. Mik., vii, 2, 1890, p. 151, and viii, 1, 1891, p. 52. WOODWARD'S Borax-Carmine (see Monthly Mic. Journ., vii, 1872, p. 38 ; Am. Quart. Mic. Journ., i, 1879, p. 220; Journ. Roy. Mic. Soc., ii, p. 613). H. GIBBES' Borax Carmine (see Journ. Roy. Mic. Soc., iii, 1883, p. 390). DELAGE'S Osmium- Carmine (Arch, de Zool. exp. et gen., iv, ser. 2, 1886 ; Zeit. f. wiss. Mik., iii, 2, 1886, p. 240). ROLLETT'S Carminroth (see Zeit. f. wiss. Mik., i, p. 91). PERL'S Soluble Carmine (see FRET, Das Mikroskop., 7 Auf., and Zeit.f. wiss. Mik., i, p. 91). Carminic Acid (see DIMMOCK, Amer. Natural., xviii, 1884, pp. 324-7 ; and Journ. Roy. Mic. Soc., 1884, pp. 471-4). Boric Acid Carmine (ARCAXGELI, see Proc.-verb. Soc. Toscana Sc. Nat., 1885, p. 283; and Zeit.f. wiss. Mik., 1885, p. 377). Boric Acid Alum- Carmine, ARCANGELI, ibid. Sali- cylic Acid Alum-Carmine, ARCANGELI, ibid. Salicylic Acid Car- mine, ARCANGELI, ibid. Picric Acid Carmine, ARCAXGELI, ibid. Picric Acid Carmine, MIXOT (see WHITMAN'S Methods in Mic. Anat., ]>. 12.i. Uranium-Carmine, GIERKE, Zeit. f. wiss. Mik., i, 1884, p. 92 ; .SCHMAUS, ibid., viii, 2, 1891 ; cf. Mdnchener med. Wochenschr., 1891, No. 8, p. 117. Carbonate of Soda Carmine, CUCCATI, Zeit.f. wiss. Mik., iv, 1, 1887, p. 50. 156 CHAPTER XIII. B. ALCOHOLIC CARMINE STAINS. 238. Alcoholic Borax-Carmine (GRENACHER, Arch. f. mik. Anat., xvi, 1879, p. 466, et seq.). Take a concentrated solution of carmine in borax solution (2 to 3 per cent, carmine to 4 per cent, borax) ; boil it for half an hour or more ; dilute it with about an equal volume of 70 per cent, alcohol, allow it to stand some time (twenty-four hours MAYER), and filter. Or the mixture of carmine and borax solution is allowed to stand for two or three days and occasionally stirred; the greater part of the carmine will dissolve. To the solution is added an equal bulk of 70 per cent, alcohol ; the mixture is allowed to stand for a week, and then is filtered. If on keeping more carmine is deposited, it must be refiltered. Preparations should remain in the stain until they are thoroughly penetrated (for days if necessary), and then be brought (without first washing out) into alcohol (of 70 per cent. ; this is absolutely necessary MAYER) acidulated with 4 to 6 drops of hydrochloric acid to each 100 c.c. of alcohol. They are left in this until the stain is differentiated, and may then be washed or hardened in neutral alcohol. Four drops of HC1 is generally enough. Three drops I find not quite sufficient. The stained objects should remain in the acidu- lated alcohol till they acquire a bright transparent look. This may require days (MAYER). For delicate objects, and for very impermeable objects, it may be well to increase the proportion of alcohol in the stain ; it may conveniently be raised to about 50 per cent. It should not exceed 60 per cent, in any case (MAYER). This stain is probably by far the most popular of any for staining in the mass. It is easy to use, and gives a most splendid coloration. But it is not so penetrating as is commonly supposed, and has the defect of sometimes forming precipitates in the cavities of bulky objects which cannot be removed by washing out. And it must be remembered that the fluid is alkaline, and therefore not suitable for delicate cytological work. I believe that some otherwise excellent cytological work has been vitiated by over-confidence in this reagent, and will have to be done over again. 239. MAYER'S Paracarmine (Mitth. Zool. Stat. zu Neapel, x, 3, 1892, p. 491). Carminic acid, 1 grin. ; chloride of alu- CAKMLM: AND COCHINEAL STAINS. 157 minium, 0'5 grm. ; chloride of calcium, 4 grins. ; 70 per cent, alcohol, 100 c.c. Dissolve cold or warm, allow to settle, and filter. A light red liquid, specially adapted for staining in bulk, and the nearest approach to a substitute for Grenadier's alcoholic borax-carmine that has yet been discovered. Objects to be stained should not have an alkaline reaction, nor contain any considerable amount of carbonate of lime (spicules or skeletal parts of corals, &c.), which would give rise to precipitates (MAYER). Wash out sections, or objects intended to be sectioned, with pure 70 per cent, alcohol. Objects intended to be mounted whole may be washed out with a weak solution of aluminium chloride in alcohol, or if this be not sufficient, with 5 per cent, common acetic acid (or 2*5 per cent, glacial acetic acid) in alcohol. For staining bulky objects with large cavities, such as Salpa, the solution should be diluted (with alcohol) ; and as this may cause precipitates to form during the staining, especially if the objects are not very clean, it is advisable to slightly acidify the dilute solutions. Paracarmine gives a nuclear stain of a red colour, though not so fiery red as that of borax-carmine. Its points of superiority over borax-carmine are that it is not alkaline, therefore less hurtful to tissues ; that it is more highly alcoholic, therefore more penetrating ; that it has less tendency to form granular precipitates in the interior of objects, and that it generally keeps perfectly without precipitating (mine has precipitated somewhat, though not to an injurious extent). 240. Alcoholic Hydrochloric-Acid Carmine. In view of the above- mentioned defects of borax-carmine defective penetration, and a tendency to form insoluble granular precipitates in the interior of objects it is desirable to possess a powerful staining medium more highly alcoholised and of acid reaction. Hydrochloric acid carmine possesses these qualities, and may be useful in the case of objects for which Mayer's paracarinine may not suffice. It may, for instance, be frequently useful in work on Arthropoda, especially the marine forms. GBEXACHEE'S receipt (Arch. f. mik. Anat., xvi, 1879, p. 468) will be found extremely troublesome by those who are not expert at neutralising. The following method, due to PAUL MAYER (quoted from GABBIKI'S Manuale per la Technica moderna del Microscopic, first ed., p. 46), is easy and gives excellent results. Take 100 grms. of alcohol (either absolute or of any weaker grade), 1 or 2 drops of HC1, and an excess of carmine, and boil until you get a clear solution, taking care that there remains an excess of carmine. This ought to give a nuclear stain, without the aid of HC1 for 158 CHAPTER XIII. washing out. If overstaining or diffusion should occur, wash out with alcohol, very slightly acidulated with HC1. BEASS (Zeit.f. wiss. Mik., ii, 1885, p. 303) takes 100 c.c. of 70 per cent, alcohol, 15 drops of HC1, and an excess of carmine. An old formula of PAUL MAYER'S (Mittli. Zool. Stat. Neapel, iv, 1883, p. 521 ; Journ. Roy. Mic. Soc. [N.S.], iv, 1884, p. 317), which gives a more powerful stain than the preceding, is somewhat inconvenient to carry out. A more recent formula of MAYER'S (Intern. Monatsschr. f. Anat., &c., 1887, p. 43) is as follows : Carmine, 4 grms. ; water, 15 c.c. ; hydrochloric acid, 30 drops. Boil till the carmine is dissolved, add 95 c.c. of 85 per cent, alcohol, and neutralise by adding ammonia until the carmine begins to precipitate. If it be desired to dilute any of these solutions, it should be done with alcohol, not water, and alcohol should be taken for washing out. 241. Alcoholic Cochineal (MAYEE'S Old Formula, Mittli. Zool. Stat. Neapel } ii, 1881, p. 14). Cochineal in coarse powder is macerated for several days in alcohol of 70 per cent. For each gramme of the cochineal there is required 8 to 10 c.c. of the alcohol. Stir frequently. Filter, and the resulting clear, deep red solution is fit for staining. The objects to be stained must previously be imbibed with alcohol of 70 per cent, and alcohol of the same strength must be used for washing out or for diluting the staining solution. The washing out must be repeated with fresh alcohol until the latter takes up no more colour. Warm alcohol acts more- rapidly than cold. Overstaining seldom happens ; it may be- corrected by means of 70 per cent, alcohol, containing yL- per cent, hydrochloric or 1 per cent, acetic acid. Small objects and thin sections may be stained in a few minutes, larger animals require hours or days. A nuclear stain, slightly affecting protoplasm. The colour varies with the reaction of the tissues, and the presence or- absence of certain salts in them. Crustacea with thick chi- tinous integuments are generally stained red, most other- organisms blue. The stain is also often of different colours in different tissue elements of the same preparation. Glands or their secretion often stain grey-green. In embryos of Lumbricus Kleinenberg found the vessels to stain red, their contents of an intense blue. Acids lighten the stain and make it yellowish red. Caustic alkalies turn it to a deep purple. The best stains are obtained in the case of objects that have been prepared with chromic or picric acid combinations, or CARMINE AND COCHINEAL STAINS. 159 with absolute alcohol. Osmic acid preparations stain very weakly unless they have been previously bleached. The acid must be carefully washed out before staining, or a diffuse stain will result. The stain is permanent in oil of cloves and balsam. The object for which this stain was imagined is to obtain an alcoholic staining fluid whose high penetrating power allows it to be employed in the case of organisms, such as Arthropoda, whose chitinous investments are but very slightly permeable by aqueous solutions of carmine. I have treated this stain at considerable length because I am convinced that it ought to be better known. It is very useful in many cases (Annelids, for instance), and indispen- sable for Arthropoda. On the other hand, it gives a very poor stain with unsuitable objects, and the majority of the objects with which the histologist has to do are in this case. For this reason MAYEE has been led to devise the fluid de- scribed in the following paragraph, which, containing in itself the salts necessary for producing a rich and energetic selectivity of stain, gives good results with all classes of ob- jects. He almost appears to intend it entirely to take the place of the old fluid. But I think the old fluid cannot yet be discarded. It has over the new fluid the (for some cases considerable) advantage of being more highly alcoholic; and it does not contain free acid, so that it can be used with cal- careous structures which it is wished to preserve which the new fluid cannot. For specimens of Pluteus, for instance, I find it excellent. 242. MAYER'S Alcoholic Cochineal, New Formula (Mitth. ZooL Stat. Neapel, x, 3, 1892, p. 498). Cochineal, 5 grms.; chloride of calcium, 5 grms.; chloride of aluminium, 0"5 grm.; nitric acid of 1'20 sp. gr., 8 drops; 50 per cent, alcohol, 100 c.c. Powder the cochineal finely and rub up in a mortar with the salts, add the alcohol and acid, heat to boiling-point, leave to cool, leave for some days standing with frequent agitation, filter. Use as the old tincture, the objects being prepared and washed out with 50 per cent, alcohol. The stain is like that of paracarmine, but not quite so strong and not so sharp. Mayer only recommends it as a succedaneum of paracarmine* 160 CHAPTER XIII. The object for which it was devised has been explained in the last paragraph, where also some limitations to its useful- ness have been indicated. For the chemical theory involved in the composition of these tinctures see also the last paragraph, and supra, 219. CHAPTER XIY. , HJIMATEIN (HAEMATOXYLIN) STAINS. v 243. Theory of Haematoxylin Staining. It appears to be now thoroughly well established (see NIETZKI, Chemie der organ- isclien Fdrbstoffe, Berlin, Springer, 1889, pp. 215 217) that the active colouring principle of haematoxylin dyes is hsema- tein ; and further, that the haematein of the usual histological staining solutions is a product of the oxidation of their con- tained haematoxylin by means of the air to which they are exposed (see MAYER, " Ueber das Farbeii mit Hamatoxylin" in Mitth. a. d. Zool. Station zu Neapel, Bd. x, Heft 1, 1891, pp. 170 186; UNNA, " Ueber die Reifung unserer Fdrbstojfe," in Zeit.f. iviss. Mik., viii, 4, 1892, p. 483). This change is known as " ripening/' and until it has taken place the solu- tions are not fit to use for staining. Hitherto it has been the practice to rely (quite uncon- sciously so far as the chemical theory is concerned) on the spontaneous absorption by the solutions of oxygen from the air to effect this "ripening," but it has now been discovered (by both MAYER and UNNA independently) that nothing is easier than to bring about the reaction artificially ; all that is necessary being, for instance, to add to a solution of haema- toxylin containing alum a little neutralised solution of per- oxide of hydrogen. The solution becomes almost instanta- neously dark blue, (( ripe," and fit for staining, thus definitely confirming the truth of the hypothesis. Mayer goes further. A solution of pure uncombined haematein would not afford a selective stain such as we require in histology ; it would be at most a dye. The usual solutions (I am not here speaking of Weigert's or similar processes) all contain alum, and Mayer holds that the active agent in them is a compound of haematein with alumina (" Haamatein- Thonerde" haemateateof alumina). He holds that this salt is precipitated in the tissues (chiefly in the nuclei) by certain 11 162 CHAPTER XIV. organic and inorganic salts there present (for instance, phos- phates) ; perhaps also by other organic bodies belonging to the tissues. Such salts are known to exist in living tissues. By the process of " fixing " they are fixed there, either in the same chemical state, as when such an agent as alcohol is used for fixing; or as fresh compounds formed by the action of the fixing agent itself, as when such a fixing medium as sublimate is used. The chief factor in the composition of these stains is, therefore after the colouring substance itself, alumina, to which, however, may be added some other bodies, such as lime, which may play a secondary role (as, for instance, chlo- ride of calcium does in Kleinenberg's solution). It follows that the first difficulty with which the worker with haematoxylin stains has to contend is that of getting his hssmatoxylin duly oxidised into hsematein, in order to the for- mation of the desired haematein-alumma compound, or lake. If this be done by the hitherto customary process of leaving the solutions to " ripen " by the action of the air, it is necessary to wait for a long time before the reaction is obtained. During all this time, ifc may be weeks or months, there is no means, except repeated trial, of ascertaining whether the solution at any moment contains sufficient haematein to afford a good stain. And here a second difficulty arises ; the oxidising pro- cess continuing, the solutions become " over-ripe ; " the haema- tein, through further oxidation, passes over into colourless compounds, and the solutions begin to precipitate. They are therefore, in reality, a mixture in constantly varying propor- tions of " unripe," " ripe/' and " over-ripe " constituents (the first and last being useless for staining purposes), and, incon- sequence, their staining power is very inconstant. These difficulties are only very imperfectly got over by any of the very various methods that have been proposed until lately. Now, however, thanks to the important dis- coveries of MAYER and UNNA, a great step in advance has been taken. The great point in Mayer's work is that not hasmatoxylin, but htematein, should be taken in the first instance for making the staining solutions. This at once relieves us from the tedious and uncertain process of " ripening " in the old way. We have a ripe solution to begin with, and we know that it H31MATEIN (H.EMATOXYLIN) STAINS. 163 must be ripe. A discovery of Unna's, to be mentioned below, affords a means of preventing the " over-ripening " brought about by excessive oxidation. 244. UNNA'S Half-ripe Constant Stock Solution (Zeit. f. wise. Mile., viii, 4, 1892, p. 483). This result may be attained by simply adding a reducing agent to tbe solution. Various reducing agents are available for this purpose (see the original paper) ; the most convenient method is the simple addition of a little sulphur. The following formula is recommended : Hseinatoxylin 1 Alum 10 Alcohol . . . .100 Water .... .200 Sublimed sulphur . . . . . . .2 If the sulphur be added to the hiematoxylin solution only when the latter has become somewhat strongly blue, i. e. after two or three days' time, the ^tage of oxidation attained by the solution will be fixed by the sulphur. The solution in this state may be used for staining. It will not give so energetic a stain as the solutions totally ripened with peroxide of hydrogen (see the paper quoted), and the stain will be somewhat more diffuse, but washing out with acids will not be necessary. If an energetic and more purely nuclear stain be desired, the solution should be treated with neutralised peroxide of hydrogen, as described in the paper quoted ; but this process is cumbrous and unnecessary, and I suppress it. MAYEE (Mittli. Zool. Stat. Neapel, xii, 2, 1896, p. 309) finds that the sulphur process does not preserve the solutions for long, whilst for some unexplained reason the simple addition of glycerin -does ; see below, " GLYCH.EMALUM." 245. Concerning Haematein. The following is taken from the two papers of MAYER quoted above, 217, 243. Hsematein occurs as a dark green mass, red by transmitted light, which may be rubbed up into a violet powder. It is entirely, though with difficulty, soluble in distilled water and in alcohol, giving a yellowish-brown solution, which remains clear on addition of acetic acid. Alkalies dissolve it with a blue-violet tint. It is now found in commerce ; but MAYER has hitherto only been able to procure it in a pure -state from GEIGY AND Co v in Bale. But there is also found in commerce an ammonia- compound of haematein Hsemate'in-Ammonicik, also known in commerce as Haemateinum crystallisatum ; this may be obtained in a sufficiently pure state from E. MERCK, of Darmstadt. This is somewhat more easily soluble in both water and alcohol than haematein is, and does quite as well for staining 164 CHAPTER XLV. purposes. The histologist can easily prepare it for himself as follows : 246. Haemateate of Ammonia \ (MAYER, ibid.). Dissolve 1 grm. of hsematoxylm with the aid of heat in 20 c.c. of distilled water, filter if necessary, add 1 c.c. of caustic ammonia (of 0-875 sp. gr.), and bring the purple liquid into a capsule of such dimensions that its bottom be not covered to a depth of more than half a centimetre. Let the liquid evaporate at the ordinary temperature and protect it from dust. The dry product will consist of hasmateate of ammonia, about equal in weight to the haematoxylin taken in the first instance. The evaporation should not be hastened by heat, as this may give rise to the formation of substances that are insoluble in alcohol. The preparation should not be touched, until it is dry, with any other instruments than such as are made of glass, porcelain, or platinum. 247. Characters of Hsematein Stains (or of Haematoxylin Stains). It is evident, from what has gone before, that all so- called haematoxylin stains are really produced by means of haematein. The alum-haematem lakes stain in different tones of blue or of red, according to the composition of the staining solution. Neutral or alkaline solutions give a blue stain ; acid solutions give a red one. In order to get a blue stain in preparations that have come out red through the acidity of the staining bath, it is a common practice to treat them with weak ammonia, in the belief that the blue colour is restored by neutralisation of the acid that is the cause of the redness. According to MAYER, the ammonia acts not by neutralising the acid, but by precipitating the alumina, which carries down the hasmatem with it (if no alumina were present the colour would be purple, not blue) . The same result can generally be obtained by merely washing out with common tap-water,, which is usually sufficiently alkaline (SquiRE has obtained the reaction with distilled water, free from even a trace of ammonia), and can be obtained with certainty by treatment with bicarbonate of soda or acetate of soda or potash. And this is the preferable course, as ammonia is certainly a danger- ous thing to treat delicate tissues with. Of course this is a different question from that of neutralising with an alkali H KMATEIN (H 31MATOXYLIN) STAINS. 165 tissues that have been treated with an acid to correct over- staining. Here the neutralisation may be indicated in the interest of the preservation of the stain, or of the tissues them- selves. SQUIRE (Methods, p. 22) finds that sections can be blued in a few seconds by treatment with a 1 : 1000 solution of bicar- bonate of soda in distilled water. MAYER holds that acetate of potash is the most inoffensive reagent to take ; a strength of 0-5 to 1 per cent, may be taken. According to WATNEY (see Phil. Trans., 1812, p. 1085 ; KRAUSE, Intern. Zeit.f. Anat. u. Hist., i, p. 154 ; and M. FLESCH, Zeit.f. wise. Mik., 1885, p. 358, from whoin I quote) the colour is an intense blue if the solution has been made with freshly prepared alum, whilst a red tone is obtained if the solution has been made -with old alum. The reason of this is that alum that has been long kept almost always contains free acid. The red solutions are said to exhibit a great affinity for connective tissue, and for the granules of "plasma-cells;" whilst the blue solutions show a special affinity for unicin and chromatin. But see on this point the remarks of MAYER quoted under " Mucin." It has been discovered by LAXGHANS (see MAX FLESCH, loc. cit.) that it is possible to obtain these two elective reactions with one and the same solution. All that is necessary is to stain with the solution of Delafield, mount the preparations in balsam, and expose them for some time to the light. The reaction is not obtained with glycerin mounts. None of the purely aqueous solutions are perfectly stable ; only one or two are fairly so. They can, however, be made to keep for a sufficient time, many months, by the addition of glycerin (EHRLICH); but the addition of the glycerin, as is well known, slows the stain greatly, and makes it somewhat less precise. (See below, Glychssmalum) . It has been explained above that the old haeinatoxylin solutions when freshly prepared stain badly and diffusely ; they ought either to be fi ripened " by Unna's method ( 243) or to be allowed to " ripen " spontaneously before use. This takes, according to the nature of the solution, a few hours, or days, or months. In my opinion there is no legitimate excuse for not making up stains with hasmatein, and the old formulae for haematoxylin solutions ought to be discarded. All of the solutions, when in good staining order, have a great tendency to over-stain. Over- stains may be corrected by washing out with weak acids (e. g. 0*1 to 0'2 or even 0*5 per cent, of hydrochloric acid), but this is not favorable to 166 CHAPTER XIV. the permanence of the stain. If acids be used, it is well to re-neutralise afterwards with ammonia or bicarbonate of soda (O'l per cent.). Bicarbonate of soda may be used for neutralisation with alcohol as the vehicle. It should be dissolved by the aid of heat in 70 per cent, alcohol (very little will dissolve), and the solution filtered after cooling. For neutralising tissues, three to five drops of the solution are added to a watch-glass full of 70 per cent, alcohol in which the tissues are soaked (voisr WISTINGHAUSEN, Mittli. Zool. Stat. Neap el 3 x, 1891, p. 41 ; Zeit. f. wiss. Mik., x, 4, 1893, p. 480). The best plan of all is to avoid over-staining by staining very carefully in extremely dilute solutions. RAWITZ (Anat. Anz.j xi, 10, 1895, p. 301) gives the proportion of one to three drops of a strong alum-hsematein solution to 25 to 50 c.c. of distilled water. Sections of picric or sublimate material may remain in this without over-staining for twenty-four to forty- eight hours. Chrom-osmium material is of course much less likely to over-stain, but even in this case the best results are obtained with very dilute solutions. It should be noted that the purest chromatin stains are obtained by staining for a short time (sublimate sections half an hour, say) in solutions of medium strength, such as hseinalum diluted ten to twenty- fold with water. The stain obtained either with very strong solutions, or with the slow stain of the dilute solutions, is at the same time a plasma- stain, which of course may or may not be desired. Chrom-osmium material will not yield a pure chromatin stain unless it is very fresh ; it is consequently next to impossible to obtain the reaction with paraffin sections of such material; they constantly give a plasma-stain in addition to the chromatin stain, which is not the case with sublimate material. Ghrom-osmium material, if not quite fresh, will require at least as many hours' staining as sublimate material does minutes. The stain is fairly permanent in balsam, but is sure to fade a little, and may fade a great deal. If acids have been used after staining, great care should be taken to wash them out thoroughly before mounting. In aqueous media the stain cannot be relied on to keep (this refers to the old solutions : MAYEE finds that his hsematein preparations have kept well both in aqueous media and, with certain precautions, in H^MATEIN (HJUMATOXYLTN) STAINS. 167 balsam). Turpentine-balsam should not be used (Mayer, in Utt.}, nor, I find, should alcohol-balsam. 248. General Remarks. We have the coal-tar colours for staining sections, and we have carmine and cochineal for staining in the mass. What, then, do we want haematein for ? The answer is that we sometimes want it for staining, either sections or in the mass, on -account of the faculty it has of staining tissues that have been treated with chromic and osmic mixtures. This it does in general better than any carmine or cochineal. We want it also for some special pur- poses, such as staining the NebenJcern and achromatic figures of nuclei, and for nerve researches and other special histo- logical objects. It is also a more powerful stain than carmine ; and according to the mode of employment affords either a chro matin stain or a sometimes valuable plasma-stain. The chief haematein plasma-stains are found not amongst the alum -haematein lakes, but amongst the other lakes, iron or chrome lakes, &c. The alum lakes will be treated of first ; then the other lakes, partly in this chapter and partly in Part II (Neurological Methods, Methods of WEIGERT, PAL, &c.). In view of the very great advantages offered by the mode of preparation worked out by MAYER I have no hesitation in saying that the old methods for preparing alumina-haematein lakes ought to be entirely discarded, and Mayer's formulae only, or at least only such formulae as conform to the principles laid down by him, used instead. For not only are his modes of preparation the most scientific, but, according to my experience, his solutions are found in practice to be greatly superior to the old ones. A. Alumina-hsematein Lakes. (24iJ MAYER'S Haemalum (Alum-hsematein Solution, Haema- laun ; MAYER, Mitth. a. d. Zool. Stat. zu Neapel, x, 1, 1891, p. 172; see also Zeit. f. wiss. Nik., viii, 3, 1891, p. 338, a very exact report, but somewhat short considering the im- portance of Mayer's paper). One grm. of the colouring matter (either haematein or the ammonia salt, 245, 246) dissolved with heat in 50 c.c. of 90 per cent, alcohol, and added to a solution of 50 gr. of alum in a litre of distilled water. Allow 168 CHAPTER XIV. the mixture to cool and settle, filter if necessary, and add a crystal of thymol to preserve from mould. A dark liquid of about the tint of Grenadier's borax-car- mine. It answers to Bohmer's haematoxylin,* stains at least as well, either at first, for it is ripe from the beginning, or later. It may be used either in the concentrated form or diluted. Concentrated, it stains almost instantaneously (sections have been stained by merely pouring- the liquid over them). Diluted twentyfold with distilled water it will still stain through the tentacles of a Tubularia in an hour. (Spring water or tap water containing lime must not be used for diluting ; perhaps weak solution of alum in distilled water is the best means of all.) After staining, sections may be washed out either with distilled or common water. The solution is admirable for staining in bulJc. Large objects will, however, require twenty-four hours' staining, and should be washed out for the same time (this should be done with 1 per cent, alum solution if a sharp nuclear stain be desired). The solution unfortunately does not keep perfectly, but becomes weak with age (see 251). Mayer's preparations have kept well in glycerin (care being taken not to have it acid), also in balsam. It is to be noted that if oil of bergamot be used for clearing, it must be thoroughly removed by means of oil of turpentine before mounting ; and that oil of cloves is danger- ous. It is best (Mayer, in litt.) to use only xylol, benzol, or chloroform, and to mount in xylol-balsam or chloroform- balsam. Haemalum may be mixed with alum-carmine, Saurefuchsin, or the like, to make a double-staining mixture ; but it seems preferable to use the solutions in succession. 250. MAYER'S Acid Haemalum (ibid., p. 174, note). This is haemalum with 2 per cent, glacial acetic acid (or 4 per cent, common acetic acid). To be used as the last, wash- ing out with ordinary water in order to obtain a blue- violet tint of stain. It has the advantage of being a perhaps even more precise nuclear stain than the last formula; and further, the solution does not precipitate through decompo- * As with Bohmer's formula, it is not necessary to conform exactly to the proportions given, and a rough and ready haemalum solution may be at any time extemporised by adding a few drops of alcoholic solution of haematein to an alum solution of any desired strength. H.KMATEIN (H^EMATOXYLIN') STAINS. 169 sition caused by the ammonia of the air and the alkali of the glass of bottles, which the simple haemalum solution does to a certain extent (though this does not affect its staining power). 251. MAYER'S Glychaemalum (Mitth. Zool. Stat. Neapel, xii, 2, 1&96, p. 310). Mayer has found that haemalum does not keep perfectly (I have also found that my solution has not. kept in a sufficiently satisfactory manner). By adding glycerin it may be made to keep perfectly clear for over a year; but as this slows the stain, more haematein should be added. The following formula is recommended : Haematein 0*4 grm. (to be rubbed up in a few drops of glycerin in a rnortar till it dissolves) ; alum, 5 grms. ; glycerin, 30 ; dis- tilled water, 70. The stain is not quite so purely nuclear as that of the simple haemalum, ground substance of cartilage, for instance, being stained. 252. HANSEN'S Solution (Zool. Anz., 1895, p. 158). Hansen having found that commercial haamatein is not of constant quality, sought for a convenient means of oxidising haema- toxylin into haematein,and recommends the following process. One grm. of hasmatoxylin crystals is dissolved in 10 grms. of absolute alcohol. Twenty grammes of potash alum are dissolved with the aid of heat in 200 grms. of distilled water, and the solution filtered after cooling. After twenty- four hours the two solutions are mixed. Three c.c. of concentrated aqueous solution of perman- ganate of potash are put into a porcelain, capsule (one part of permanganate 'dissolves in 16 of water at 15 C.). The haematoxylin solution is added, and the whole is gradually heated up to boiling-point with constant stirring. The solu- tion turns to a dark reddish violet. The boiling is continued for half a minute or a minute. The solution is then cooled, which is best done by floating the capsule on to cold water. It may be filtered after cooling if desired, but this is seldom necessary. The solution is now ripe and ready for use. The object of the permaganate in this process is of course to effect the instantaneous oxidation of the haematoxylin. Hansen fancies that it has another point of utility : that by destroying all germs that may be present in the ingredients it should make the solution keep better than the usual sola- 170 CHAPTER XIV. tions. As a matter of fact it does not ; mine formed a pellicle and strong precipitate in a few days, and the same was found at the Naples station (MAYER, in Hit.). With this exception I find the solution excellent; the stain is most beautiful, and cannot be distinguished from that of heemalum. MAYER (ffitth. Zool. 8 tat. Neapel, xii, 2, 1896, p. 309) thinks the presence of the manganese in the solution favours the formation of the precipitate. He suggests that the oxidation might be brought about by taking an alcoholic solution of ha3matoxylin, leaving it for a short time in contact with the permanganate, and then adding it to 5 per cent. alum solution. The alcohol would then not bring over mananese into the stain. 253. Bohmer's Hsematoxylin (Arch.f. mik. Anat., iv, 1868, p. 345; Aerzt. IntelligenzbL, Baiern, 1865, p. 382). Make (A) a solution of hseniatox. cryst. 1 part, alcohol (absolute) 12 parts, and (B) alum 1 part, water 240. For staining, add two or three drops of A to a watch-glassful Of B. The staining solution ought not to be made up at the moment of using, but should be made up beforehand and allowed to ripen for some days (Mayer says at least two months). The alcoholic solution of haBmatoxylin may be kept in stock; it becomes brown, but does not lose its properties. Washing out may be done with a O'o per cent, solution of alum in water, or with acids if desired. I consider this to be of merely historical interest. 254. Delafield's Hsematoxylin (Zeit.f. wiss. Mik., ii, 1885, p. 288).- The history of this formula is as follows : It had long been in use in the Institute of Pathology at Heidelberg, when it was communicated by Pfitznei to Flemming, who published it and particularly recommended it in his Zellstz., &c., p. 388, 1882. Flemming then attributed the formula to Grenadier, and in consequence the stain went for years by the name "Grenadier's hsematoxylin." Later on Flemming discovered that this attribution was erroneous, and attributed the formula to Prudden ; and in cconsequence it was thenceforth known for some time as " Prudden's heema- toxylin." In 1885 matters were set right by Prudden's explaining that tin stain was the invention of Delafield,and publishing the correct formula here quoted (in the formula as published by Flemming the proportions are some what different). To 400 c.c. of saturated solution of ammonia-alum* add 4 grins, o: hrematox. cryst. dissolved in 25 c.c. of strong alcohol. Leave it exposed to * Ammonia-alum dissolves in about 11 parts of water. H.T.MATEIN (flJIMATOXYLIK) STAINS. 171 the light and air in an unstoppered bottle for three or four days. Filter, and add 100 c.c. of glycerin and 100 c.c. of rnethylic alcohol (CH 4 0). Allow the solution to stand until the colour is sufficiently dark, then filter and keep in a tightly stoppered bottle. This solution keeps well, it may be said to keep for years. It is well to allow it to ripen for at least two months before using it. For staining, enough of the solution should be added to pure water to make a yery dilute stain ; and even then care should be taken not to leaye objects too long in the fluid. It is an extremely powerful stain. BUTSCHLI (Unters. iib. mikroscopische Schiiume u. das Protoplasma, &c., 1892 ; Zeit. f. wiss. Mik., ix, 2, 1892, p. 197) recommends, under the name of "acid hsematoxylin," solution of Delafield very strongly diluted, and with enough acetic acid added to it to give it a decidedly red tint. This giyes a sharper and more differentiated nuclear stain than the usual solution. I used Delafield's solution a good deal a few years ago, in view of its faculty for affording a plasma-stain at the same time as a nuclear stain. It sometimes gives good images of spindles, rication, Berlin ; it may be obtained from GRUBLER, 216). This is not a triple stain in the sense of giving three different colours in the result; the orange does not act as a separate stain, but as an agent for the differentiation of the gentian stain. Chromatin and nuclei are purple-red ; achro- matic spindle-fibrils grey to grey-brown, or, in very favorable cases, violet, and are very clearly brought out; attractive spheres, centrosomes, polar corpuscles, and " Zwischenkorper " reddish in light stains, brown- violet to black-brown in strong ones. N.B. The description of the process given by 218 CHAPTER XVIIF. above presumably applies to the case of loose sections. Working with sections fixed to the slide, I find that it does not apply quite exactly. An important difference is that I do not find that the orange washes out any of the gentian ; at all events, no perceptible clouds of colour come away. I have found it well to prolong the treatment with the orange to a.t least a quarter of an hour. The duration of the staining bath in the gentian, as given by Flemming, seems to me much too long for any objects ; half an hour seems quite enough. I consider this a very fine stain when successful. The re- sults are as described by Flemrning. But it is very capricious, one can never be certain of success. For this reason, and for others, I prefer for most objects the safranin and Kernschwarz stain described in the next chapter. 302. REINKE'S Modification of FLEMMING'S Orange Method (Arch. f. mik. Anat, xliv, 2, 1894, p. 262). Sections of material fixed in liquid of HERMANN are put for twenty-four hours into a concentrated solution of potassium sulphite. They are washed with water and stained for an hour or two in safranin. They are then well washed with water, and stained for twenty-four hours in a ' ' neutral " mixture of gentian and orange, prepared as follows. To a concentrated aqueous solution of gentian violet are added "a few drops" of a like solution of orange G. The solution precipitates in part, owing to the formation of an im- perfectly soluble " neutral 3 ' colour; but becomes almost clear again if an excess of water be added. A drop of the mixture placed on blotting-paper should form a violet or brown spot with a narrow orange border. The solution is not to be filtered, but the sections are to be stained in it as it is (it is impossible to make out whether Reinke means the undiluted mixture, or the mixture made almost clear by addition of water). It is said that the " neutral " solution maybe pre- served for future use by adding to it one third of alcohol. After staining, you differentiate rapidly with alcohol, and clear with clove oil. I have tried this process, and obtained exactly the same final results as with Flemming's process. I have not suffi- cient experience of it to be able to say whether it is any less capricious than Flemming's process. THE COAL-TAR PLASMA STAINS. 219 303. Metanil Yellow (Metanilgelb). See GEIESBACH (Zeit.f. wise. Mik., iv, 4, 1887, p. 448 ; see also Journ. Roy. Mic. Soc., 1889, p. 464). It is sanl to have a certain affinity for various elements belonging to the group of the connective tissues. 304. Sauregelb (Echtgelb), Tropaeolin O., Crocein, Gold Orange, tsee GKIESBACH, Arch.f. mik. Anat., xxii, p. 132). 305. Saurefuchsin (Acid Fuchsin, Fuchsin S, Acid Rubin, Rubin S, Saurerubin, Acid Magenta, Magenta S) The denomination " Patentsaurerubin " attributed to this colour by KCLTSCHIZKY (Anat. Anz., viii, 1893, p. 357) is erroneous; the proper name of the colour used by Kultschizky is "Rubin S, rein pat.," see Ergebnisse der Anatomic, iii, 1893, pp. 18, 19). The chemical description of this acid colour has been given ( 268) : it is im- portant not to confound it with basic t'uchsin, as seems to have been done by some writers. This is one of the best cytoplasm stains that I know of. I use a 05 per cent, solution in water, and allow it to act on sections for about five minutes. But it is a matter of chance whether one hits off exactly the desired effect in this way. The chief use of Saurefuchsin is as an ingredient in the EHRLICH-BIONDI-HEIDENHAIN stain, and EHRLICH'S Triacid, which see. It is also used as a specific stain for nerve tissue (see " Neurological Methods" in Part II). 306. The EHRLICH-BIONDI Mixture (or EffRLlCH-BiONDi-HjEl- DEXHAIX Mixture) (PfliigeSs Arch., xlii, 1888, p. 1; Zeit.f. wiss. Nik., v, 4, 1888, p. 520). This well-known stain is somewhat troublesome to prepare. It may be obtained ready made from GRUBLER and Co. The receipt is as follows : To 100 c.c. saturated aqueous solution of Orange add with continual agitation 20 c.c. satu- rated aqueous solution of Saurefuchsin (Acid Fuchsin) and 50 c.c. of a like solution of Methyl Green. (According to Krause, 100 parts of water will dissolve about 20 of Saure- fuchsin "Rubin S.], 8 of Orange G, and 8 of Methyl Green.) The solutions must be absolutely saturated, which only happens after several days. Dilute the mixture with 60 to 100 volumes of water. The dilute solution ought to redden if acetic acid be added to it; and if a drop be placed on blotting-paper it should form a 220 CHAPTER XVIII. spot bluish green in the centre, orange at the periphery. If the orange zone is surrounded by a broader red zone, the mixture contains too much fuchsin. According to M. HEIDENHAIN' s instructions (" Ueber Kern u. Protoplasma," in Festschr. Herrn. Geb. A. v. Kolliker gewidm., &c., 1892, p. 115; see Zeit. f. wiss. Mik., ix, 2, 1892, p. 202) the orange to be used should be " Orange Gr ; " the Acid Fuchsin or Saurefuchsin should be " Rubin S " (" Kubin " is a synonym of Fuchsin); and the Methyl Green should be " Methylgriin OO." And it is absolutely necessary that these ingredients be those prepared under those names by the Actienfabrik fur Anilinfabrication in Berlin. The strong solutions directed to be taken readily precipi- tate on being mixed. To avoid this it is recommended by SQUIRE (Methods and Formulae, &c., p. 37) to dilute them before mixing. Stain sections (N.B., sections only) for six to twenty-four hours. Dehydrate with alcohol, clear with xylol, and mount in xylol balsam. In the intention of the observers who have elaborated this stain, it is a progressive stain, and not a regressive one. It does not require any differentiation, and the sections should be got through the alcohol into xylol as quickly as possible iu order to avoid any extraction of the colour. The great point of difficulty in working with this stain is to prevent the colour of the methyl green from coming out in the alcohol. If (HEIDENHAIN, Arch. f. mik. Anat., xxxv, 1890, p. 173 ; Zeit. f. wiss. Mik., vii, 3, 1890, p. 357) the alcohol used for washing out be slightly alkaline, the stain of the fuchsin will become relatively pale in the result, and the stain of the methyl green relatively strong ; whilst a slightly acid reaction of the alcohol will produce the opposite effect. The energy of coloration of the fuchsin is often found to become weakened in kept solutions ; it may be restored by adding very dilute acetic acid until a marked intensification of the reel tint of the mixture is perceived. The precautions detailed above are not sufficient to ensure the correct working of the stain, particularly as regards the study of " attraction-spheres " and other achromatic elements. M. HEIDENHAIN (Ueber Kern und ProopZa$ma,Engelmaim, 1892, p. 116 ; see also Zeit. f. wiss. Mik., ix, 2, 1892, p. 202, a very THE COAL-TAR PLASMA STAJNS. 221 full report) gives the following instructions : Preparations made with the usual mixture are liable to fade; by acidifying tlu 1 mixture a stronger and more sharply selective stain is obtained, which does not fade. But too much acid must not be added, as this would cause a staining of the interfilar substances. Heidenhain's directions are as follows : Take some of the " commercial stock solution " (by which appears to be meant the undiluted mixture as given above), and dilute it with distilled water in the proportion of about 6 parts of the mixture to 400 of water. Fill two beakers with distilled water, and add to each a few drops of the diluted solution, so as to bring them both up to the same tint. The coloured liquid thus obtained will show, at the same time, a red tint owing to the rubin, a yellow tone owing to the orange, and a greyish* tone derived from the methyl green. Now add to one of the beakers, with continual agitation, drop by drop, a 1 : 500 solution of acetic acid until the colour of the liquid turns to a strong crimson, the former yellow tone disappearing, and the grey of the methyl green becoming less marked. The two beakers serve as standards for the degree of acidity that should be given to the staining bath. The dilute solution that you made by diluting the original stock solution * is now acidified with the dilute acetic acid, added drop by drop with continual agitation, and from time to time a few drops are taken from it and added to a beaker of distilled water until there is obtained the crimson tint of the first test-beaker. Addition of acid should then cease, and the staining bath is ready. If the preparations should not turn out quite satisfactory a small further quantity of acid may be added. Before staining, sections should be treated for a couple of hours with 01 per cent, acetic acid, then for ten to fifteen minutes with officinal tincture of iodine, and be rinsed with alcohol before bringing into the stain, in which they should remain for twelve to eighteen hours. The treatment with acid is necessary in order to ensure having the sec- tions acid on mounting in balsam. The primary object of the iodine is to remove any sublimate from the preparations (Heidenhain's descriptions refer to sublimate objects, but Ehrlich-Biondi mixture will work with chrom-osmium ob- * " Das aus der Stammlosung durch Verdiinnung gewonnene Quantum." 222 CHAPTER xvnr. jects) ; but it also enhances the power of staining of the chromatin with methyl green, and produces a more selective staining of protoplasmic elements. Nor will these precautions always suffice to ensure correct results. In Arch. f. mik. Anat., xliii, 3, 1894, p. 430,, M. j HEIDENHAIN adds that it is so highly important to attain and I maintain exactly the right degree of acidity, that further > precautions are necessary. The solution must not be filtered, for filtration may render it less acid. If it has been kept for some time a little more acid must be added; for it will have dissolved traces of glass, which is an alkaline body. HEIDEN- ! HAIN therefore recommends that it be preserved in rubber bottles. The following instructions for acidifying the EHKLICH- BIONDI mixture, also due to M. HEJDENHAIN ; are from a paper of WARBURG'S (quoted from Zeit. f. wiss. Mik., xi, 3, 1894, p. 383). To 2 c.c. of the Biondi mixture (1 : 30) (by this is presumably meant the original mixture as given above, but diluted with only 30 volumes of water instead of 60) add 40 c.c. of distilled water, 3 c.c. of 0'5 per cent, solution of Saurefuchsin, and 0'2 c.c. of one-fifth per cent, solution of acetic acid (or. according to GROUVEN, op. cit., xii, 3, 1896, p. 379, four drops). [Grouven here speaks of the mixture as "Triacid" (see next section) : there is a deplorable confusion in the nomenclature of these stains.] The characters of the stain when successful are much as follows : The nuclear staining is very sharp and good, the chromatic elements being coloured of a somewhat slaty blue. Cytoplasm is of a more or less violet or more or less orange red, and caryoplasui is of the same colour as the cytoplasm, but of a lighter tone. Cell membranes, nuclear membranes, achromatic fibrillar structures, the Nebenkern when present in a word, all the denser protoplasmic structures, are stained of the same colour as the hyaloplasm, but darker. After a very careful study of this admired stain, I find I cannot recommend it for any but the most special objects. It must in the first place, I think, be acknowledged that a reagent that can neither be prepared nor preserved without the minute precautions detailed above, is something much too great and good for human nature's daily food. The stain is a very fine one when successful, but the most minute pre- THE COAL-TAR IT.ASMA STAINS. 223 cautions will not ensure its being successful. It is very capricious, it seldom gives the same result twice running. The correct result should be a precise chromatin stain, com- bined with a precise stain of the plastin element of cytoplasm by the Saurefuchsin. Now the least defect or excess of acidity causes the plasma stain of the Saurefuchsin to be- come a diffuse one, instead of being sharply limited to the plastin element. And the methyl green, being very little resistent to alcohol, and ix, 3, 1893, p. 316) do not appear to me to constitute a useful contribu- tion to the subject. 378. The method of BROSICKE (Centralb.f. d. med. Wiss., 1879, p. 873 ; Zeit.f. wiss. Mik., i, 1884, p. 409) consists in treating osmium objects for twenty-four hours with a solution of one part of oxalic acid in fifteen parts- of water. This gives a Burgundy-red stain. It is necessary that the objects be washed and brought into the oxalic acid as soon as possible after the treatment with osmium, as if the osmium has once begun to blacken them, the oxalic acid is powerless afterwards to redden them. I consider this method to be superseded by the pyrogallol process. 379. Perchloride of Iron. This reagent, introduced by POLAILLON (Journ. de VAnat., iii, 1866, p. 43), sometimes gives most useful results, especially in the study of peripheral nerve-ganglia, in which it stains the nervous tissue alone, the connective tissue remaining colourless. The method consists in impregnating in perchloride of iron, and reducing in tannic, gallic, or pyrogallic acid. The HOGGANS, who have done very good work with this reagent, proceed as follows (Journ. Quekett Club, 1876 ; Journ. Roy. Mic. Soc., ii, 1879, p. 358) : The tissue (having been first fixed with silver nitrate, which is somewhat reduced by a short exposure to diffused light) is dehydrated in alcohol, and treated for a few minutes with 2 per cent, solution of per- chloride of iron in spirit. It is then treated with a 2 per cent, solution of pyrogallic acid in spirit, and in a few minutes more, according to the depth of tint required, may be washed in water and mounted in glycerin. FOL (see ante, 67) fixes in perchloride solution, and treats the prepara- tions for twenty-four hours with alcohol containing a trace of gallic acid. POLAILLON (I. c.) reduces in tannic acid. This method is not applicable to chromic objects. I should add that in my own experience I have found it very useful in certain special cases. 380. Pyrogallate of Iron (ROOSEVELT, Med. Rec., ii, 1817, p. 84 ; Journ. Roy. Mic. Soc., 1888, p. 157). A stain compound of 20 drops of saturated solution of iron sulphate, 30 grms. water, and 15 to 20 drops pyro- gallic acid. METALLIC STAINS (IMPREGNATION METHODS). 261 381. Palladium Chloride (F. SCHTJLTZE, see ante, 66 and 102). Prussian Blue (see LEBER, Arch.f. Ophthalm., xiv, p. 300; RANVIER, Traite, p. 108). Cupric Sulphate (see LEBEE, ibid.). Lead Chromate (see LEBEE, ibid.). Sulphides (see LANDOIS, Centralb.f. d. med. Wiss., 1885, No. 55 ; and GIEBKE, in Zeit. f. wiss. Hik., i, 1884, p. 497). Molybdate of Ammonium (MEEKEL ; KBAUSE) (see GIEBKE, Zeit. f. wiss. Mik., i, 1884, p. 96). Oxychloride of Ruthenium (NicoLLE and CANTACUZENE) (see Ann. Inst. Pasteur, vii, 1893, p. 331). Impregna- tion with Fats, Altmann's Method (see^ostf, " CoiTosion"). CHAPTER XXL EXAMINATION AND PRESERVATION MEDIA. 382. Introductory. I comprehend under this heading all the media in which an object may be examined. The old distinction of " indifferent " liquids, and those which have some action on tissues, appears to be misleading more than helpful ; inasmuch as it is now well understood that no medium is without action on tissues except the plasma with which they are surrounded during the life of the organism ; and this plasma itself is only " indifferent " whilst all is in situ ; as soon as a portion of tissue is dissected out and trans- ferred to a slide in a portion of plasma the conditions become evidently artificial. It does not appear necessary to create a separate group for mounting media, as all preservative media may be used for mounting. For directions as to making permanent mounts in fluid media see the early sections of Chap. XXII. 383. Water. To preserve it from mould, a lump of thymol or camphor should be kept in the supply. Water may be employed without inconveni- ence, and sometimes (on account of its low index of refraction) with great advantage for the examination of all structures that have been fixed with osmic or chromic acid, or some salt of the heavy metals; but it is by no means applicable to the examination of fresh tissues, that is, tissues that have not been so fixed. It is important that the beginner should bear in mind that water is very far from being an " indifferent " liquid ; many tissue-ele- ments are greatly changed by it (nerve-end structures, for instance), and some are totally destroyed by its action if prolonged (for instance, red blood-cor- puscles). 384. Theory of Indifferent Liquids. In order to render water inoffensive to such tissues as these it must, firstly, have dissolved in it some substance that will give it a density equal to that of the liquids of the tissue, so as to prevent the occurrence of osmosis, to which process the destructive action of pure water is mainly due. Salt solution is a medium sug- gested by this necessity. But salt solution by no means EXAMINATION AND PRESERVATION MEDIA. 263 fulfils all the conditions implied in the notion of an " indif- ferent " liquid. In so far as it possesses a density approach- ing to that of the liquids of the tissues, one cause of osmosis is eliminated; but there remains another, due to the dif- ference of composition of the liquids within the tissues and that without. Cell contents are a mixture of colloids and crystalloids; salt solution contains only a crystalloid, whose high diffusibility causes it to diffuse over into the colloids of the tissues. In order to reduce the consequent osmotic pro- cesses to a minimum, it is necessary that the examination medium contain, in addition to a due proportion of salt or other crystalloid, also a due proportion of colloids. By adding, for instance, white of egg to salt solution this end may be attained ; and, as a matter of fact, the liquids re- commended as indifferent are found invariably to contain both crystalloids and colloids. Thus (as stated by Frey) vitreous humour contains 987 parts of water to about 4' 6 of colloid matters and 7*8 of crystalloids (common salt). In 1000 parts of liquor amnii are contained about 3*8 parts of colloid matter (albumen), 5'8 of salt, and 3'4 of urea. In blood-serum, 8*5 of colloids and 1 of crystalloid substance are found. 385. Salt Solution (" normal salt solution," " physiological salt solution "). 0'75 per cent, sodium chloride in water. Carnoy recommends the addition of a trace of osmicacid. 386. PICTET'S Liquid (Mitth. Zool. Stat. Neapel, x, 1, 1891, p. 89). Five to ten per cent, solution of chloride of man- ganese. According to my experience, this solution is ex- cellent, and very often advantageously takes the place of "normal salt solution." The proportions given are for marine animals, and for terrestrial animals will generally be found much too high. For these from 1 per cent, to 3 per cent, will be nearer the mark. 387. Iodised Serum. Iodised serum was first recommended by Max Schultze (Virchow's Archiv, xxx, 1864, p. 263). I take the following instructions concerning it from Ranvier (Traite, p. 76). The only serum that gives really good results is the amniotic 264 CHAPTER XXI. liquid of mammals. A gravid uterus of a sheep or cow having been obtained (in large slaughterhouses such can be obtained without difficulty), an incision is made through the wall of the uterus and the foetal membranes. A jet of serum issues from the incision, and is caught in a flask prepared for the purpose. Flakes of iodine are then added, and the flask is frequently agitated for some days. Two points should be noted. A perfectly fresh amnios must be taken, for the merest incipience of putrefaction will spoil the preparation. The flask should have a wide bottom, so that the serum may form only a shallow layer in it ; otherwise the upper layers will not be sufficiently exposed to the action of the iodine. Another method is as follows : Serum is mixed with a large proportion of tincture of iodine ; the precipitate of iodine that forms is removed by filtration, and there remains a strong solution of iodine in serum. This should be kept in stock, and a little of it added every two or three days to the serum that is intended for use. Ranvier explains that at the outset serum dissolves very little iodine ; but if an excess of iodine be kept constantly present in the solution, it will be found that after two or three weeks iodides are formed, and allow fresh quantities of iodine to dissolve ; so that after one or two months a very strongly iodised serum is obtained. It should be dark brown. Such a solution is the most fitting for the purpose of iodising fresh serum in the manner directed above, and for making the different strengths of iodised serum that are required for different purposes. In general, for maceration purposes, a serum of a pale brown colour should be employed. 388, Aqueous Humour, Simple White of Egg. Require no preparation beyond filtering. They may be iodised if desired. 389. Artificial Iodised Serum (FEEY, Le Microscope, p. 131). Distilled water . . . . .135 grms. White of egg ..... 15 Sodium chloride ..... 0*20 grin. Mix, filter, and add Tincture of iodine .... 3 grms. EXAMINATION AND PRESERVATION MEDIA. 265 There is formed a precipitate, which is removed by filtering through flannel ; and a little iodine is added to the filtrate. 390. KRONECKER'S Artificial Serum (from YOGT et YUNG, Traite d'Anat. comp. prat., p. 473 : I have been unable to discover the original source). Common salt ...... 6 grms. Caustic soda 0'06 grm. Distilled water .... 1000 grms. 391. MIGULA'S Glycerized Blood-serum (see the paper in Zeit. f. wiss. Mik., vii, 2, 1890, p. 172; also Journ. Roy. Hie. Soc., 1890, p. 804). 392. Syrup. An excellent medium for examining many structures in the fresh state. To preserve it from mould, chloral hydrate may conveniently be dissolved in it (1 to 5 per cent.). I have used as much as 7 per cent., and found no disadvantage. Carbolised Syrup, Carbolic acid may be employed instead of chloral ; 1 per cent, is sufficient. Either of these syrups maybe used as a mounting medium, but they are not to be recommended for that purpose, as there is always risk of the sugar crystallising out. A good strength for syrup is equal parts of loaf sugar and water. Dissolve by boiling. 393. Acetate of Alumina (GANNAL'S Solution, BEALE, How to Work, Ac., p. 58). Acetate of alumina . . . . .1 part. Water . 10 parts. 394. Acetate of Potash (MAX SCHULTZE, Arch. mik. Anat., vii, 1872, p. 180). A nearly saturated solution in water. It is used by letting a drop run in under the cover-glass to the object, which is in water. After twenty- four hours the mount may be closed. The index of refraction is lower than that of glycerin. This medium has been frequently recommended as having the property of preventing the blackening of objects that have been treated with osmium ; but it seems extremely doubtful whether this is really the 395. Chloral Hydrate. 5 per cent, in water (LADOWSKY, Arch.f. mile. Anat., 1876, p. 359). 266 CHAPTER XXI. Or, 2'5 per cent, in water (BEADY, British Oopepods). Or, 1 per cent, in water (MUNSON, Journ. Roy. Mic. Soc. f 1881, p. 847). 396. Alcohol. Not very recommendable for mounting, as if taken weak it is not a very efficient preservative, and if taken strong it attacks the cement of mounts. CARPENTER (The Microscope) recommends a strength of 1 part to 5 of water. The chief use of alcohol for preservation purposes is of course for preserving specimens in till wanted for further preparation and study. See, on this point, the remarks in Chap. I, 3. 397. Formaldehyde. See 86 and 109. Mercurial Liquids. 398. GILSON'S Fluid (CAKNOY'S Biologie cellulaire, p. 94). Alcohol of 60 per cent. . . .60 c.c. Water 30 Glycerin 30 Acetic acid (15 parts of the glacial to 85 of water) . . . 2 Bichloride . . . . .0*15 grm. A really excellent medium for the study of fine cellular detail with well-fixed objects. 399. GAGE'S Albumen Fluid (Zeit. f. wiss. Mik., 1886, p. 223). White of egg . . . .15 c.c. Water 200 Corrosive sublimate . . 0'5 grm. Salt ...... 4 grins. Mix, agitate, filter, and preserve in a cool place. Recom- mended for the study of red blood-corpuscles and ciliated cells. 400. PACINI'S Fluids (Journ. de Mic., iv, 1880 ; Journ. Roy. Mic. Soc. [N.S.], ii, 1882, p. 702, and previous editions of this work). These anti- quated formulae are quite superfluous for the study of fixed tissues. They consist essentially of corrosive sublimate of from one half to one third per cent, strength, with the addition of a little salt or acetic acid. 401. HABTING'S Fluid. See Micro. Diet., art. " Preservation," p. 640. EXAMINATION AND PRESERVATION MEDIA. 267 402. GOADBY'S Fluids (Micro. Diet., art. "Preservation," or pre- Tious editions of this work). They are quite unsuited for histological pur- poses. 403. OWEN'S Fluid (see VOQT et YUXG, Traite d'Anat. comp. pratique, p. 19, or previous editions of this work). It is quite superfluous for tissues that have been duly fixed. Other Fluids. 404. Chloride and Acetate of Copper (RIPART et PETIT' s fluid, Brebissonia, 1880, p. 92; CARNOY'S Biol. cell., p. 95). Camphor water (not saturated) . 75 grms. Distilled water . . . .75 ,, Crystallised acetic acid ... 1 grm. P Acetate of copper .... 0*30 Chloride of copper .... 0'30 This is certainly a most valuable medium for work with delicate fresh tissues. It may be used in combination with methyl green, which it does not precipitate. The most delicate elements are perfectly preserved in it ; the addition of a drop of osmic acid or corrosive sublimate does not cause the least turbidity, and enhances its fixing action. 405. Tannin (CAENOY, Z. c.). Water 100 grms. Powdered tannin . . . 0'50 grm. 406. Picro-carmine. Picro-carmine has been recommended by Eanvier as a medium for teasing fresh tissues in, in the belief that it possesses suffi- cient fixing action to preserve the form of cells. Carnoy finds that cells live in it for a considerable time, and become gorged with water and deteriorated to a considerable degree. Unfortunately, too, picro-carmine cannot be com- bined with a good fixing agent, as it is precipitated by alcohol and by acids, and especially by osmic acid. 407. Methyl Green. See under STAINING AGENTS. The aqueous solution is very useful as an examination medium for fresh tissues. It should be taken fairly concentrated, in which state it has sufficient fixing power, which is enhanced by the addition of a trace of osmic acid. 408. WICKEESHEIMER'S Fluid (Zool. Anz., 1879, p. 670; cf. Journ. Boy. Mic. Soc., 1882, p. 427; id., 1880, p. 355; and Entomol. Nachr. 1880. p. 129). This once famous fluid appeal's to be quite unsuccessful for histological purposes. 409. MEYER'S Salicylic Vinegar Preservative Solutions (Arch, mik. An at., xiii, 1876, p. 868). "Salicylic vinegar" is a 2 68 CHAPTER xxr. solution of 1 part of salicylic acid in 100 parts of pyroligneous acid. The pyroligneous acid should be of 1*04 specific gravity, and should be of a pale yellow colour. This product is found in commerce, and maybe obtained from Herrn J. M. Andreas, Droguerie-Handlung, Frankfurt-a.-M. IST FLUID : One vol. salicylic vinegar to 10 vols. of the following dilute glycerin, viz. glycerin 1 vol., water 2 vols. For various larvae, Hydras, Nematodes, &c. 2ND FLUID : One vol. salicylic vinegar to 10 vols. of the following dilute glycerin, viz. glycerin 1 vol., water 4 vols. For Infusoria. 410. NOLL'S Salicylic Vinegar and Gum Medium (Zool. Anz., 1883, p. 472). A mixture of equal vols. of Meyer's second fluid (ante, last formula) and Farrant's medium (post, 413.) This mixture never becomes turbid, and does not dry up. The covers may be luted with asphalt or any other cement. The fluid answers admirably for delicate Crustacea and their larvae; the preparations do not shrink, and are not too much cleared. It also answers well for hardened and stained pre- parations of Hydroids, small Medusae, and other Coelen- terates. 411. DEANE'S Medium (see Micro. Diet., art. " Preservation "). Appears to be now superfluous. 412. HOYER'S Gum with Chloral Hydrate or Acetate of Potash (Biol. Centralb., ii, 1882, pp. 23-4; Journ. Roy. Mic. Soc. [N.S.], iii, 1883, pp. 144-5). A high 60 c.c. glass with a wide neck is filled two thirds full with gum arabic (in pieces), .and then either a solution of chloral (of several per cent.) containing 5 10 per cent, of glycerin is added, or acetate of potash or ammonia. The gum with frequent shaking dissolves in a few days, and forms a syrupy fluid, which is slowly filtered for twenty-four hours. The clear filtered fluid will keep a long time, but if spores of fungi begin to develop a little chloral can be added and the fluid refiltered. The solution with chloral is for carmine or haematoxylin objects that with acetate for anilin objects. 413. FARRANT'S Medium (BEALE, How to Work, &c., p. 58). EXAMINATION AND PRESERVATION MEDIA. 269- Picked gum arabic . . .4 ounces. Water 4 Glycerin . . . . . . 2 To be kept in a stoppered bottle with a lump of camphor. This medium is quoted by Frey as consisting of equal parts of gum, glycerin, and saturated aqueous solution of arsenious acid. The Micrographic Dictionary gives the following directions : Gum arabic 1 ounce, glycerin 1 ounce, water 1 ounce,, arsenious acid H grains ; dissolve the arsenious acid in the water, then the gum (without heat), add the glycerin, and incorporate with great care to avoid forming bubbles. Another method for making this medium is given by A. F. STANLEY KENT in Journ. Roy. Mic. Soc., 1890, p. 820. 414. Gum and Glycerin Medium (LANGERHANS* formula, a modification of FARRANT'S medium, Zool. Anzeig., ii, 1879,. p. 575). Gummi arab. ...... 5'0 Aquae ....... 5'0 To which after twelve hours are added Glycerini ....... 5*0 Sol. aquosa acid, carbol. (5*100) . . 10*0 Marine animals may be preserved in this by simply run- ning in a drop under the cover, and next day or later adding what is necessary to make up for evaporation, and closing the mount. Shrinkage is very slight, and most colours keep well. 415. FARIS'S Glycero-gum (The Microscope, x, 1890, p. 59 ; Journ. Roy. Mic. Soc., 1890, p. 414). Gum arabic 2 ounces, glycerin 1*5 ounces, water 1*5 ounces, thymol 1 grm. Mix,. dissolve with heat, and filter. 416. Gum and Glycerin Jelly (SHIMER, The Microscope, ix, 1889, p. 138; Journ. Roy. Mic. Soc. f 1890, p. 411). Equal parts of glycerin jelly (Foi/s second formula, post, 432), Farrant's solution, and glycerin. 417. COLE'S Gum and Syrup Medium. See 179. 418. APATHY'S Gum and Syrup Medium (see Chap. IX, 293). This medium is recommended by Apathy in a general 270 CHAPTER XXI. way, and not merely for the special purpose for which it is quoted in 293. It sets very hard, and, combined with a paper cell (see 453), may be used for ringing glycerin mounts. 419. FABRE-DOMERGUE'S Glucose Medium (La Nature, No. 823, 9, Mars, 1889, supp.). Glucose syrup diluted to twenty- five degrees of the areometer (sp.gr. 1-1968) . . . 1000 parts. Methyl alcohol . . . . 200 Glycerin 100 Camphor, to saturation. The glucose is to be dissolved in warm water, and the other ingredients added. The mixture, which is always acid, must be neutralised by the addition of a little potash or soda. This medium is said to preserve without change almost all animal pigments. 420. BRUN'S Glucose Medium (from FABRE-DOMERGUE'S Pre- miers Principes du Microscope et de la Technique micros copique, Paris, 1889, p. 123). Distilled water .... 140 parts. Camphorated spirit . . . 10 Glucose . . . . . . 40 Glycerin . . . . . 10 Mix the water, glucose, and glycerin, then add the spirit, and filter to remove the excess of camphor which is pre- cipitated on mixing. I am indebted to Dr. HENNEGUY for calling my attention to this liquid, which is an important one. It is preferable to glycerin because it preserves the colour of preparations stained with anilin dyes, methyl green included. 421. Levulose as a Mounting Medium. Levulose is recommended as a mounting medium by BEHKENS, KOSSEL, u. SCHIEFFEEDECKEK (Das Mikroskop u. d. Meth. d. mile. TTnters., Braunschweig, 1889). It is un- crystallisable, and preserves well carmine and coal-tar stains (ha3matoxylin stains fade somewhat in it). The index of refraction is somewhat higher than that of glycerin. Objects may be brought into it out of water. Glycerin Media. 422. Glycerin. Glycerin diluted with water is frequently employed as an examination and mounting medium. Dilution EXAMINATION AND PRESERVATION MEDIA. 271 ith water is sometimes advisable from an optical point of view, on account of the increased visibility that it gives to many structures by lowering the index of refraction of the glycerin. But from the point of view of efficadious preserva- tion it is always advisable to use undiluted glycerin, the strongest that can be procured. Long soaking of tissues in glycerin of gradually increased strength is a necessary preliminary to mounting in all cases in which it is desired to obtain the best possible preparations, and to ensure that they shall keep well. If this soaking is done on the slide (the cover being removed and the object treated with fresh glycerin every one or two days), it is well to take the precaution recommended by Beale, of luting the edges of the cover so as to make the preparation air-tight, as glycerin is so highly hygroscopic that a drop of it exposed to the air rapidly diminishes in strength to a very considerable degree. In order to facilitate the removal of the cover in this process, the slide may be gently warmed by passing it two or three times through the flame of a spirit lamp. No preparation can be considered to be made secundum artem until every part of the object has been thoroughly impregnated with strong pure glycerin. The shrinking that frequently occurs when delicate struc- tures are brought into glycerin may generally be cured by this treatment ; cells which first appear hopelessly collapsed gradually swell out to their normal forms and dimensions. For closing glycerin mounts, the edges of the cover should first (after having been cleansed as far as possible from superfluous glycerin) be painted with a layer of glycerin jelly ; as soon as this is set a coat of any of the usual cements may be applied. This has of course been for the last twenty years one of the commonplaces of histological technic, but that has not prevented somebody from recently describing the process at great length as new. Glycerin dissolves carbonate of lime, and is therefore to be rejected in the preparation of calcareous structures that it is wished to preserve. 423. Extra-refractive Glycerin. The already high index of refraction of glycerin (Price's glycerin, n = 1'46) may be raised to about that of crown glass by dissolving suitable substances in the glycerin. Thus the 272 CHAPTER XXI. refractive index of a solution of chloride of cadmium (CdCL)* in glycerin may be 1'504 ; that of a saturated solution of sulphocarbolate of zinc in glycerin maybe I'oOl ; that of a saturated solution of Schering'sf chloral hydrate (in crusts) in glycerin is I'olO ; that of iodate of zinc in glycerin may be brought up to I'oB.J The clearing action of glycerin may thus be greatly increased, and the full aperture of homogeneous objectives brought to bear on objects mounted in one of the above-named solutions. The sulphocarbolate of zinc solution may be prepared by taking equal parts by weight of Price's glycerin and sulphocarbolate of zinc crystals, mingling the two, and applying sufficient heat to boil the glycerin. The solution can be made in about an hour, but no fear need be had about boiling too long, as the longer this is done the less liability will there be for the solution to deposit crystals on the bottom of the bottle when cooled, which it will do if the temperature is only kept up long enough to dissolve the crystals. Filter while hot. The index may be brought up to T525 if desired by evaporating the solution somewhat, or by adding more carbolate. 424. BAEFF'S Boroglyceride (seeJourn. Roy. Mic. Soc., 1882, p. 124). This preparation may be obtained (price Is. per bottle) from The Kreo- chyle Company, Viaduct House, Farringdon Street, E.C., or all whole- sale chemists. 425. Glycerin and Alcohol Mixtures. These most useful fluids afford one of the best means of bringing delicate objects gradually from weak into strong glycerin. The object is mounted in a drop of the liquid, and left for a few hours or days, the mount not being closed. By the evaporation of the alcohol the liquid gradually increases in density, and after some time the mount may be closed, or the object brought into pure glycerin or glycerin jelly. 1. CALBERLA'S LIQUID : Glycerin ...... 1 part. Alcohol . . . . . . 1 Water 1 A most valuable examination fluid. As already pointed out (p. 5), this liquid is in many cases to be preferred to alcohol for keeping fixed objects in until required for dissection or other further preparation. 2. I strongly recommend the following for very delicate- objects : * Journ. Roy. Mic. Soc., ii, 1879, p. 346. f Ibid. (N.S.), i, 1881, p. 943. J Ibid., p. 366. Ibid., iii, 1880, p. 1051. EXAMINATION AXD PRESERVATION MEDIA. 273 Glycerin ...... 1 part. Alcohol ...... 1 Water 2 parts. 3. H^ENTSCH'S LIQUID : Glycerin ...... 1 part. Alcohol ...... 3 parts. Water . . . . . . 2 4. JAGER'S LIQUID (quoted from VOGT and YUNG'S Traite d'Anat. comp. prat., p. 16) : Glycerin . . . . . .1 part. Alcohol . . . . . . 1 Sea water . . . . . .10 parts. 426. DEANE'S Glycerin Jelly (from FEET'S Le Microscope, p. 231). 120 grammes glycerin, 60 grammes water, 30 grammes gelatin. Dissolve the gelatin in the water, and add the gly- cerin. This, and the following glycerin jellies, must of course be used warm. 427. LAWRENCE'S Glycerin Jelly (DA VIES, Preparation and Mounting of Microscopic Objects, p. 84). " He takes. a quan- tity of Nelson's gelatin, soaks it for two or three hours in cold water, pours off the superfluous water, and heats the soaked gelatin until melted. To each fluid ounce of the gelatin, whilst it is fluid but cool, he adds a fluid drachm of the white of an egg. He then boils this until the albumen coagulates and the gelatin is quite clear, when it is to be filtered through fine flannel, and to each ounce of the clarified solution add 6 drachms of a mixture composed of 1 part of glycerin to 2 parts of camphor water." 428. BEALE'S Glycerin Jelly (Row to Work, &c., p. 57). Gelatin or isinglass, soaked, melted, and clarified if desired, as in the last formula. To the clear solution add an equal bulk of strong glycerin. 429. BRANDT'S Glycerin Jelly (Zeit.f. wiss. Mik., ii, 1880, p. 69; Journ. Roy. Mic. Soc., iii, 1880, p. 502). Melted gelatin 1 part, glycerin 1 1 parts. The gelatin to be soaked in water and melted in the usual way. After incorporating the glycerin, the mixture is to be 18 274 CHAPTER XXI. filtered. This is a point of vital importance, as the gelatin of commerce is always mixed with particles of dust and minute threads. Swedish filtering-paper does not allow the fluid to pass through sufficiently, and flannel produces more threads than before. The following simple apparatus is found effec- tive. A wide-necked bottle is broken in two, and the upper part taken. The neck is stopped with a cork having two holes bored in it. In the first hole a glass tube, about 20 cm. long, is inserted so as to project a little into the inside of the bottle, and on the outside it is bent sharply to one side and drawn out into a point of about 14 to 2 mm. diameter. In the second hole a funnel-shaped filter is inserted so that the coni- cal part is inside the bottle and the tube projects a few centi- metres beyond the cork and the neck of the bottle. The apparatus is then placed so that the wide opening of the bottle and of the funnel is uppermost, and some spun glass is pressed into the lower conical part of the filter. In using the apparatus the funnel is filled with glycerin gelatin, and the bottle with hot water, which runs off slowly through the tube in the first hole and is constantly replenished. Some drops of carbolic acid should be added to the fluid product of the filtering. For mounting, use warm by melt- ing a small portion on the slide, the object having been pre- viously soaked for some time in a small bottle of the medium warmed with a suitable apparatus. 430, KAISER'S Glycerin Jelly (Bot. Cent., i, 1880, p. 25 Journ. Roy. Mic. Soc., iii, 1880, p. 504). One part by weight finest French gelatin is left for two hours in 6 parts by weight distilled water, 7 parts of glycerin are added, and for every 100 grammes of the mixture 1 gramme of concentrated car- bolic acid. Warm for ten to fifteen minutes, stirring all the while, until the whole of the flakes produced by the carbolic acid have disappeared. Filter whilst warm through the finest spun glass laid wet in the filter. Use for mounting as above. I prepared some of this jelly many years ago, and find it is still perfectly clear. 431. SEAMAN'S Glycerin Jelly (Amer. Mon. Mic. Journ., ii, 1881, p. 45 ; Journ. Roy. Mic. Soc. [N.S.], i, 1881, p. 534). Dissolve isinglass in water, so that it makes a stiff jelly when at the ordinary temperature of the room, add one tenth as EXAMINATION AND PRESERVATION MEDIA. 275 much glycerin, and a little solution of borax, carbolic acid, or camphor water. Filter whilst warm through muslin, and add a little alcohol. 432. FOL'S Glycerin Jellies (Lehrb., p. 138). 1. Melt together one volume of Beale's jelly (428) and one half to one volume of water, and add 2 to 5 per cent, of .salicylic acid solution, or carbolic acid or camphor. 2. Gelatin 30 parts. Water 70 Glycerin 100 Alcoholic solution of camphor . 5 Prepare as before, adding the camphor last. 3. Gelatin 20 parts. Water 150 Glycerin 100 Alcoholic solution of camphor . . 15 433. SQUIRE'S Glycerin Jelly (SQUIRE'S Methods and Formulae, &c., p. 84). Soak 100 grms. of French gelatin in chloroform water, drain when soft, and dissolve with heat in 750 grms. of glycerin. Add 400 grms. of chloroform water with which has been incorporated about 50 grms. of fresh egg-albumen ; mix thoroughly, and heat to boiling-point for about five minutes. Make up the total weight to 1550 grms. with chloroform water. Filter in a warm chamber. 434. GILSON'S Chloral Hydrate Jelly (kindly communicated by Prof. GILSON). 1 vol. of gelatin, melted secundum artem, and 1 vol. of Price's glycerin. Mix, and add 1 vol. of chloral hydrate (i. e. add crystals of chloral until the volume of the mixture has increased by one half) ; warm till dissolved. This gives a very highly refractive aqueous mounting medium, which is found useful for opaque tissues that it is desired not to dehydrate. A similar medium is published by GEOFFROY, Journ. de Eotan., 1893, p. 55 (see Zeit. f. iviss. Mik., ix, 4, 1893, p. 476). He dissolves, by the aid of as little heat as possible, 3 to 4 grms. of gelatine in 100 c.c. of 10 per cent, aqueous solution -of chloral hydrate. 435. STEPHENSON'S Biniodide of Mercury and Iodide of Potas- 276 CHAPTER XXI. slum (Journ. Roy. Hie. Soc. N.S., ii, 1882, p. 167). A solution of the two salts in water. " This is very easily prepared by adding the two salts to the water until each shall be in excess ; when this point of saturation has been reached the liquid will be found to have a refractive index of 1*68, by far the highest aqueous solution known to me. Its advantages from an optical point of view are considerable, and it may be used of any strength ; commencing with pure water, with a refractive index of 1*33, we can go on progressively to 1*465, which represents glycerin, still on to 1'54 (Canada balsam), again onwards to 1*624, which represents bisulphide of carbon, to 1*658, which represents the monobromide of naphthalin, to 1*662, the equivalent of a solution of sulphur in bisulphide of carbon, until, undiluted, it finally reaches its own maximum of 1*680; thus we have the representatives of all these media and an infinite number of others in this one fluid. " This fluid is very dense, its specific gravity being 3*02. It is highly antiseptic. " Its refractive index being 1*68, the visibility of diatoms, when mounted in it, is represented by the number 25 as com- pared with 11 in Canada balsam, in other words, the image is nearly two and a half times as strong For muscular fibre, on the other hand, a strong solution is not suitable, since the high refractive power of the object ap- proaches that of the medium, but as every other medium, of | a lower index than 1*68 can, by dilution, be represented by it, any degree of visibility down to that of water can be obtained. " For marine animals a weak solution is probably well adapted, as about a 1 per cent, solution (5 minims to the ounce) will give the specific gravity of sea water, with no appreciable difference in the refractive index." Covers should be sealed with white wax, and the mounts finished with two or three coatings of gold-size and one of shellac. I have experimented both with strong and with weak solutions. They are not adapted, I find, for the purposes of a permanent mounting medium. Tissues are well preserved, but the preparations are ruined by a precipitate which forms in the fluid. But as a temporary examination medium I have occasionally found this solution valuable. Its optical properties are wonder- ful ; it allows of the examination of watery tissues, without any dehydra- EXAMINATION AND PRESERVATION MEDIA. 277 tion, in a medium of refractive index surpassing that of any known resinous medium. 436. Monobromide of Naphthalin. See Journ. Roy. Hie. Soc., 1880, p. 1043 (ABBE and VAN HEUBCK), and Zool. Anz., 1882, p. 555 (MAX FLESCH). 437, THOMPSON'S High Refractive Medium. See Journ. Roy. Hie. Soc., 1892, p. 902. Resinous Media. 438. Resins and Balsams. Resins and balsams consist of a vitreous or amorphous substance held in solution by an essen- tial oil. By distillation or drying in the air they lose the essential oil and pass into the solid state. It is these solidi- fied resins that should, in my opinion (and that, I believe, of the best microscopists), be employed for microscopical pur- poses ; for the raw resins always contain a certain proportion of water, which makes it difficult to obtain a clear solution with the usual menstrua, is injurious to the optical properties of the medium and to its preservative qualities, and, further, especially hurtful to the preservation of stains. I therefore recommend that all solutions be made by heating gently the balsam or resin in a stove until it becomes brittle when cold, and then dissolving in an appropriate menstruum. Solid resins are now easily found in commerce. FOL (Lehrb.f pp. 1389) is of a different opinion. Solutions made with volatile menstrua, such as xylol and chloroform, set rapidly, but become rapidly brittle. Solutions made with non- volatile media, such as turpentine, set much less rapidly, and pass much less rapidly into the brittle state. The former should, therefore, be employed whenever it is desired to have a mount that sets hard rapidly; but the latter should be employed whenever it is above all desired to have a mount that will prove as durable as possible. According to my experience, there is no such thing as a faultless resinous mounting medium for histological purposes. Solutions of gum Damar in xylol are very beautiful from the physical point of view, and frequently afford a better defini- tion of delicate detail than Canada balsam does. But I am convinced that no Damar solution is perfectly stable. A review 278 CHAPTER XXI. of some old Damar mounts has shown that the majority of them have developed granules that have deteriorated the preparations to a greater or less extent. (These granules are in the worst cases large enough to at once attract attention even with low powers ; at other times they are so small that they can only be seen with the highest powers, and in this case may be mistaken for normal elements of cells.) Xylol balsam and benzol balsam mounts are in the same case, but to a less degree. Chloroform balsam keeps much better, so far as granules are concerned. But it becomes very brown with age, and has the defect that it is injurious to stains made with coal-tar colours. Seiler's alcohol-balsam keeps remark- ably well, but it also will not preserve the coal-tar stains or haamatein stains. I use it for carmine stains. For coal-tar stains I now generally use turpentine colophonium. It gives very good definition of delicate detail, and keeps perfectly. (Dr. PAUL MAYER, however, writes me that turpentine solu- tions are not at all good for hsematein stains). Turpentine colophonium has a rather low index of refraction for objects that require much clearing. For these I very frequently use oil of cedar wood in preference to any resinous medium. It gives perfect definition of all elements of the right index of visibility, and it keeps perfectly. With time it thickens sufficiently to hold the cover in place ; or, if desired, prepara- tions may be luted with Bell's cement. After using an oil immersion objective on a fresh mount, it is always easy to change the cover by floating it up with a drop of the oil placed at the side. Another reason for preferring turpentine-colophonium where possible is that it does not shrink in drying nearly so much as the media made with volatile solvents. Workers who use benzol balsam, for instance, generally mount sections with strips of paper interposed between the slide and the cover, in order to prevent the sections from being crushed by the cover as it is drawn down in the process of drying. With turpentine media this is not necessary. Still another motive is that turpentine media preserve the index of visibility of the preparations much longer than do media made with volatile menstrua. Preparations made with these last become so transparent in course of time that much fine detail is often lost. (Such mounts may, however, be EXAMINATION AND PRESERVATION MEDIA. 279 revivified without removing the cover by putting them for a day or two into a tube of benzol ; the benzol penetrates the balsam, and brings it down to a lower refractive index.) The visibility of minute structures is proportional to the difference between the refractive indices of the object and of the medium in which it is mounted. The majority of the elements of soft tissues are of an index of refraction somewhat superior to that of Canada balsam. It follows that by lowering the index of the balsam, increased visibility is obtained, and the desideratum in any case is to find a medium just low to give good visibility, and yet not so low as to serioi|sljj"jsp^ ; down the X.A. of the objectives employed. 439. Choice of a Mounting Medium. For the foregoing reasons I recommend turpentine-colophonium for general work ; whilst for cases in which a more highly refractive medium is desired, I would recommend oil of cedar for coal- tar stains, and SEILER'S alcohol-balsam for carmine stains. For haematein stains perhaps xylol-balsam, though those of my haematein preparations of which I have full notes have kept very well in either cedar oil or turpentine-colophonium. Xylol-balsam is certainly a very fine medium. I have merely wished to point out that it is not perfectly safe on the score of the possible formation of granules. (P. MAYER, in litt., is of the same opinion.) 440. Canada Balsam. Prepare with the solid balsam as above described, 438. The usual menstrua are xylol, benzol, chloroform, and turpentine. Dissolve the solid balsam in one of these to the required consistence. The turpentine solution is to be preferred only in cases where it is desired to have a medium that sets very slowly, or in view of the better pre- servation of certain stains. (The objection to turpentine as a solvent is that it does not always give a homogeneous solution with Canada balsam as it does with colophonium.) For most other purposes the xylol solution is the best. If time be an object, a benzol solution should be preferred, as it sets much quicker than the xylol solution. HEYS states that if the chloroform solution be poured into long, thin, half-ounce phials, corked up, and set aside for at least a month, the medium will be clearer and set much 280 CHAPTER XXI. quicker than if the balsam is mixed with the chloroform at the time it is required for use (Trans. Mic. Soc., Jan., 1865, p. 19 ; BEALE, p. 51). SAHLI (Zeit. f. wiss. Mik., 1885, p. 5) recommends oil of cedar as a menstruum. MAETINOTTI (Zeit. f. wiss. Mik., iv, 2, 1887, p. 159) says that he has obtained some beautiful solutions with oil of spike ("essence d'aspic rectifiee," of Duroriez, Paris). Un- fortunately, he says, this medium will not preserve safranin stains. 441. SEILEE'S Alcohol Balsam (Proc. Amer. Soc. Mic., 1881, pp. 60-2 ; Journ. Roy. Mic. Soc. [N.S.], ii, 1882, pp. 126-7). " Take a clear sample of Canada balsam and evaporate it in a water- or sand-bath to dryness ; i. e. until it becomes brittle and resinous when cold. Dissolve this while warm in warm absolute alcohol and filter through absorbent cotton." The advantage of this medium is stated to be that objects may be mounted in it direct from absolute alcohol, without previous treatment with an essential oil or other clearing agent ; Seiler considers that by this means " shrivelling is avoided, as well as the solution of fat in the cells. }) The process of mounting direct from alcohol is not very easy to carry out, and I cannot recommend it for general work. But used in the ordinary way, after clearing by an essence, only xylol or the like, Seiler's solution is for some purposes admirable. As stated above, I find that it is one of the most stable solu- tions known to me. (My stock, made up fifteen years ago, is still perfectly limpid, and has not sensibly darkened in colour.) It works pleasantly enough (if care be taken not to breathe on it during the process of mounting, as this may easily cause cloudiness). The definition is very fine, and the preservation of the preparations almost invariably perfect; my oldest pre- parations only show a few granules of little importance. Of course it has serious limitations. It cannot be used with the soluble coal-tar colours, and I find that it does not always preserve haematein stains, so that its chief employment is for carmine stains. 442. Damar (Gum Damar, or Dammar, or d'Ammar) . The EXAMINATION AND PRESERVATION MEDIA. 281 menstrua are the same as for balsam, and the solution should be prepared in the same way. The most beautiful of all these mounting media is the solution of damar in xylol. Heat is not necessary to make the solution. Minute directions (which I think unnecessary) for preparing a working solution are given by MARTINOTTI in Zeit. f. wiss. Hik., iv, 2, 1887, p. 156, and in Malpighia, ii, 1888, p. 270; cf. also Journ. Roy. Mic. Soc., 1889, p. 163. FLEMMING, PFITZNER, and a writer signing C. J. M., all employ a mixture of benzol and turpentine (see Arch. mik. Anat., xix, 1881, p. 322 ; Sci. Gossip, 1882, p. 257 ; Journ. Roy. Mic. Soc. [N.S.], iii, 1883, p. 145; Morphol. Jahrb., vi, 1880, p. 469 ; Journ. Roy. Mic. Soc. [N.S.], ii, 1882, p. 583). MAX FLESCH notes hereon (Zool. Jahresber. f. 1880, p. 51) that at Wiirzburg the ordinary dammar varnish of artists is employed. JAMES (Engl. Mech., 1887, p. 184; Journ. Roy. Mic. Soc., 1887, p. 1061) also gives some, I think, superfluous formulas for damar solutions; and still another new method is given, op. cit., 1890, p. 680. A formula for a damar and mastic medium is given by SQUIRE in his Methods and Formulae, &c., p. 84. See further details concerning these solutions in former editions- I quite acknovvledge the special beauty of definition ob- tained by means of damar solutions ; but I am convinced that not one of these solutions can be depended on for really per- manent preservations. Sooner or later, sometimes after a few weeks or days, or it may be only after months or years, the granules mentioned in 438 will make their appearance. 443. Colophonium. A solution of colophonium in turpen- tine was first recommended by Kleinenberg. I find it to be ust highly recommendable. This medium sets very slowly, so that ample time is afforded for arranging objects in it. Kleinenberg warns against the employment of absolute alcohol as a solvent ; the prepara- tions are beautiful at first, but soon become spoiled by the precipitation of crystals or of an amorphous substance. The turpentine solution keeps perfectly limpid, gives very good definition, and is altogether so excellent a medium that 282 CHAPTEli XXI. I am surprised that it is not more used. It should be recom- mended to beginners. And, as stated in 438, I consider that for many purposes it is perhaps the best and most reliable medium known. To make the solution, I add small lumps of colophoniuin to a quantity of rectified oil of turpentine kept in a stove, and when a sufficiently thick solution has been obtained, filter twice, the filtering being done in the stove. About a fortnight is required for the whole process. The solution should not be too thick, as it thickens somewhat with age. The palest sorts of colophonium should of course be selected. 444. Venice Turpentine for Mounting (VOSSELBB, Zeit. f. wiss. Mik., vi, 3, 1889, p. 292, et seq.). Yosseler strongly recom- mends this medium as having considerable advantages over Canada balsam or damar. Commercial Venice turpentine is mixed in a tall cylinder glass with an equal volume of 96 per cent, alcohol, allowed to stand in a warm place for three or four weeks, and decanted. It is stated that preparations may be mounted in this medium without previous clearing with essential oils or the like. The index of refraction being lower than that of the above-named balsams, delicate details are more distinctly brought out. Stains keep well in the medium, and Yosseler states that he possesses preparations made fifteen years ago that are perfectly well preserved. This medium is also recommended by SUCHANNEK (ibid., vii, 4, 1891, p. 463). He advises that it be prepared with equal parts of Venice turpentine and neutral absolute alcohol (ob- tained by treating commercial absolute alcohol with calcined cupric sulphate and quicklime). The mixture should be agitated frequently and kept in a tile stove for a day or two until clear and sufficiently inspissated. 445. Oopal Varnish. I have seen tissues very instructively mounted in this medium. I have tried it myself, and failed. " Berry's Hard . Finish," which is an easily obtainable copal varnish, has been highly praised for mounting purposes (see Journ. Roy. Mic. Soc., 1887, p. 1064). 446. Cedar Oil. I most highly recommend this oil, both as a temporary examination medium, and as a mounting medium. See 438. 447. Castor Oil. This was recommended as a mounting medium for EXAMINATION AND PRESERVATION MEDIA. 283 certain delicate tissues (sections of eyes of Cephalopods) by GBENACHER (Abhandl naturf. Ges. Halle-a.-S., Bd. xvi ; Zeit. f. wiss. Mik., 1885, p. 2il). This was with the idea that its low refractive index n = 1'49, whilst Canada balsam n = T54) would give a useful augmentation of visi- bility for the more refractive elements of the tissues. "With the objects with which I have experimented I have not found this to be the case. 448. Photographic Negative Varnish (for mounting large sections without cover-glasses). See WEIQEBT, Zeit. f. wiss. Mik. y iv, 2, 1887, p. 209. 449. Styrax and Liquidambar. See Journ. Roy. Mic. Soc., 1883, p. 741 ; ib., 1884, pp. 318, 475, 655, and 827 ; and the places there quoted. Also Bull. Soc. Beige de Mic., 1884, p. 178 ; and FOL, Lehrb., p. 141. These are very highly refractive media, which is just what is not wanted in general in histology. 450. Sandarac (LAVDOWSKY. from Ref. Handbook Med. Sci., Supp. p. 438). Gum sandarac 30 grs., absolute alcohol 50 c.c. This may, if desired, be diluted with an equal volume of absolute alcohol, and used for clearing sections. CHAPTER XXII. CEMENTS AND VARNISHES. 451. Introduction. Thanks to the efforts of the dilettanti to outshine one another with neatly gaudy "rings," micro- scopical literature contains a goodly show of receipts for cements and varnishes. I have collected such as appear, likely to be useful, rejecting all that relates merely to ornament. Two, or at most three, of the media given below will certainly be found sufficient for all useful purposes. For many years I have used only one cement (Bell's). I recom- mend this as a c.ement and varnish ; gold size may be found useful for turning cells ; and Miller's caoutchouc cement may be kept for occasions on which the utmost solidity is required. Marine glue is necessary for making glass cells. Carpenter lays great stress on the principle that the cements or varnishes used for fluid mounts should always be such as contain no mixture of solid particles / he has always found that those that do, although they might stand well for a few weeks or months, yet always become porous after a greater lapse of time, allowing the evaporation of the liquid and the admission of air. All fluid mounts should be ringed with glycerin jelly before applying a cement ; by this means all danger of running in is done away with. See 453 and 454. The above passage stands as it stood, italicised as here, in the first and second editions. It was translated and amplified, in a special paragraph, in the Traite des Meth. techniques. I may therefore be excused from hunting up the name of the anatomist who recently published as new this old old method, or the pages of the journals which reproduced his paper without protest. The reader who requires more information concerning microscopical cements and varnishes than can be given in this chapter may consult with advantage the papers of CEMENTS AND VARNISHES. 285 AUBERT, The Microscope, xi, 1891, p. 150, and Journ. Roy. Mic. Soc., 1891, p. 692; BECK, The Microscope, xi, 1891, pp. 338, 368, and Journ. Roy. Mic. Soc., 1892, p. 293; and the last edition of BEHRENS' Tabellen zum Gebrauch bei mikro- skopischen Arbeiten (Bruhn, Braunschweig, 1892). 452. Comparative Tenacity of Cements (see BEHRENS, Zeit. f. wiss. Mik., ii, 1885, p. 54, and AUBERT, Amer. Mon. Mic. Journ., 1885, p. 227; Journ. Roy. Mic. Soc., 1886, p. 173). Behrens gives the palm to amber varnish ; Anbert places Miller's caoutchouc cement at the head of the list, Lovett's cement coming halfway down, and zinc white cement at the bottom, with less than one quarter the tenacity of the caout- chouc cement. 453. The Paper Cell Method. According to my experience, the best way to make a fluid mount safe is the following : By means of two punches I cut out rings of paper of about a millimetre in breadth, and of about a millimetre smaller in diameter than the cover-glass. Moisten the paper ring with mounting fluid, and centre ifc on the slide. Fill the cell thus formed with mounting fluid ; arrange the object in it ; put the cover on ; fill the annular space between the paper and the margin of the cover with glycerin jelly (a turn-table may be useful for this operation) ; and as soon as the gelatin has set turn a ring of Bell's or other cement on it. For greater safety, the gelatin may of course be treated with bichromate according to Marsh's plan, next . 454. Gelatin Cement (MARSH'S Section-cutting, 2nd ed., p. 104). Take half an ounce of Nelson's opaque gelatin, soak well in water, melt in the usual way, stir in 3 drops of krea- sote, and put away in a small bottle. It is used warm. When the ring of gelatin has become quite set and dry, which will not take long, it may be painted over with a solution of bichromate of potash made by dissolving 10 grains of the salt in an ounce of water. This should be done in the daytime, as the action of daylight is necessary to enable the bichromate to render the gelatin insoluble in water. The cover may then be finished with Bell's cement. 286 CHAPTER XXII. This process is particularly adapted for glycerin mounts. 455. BELL'S Cement. Composition unknown. May be ob- tained from the opticians or from J. Bell and Co., chemists, 338, Oxford Street, London. This varnish flows easily from the brush, and sets quickly. For glycerin or other fluid mounts, the cover should be ringed as above described with glycerin jelly before applying the varnish. This precaution is especially necessary with glycerin. This is the best varnish for fluid mounts known to me. It is soluble in ether or chloroform. It is not attacked by oil of cedar. 456. MILLER'S Caoutchouc Cement. Composition unknown. May be obtained from the opticians. A very tenacious and, which is frequently an important point, a quickly drying cement. It may be diluted by a mixture of equal parts of chloroform and strong alcohol (see ROUSSELET, Journ. Quek. Club, v, ii, 1895, p. 8). 457. CLAEKE'S Spirit-proof Cement. Mr. CH. EOUSSELET has highly recommended this to me. It may be procured from Mr. J. BOLTON, 25, Balshall Heath Road, Birmingham. 458. Asphalt Varnish (Bitume de Judee). Unquestionably one of the best of these media, either as a cement or a varnish provided it be procured of good quality. It can be procured from the opticians or from the oil-shops. KITTON (Month. Hie. Journ., 1874, p. 34) recommends asphalt dissolved in benzol with the addition of a small quantity of gold size. 459. Brunswick Black. Best obtained from the opticians. A receipt for preparing it is given in BEALE, How to Work, &c., p. 49. " If a little solution of india-rubber in mineral naphtha be added to it, there is no danger of the cement cracking when dry." Carpenter states that without this addition it is brittle when dry. Brunswick black is soluble in oil of turpentine. A most useful cement, works easily and dries quickly. It can be recommended for turning cells. 460. Brunswick Black and Gold Size (EULENSTEIN, BEALE, CEMENTS AND VARNISHES. 287 How to Work, &c., p. 49). Equal parts of Brunswick black and gold size with a very little Canada balsam. 461. Gold Size. Receipts for preparing it may be found in the Micrographic Diet, or in COOLEY'S Cyclopaedia ; but it is certainly best to obtain it from the opticians or oil-shops. It is soluble in oil of turpentine. A good cement, when of good quality, and very useful for turning cells. 462. Marine Glue. Found in commerce. Carpenter says the best is that known as G K 4. It is soluble in ether, naphtha, or solution of potash. Its use is for attaching glass cells to slides, and for all cases in which it is desired to cement glass to glass. Receipts for preparing it may be found in BEALE, p. 40, or in COOLEY'S Cyclopaedia. 463. HARTIXG'S Gutta-percha Cement (see BEALE'S How to Work, &c., p. 49). Marine glue serves the same purpose, viz. that of attaching cells to slides. 464. India-rubber and Lime French Cement. See 58. 465. Knotting (Journ. Roy. Mic. oc.,1882,p. 745). "Patent knotting" from oil and colour stores, exposed to the air until it has become of the proper consistency; for mending cells and for preventing running-in of the finishing varnish (Northern Microscopist, ii, 1882). 466. Turpentine, Venice Turpentine (CsoKOR,^lrc/L mik. Anat., xxi, 1882, p. 353 ; PAEKER, Amer. Mon. Mic. Journ., ii, 1881, pp. 229-30; Journ. Roy. Mic. Soc. [N.S.], ii, 1882, p. 724). Venice turpentine (Terebinthina veneta) is the liquid resinous exudation of Abies larix. It is seldom met with in a pure state. The following are the directions for preparing and using it given by Parker : Dissolve true Venice turpentine in enough alcohol, so that after solution it will pass readily through a filter, and, after filtering, place in an evaporating dish, and by means of a 288 CHAPTER XXII. sand-bath evaporate down to about three quarters of the quantity originally used. (The best way to tell when the evaporation has gone far enough is to drop some of the melted turpentine, after it has evaporated down to about three quarters of its original volume, into cold water ; if on being taken out of the water it is hard and breaks with a vitreous fracture on being struck with the point of a knife, cease evaporation and allow to cool.) Or (CsoKOR), common resinous turpentine of commerce is put in small pieces to melt over a water-bath, then poured into a suitable vessel and allowed to cool. It should form a brittle, dark brown mass, not yielding to the pressure of a finger. It is sometimes useful, in order to attain the right degree of hardness in the cold mass, to add a little resinous oil of turpentine to the melted mass, and then to evaporate for several hours over the water-bath. This cement is used for closing glycerin mounts ; it is applied in the following manner : Square covers are used, and superfluous glycerin is cleaned away from the edges in the usual way. The cement is then put on with a piece of wire bent at right angles (No. 10 12 wire is taken, and copper is the best, as it gives to the turpentine a greenish tinge) ; the short arm of the wire should be just the length of the side of the cover-glass. The wire is heated in a spirit lamp, plunged into the cement, some of which adheres to it, and then brought down flat upon the slide at the margin of the cover. The turpentine distributes itself evenly along the side of the cover, and hardens immediately, so that the slide may be cleaned as soon as the four sides are finished. It is claimed for this cement that it is perfectly secure, very handy, and never runs in. Parker saw this cement, or a similar one known as Vendischer Damarlack, exclusively used for gly- cerin mounts in the Pathological Laboratory at Vienna. This is an extremely valuable method. It is very rapid and very safe. The cement sets hard in a few seconds. 467. Colophonium and Wax (KEONIG, Arch. f. mik. Anat., 1886, p. 657; Journ. Roy. Mic. Soc., 1887, p. 344). Seven to nine parts of colophonium are added piecemeal to two parts of melted wax, the whole filtered and left to cool. For use, CEMENTS AND VARNISHES. 289 the mass is melted by placing the containing vessel in hot water. The cement is not attacked by water, glycerin, or caustic potash. 468. APATHY'S Cement for Glycerin Mounts (Zeit. f. wiss. Mik., vi, 2, 1889, p. 171). Equal parts of hard (60 C. melt- ing-point) paraffin and Canada balsam. Heat together in a porcelain capsule until the mass takes on a golden tint, and no longer emits vapours of turpentine. On cooling, this forms a hard mass, which is used by warming and applying with a glass rod or brass spatula. One application is enough. The cement does not run in, and never cracks. 469. JULIEN'S Balsam-Paraffin, see Journ. New York Mic. Soc., ix, 1893, p. 39, or Journ. Roy. Mic. Soc. t 1893, p. 567. 470. Paraffin. Temporary mounts may be closed with pure paraffin, by applying it with a bent wire, as described, 466. 471. Canada Balsam, or Damar. Cells are sometimes made with these. They are elegant, but in my experience are not reliable for per- manent mounts. 472. Amber Varnish. As above mentioned, BEHRENS finds this cement to possess an extreme tenacity. He does not give the composition of his varnish, which was procured from E. Pfannenschmidt at Dantzic. The following is from COOLEY'S Cyclopaedia, art. "Varnish :" " Take of amber (clear and pale) 6 Ibs., fuse it ; add of hot clarified linseed oil 2 gallons, boil it until it " strings " well, then let it cool a little and add of oil of turpentine 4 gallons or q. s." Other receipts, 1. c. 473. Amber and Copal Varnish (HEYDENEEICH, Zeit.f. wiss. Mik., 1885, p. 338). An extremely complicated mode of preparation. The varnish may be obtained from Ludwig Marx, at 110, Moskowskaja Sastawa, St. Petersburg; or 79, Gaden, Vienna ; or 1, Romerthal, Mayence. 474. Shellac Varnish (BEALE, p. 28). Shellac should be broken into small pieces, placed in a bottle with spirit of wine, and frequently shaken until a thick solution is obtained. The Micro. Dictionary says that the ^addition of 20 drops of castor oil to the ounce is an improvement. 19 290 CHAPTER XXII. Untrustworthy, but useful for protecting balsam mounts from the action of oil of cedar. For a method of preparing chemically pure shellac (a somewhat important matter), see WITT, Zeit.f. wiss. Mik., 1886, p. 199. For SEAMAN'S shellac cement for attaching metal to glass, see Journ. \ Roy. Mic. Soc., 1888, p. 520. 475. Sealing- Wax Varnish. (Micro. Diet., " Cements "). Add enough spirit of wine to cover coarsely powdered sealing-wax, and digest at a gentle heat. This should only be used as a varnish, never as a cement, as it is apt to become brittle and to lose its hold upon glass after a time. 476. Tolu Balsam Cement (CAENOY'S Biol. Cell, p. 129). Tola balsam . . . . .2 parts. Canada balsam . . . . .1 part. Saturated solution of shellac in chlo- roform . . . . .2 parts. Add enough chloroform to bring the mixture to a syrupy consistence. Carnoy finds this cement superior to all others. I 477. STIEDA'S Zinc White or Red Cement (Arch. f. mile. Anat., 1866, p. 435). Rub up oxide of zinc with turpentine, and add, stirring con- tinually, for every drachm of the zinc oxide 1 ounce of a solution of damar in turpentine (of the consistency of thick syrup). This gives a white cement like Ziegler's. For a red cement take, instead of zinc, cinnabar A and take 2 drms. of the metal for each ounce of the damar solution. If the] cement has become too thick with age, dilute with turpentine, ether, or] chloroform. Eecent authors who have discussed this cement are agreed that \ it is quite untrustworthy as a cement, though it may be useful as a finish. 478. ZIEGLER'S White Cement. Composition unknown. Is very much i used on the Continent. 479. KITTON'S White Lead Cement (Month. Mic. Journ., 1876, pJ 221). Equal parts of white-lead, red-lead, and litharge (all in powder),; ground together with a little turpentine until thoroughly incorporated, then mixed with gold size. The mixture should be thin enough to work with a] brush. No more of the cement should be made than is required for present! use, as it soon sets and becomes unworkable ; but a stock of the materials; may be kept ready ground in a bottle. 480. LOVETT'S Cement (Journ. Roy. Mic. Soc., 1883, p. 786). Two parts white-lead, two parts red oxide of lead (minium), three parts litharge. To be ground very fine, mixed dry, and kept so in a bottle. When required for use mix a little of the powder with gold size to the consistency of paint, taking care that no grit gets into it. 481. ASPINALI/S Enamel. STANLEY KENT (Journ. Roy. Mic. Soc., 1890, p. 821) finds this of great use, both for ringing slides and making cells. PAET II. SPECIAL METHODS AND EXAMPLES. CHAPTER XXIII. INJECTIONS GELATIN MASSES. 482. Introduction. Injection masses are composed of a coloured substance, technically termed the colouring mass, and of a substance with which that is combined, technically termed the vehicle. The following formulas are grouped mainly according to the nature of the vehicle. For injections made for the study of the angiology of verte- brates, the student will do well to follow the masterly practice of Robin and Ranvier, consulting also, if necessary, the excellent instructions given in the Lehrbuch der vergleich- enden mikroskopischen Anatomie of Fol. For injections of Invertebrates (and, indeed, for Vertebrates if it is desired to demonstrate the minute structure of environing tissues at the same time as the distribution of vessels) glycerin masses are frequently preferable to the gelatin masses chiefly employed by these authors; and I would recommend as particularly convenient the Prussian blue glycerin masses of Beale. Glycerin masses have the great advantage that they are used 483. Nitrite of Amyl as a Vaso-dilator. As stated above, glycerin masses are certainly very convenient, and give very good results from the scientific not from the aesthetic point of view. They have a great defect for the injection of fresh specimens that is, those in which rigor mortis has not set ID : they stimulate the contraction of arteries. In these cases it may be advisable to use nitrite of amyl as a vaso-dilator. The animal may be anaesthetised with a mixture of ether and nitrite of amyl, and finally killed with pure nitrite. Or, after killing by nitrite, a little nitrite of amyl in salt solution may be injected before the injection mass is thrown in. In any case it is advisable to add a little nitrite to the mass just 294 CHAPTER XXIII. before using. The relaxing power is very great (see OYIATT and SARGENT, in 8t. Louis Med. Journ., 1886, p. 207 ; and Journ. Roy. Hie. Soc., 1887, p. 341). ROBIN'S Masses. 484. EOBIN'S Gelatin Vehicle (Traite, p. 30). Take some gelatin, of the sort known as "colle de Paris." (This gelatin is found in commerce in the form of thin sheets, marked with lozenge- shaped impressions of the cords which supported them whilst drying.) Soak it in cold water, then heat in water over a water-bath. One part of gelatin should be taken for every 7, 8, 9, or even 10 parts of water ; it is a common error to employ solutions containing too much gelatin. The solution is now to be combined with one of the colouring masses given below. This vehicle, like all gelatin masses, is liable to be attacked by mould if kept long ; camphor and carbolic acid do not suffice to preserve it. Chloral hydrate added to the mass will preserve it (HOYER). A sufficient dose, at least 2 per cent., should be employed (see below, 493, 494), 485. ROBIN'S Glycerin- gelatin Vehicle (Traite, p. 32). Dis- solve in a water-bath 50 grms. of French gelatin (" colle de Paris ") in 300 grms. of water, in which has been dissolved some arsenious acid ; add of glycerin 150 grms., and of car- bolic acid a few drops. Unlike the pure gelatin vehicles, this mass does keep indefinitely. The colouring masses recommended for combination with the vehicles above described are made as follows : 486. Carmine Colouring Mass (Traite, p. 33). Rub up in a mortar 3 grms. of carmine with a little water and enough ammonia to dissolve the carmine. Add 50 grms. of glycerin, and filter. Prepare 50 grins, of acid glycerin (containing 5 grms. of acetic acid for every 50 grms. of glycerin), and add it by degrees to the carmine-glycerin, until a slightly acid reaction is obtained (as tested by very sensitive blue test-paper, moist- ened and held over the mixture). INJECTIONS GELATIN MASSES. 295 One part of this mixture is to be added to 3 or 4 parts of the gelatin injection vehicle (ante, Formula 484), or of the glycerin-gelatin ( 485). 487. Ferrocyanide of Copper Colouring Mass (ibid., p. 34). Take (1) Ferrocyanide of potassium (concentrated solution) ...... 20 c.c. Glycerin . . . . . . 50 (2) Sulphate of copper (concentrated solu- tion) . . . . . . 35 ,, Glycerin 50 Mix (1) and (2) slowly, with agitation ; at the moment of injecting combine with 3 volumes of vehicle. 488. Blue Colouring Mass (Prussian Blue) (Robin's modifica- tion of Beetle's formula, ibid., p. 35). Take (A) Sulphocyanide of potassium (sol. sat.) 90 c.c. Glycerin . . . . . . 50 (B) Liquid perchloride of iron at 30 . 3 Glycerin 50 Mix slowly and combine the mixture with 3 parts of vehicle. It is well to add a few drops of HC1. 489. Cadmium Colouring Mass (ibid., p. 36). Take- Sulphate of cadmium (sol. sat.) . . 40 c.c. Glycerin . . . . . . . 50 and Sulphide of sodium (sol. sat.) . . . 30 Glycerin 50 Mix with agitation and combine with 3 vols. of vehicle. 490. SCHEELE'S Green Colouring Mass (ibid., p. 37). Take- Arseniate of potash (saturated solution) 80 c.c. Glycerin . . . . . . 50 and Sulphate of copper (saturated solution) 40 Glycerin 50 Mix and combine with 3 vols. of vehicle. 296 CHAPTER XXIII. Carmine-gelatin Masses. 491. RANVIER'S Carmine -Gelatin Mass (Traite technique, p. . 110). Take 5 grins. Paris gelatin, soak it in water for half an hour, or until quite swollen and soft ; wash it ; drain it ; put it into a test-tube and melt it, in the water it has absorbed, over a water-bath. When melted add slowly, and with con- tinual agitation, a solution of carmine in ammonia, prepared as follows: 2^ grms. of carmine are rubbed up with a little water, and just enough ammonia, added drop by drop, to dis- solve the carmine into a transparent solution. When the carmine has been added to the gelatin, you will have about 15 c.c. of ammoniacal solution of carmine in gelatin, if the operations have been properly performed. This solution is to be kept warm on the water-bath, whilst you proceed to neutralise it by adding cautiously, drop by drop, with continual a.gitation, a solution of 1 part of glacial acetic acid in 2 parts of water. (When the mass is near neutrality, dilute the acetic acid still further.) The instant of saturation is determined by the smell of the solution, which gradually changes from ammoniacal to sour. As soon as the sour smell is perceived, the addition of acetic acid must cease, and the liquid be examined under the microscope. If it contains a granular precipitate of carmine, too much acid has been added, and the mass must be thrown away. Ranvier states that by practice the operator learns to attain to perfect neutralisation almost infallibly in this way, and that this is the only way to attain to it. Trust must not be pub in certain formulae that profess to indicate the propor- tions of ammonia and acetic acid necessary for neutralisation, on account of the variation in strength of the solutions of am- monia kept in laboratories. The method proposed by Frey of determining beforehand the quantity of a known acetic solution that is necessary for neutralisation of a given quantity of the ammonia employed, is not infallible because it often happens that commercial gelatin is acid ; in which case the proposed method would cause the operator to overpass the point of saturation. The mass having been perfectly neutralised is strained through new. flannel. 492. How to Neutralise a Carmine Mass (ViLLE, Gaz. held. INJECTIONS GELATIN MASSES. 297 d. Sci. med. de Montpellier, Fev., 1882 ; may be had separately from Delahaye et Lecrosnier, Paris). Yille is of Ranvier's opinion that the method of titration recommended by Frey is defective, but for a different reason. When carmine is treated with ammonia a certain proportion of the ammonia combines with the carmine to form a transparent purple compound, and the rest of the ammonia remains in excess. It is this excess that it is required to neutralise precisely. In Frey's method a quantity of acid sufficient for the neutralisation of the whole of the ammonia employed is taken ; hence, naturally, the point of neutralisation is overstepped, and a granular mass is the result. As to the acidity accidentally found in commercial gelatin, that source of error is easily eliminated. Instead of soaking the gelatin in water, it should be placed in a large funnel with a narrow neck, or better, in a stopcock funnel, and the whole should be placed under a tap, and a stream of water arranged in such a manner that the gelatin be constantly completely immersed. Washing for an hour or so in this way will remove all traces of acids mechanically retained in the gelatin. As to the neutralisation of the colouring mass, Yille is of opinion that the criterion of neutrality given by Ranvier the sour smell that takes the place of the ammoniacal odour cannot be safely relied on in practice. He considers it greatly preferable to employ dichroic litmus paper (litmus paper sensitized so as to be capable of being used equally for the demonstration of acids and bases). To prepare such a paper, the tincture obtained by decoction of cake litmus is slightly acidified by an excess of sulphuric acid. By this means the excess of alkali, or of alkaline carbonate, that is always present in litmus decoction, and which diminishes its sensibility as a reagent, is neutralised. The decoction is then heated and agitated with an excess of precipitated carbonate of baryta, and filtered. The solution of litmus thus obtained is exposed to the air in wide vessels until its intense blue colour has given place to a reddish tint. Strips of white unsized paper are then dipped in it, and dried in the shade on stretched threads, in a place free from vapour of ammonia. A shorter method consists in adding very dilute sulphuric acid, drop by drop, to the ordinary laboratory tincture of 298 CHAPTER XXIII. litmus, until the colour changes to red. Then, by adding successively traces of alkali and very dilute sulphuric acid, the reddish dichroic tint may be obtained, and the paper pre- pared with the solution as before. The paper is used in the same way as ordinary litmus paper. A strip is moistened with distilled water and held as close as possible to the injection mass kept melted on a water- bath. It becomes blue at first, very rapidly and decidedly ; but as fast as fresh quantities of acid are added this reaction becomes less evident, and at a certain moment the change of colour becomes very slow in making its appearance. It is then that the addition of acid should cease, and the operation is ended. Very delicate sensitized paper may also be prepared with other reagents than litmus for instance, with Nessler's reagent* or with alcoholic solution of hgematoxylin, or with a solution made by adding a trace of dilute sulphuric acid to " liqueur orange No. 3 " (a liquid found in commerce, and - used for detecting acids) ; the solution takes on a gooseberry red colour. The preparation of the injection mass is facilitated by em- ploying acetic acid and ammonia of known strength. For j the acetic acid it is sufficient to keep the glacial acid in a I well-stoppered bottle. But this will not suffice for the! ammonia, which is notably lowered in strength through the i mere pouring from one bottle into another. Ville has ' imagined an apparatus which allows of withdrawing a known j quantity without permitting any access of air to the stock solution. Description and figures, 1. c. With the exception of the processes above described, Ville prepares the injection mass exactly as Kanvier. 493. HOYER'S Carmine-Gelatin Mass (Biol. Centralb., 1882, : p. 21). Take a concentrated gelatin solution and add to it a i corresponding quantity of neutral carmine staining solution (1. c., p. 17). Digest in a water-bath until the dark violet-red colour begins to pass into a bright red tint. Then add 5 10 * Nessler's reagent may be prepared as follows : Mercuric chloride, in powder, 35 grms. ; iodide of potassium, 90 grms. ; water, 1750 c.c. Heat gently till dissolved in a large basin ; then add of stick caustic potash 320 grms., and 50 c.c. of saturated solution of mercuric chloride (WANKLYN). From COOLEY'S Cyclopaedia, s. v. " Nessler's Test." INJECTIONS GELATIN MASSES. 299 per cent, by volumes of glycerin, and at least 2 per cent, by weight of chloral, in a concentrated solution. After passing through flannel it can be kept in an open vessel under a bell- glass. 494. FOL'S Carmine-Gelatin Mass (Zeit.f. wiss. Zool., xxxviii, 1883, p. 492). The following method of preparation has the advantage of producing masses that can be kept in the dry state for an indefinite length of time. (Fol finds that the addition of chloral hydrate to wet masses is not an efficient preservative.) One kilog. of Simeon's photographic gelatin* is soaked for a couple of hours, until thoroughly soft, in a small quantity of water. The water is then poured off and the gelatin melted over a water-bath, and one litre of concentrated solu- tion of carmine in ammonia is poured in with continual stirring. (The carmine solution is prepared by diluting strong solution of ammonia with three or four parts of water and adding carmine to saturation ; the undissolved excess of carmine is removed by filtration just before the solution is added to the gelatin.) To the mixture of gelatin and carmine, which should have a strong smell of ammonia, sufficient acetic acid is added to turn the dark purple colour of the mixture into the well-known blood-red hue. Exact neutralisation is not necessary. The mass is set aside until it has become firm, and is then cut up into pieces, which are tied up in a piece of tulle or fine netting. By means of energetic compression with the hand under water (it must be acidulated water, O'l per cent, acetic acid, other- wise the carmine will wash out : cf. Journ. Roy. Mic. Soc., iv, part 3, 1884, p. 474) the mass is driven out through the meshes of the stuff in the shape of fine strings, which are washed for several hours in a sieve placed in running water in order to free them from any excess of acid or ammonia. The strings are then again melted, and the molten mass is poured * This gelatin may be obtained eitber from tbe ordinary providers of articles used in photography, or direct from Simeon's Gelatin-fabrik, Winterthur, Switzerland. Two sorts, a hard and a soft, are sold ; the softer is to be preferred on account of its lower point of fusion. Probably the photographic gelatins of Hinrichs, of Frankfurt, and of Coignet, of Paris, would answer equally well ; as also the best English preparations. 300 CHAPTER XXIII. on to large sheets of parchment paper soaked with paraffin, and the sheets are hung up to dry in an airy place. When dry the gelatin can easily be separated from the sheets, and may be cut into long strips with scissors and put away, pro- tected from dust and damp, until wanted for use. In order to get the mass ready for use, all that is necessary is to soak the strips for a few minutes in water and melt them over a water-bath. The process may be simplified, without giving very greatly inferior results, as follows (Lehrb., p. 13). Gelatin in sheets is macerated for two days in the above-described carmine solution, then rinsed and put fora few hours into water acidu- lated with acetic acid. It is then washed on a sieve for several hours in running water, dried on parchment paper, and pre- served as above. This injection mass is very well spoken of, 495. Other Carmine Gelatin Masses. GEELACH'S Car mine -Gela- tin Mass (see Arch. f. mik. Anat., 1865, p. 148 ; and Eanvier's Traite, p 113). THIEESCH'S Carmine-Gelatin Mass (see ibid.). CAETEE'S Car- mine-Gelatin Mass (see BEALE, p. 113). DAVIES' Carmine-Gelatin Mass (see his Prep, and Mounting of Mic. Objects, p. 138). Slue Gelatin Masses. 496. ROBIN'S Prussian Blue Gelatin Mass (see above, 488). 497. RANVIEK'S Prussian Blue Gelatin Mass (Traite, p. 119). Twenty-five parts of a concentrated aqueous solution of soluble Prussian blue (prepared as directed below) mixed with 1 part of solid gelatin. The mixture of the Prussian blue with the vehicle is effected in the following manner : Weigh the gelatin, soak it in water for half an hour or an hour, wash it, and melt it in a test-tube, in the water it has absorbed, by heating over a water-bath. Put the solution of Prussian blue into another test-tube, and heat it on the same water- bath as the gelatin, so as to have the two at the same temperature. Pour the gelatin gradually into the Prussian blue solution, stirring continually with a glass rod. Continue stirring until the disappearance of the curdy pre- cipitate that forms at first. (Some gelatins produce a per- INJECTIONS GELATIN MASSKS. 301 sistent precipitate ; these must be rejected ; but it must be borne in mind that the precipitate that invariably forms in even the best gelatins disappears if the heating be continued. It is essential to remember this when preparing Prussian blue and gelatin mass.) As soon as the glass rod has ceased to show blue granulations on its surface on being withdrawn from the liquid, it may be concluded that the Prussian blue is completely dissolved. Filter through new flannel, and keep the filtrate at 40 over a water-bath until injected. The soluble Prussian blue for the above mass is prepared as follows : 498. Soluble Prussian Blue for Injection Masses (RANVIER, ibid.} . Make a concentrated solution of sulphate of peroxide of iron in distilled water, and pour it gradually into a con- centrated solution of yellow prussiate of potash. There is produced a precipitate of insoluble Prussian blue. (An excess of prussiate of potash ought to remain in the liquid ; in order to ascertain whether this is the case take a small quantity of the liquid and observe whether a drop of sulphate of iron still precipitates it.) Filter the liquid through a felt strainer, underneath which is arranged a paper filter in a glass funnel. The liquid at first runs clear and yellowish into the lower funnel ; distilled water is then poured little by little on to the strainer ; gradually the liquid issuing from the strainer acquires a blue tinge, which, however, is not visible in that which issues from the lower filter. Distilled water is continually added to the strainer for some days until the liquid begins to run off blue from the second filter. The Prussian blue has now become soluble. The strainer is turned inside out and agitated in distilled water ; the Prussian blue will dissolve if the quantity of water be sufficient. The solution may now be injected just as it is, or it may be kept in bottles till wanted, or the solution may be evaporated in a stove, and the solid residuum put away in bottle. For injections, if a simple aqueous solution be taken, it should be saturated. Such a mass never transudes through the walls of vessels. Or it may be combined with one fourth of glycerin, or with the gelatin vehicle above described. 499. Soluble Prussian Blue (GuiGNET, see Journ. de Microgr.^. 302 CHAPTEE XXIII. 1889, p. 94 ; Journ.Roy. Mic. Soc., 1889, p. 463; or previous editions of this work). 500. BRUCKE'S Soluble Berlin Blue (Arch. f. mifc. Anat., 1865, p. 87). Briicke first prepared it by taking a 10 per cent, solution of ferrocyanide of potassium, and precipitating by means of a dilute solution of sesquichloride of iron (taken in such a quantity as to contain just half as much chlorine as is necessary for the decomposition), and washing the precipitate on the filter until solubility is attained. Later on he employed a greater excess of ferrocyanide, and took just so much dilute solution of chloride of iron that the weight of the dry chloride employed came to y 1 ^ or of that of the ferrocyanide. The precipitate was washed on a filter (using the filtrate to wash with) until nothing but a clear yellow liquid filtered off, then washed with water until the water began to run off blue, then dried, pressed between blotting-paper in a press, the resulting mass broken in pieces and dried by exposure to the air. A cheaper method is the following : Make a solution of ferrocyanide of potassium containing 217 grammes of the salt to 1 litre of water. Make a solution of 1 part commercial chloride of iron in 10 parts water. Take equal volumes of each, and add to each of them twice its volume of a cold saturated solution of sulphate of soda. Pour the chloride solution into the ferrocyanide solution, stirring continually. Wash the precipitate on a filter until soluble, and treat as above described. The concentrated solution of the colouring matter is to be gelatinised with just so much gelatin that the mass forms a jelly when cold. 501. THIERSCH'S Prussian Blue Gelatin Mass (Arch. f. mik. Anat., i, 1865, p. 148). Take (1) A solution of 1 part gelatin in 2 parts water. (2) A saturated aqueous solution of sulphate of iron. (3) A saturated aqueous solution of red prussiate of potash. (4) A saturated aqueous solution of oxalic acid. INJECTIONS GELATIN MASSES. 303 ^Now (A) mix 12 c.c. of the iron solution with one ounce of e gelatin solution at the temperature of 25 R. Then (B) mix, at the same temperature, 24 c.c. of the prus- siate solution with two ounces of the gelatin solution. (c) To the latter mixture add first 24 c.c. of the oxalic acid solution, stir well, and then add the gelatin and iron mixture (A). Stir continually, keeping the temperature at from 20 to 25 R. until the whole of the Prussian blue is precipitated. Finally, heat over a water-bath to about 70 R. and filter through flannel. 502. FOL'S Berlin Blue Gelatin Mass (Zeit. f. wiss. ZooL, xxxviii, 1883, p. 494). A modification of Thiersch's formula, last . 120 c.c. of a cold saturated solution of sulphate of iron are mixed with 300 c.c. of the warm gelatin solution. In a separate vessel 600 c.c. of the gelatin solution are mixed with 240 c.c. of a saturated solution of oxalic acid, and 240 c.c. of a cold saturated solution of red prussiate of potash are added to the mixture. The first mixture is now gradually poured into the second, with vigorous shaking, the whole is warmed for a quarter of an hour over a boiling water-bath, the mass is allowed to set, is pressed out into strings through tulle or netting, as described for the carmine mass, supra, 494, and the strings are washed and spread out to dry on the prepared paper. (It is necessary to dry the strings without re-inelting in this case, because the mass does not readily melt without the addition of oxalic acid.) In order to prepare the mass for injection, the strings are put to swell up in cold water, and then warmed with the addition of enough oxalic acid to allow of complete solution. 503. HOYER'S Soluble Berlin Blue Gelatin Mass (Arch. f. mik. Anat., 1876, p. 649). The filtered and not too much washed precipitate of soluble Berlin blue is brought in a little water on to a Graham's dialyser, and the external water changed until the solution begins to pass through the parchment. Dilute the solution and filter through filter-paper, an opera- tion which becomes easy after dialysis. The solution may be injected pure (for lymphatics, for instance) or may be com- bined with gelatin. To do this, warm the solution almost to boiling-point, and add gradually a warm, thin solution of 304 CHAPTER XXIII. gelatin until coagulation begins to set in. Strain through, wetted flannel. Gelatin Masses of other Colours. 504. KOBIN'S Cadmium Gelatin Mass (see 489). 505. THIERSCH'S Lead Chromate Gelatin Mass (Arch. f. mik. Anat., 1865, p. 149). Make (A) A solution of 1 part gelatin in 2 parts water. (B) A solution of 1 part neutral chroniate of potash in 11 parts water. (c) A solution of 1 part nitrate of lead in 11 parts water. Mix 4 parts of the gelatin solution with 2 parts of the lead solution, and in another vessel mix 4 parts gelatin solution with 1 part of the chromate solution. Heat both the mixtures to 25 R. ; mix them together with continual stirring until all the chromate of lead is precipitated ; heat over a water-bath to 70 E., and filter through flannel. FOL remarks that this is the most beautiful of yellow masses, but will not keep, as the chromate of potash causes the gelatin to pass over gradually into the insoluble state. 506. HOYER'S Lead Chromate Gelatin Mass (ibid., 1867, p. 136). Take- One volume of a solution of gelatin containing 1 part of gelatin to 4 of water. One volume of cold saturated solution of bichromate of potash. And one volume cold saturated solution of sugar of lead (neutral plumbic acetate). Filter the gelatin solution through flannel, and mix in the bichromate solution. Then warm almost to boiling-point, and add gradually the (warmed) sugar of lead solution. Allow the mass to cool down to body temperature, and inject at once. Another mode of preparation is as follows : Mix the sugar of lead solution with part of the gelatin solution, mix the bichromate solution with the remaining gelatin solution, heat the latter mixture, and pour into it the former mixture (gradually), stirring continually. INJECTIONS GELATIN MASSES. 305 If the solutions are mixed at a low temperature a lumpy granular precipitate is formed. Further, when solution of sugar of lead is added to a hot solution of bichromate of potash a rich orange-red precipitate is obtained ; whilst if the solutions be mixed cold the precipitate is bright yellow. If the solutions of the two salts be kept ready prepared, the injection mass may be mixed in less than a quarter of an hour. Its advantages are that, on account of the extremely fine state of division of the precipitate, the mass is almost transparent, and runs so freely that even lymphatics may be perfectly injected with it, whilst its intensity of colour makes the vessels much more distinct than the very pale mass of Thiersch (last Section). It is also easier to manage than Thiersch's mass, as it does not solidify so quickly. It shows well in the vessels by reflected, as well as by transmitted light. 507. Foi/s Lead Chromate Gelatin Mass (Lehrb., p. 15). 508. HOYER'S Silver Nitrate Yellow Gelatin Mass (Biol. Cen- tralbl., ii, 1882, pp. 19, 22; Journ. Roy. Hie. Soc. [N.S.], iii, 1883, p. 142). "A concentrated solution of gelatin is mixed with an equal volume of a 4 per cent, solution of nitrate of silver and warmed. To this is added a very small quantity of an aqueous solution of pyrogallic acid, which reduces the silver in a few seconds; chloral and glycerin are added as before " (see ante, HOYER'S formula for carmine-gelatin, No. 493). This mass is yellow in the capillaries and brown in the larger vessels. It does not change either in alcohol, chromic or acetic acid, or bichromate of potash, &c. 509. Other Colours. HOYEE'S Green Gelatin Masses (ibid.). Made by mixing a blue mass and a yellow mass. THIEBSCH'S Green Gela- tin Mass (Arch. f. mile. Anat., 1865, p. 149). Made by mixing the blue mass, 501, and the yellow mass, 505. ROBIN'S SCHEELE'S Green Gela- tin Mass (see 490). HAETIO'S White Gelatin Mass (see FEET, Le Microscope, p. 190). FEET'S White Gelatin Mass (ibid.}. TEICHMANN'S White Gelatin Mass (ibid., p. 191). FOL'S Brown Gelatin Mass (Zeit. f. wiss. Zool., xxxviii, 1883, p. 494). MILLEE'S Purple Silver Nitrate Gelatin Mass (see Amer. Hon. Mic. Journ., 1888, p. 50; Journ. Roy. Mic. Soc., 1888, p. 518 ; Zeit.f. wiss. Mik.,v, 3, 1888, p. 361). ROBIN'S Mahogany Gelatin Mass (see 487). 20 306 CHAPTER XXIII. 510. RANVIEK'S Gelatin Mass for Impregnation ( Traite,-p. 1 23) . Concentrated solution of gelatin, 2, 3, or 4 parts; 1 per cent, nitrate of silver solution, 1 part. 511. FOL'S Metagelatin Vehicle (Lehrb., p. 17). The opera- tion of injecting with the ordinary gelatin masses is greatly complicated by the necessity of injecting them warm. FOL proposes to employ metagelatiu instead of gelatin. If a slight proportion of ammonia be added to a solution of gelatin, and the solution be heated for several hours, the solution passes into the state of metagelatin, that is, a state in which it no longer coagulates on cooling. Colouring masses may be added to this vehicle, which may also be thinned by the addition of weak alcohol. After injection, the prepara- tions are thrown into strong alcohol or chromic acid, which sets the mass. CHAPTER XXIV. INJECTIONS OTHER MASSES (COLD). 512. JOSEPH'S White-of-Egg Injection Mass (Carmine) (Ber. naturw. Sect. Schles. Ges. 1879, pp. 36 40; Journ. Roy. Hie. Soc. [N.S.J, ii, 1882, p. 274)." Filtered white of egg, diluted with 1 to 5 per cent, of carmine solution This mass remains liquid when cold ; it coagulates when immersed in dilute nitric acid, chromic or osmic acid, remains trans- parent, and is sufficiently indifferent to reagents/' For Invertebrates. 513. BJELOUSSOW'S Gum Arabic Mass (Arcli.f. Anat. u. Phys. } 1885, p. 379). Make a syrupy solution of gum arabic and a saturated solution of borax in water. Mix the solutions in such proportions as to have in the mixture 1 part of borax to 2 of gum arabic. Hub up the transparent, almost insoluble mass with distilled water, added little by little, then force it through a fine-grained cloth. Kepeat these operations until there is obtained a mass that is free from suspended gela- tinous clots. (If the operation has been successful, the mass should coagulate in the presence of alcohol, undergoing at the same time a dilatation to twice its original volume.) The vehicle thus prepared may be combined with any colouring mass except cadmium and cobalt. After injection the preparation is thrown into alcohol, and the mass sets immediately, swelling up as above described, and consequently showing vessels largely distended. Cold-blooded animals may be injected whilst alive with this mass. It does not flow out of cut vessels. Injections keep well in alcohol. Glycerin may be used for making them transparent. If it be desired to remove the mass from any part of a pre- paration, this is easily done with dilute acetic acid, which dissolves it. 308 CHAPTER XXIV. Glycerin Masses.* 514. BEALE'S Carmine Glycerin Mass (How to Work, &c., p. 95). Five grains of carmine are dissolved in a little water with the aid o about five drops of ammonia, and added to half an ounce of glycerin. Then add half an ounce of glycerin with eight or ten drops of acetic or hydrochloric acid, gradu- ally, with agitation. Test with blue litmus paper, and necessary add more acid till the reaction is decidedly acid Then add half an ounce of glycerin, two drachms of alcohol, and six drachms of water. I have found this useful, but not so good as the Prussian blue injections. 515. BEALE'S Prussian Blue Glycerin Mass (How to Work, &C.,. p. 93). Common glycerin ... 1 ounce. Spirits of wine . . . . 1 ,, Ferrocyanide of potassium . . 12 grains. Tincture of perchloride of iron . 1 drachm. Water ..... 4 ounces. Dissolve the ferrocyanide in one ounce of the water and glycerin, and add the tincture of iron to another ounceJ " These solutions should be mixed together very gradually,, and well shaken in a bottle, the iron being added to the soluA tion of the ferrocyanide of potassium. Next, the spirit and the water are to be added very gradually, the mixture being constantly shaken." " The water " spoken of in the last sentence appears to- mean the remaining three ouuces of water that were not mixed with the glycerin at first. Injected specimens should be preserved in acidulated gly- cerin, otherwise the colour may fade. 516. BEALE'S Acid Prussian Blue Glycerin Mass (ibid., p. 296) J Price's glycerin .... 2 fluid ounces. Tinct. of sesquichloride of iron . 10 drops. Ferrocyanide of potassium . . 3 grains. Strong hydrochloric acid . . 3 drops. Water ..... 1 ounce. * See the remarks on Glycerin Masses, 483. INJECTIONS OTHER MASSES (COLD). 309 Proceed as directed above, dissolving the ferrocyanide in one half of the glycerin, the iron in the other, and adding the latter drop by drop to the former. Finally add the water and HC1. Two drachms of alcohol may be added to the whole if desired. I consider this a most admirable formula. I possess some of this mass prepared many years ago, in which not the smallest flocculus has made its appearance. The Prussian blue appears to be in a state of true solution. The mass runs well, and has not so much tendency to exude from cut capil- laries as might be supposed. Unfortunately it is a rather expensive preparation. 517. RANVIEE'S Prussian Blue Glycerin Mass (Traite, p. 120). Consists of the Prussian blue fluid, 498, mixed with one fourth of gly- cerin. 518. Other Colours. Any of the colouring masses, 486 to 490, or other suitable colouring masses, combined with glycerin, either dilute or pure. Aqueous Masses. 519. RANVIER'S Prussian Blue Aqueous Mass (Traite, p. 120). The soluble Prussian blue, 498, injected without any vehicle. It does not extravasate. 520. MULLER'S Berlin Blue (Arch.f. mik. Anat., 1865, p. 150). Precipitate a concentrated solution of Berlin blue by means of 90 per cent, alcohol. The precipitate is very finely divided ; the fluid is perfectly neutral, and much easier to prepare than the formula of Beale. 521. MAYER'S Berlin Blue (Mitth. zool. Stat. Neapel, 1888, p. 307). A solution of 10 c.c. of tincture of perchloride of iron in 500 c.c. of water is added to a solution of 20 g*r. of yellow prussiate of potash in 500 c.c. of water, allowed to stand for twelve hours, decanted, the deposit washed with distilled water on a filter until the washings come through dark blue (one to two days), and the blue dissolved in about a litre of water. 522. EMEKY'S Aqueous Carmine (ibid., 1881, p. 21). To a 10 per 310 CHAPTER XXIV. ' cent, ammoniacal solution of carmine is added acetic acid, with continual stirring, until the colour of the solution changes to blood-red through incipient precipitation of the carmine. The supernatant clear solution is poured off, and injected cold without further preparation. The injected organs are thrown at once into strong alcohol to fix the carmine. For injec- tion of fishes. 523. LETELLIER'S Vanadate of Ammonia and Tannin. See Journ. Roy. Mic. Soc., 1889, p. 151, or previous editions. 524. TAGUCHI'S Indian Ink (Arch. f. mile. Anat., 1888, p. 565; Zeit. f. wiss. Mik., 1888, p. 503). Chinese or (better) Japanese ink well rubbed up on a hone until a fluid is obtained that does not run when dropped on thin blotting-paper, nor form a grey ring round the drop. Inject until the prepara- tion appears quite black, and throw it into some hardening liquid (not pure water). I believe this will be found useful for many purposes, especially for work amongst Invertebrates, as well as for lymphatics, juice-canals, and the like. Cello i din Masses. 525. SCHIEFFEEDECKER'S Celloidin Masses (Arch. Anat. u. Phys., 1882 [Anat. Abth.'], p. 201). (For Corrosion preparations.) See previous editions of this work, or WHITMAN'S Methods in Microscopical Anatomy. 526. HOCHSTETTER'S Modification of SCHIEFFERDECKER'S Mass (Anat. Anz., 1886, p. 51 ; Journ. Roy. Mic. Soc., 1888, p. 159). Other Masses. 527. BUDGE'S Asphaltum Mass. See Arch.f. mik. Anat.,xiv, 1877, p. 70, or previous editions. 528. HOYER'S Shellac Mass (Arch.f. mik. Anat., 1876, p. 645). See previous editions. This method, with some slight modifications of detail, has been recom- mended by BELLARMINOW (Anat. Anz., 1888, p. 650; see also Zeit.f. wiss. Mile., v, 4, 1888, p. 523, and Journ. Boy. Mic. Soc., 1889, p. 150). 529. HOYER'S Oil-colour Masses (Internat. Monatschr.f. Anat., 1887, p. 341 ; see also Zeit.f. wiss. Mik., 1888, p. 80, and Journ. Roy. Mic. Soc., 1888, p. 848). PANSCH'S Starch Mass (see Arch.f. Anat. u. Entw., 1877, p. 480; 1880, pp. 232, 371 ; 1881, p. 76; 1882, p. 60; 1883, p. 265 ; and a modification of the same by GAGE, Amer. Mon. Mic. Journ., 1888, p. 195 ; and Journ. Roy. Mic. Soc., 1888, p. 1056). Teichmann's Linseed-Oil INJECTIONS OTHER MASSES (COLD). 311 Masses (see 8. S. Math. Kl. Krakau Akad., vii, pp. 108, 158 ; Journ. Roy. Mic. 8oc. t 1882, pp. 125 and 716, and 1895, p. 704). 530. Natural Injections (RosiN, Traite, p. 6). To preserve these throw the organs into a liquid composed of 10 parts of tincture of perchloride of iron and 100 parts of water. RETTERER and ZELLNER use solution of Miiller, see Journ. Roy. Mic. Soc., 1894, p. 641. CHAPTER XXY. MACERATION AND DIGESTION. Maceration. 531. Methods of Dissociation. It is sometimes necessary, in order to obtain a complete knowledge of the forms of the elements of a tissue, that the elements be artificially separated from their place in the tissue and separately studied after they have been isolated both from neighbouring elements and from any interstitial cement-substances that may be present in the tissue. Simple teasing with needles is often insufficient to effect the desired isolation, as the cement-substances are often tougher than the elements themselves, so that the latter are torn and destroyed in the process. In this case recourse must be had to maceration processes, by which is here meant treatment with media which have the property of dissolving or at least softening the cement-substances or the elements of the tissue that it is not wished to study, whilst preserving the forms of those it is desired to isolate. When this soften- ing has been effected the isolation is completed by teasing, or by agitation with liquid in a test-tube, or by the method of tapping, which last gives in many cases (many epithelia, for instance) admirable results which could not be attained in any other way. The macerated tissue is placed on a slide and covered with a thin glass cover supported at the corners on four little feet made of pellets of soft wax. By tapping the cover with a needle it is now gradually pressed down, whilst at the same time the cells of the tissue are segregated by the repeated shocks. When the segregation has pro- ceeded far enough, mounting medium may be added, and the mount closed. The student will do well not to neglect this simple method, which is one that it is most important to be acquainted with. A good material for making ivaxfeetis obtained (VossELEK, Zeit.f. wiss. Mik., vii, 4, 1891, p. 461) by melting white wax MACERATION AND DIGESTION. 313 and stirring into it one half to two thirds of Venice turpen- tine. Care must be taken if the operation be performed over a naked flame, as the turpentine vapours are inflammable. 532. Iodised Serum. The preparation of this reagent has been given in Chap. XXI. The manner of employing it for maceration is as follows : A piece of tissue smaller than a pea must be taken, and placed in 4 or 5c.c. of weakly iodised serum in a well-closed vessel. After one day's soaking the maceration is generally sufficient, and the preparation may be completed by teasing or pressing out, as indicated above ; if not, the soaking must be continued, fresh iodine being added as often as the serum becomes pale by the absorption of the iodine by the tissues. By taking this precaution, the macera- tion may be prolonged for several weeks. It is obvious that these methods are intended to be applied to the preparation of fresh tissues, the iodine playing the part of a fixing agent with regard to protoplasm, which it slightly hardens. 533. Artificial Iodised Serum (FEEY, Le Microscope, p. 131 ; RAXVIEE, Traite, p. 77). The formula has been given in Chap. XXI. Ranvier states that he has been unable to obtain good results, for purposes of maceration, by this method. 534. Alcohol. RAXVIEE employs one-third alcohol (1 part of 36 alcohol to 2 parts of water) . Epithelia will macerate well in this in twenty-four hours. Ranvier finds that this mixture macerates more rapidly than iodised serum. Other strengths of alcohol may be used, either stronger (equal parts of alcohol and water) or weaker (i alcohol, for isolation of the nerve-fibres of the retina, for instance TBIN) . All observers are agreed that one-third alcohol is a mace- rating medium of the highest order; LIST (Zeit.f. wiss. Mik. 3 1885, p. 511) states that for glandular structures it should be used with precaution, on account of swellings that it produces in the cells, and that Miiller's solution, or osmic acid, should be preferred for such objects. 535. Salt Solution. 10 per cent, solution of sodium chloride is a well-known and valuable macerating medium. 314 CHAPTER XXV. 536. MOLESCHOTT and Piso BORME'S Sodium Chloride and Alcohol (MOLESCHOTT'S Untersuchungen zur Naturlehre, xi, pp. 99107; RANVIER, Traite,p. 242). -10 per cent, solution of sodium chloride, 5 volumes ; absolute alcohol, 1 volume. For vibratile epithelium, Ranvier finds the mixture inferior to one-third alcohol. 537. Formaldehyde. GAGE has tested formaldehyde as a dissociating agent. He recommends the addition of two parts of formalin (40 per cent, solution of formaldehyde) to 1000 parts of normal salt solution. He states that the results are highly satisfactory, the mixture acting quickly and yet retarding deterioration for some time (quoted from FISH, Proc.Am. Hie. Soc., xvii, 1895, p. 328). 538. Chloral Hydrate. In not too strong solution, from 2 to 5 per cent., for instance, chloral hydrate is a mild macerating agent that admirably preserves delicate elements. LAVDOWSKY (Arch, f. mik. Anat., 1876, p. 359) recommends it greatly for salivary glands. HICKSON (Quart. Journ. Mic. Sci., 1885, p. 244) recommends it for the study of the retina of Arthropods. 539. Caustic Potash, Caustic Soda. These solutions must be employed strong, 35 to 50 per cent. (Moleschott) ; so employed they do not greatly alter the forms of cells, whilst weak solu- tions destroy all the elements. (Weak solutions may, how- ever be employed for dissociating the cells of epidermis, hairs, and nails.) The strong solutions may be employed by simply treating the tissues with them on the slide. To make permanent preparations, the alkali should be neutralised by adding acetic acid, which forms with caustic potash acetate of potash, a well-known mounting medium (see BEHRENS, KOSSEL, and SCHIEFFERDECKER, Das Mikroskop, i, 1889, p. 156). It has been found by S. H. and S. P. GAGE (Proc. Amer. Soc. of Microscopists, 1889, p. 35; Zeit. f. wiss. Mik., vii, 3, 1890, p. 349) that instead of acetic acid, 60 per cent, acetate of potash solution, employed in considerable quantity and if desired with addition of 1 per cent, of acetic acid, may be used, the preparations either being mounted therein, or in glycerin or glycerin jelly. They may be stained if the acetate be first MACERATION AND DIGESTION. 315 washed out by treatment for twenty-four hours with aluin solution. 540. Sulphocyanides of Ammonium and Potassium (STIRLING, Journ. Anat. and Phys., xvii, 1883, p. 208). 10 per cent, solution of either of these salts is, according to Stirling, an admirable dissociating medium for epithelium. Macerate small pieces for twenty-four to forty-eight hours. If a crystalline lens be macerated as above its fibres become beaded or moniliform. 541. SOULIER'S Sulphocyanide Mixtures (Travaux de VInst. zool. de Hontpellier, Nouv. Ser., 2, 1891, p. 171). Soulier has found that Stirling's solution greatly deteriorates cellular elements, but that good results are obtained by combining it with a fixing agent. He prepared the following series of mixtures : Nos. 1. 2. 3. 4. 5. 6. 7. 8. Sulphocyanide of ammonium or potassium of 10 per cent., -20c.c. 30c.c. 35c.c. 36c.c. 37 c.c. 38c.c. 39c.c. 39'5 c.c. 5 per cent., 2'5 per cent., or 1-25 per cent, strength Solution of ") Ripart and [ 20 c.c. lOc.c. 5 c.c. 4c.c. 3 c.c. 2c.c. 1 c.c. O'o c.c. Petit . . . J The best results were obtained with series made with a 2 per cent, solution of Sulphocyanide. Soulier also obtained good results by combining liquid of Ripart and Petit with artificial serum of Kronecker instead of Sulphocyanide, or with pepsin, eau de Javelle, 10 per cent, sulphate of soda, or 1*5 per cent, solution of caustic soda. And he further found that good results are obtained by combining solutions of chloride of sodium, or solutions of caustic potash or soda, with any of the usual fixing agents. 542. Saliva, Artificial (for embryology of nerve and muscle) (CALBERLA'S formulae, Arch. f. mik. Anat., xvi, 1879, p. 471, et seq.). After having made trial of various different mace- rating agents, with the object of obtaining isolation of tha developing muscle and nerv of embryos of Amphibia and 316 CHAPTER XXV. Ophidia, CaH^rla found that the best results were obtained by means of Czerny's mixture of saliva and solutio Miilleri. This led him to imagine an artificial saliva, which on trial gave results as good as those obtained by natural saliva, or even better. Second formula (the first formula is suppressed, as being more complicated, and not giving better results) : Potassium chloride . . . . 0'4 Sodium chloride . . . . . 0'3 Phosphate of soda .... 0'2 Calcium chloride . . . . . 0*2 M This is dissolved in 100 parts of water, saturated with carbonic acid, and the solution combined with water and solutio Miilleri, one volume of the solution being combined with half a volume of Miiller's solution and a volume of water. In either case the Miiller's solution may be replaced by a 2 per cent, solution of chromate of ammonia. The best results were obtained when the solutions were saturated with the C0 2 just before using. The tissues are isolated by teasing and shaking, and speci- mens mounted in concentrated acetate of potash. 543. LANDOIS'S Solution (Arch. f. mik. Anat., 1885, p. 445). Saturated sol. of neutral chromate of ammonia 5 parts. Saturated sol. of phosphate of potash . . 5 Saturated sol. of sulphate of soda . . 5 Distilled water 100 To be used in the same way as chromic acid : Small pieces of tissue are macerated for one to three, or even four or five days, in the liquid, then brought for twenty-four hours into ammonia carmine diluted with one volume of the macerating liquid. GIEKKE particularly recommends this liquid for all sorts of macerations, but especially for the central nervous system, for which he finds it superior to all other agents. It is also re- commended for the same purpose by NANSEN (v. Zeit. f. wiss. Mik.,v,2, 1888, p. 242). 544. Permanganate of Potash. Has an action similar to that MACERATION AND DIGESTION. 317 of osmic acid, but more energetic. Is recommended, either alone or combined with alum, as the best dissociating agent for the fibres of the cornea (ROLLETT, Strieker's Handbuch, p. 1108). 545. Chromic Acid. Generally employed of a strength of about 0'02 per cent. Specially useful for nerve tissues and smooth muscle. Twenty-four hours' maceration will suffice for nerve-tissue. About 10 c.c. of the solution should be taken for a cube of 5 mm. of the tissue (RANVIER). 546. Bichromate of Potash. 0*2 per cent. 547. Miiller's Solution. Same strength. 548. Midler's Solution and Saliva (see above, 542). 549. BROCK'S Medium (for nervous system of Mollusca, Intern. Monatsch. f. Anat., i, 1884, p. 349). Equal parts of 10 per cent, solution of bichromate of potash and visceral fluid of the animal. 550. MOBIUS'S Media (quoted from Zeit. f. wiss. Mik., iii, 3, 1886, p. 402). 1. One part of sea water with 4 to 6 parts of 0*5 per cent, solution of bichromate of potash. 2. 0-25 per cent, chromic acid, O'l per cent, osmic acid, 0*1 per cent, acetic acid, dissolved in sea water. For Lamelli- branchiata. Macerate for several days. 551. GAGE'S Picric Alcohol (Proc. Amer. Soc. of Microscopists, 1890, p. 120; Zeit.f. wiss. Mik., ix, 1, 1892, pp. 87, 88). 95 per cent, alcohol, 250 parts; water, 750; picric acid, 1. Recommended for most tissues, but especially for epithelia and smooth and striated muscle. This is also much recom- mended by HOPKINS in the same place. A few hours' macera- tion is generally sufficient. 552. Osmic Acid. 0*1 per cent., for from a few minutes to a fortnight (cortex of cerebrum RINDFLEISCH). May be fol- lowed by maceration in glycerin. 318 CHAPTER XXV. 553. Osmic and Acetic Acid (the HEETWJGS' Liquid, Das Ner- vensystem u. die Sinnesorgane der Medusen, Leipzig, 1878, and Jen. Zeitschr., xiii, 1879, p. 457 ; Journ. Roy. Mic. Soc., iii, 1880, p. 441, and [N.S.] iii, 1883, p. 732). O05 per cent, osmic acid . . .1 part. 0*2 acetic acid . . . 1 Medusx are to be treated with this mixture for two or three minutes, according to size, and then washed in repeated changes of O'l per cent, acetic acid until all traces of free osmic acid are removed; they then remain for a day in O'l per cent, acetic acid, are washed in water, stained in Beale's carmine (in order to prevent the osmium from over-blacken- ing, and to assist the maceration), and are preserved in glycerin. For Actinias the osmic acid is taken weaker, 0*04 per cent. ; both the solutions are made with sea water ; and the washing out is done with 0'2 per cent, acetic acid. If the maceration is complete, stain with picro-carmine ; if not, with Beale's carmine. 554. BELA HALLEE'S Mixture (Morplwl. Jahrb., xi, p. 321). One part glacial acetic acid, 1 part glycerin, 2 parts water. Specially recommended for the central nervous system of Mollusca (Rhipidoglossa). A sufficient degree of maceration is obtained in thirty to forty minutes, the cells showing less shrinkage than with other liquids. 555. Nitric Acid. Most useful for the maceration of muscle. The strength used is 20 per cent. After twenty-four hours' maceration in this, isolated muscle-fibres may generally be obtained by shaking the tissue with water in a test-tube. Preparations may afterwards be washed with water and put up in strong solution of alum, in which they may be pre- served for a long time (HOPKINS, Proc. Amer. Soc., of Micro- scopists, 1890, p. 165; Zeit.f. iviss. Mik., ix, 1, 1892, p. 86). Maceration is greatly aided by heat, and at a temperature of 40 to 50 C. may be sufficiently complete in an hour (GAGE). 556. Nitric Acid and Chlorate of Potash (KUHNE'S method, UeberdieperipherischenEndoryane, &c., 1862; RANVIEE, Traite, MACERATION ANb DIGESTION. 319 p. 79). Chlorate of potash is mixed, in a watch-glass, with four times its volume of nitric acid. A piece of muscle is buried in the mixture for half an hour, and then agitated with water in a test-tube, by which means it entirely breaks up into isolated fibres. V J 557. Sulphuric Acid (RANVIEE, Traite, p. 78). Sulphuric acid has been employed by Max Schultz for isolating the fibres of the crystalline. Macerate for twenty-four hours in 30 grms. of water, to which are added 4 to 5 drops of concentrated sulphuric acid. Agitate. ODENIUS found very dilute sulphuric acid to be the best reagent for the study of nerve-endings in tactile hairs. He macerated hair-follicles for from eight to fourteen days in a solution of from 3 to 4 grains of " English sulphuric acid " to the ounce of water. Hot concentrated sulphuric acid serves to dissociate horny epidermic structures (horn, hair, nails). 558. Oxalic Acid. Maceration for many days in concen- trated solution of oxalic acid has been found useful in the study of nerve-endings. 559. SCHIEFFEKDECKER'S Methyl Mixture (for the retina) (Arch. f. mik. Anat., xxviii, 1886, p. 305). Ten parts of gly- cerin, 1 part of methyl alcohol, and 20 parts of distilled water. Macerate for several days (perfectly fresh tissue). 560. Lysol (REINKE, Anat. Anz., viii, 1892, p. 582; Zeit. f. wiss. Mik., x, 2, 1893, p. 224). Lysol is an industrial product consisting in a solution of cresol in a neutral soap, and is used as an antiseptic. According to Reinke, it possesses a very rapid macerating action. He uses a 10 per cent, solu- tion in distilled water or in water containing alcohol and glycerin. Spermatozoa of the rat are said to be resolved into fibrils in a few minutes. The cortical cells of hairs are likewise resolved into fibrils in a few minutes. Epithelial cells of Salamandra are said to be dissociated instantaneously. Nuclear chromatin is destroyed, bringing into evidence the reticulated caryoplasm. 320 CHAPTER XXV. For some conclusions founded on this reaction, see Arch. f. mik. Anat., xliii, 3, 1894, p. 398, et seq. Digestion. 561. BEALE'S Digestion Fluid (Archives of Medicine, i, 1858 pp. 296 316). The mucus expressed from the stomach glands of the pig is rapidly dried on glass plates, powdered and kept in stoppered bottles. It retains its properties years. Eight tenths of a grain will dissolve 100 grains o coagulated white of egg. To prepare the digestion fluid, the powder is dissolved in distilled water, and the solution filtered. It filters readily Or the powder may be dissolved in glycerin. The tissues to be digested may be kept for some hours in the liquid at a temperature of 100 F. (37 C.). 562. BRUCKE'S Digestion Fluid (from CARNOY'S Biologie cellu laire, p. 94). Grlycerinated extract of pig's stomach . . 1 vol. 0*2 per cent, solution of HC1 . . . :. 3 vols. Thymol, a few crystals. 563. BICKFALVI'S Digestion Fluid (Centrabl.f. d. med. Wiss. 1883, p. 833). One grm. of dried stomachal mucosa is mixec with 20 c.c. of 0*5 per cent, hydrochloric acid, and put into an incubator for three or four hours, then filtered. Macerate the tissue in the solution for not more than half an hour to an hour. 564. KUSKOW'S Digestion Fluid (Arch. f. mik. Anat., xxx p. 32 ; cf. Zeit.f. wiss. Mik., iv, 3, 1887, p. 384). One part o: pepsin dissolved in 200 parts of 3 per cent, solution of oxalic acid. The solution should be freshly prepared, and the objects (sections of hardened Ligamentum Nuchae) remain in it at the ordinary temperature for ten to forty minutes. 565. SCHIEFFERDECKER'S Pancreatin Digestion Fluid (Zeit. f wiss. Mik., iii, 4, 1886, p. 483). Solution of pancreatin in water. Schiefferdecker employs the "Pankreatinum siccum" prepared by Dr. Witte, Rostock. A saturated solution is MACERATION AND DIGESTION. 321 made in distilled water, cold, and filtered. Pieces of tissue (epidermis) are macerated in it for three to four hours at about body temperature. Nuclei are preserved, and the forms of prickle-cells well shown. 566. KCHNE'S Trypsin Methods (see Unters. a. d. Phys. Inst. Univ. Heidelberg, i, 2, 1877, p. 219). Very complicated. 567. GEDOELST'S Methods (see La Cellule, iii, 1887, p. 117, and v, 1889, p. 126 ; also Zeit.f. wiss. Mile., vii, 1, 1890, p. 57). 21 CHAPTER XXVI. CORROSION, DECALCIF1CATION, AND BLEACHING. Corrosion. 568. Caustic Potash, Caustic Soda, Nitric Acid. Boiling, or long soaking in a strong solution of either of these, is an effi-j cient means of removing soft parts from skeletal structures (appendages of Arthropods, spicula of sponges, &c.) . 569. Eau de Javelle (Hypochlorite of Potash) (NOLL'S METHOD I Zool. Anzeig., 122, 1882, p. 528). Noll remarks that the usual method of preparing the skeleton of siliceous sponges and similar structures by corroding away the soft parts byj means of caustic potash has many disadvantages, of which a] principal one is that the spicula are not preserved in their normal positions. He therefore proceeds as follows : A pieced of sponge is brought on to a slide and treated with a few drops j of eau de Javelle, in which it remains until all soft parts are! dissolved. (With thin pieces this happens in twenty to thirty] minutes.) The preparation is then cautiously treated with, acetic acid, which removes all precipitates that may have- formed, and treated with successive alcohols and oil of cloves J and finally mounted in balsam. The same process is stated to be applicable to calcareoui structures. I feel convinced, however, that if the structures are delicate, they will suffer, or be entirely destroyed-. 570. Eau de Labarraque (Hypochlorite of Soda) may be useJ in the same way as eau de Javelle. Looss (Zool. Anz., 1885J p. 333) finds that either of these solutions will completely dissolve chitin in a short time with the aid of heat. For this purposajjhe commercial solution should be taken concentrated and boiling. A formula for making it is given in 597. If solutions diluted with 4 to 6 volumes of water be takenl and chitinous structures be macerated in them for twenty- CORROSION, DEC ALCIF! CATION, 'AND BLEACHING. 323 four hours or more, according to size, the chitin is not dis- solved, but becomes transparent, soft, and permeable to stain- ing fluids, aqueous as well as alcoholic. The most delicate structures, such as nerve-endings, are stated not to be injured by the treatment. The method is applicable to Nematodes and their ova, an object well known for the resistance they oppose to ordinary reagents, and also to the removal of the albumen from ova of Amphibia, &c. This is undoubtedly a valuable method. 571. ALTMANX'S Corrosion Method (Arch.f.mik.Anat., 1879, p. 471). Whilst almost all animal tissues are very quickly destroyed by eau de Javelle, vet fats, and particularly fats hardened by osmic acid, withstand its action for a long time. If, then, you introduce some fat or other into a tissue, harden it with osmic acid and corrode the tissue with eau de Javelle, you will obtain a mould in osmium-blackened and hardened fat, of the spaces you had filled with the fat introduced. The method may be of much use in certain special researches, such as those on the choroid, iris, and pigmented organs. I recommend the reader to carefully study the article, which does not well bear abstracting. A good abstract will be found in Journ. Roy. Mic. Soc., 1879, p. 610, with plate. Decalcification and Desilicification. 572. Decalcification. In order to obtain the best results, it is important to employ only material that has been duly fixed and hardened secundum artem; and it is well not to put too much confidence in reagents that are said to have the pro- perty of hardening and decalcifying fresh material at the same time (Fisn, Ref. Handb. Med. ScL, Supp., p. 425.) 573. Decalcification of Bone. I take the following historical sketch from Busch's article " On the Technique of the Histo- logy of Bone" (Arch. f. mik. Anat., xiv, 1877, p. 481 ; see also the paper of HAUG, in Zeit. f. wiss. Mik., viii, i, 1891, p. 1). The most widely used agent for decalcification is hydro- chloric acid. Its action is rapid, even when very dilute, but it has the disadvantage of causing serious swelling of the tissues. To remedy this, chromic acid may be combined with it, or alcohol may be added to it. Or a 3 per cent, solution of the acid may be taken and have dissolved in it 10 to 15 per cent, of common salt. Or (Waldeyer) to a yoVu P er cen ^ .solution of chloride of palladium may be added -^ of its volume of HC1. 324 CHAPTER XXVI. Chromic acid is also much used, but has a very weak decal- cifying action and a strong shrinking action on tissues. For this latter reason it can never be used in solutions of more than 1 per cent, strength, and for delicate structures much lower strengths must be taken. Phosphoric acid has been recommended for young bones. Acetic, lactic, and pyroligneous acids have considerable decalcifying power, but cause great swelling. Picric acid lias a very slow action, and is only suitable for very small structures. See further under the head of " Bone." 574. Nitric Acid (Bascn, 1. c.). To all other agents Busch prefers nitric acid, which causes no swelling and acts most efficaciously, whilst at the same time it does not injuriously attack tissue-elements. One volume of chemically pure nitric acid of sp. gr. 1*25 is diluted with 10 vols. water. It may be used of this strength for very large and tough bones ; for young bones it may be diluted down to 1 per cent. Fresh bones are first laid for three days in 95 per cent, alcohol ; they are then placed in the nitric acid, which is changed daily, for eight or ten days. They must be removed as soon as the decalcification is complete, or else they will become stained yellow. When removed they are washed for one or two hours in running water and placed in 95 per cent, alcohol. This is changed after a few days for fresh alcohol. Young and foetal bones may be placed in the first instance in a mixture containing 1 per cent, bichromate of potash and -^ per cent, chromic acid, and decalcified with nitric acid of 1 to 2 per cent., to which may be added a small quantity of chromic acid (-^- per cent.) or chromate of potash (1 per cent.). By putting them afterwards into alcohol the well-known green stain is obtained. 575. Nitric Acid and Alcohol. 3 per cent, of nitric acid in 70 per cent, alcohol. Soak specimens for several days or weeks. I do not know who first recommended this admirable medium. Pure nitric acid, even if weak, readily exercises a gelatinising action on bone; whilst the addition of alcohol (or of alum) counteracts this action (Fisn, Ref. Handb. Med. 8ci. t Supp., p. 425). CORROSION, DECALCIFICATION, AND BLEACHING. 325 THOMA (Zeit. f. wiss. Mik., viii, 2, 1891, p. 191) gives the following method. Take 5 vols. of 95 per cent, alcohol and 1 vol. pure concentrated nitric acid. Leave bones in this mix- tare, changing the liquid every two or three days, until thoroughly decalcified, which should happen even with large bones in two or three weeks. Wash out until every trace of acid is removed (i. e. for some days after no acid reaction is obtained with litmus paper) in 95 per cent, alcohol containing an excess of precipitated chalk. This may take eight to four- teen days, after which the tissues will stain well and may be treated as desired. 576. Nitric Acid and Alum (GAGE, quoted from FISH, 1. c.). A saturated aqueous solution of alum is diluted with an equal volume of water, and to each 100 c c. of the dilute so- lution is added 5 c.c. of strong nitric acid. Change every two or three days, until the decalcification is complete. For teeth this is said to be, perhaps, a better decalcifier than the alcohol mixture. 577. Hydrochloric Acid (see above, 573). RANVIEB says that it may be taken of 50 per cent, strength, and then has a very rapid action. To counteract the swelling action of the acid, sodium chloride maybe added. Two formulae of this sort have been given by VON EBNEE (see HAUG'S paper quoted in the last section). The first is 100 c.c. cold saturated solu- tion of sodium chloride in water, 100 c.c. water, and 4 c.c. hydrochloric acid. Preparations to be placed in this, and 1 to 2 c.c. hydrochloric acid added daily until they are soft. The second is, 2'5 parts of hydrochloric acid, 500 of alcohol, 100 of water, and 2'5 of sodium chloride. HAUG pre- fers the proportions of I'O to o'O of acid, 70 of alcohol, 30 of water, and 05 of salt. 578. Hydrochloric Acid and Chromic Acid (BAYEBL, Arcli.f. mik* Anat., 1885, p. 35). Equal parts of 3 per cent, chromic acid and 1 per cent, hydrochloric acid. For ossifying cartilage. HAUG recommends equal parts of 1 per cent, hydrochloric acid and 1 per cent, chromic acid (1. c.). 579. Hydrochloric Acid and Nitric Acid ( HOPE WELL SMITH, Journ. Roy. Hie. Soc., 1892, p. 433). Place teeth in 12 parts of 10 percent. HC1, and after 15 hours add 1/5 parts of HN0 3 , and after 48 hours add To parts more of HN0 3 . After 75 to 80 hours remove and wash for half an hour in a solution of 5 grms. of lithium carbonate to an ounce of water. 580. Hydrochloric Acid and Glycerin. Glycerin, 95 ; hydrochloric acid, 5. Recommended for softening teeth in SQUIBE'S Methods and For- mula?, p. 12. 326 CHAPTER XXVI. 581. Picric Acid should be taken saturated. Picro-sulphuric acid should of course be avoided on account of the forma- tion of gypsum. Picro-nitric or Picro-hydrochloric Acid. The reader will perhaps reflect that the last two fluids appear likely to be very useful for decalcifications. Mayer points out that the action is very rapid, and that the copiously evolved C0 2 often produces, mechanically, lesions in tissues; so that in many cases in which calcareous structures are concerned chromic acid is to be preferred, the more so as it more effectually hinders any collapsing of the structures that might result from the withdrawal of their supporting cal- careous elements. Picric acid fluids are good media, as though their action is slow they preserve tissues well, and leave them in a good state for subsequent staining. 582. Phosphoric Acid. 10 to 15 per cent. (HAUG, 1. c., in 573). Somewhat slow, staining not good. 583. Lactic Acid. 10 per cent, or more. Fairly rapid, preserves well, and may be recommended (HAUG, 1. c.). 584. Chromic Acid is employed in strengths of from O'l per cent, to 2 per cent., the maceration lasting two or three weeks (in the case of bone). It is better to take the acid weak at first, and increase the strength gradu- ally. In any way the action is extremely slow, and it is therefore better to take one of the mixtures of chromic acid with a more energetic agent. 585. Chromic and Nitric Acid. Dissolve 15 grms. pure chromic acid in 7 oz. of distilled water, to which 30 minims of nitric acid are after- wards to be added. Macerate for three or four weeks, changing the fluid frequently (Marsh). FOL takes 70 volumes of 1 per cent, chromic acid, 3 of nitric acid, and 200 of water (Lehrb., p. 112). FISH takes simply liquid of Perenyi. It remains to be added that even with the addition of nitric or hydro- chloric acid the action is excessively slow, frequently requiring months to be complete. 586. Arsenic Acid. 4 per cent, aqueous solution, used at a tempera- ture of 30 to 40 C. (SQUIRE'S Methods and Formula, &c.,.'p.'ll). 587. Phloroglucin (ANDEER, Centralbl. f. d. med. Wiss., xii, xxxiii, pp. 193, 579 ; Intern. Monatschr., i, p. 350 ; Zeit.f. wiss. Mik., 1885, pp. 375, 539 ; Journ. Roy. Mic, Soc., 1887, p. 504 ; HAUG, Zeit.f. wiss. Mik., viii, 1, 1891, p. 8; FEREERI, ibid.,ix, 2, 1892, p. 236; Bull R. Accad. Med. di Boma, 1892, p. 67). This is the most recent of the decalcification methods. It has the advantages of being the most rapid of any, and of preserving the tissues very well (with the exception of blood). CORROSION, DECALCIFICATIOX, AND BLEACHING. 327 Phloroglucin by itself is not a solvent of lime salts; its function in the mixtures given below is so to protect the organic elements of tissues against the action of the miueral acids that these can be used in a much more concentrated form than would be otherwise advisable. HAUG advises the following procedure : Bring 1 grm. of phloroglucin into 10 c.c. of pure, not fuming nitric acid (1'4 sp. gr.), and warm very slowly and carefully with gentle agitation. There is formed a clear solution of (presumably) a nitrate of phloroglucin. Dilute the solution with 100 e.c- of distilled water, and add 10 c.c. of nitric acid. This gives a solution containing 20 per cent, of acid, which is the proper proportion. More water may be added to the solution, to make it up to 300 c.c., if nitric acid be also added in the proportion given. But the dilution must not be carried beyond this point, in order that the preservative action of the phloroglucin be not overmuch weakened. The process of decalcifi cation in this solution is extremely rapid, and there- fore should be carefully watched. Foetal and young bones become quite soft in half an hour ; small pieces of old and hard bones (femur, temporal bone) in a few hours. Teeth take longer, and may require, if time be an object, a solution made with 35 to 45 per cent, of nitric acid. Wash out for two days in running water. The tissues stain well. The solution may be made with hydrochloric acid instead of nitric acid, 30 per cent, of acid being taken, and 0'5 per cent, of sodium chloride added. For slow decalcification a 2 to 5 per cent, nitric acid solu- tion -may be used, or a mixture containing of phloroglucin 1 part, nitric acid 5, alcohol 70, and distilled water 30 parts. For the labyrinth, FEREERI advises a mixture containing 1 grm. of phloroglucin, dissolved with the aid of heat in 10 grms. of hydrochloric acid with 100 of water, 200 of 70 per cent, alcohol being added after cooling. The mixture should be changed once a week during thirty to forty days. Desilicification . 588. Hydrofluoric Acid (MAYEE'S method, Zool. Anz. t 1881, No. 97, p. 593). The objects from which it is desired to remove siliceous parts are brought in alcohol into a glass vessel coated internally with paraffin (other- wise the glass would be corroded by the acid). Hydrofluoric acid is then added drop by drop (the operator taking great care to avoid the fumes, 328 CHAPTER XXVI. which attack mucous membranes with great energy). A Wagnerella borealis may thus be completely desilicified in a few minutes. Small pieces of sili- ceous sponges will require a few hours, or at most a day. The tissues do not suffer ; and if they have been previously stained with acetic acid carmine the stain does not suffer; at least, this was so in the case of Wagnerella. This dangerous method is best avoided. As regards sponges, I would point out that if well imbedded, good sections may be made from them without previous removal of the spicula. The spicula appear to be cut ; probably they break very sharply when touched by the knife. Knives are of course not improved by cutting such sections. Bleaching. 589. MAYEE'S Chlorine Method (Mitth. Zool. Stat. Neapel, ii, 1881 , p. 8). This is a process imagined for the purpose of getting rid of the blackening that often occurs as a conse- quence of treatment by osmic acid. The specimens are put into alcohol (either 70 or 90 per cent.). Crystals of chlorate of potash are added until the bottom of the vessel is covered with them. A few drops of concentrated hydrochloric acid are then added by means of a pipette, and mixed in by shaking the vessel as soon as the green colour of the evolving chlorine has begun to show itself. Warm if necessary ; but most objects, even large ones, may be bleached in half a day without the employment of heat. The tissues do not suffer. Instead of hydrochloric acid, nitric acid may be used ; in which case the bleaching agent is the freed oxygen, instead of chlorine. The first method may be used for the purpose of removing pigment from the eyes of insects. 590. MABSH'S Chlorine Method (Section Cutting, p. 89). Marsh generates chlorine in a small bottle by treating crystals of chlorate of potash with strong HC1, and leads the gas (by means of a piece of glass tubing bent twice at right angles) to the bottom of a bottle containing the sections in water. (See a fig. of the apparatus in Journ. Roy. Mic. Soc., iii, 1880, p. 854.) 591. Chlorine Solution (SARGENT'S method). Hydrochloric acid, 10 drops ; chlorate of potash, dr. ; water, 1 oz. Soak for a day or two. Wash well. Tliis method is intended for " bleaching insects; " it will be seen that it is CORROSION, DECALCIFICATIOX, AND BLEACHING. 329 only applicable to the preparation of hard parts, as soft tissues would be destroyed by the solution. 592. Kreasote (POUCHET'S method, Journ. de I'Anat., 1876, p. 8, et. seq.). I gather from the paper here quoted that, most of the granular animal pigments are soluble in kreasote. Other solvents are mentioned in this paper ("On the Change of Colouration through Nervous Influence"), but this appears to be the only one capable of general histological appli- cation. 593. Nitric Acid has a similar action. PARKER (Bull. Comp. ZooL, Cambridge, U.S.A., 1889, p. 173; see Zeit. f. ?//.>.-. Mik.j viii, 1, 1891, p. 82) says that for eyes of scorpions the usual 5 to 10 per cent, solutions are not strong enough. He treats sections, fixed to the slide with Schallibaum's medium, for about a minute with a solution of up to 50 per cent. of nitric acid in alcohol, or, still better, with a 35 per cent. solution of a mixture of equal parts of nitric and hydrochloric acid in alcohol. To make the solution, the acid should be poured slowly into the alcohol (not vwe versa), and the mixture kept cool. 594. Peroxide of Hydrogen (Oxygenated Water) (POUCHET'S method, M. DUVAL, Precis, &c., p. 234). Macerate in glycerin to which has been added a little oxygenated water (5 to 6 drops to a watch-glass of glycerin). (Oxygenated water may be procured from perfumers or hair-dressers, by whom it is sold as a hair dye under the name of "Aureoline," " Golden hair-wash," or the like.) The brownish-green colour communicated to tissues by chromic solutions may be changed to yellow by means of oxygenated water (see 41). Osmium preparations may be bleached in the same way (see 38). 595. Peroxide of Sodium (CARAZZI, ZooL Anz., 444, 1894, p. 135). Peroxide of sodium, Na 2 O 2 , is a yellowish powder, which in presence of water gives off oxygen, and communi- cates an alkaline reaction to the water. With acidified water this alkalinity is not produced. Carazzi advises that a 10 per cent, solution of tartaric or acetic acid be put into a suitable recipient, and a small quantity of the peroxide be put at the bottom of the recipient. Alcohol of 70 per cent, is then cautiously poured on to the surface of the liquid, and the 330 CHAPTER XXVI. objects to be bleached (previously saturated with alcohol) are then put into the supernatant alcohol. Oxygen is given off from the bottom of the liquid, and rises and dissolves in the alcohol, bleaching the objects that have been placed there. Mineral acids must not be employed, and large quantities of peroxide must not be added to a small quantity of liquid, as the reaction under these conditions is very violent. 596. Sulphurous Acid. Prof. GILSON writes me that he finds alcoholic solution of sulphurous anhydride (SO 2 ) very con- venient for the rapid decolouration of bichromate objects. A few drops suffice. 597. Eau de Labarraque. Eau de Javelle (see 569, 570). These are bleaching agents. For the manner of preparing a similar solu- tion see Journ. de Microgr., 1887, p. 154, or Journ. Roy. Mic. Soc., 1887> p. 518. It is, shortly, as follows : 8 parts of caustic soda are dissolved in 100 parts of distilled water, and chlorine is passed" through to saturation. During the passage of the chlorine the solution must be surrounded with a mixture of salt and ice, otherwise the temperature rises, and chloride and chlorate of soda are produced. The resulting solution contains 7'45 per cent, of hypochlorite of soda. It is green ; and the more effectual the cold, the greener is the colour. The energy of the decolourising action is pro- portional to the greenness of the solution. Of course the method cannot be used for bleaching soft parts which it is desired to preserve. 598. Chloroform helps to clear strongly pigmented chitin, and com- bined with nitric acid will decolourise it entirely (see below, in the chapter on Arthropods, Part II). 599. GRENACHER'S Mixture for Eyes of Arthropods and other Animals (Abh. nat. Ges. Halle-a.-S., xvi ; Zeit. f. wiss. Mik., 1885, p. 244). Glycerin . . . . . .1 part. 80 per cent, alcohol ... .2 parts. Mix and add 2 to 3 per cent, of hydrochloric acid. Pigments dissolve in this fluid, and so doing form a stain which suffices in twelve to twenty-four hours for staining the nuclei of the preparation. You may, if you like, first stain the objects with borax-carmine, and then put them into the liquid, the pigment being washed out more rapidly than the carmine. But the progress of the decolouration must be care- fully watched. CHAPTER XXVII. EMBRYOLOGICAL METHODS. 600. Artificial Fecundation. This practice, which affords the readiest means of obtaining the early stages of develop- ment of many animals, may be very easily carried out in the case of the Amphibia anura, Teleostea, Cyclostomata, Echino- dermata, and many Vermes and Ccelenterata. In the case of the Amphibia, both the female and the male should be laid open, and the ova should be extracted from the uterus and placed in a watch-glass or dissecting-dish, and treated with water in which the testes, or, better, the vasa deferentia, of the male have been teased. Females of Teleostea are easily spawned by manipulating the belly with a gentle pressure ; and the milt may be ob- tained from the males in the same way. (It may occasionally be necessary, as in the ease of the Stickleback, to kill the male, and dissect out the testes and tease them.) The sper- matozoa of fish, especially those of the Salmonidae, lose their vitality very rapidly in water ; it is therefore advisable to add the milt immediately to the spawned ova, then add a little water, and 'after a few minutes put the whole into a suitable hatching apparatus with running water. Artificial fecundation of Invertebrates is easily performed in a similar way. It is sometimes possible to perform the operation under the microscope, and so observe the penetra- tion of the spermatozoon and some of the subsequent phe- nomena, as has been done by Fol, the Hertwigs, Selenka, and others for the Echinodermata and other forms. 601. Superficial Examination. The development of some animals, particularly some Invertebrates, may be to a certain extent followed by observations of the living ova under the microscope. This may usefully be done in the case of various Teleosteans, such as the Stickleback, the Perch, Macropodus, and several pelagic forms, and with Chironomus, Asellus 332 CHAPTER XXVII. aquaticus, Ascidians, Planorbis, many Coelenterata, &c. I advise the student to carefully draw the different stages so observed, for such drawings are most important aids to the study of the same stages by the section method. Some ova of Insecta and Arachnida which are completely opaque under normal conditions become transparent if they are placed in a drop of oil ; if care be taken to let their surface be simply impregnated with the oil, the normal course of development is not interfered with (BALBIANI). 602. Preparation of Sections. Osmic acid, employed either alone or in combination with other reagents, is an excellent fixing agent for small embryos, but not at all a good one for large ones. It causes cellular elements to shrink somewhat, and therefore brings out very clearly the slits that separate germinal layers, and any channels or other cavities that may be in course of formation. In virtue of its property of blackening fatty matters, myelin amongst them, it is of service in the study of the development of the nervous system. Chromic acid is indispensable for the study of the external forms of embryos ; it brings out elevations and depressions clearly, and preserves admirably the mutual relations of the parts ; but it does not always preserve the forms of cells faithfully, and is a hindrance to staining in the mass. Picric liquids have an action which is the opposite of that of osmic acid ; they cause cellular elements to swell some- what, and thus have a tendency to obliterate spaces that may exist in the tissues. But notwithstanding this defect, the picric compounds, and especially Kleinenberg's picro- sulphuric acid, are amongst the best of embryological fixing agents. RABL (Zeit. f. iviss. Mik., xi, 2, 1894, p. 165) highly re- commends for embryos of Vertebrates, and also for other objects, the following platino-sublimate mixture. Platinic chloride, 1 per cent, solution 1 vol. Saturated aqueous sublimate solution 1 Distilled water .... 1 This serves for a large number of blastoderms and young embryos (Pisces, Amphibia, Aves, Mammalia). Advanced embryos of Teleostea ought to be fixed in the warmed mix- EMBUYOLOGICAL METHODS. 333 ture, in order to avoid rupture of the muscles and shrinkage of the chorda. RABL'S picro-sublimate mixture has been given 61. It is recommended especially for somewhat advanced embryos, such as embryo chicks from the third or fourth day, and other embryos of a similar size. For imbedding, the celloidin-chloroform method of Yiallanes gives excel- lent results, and so does paraffin. This latter is preferable in so far as it lends itself better to the rapid production of series of sections, and allows of the use of the Cambridge Rocking Microtome, the Minot, or the Reinhold- Giltay, which is perhaps the microtome par excellence of the embryologist. As to staining, my eminent fellow-worker, Dr. Henneguy, writing the chapter on embryological methods for the French edition of this work, advised staining in the mass with borax-carmine or alum-carmine (Henne- guy's acetic acid formula) ; or, as an alternative, the staining of sections by Flemming's method. The improvements that have in recent times been worked out in this method give still greater weight to the latter recom- mendation. 603. Reconstruction of Embryos from Sections. The study of a series of sections of any highly differentiated organism of unknown structure is so complicated that it is often necessary to have recourse to elaborate methods of geometrical or of plastic reconstruction in order to obtain an idea or a model of the whole. These methods have now been brought to so high a degree of complexity that a volume rather than a paragraph would be necessary to describe them. See BORN, "Die Plattenmodellirmethode," in Arch. f. mik. Anat., 1883, p. 591, and Zeit. f. wiss. Mik., v, 4, 1888, p. 433 ; STRASSER, in Zeit.f. wiss. Mik., iii, 2, 1886, p. 179, and iv, 2 and 3, pp. 168 and 330 ; KASTSCHENKO, in Zeit. f. iviss. Mik., iv, 2 and 3, 1887, pp. 235-6 and 353, and v, 2, 1888, p. 173 (abstracts of all these papers may be found in Journ. Roy. Mic. Soc. of the years quoted). A simple, but in many cases quite efficient plan, has been described by FOL (Lehrb.j p. 35) as follows : Before cutting your sections, you make an outline drawing of your object, under the magnification that you intend to employ for the reconstructed drawing, and in a plane perpendicular to that of the intended sections. For instance, if you intend to make trans- verse sections of an embryo, begin by making a profile drawing of it, that is, a drawing of the outline of an ideal sagittal section of it. Then make your series of sections, and make drawings of them all under the same magnification as the sagittal drawing. Then trace over your sagittal draw- 334 CHAPTER XXVII. ing a series of equidistant parallel lines in positions corresponding to the sections that have been made. If your sections are one hundredth of a millimetre thick, and your drawing be magnified one hundred times, the lines should be one millimetre apart (if you intend to reconstruct the whole of your sections, but the operation may frequently be abridged by only reconstructing say every fifth or every tenth section). You have now to fill in your outline drawing with details borrowed from the drawings of the sections. You may help yourself greatly in the follow, ing way : A plate of glass, of a size suitable to the intended drawings, is covered with a layer of gelatin, and dried. On this is ruled a series of parallel lines, very close together, and ruled with differently coloured inks, the colours recurring in regular order. The plate is then cut into two unequal parts by a diamond, on a line perpendicular to the coloured lines. Lay one of the parts of the plate on the outline drawing so that the cut edge covers the line that corresponds to the first section you are going to fill in ; then lay the other part of the plate on the drawing of the section in such a position that the limits of the drawing correspond to the same coloured lines that cover the limits of the outline drawing on the other part of the plate already placed. Trace on the plate that covers the drawing of the section the outline of the internal organs. Lay it against its fellow-plate on the outline drawing, making the coloured lines correspond, and you will easily be able to mark off accurately on the outline drawing a series of dots that correspond in position to the outlines of the internal organs. This operation having been repeated for each of the sections that you desire to bring into your recon- struction, nothing remains but to join your dots by lines, and you will have filled up your outline drawing with a representation of the internal organs in the same plane. If any reader think this process complicated, he needs but to spend five minutes in trying it with a piece of tracing paper, and will find it to be in reality extremely simple. Another simple plan is to gum the drawings of the sections on cardboard of a thickness proportional to the thickness of the section and the magnifi- cation, cut out all the cavities of the drawing with a knife or fret-saw, and gum all the fretwork thus obtained together, This gives, of course, a model of the object. In simple cases it may be sufficient to adopt the plan described by SCHAFFEK (Zeit. f. wiss. Mik., vii, 3, 1890, p. 342). Careful outlines of the sections to be reconstructed are drawn on tracing-paper with the aid of the camera lucida, super- posed, and held up against the light for examination by transparence. EYCLESHEIMEE'S method of producing orienta- tion lines in celloidin imbedding mass has been described in Chap. X, and some paraffin orientation methods in 136. EMBRYOLOGICAL METHODS. 335 Mammalia. 604. Rabbit Dissection. The rabbit may conveniently be taken as a type for this kind of work. For the study of the early stages the ova must be sought for in the tubae a certain number of hours after copulation. The dehiscence of the follicles takes place about ten hours after the first coitus. The tubse and cornua having been dis- sected out should be allowed to cool, and remain until the muscular contractions have ceased. Then, with the aid of fine scissors or a good scalpel, all the folds of the genital duct are carefully freed from their peritoneal investment. The tubae are then (if the ova are still within them, which is the case up to the end of the third day after coition) laid out on a long slip of glass, and slit up longitudinally by means of a pair of fine, sharp scissors. By means of needles and forceps the tubal mucosa is spead out so as to smooth out its folds as much as possible, and is carefully looked over with a strong lens or with a low power of the microscope. When the ova are found, a drop of some " indifferent " liquid is dropped on each, and it is carefully taken up with the point of a scalpel, a cataract needle, or a small pipette. They may be examined in the peritoneal fluid of the mother if the animal has been killed, or in its aqueous humour, or in amniotic liquid, or in blood-serum, or in Kronecker's or other artificial serum. If you have not been able to find the ova with the lens or the microscope, scrape off the epithelium of the tubal mucosa with a small scalpel, mix it with a little indifferent liquid, and look for the ova under the microscope by transmitted light. Another method, employed by KOLLIKEK, consists in in- jecting solution of Muller or weak osmic acid into the oviduct by means of a small syringe, and collecting the liquid that runs out in a series of watch-glasses, in which the ova can very easily be found by the microscope. The same doe may be made to serve for two observations, at some hours' or days' interval. A longitudinal incision of 8 to 10 centimetres length is made on the median or a lateral line of the abdomen ; an assistant keeps the intestines in their place ; a ligature is placed at the base of one of the uterine cornua, beneath the neck, and a second ligature around the mesome. 336 CHAPTER XXVI I. trium and mesovarium. The ovary, the tuba, and the cornu of that side are then detached with scissors. The abdomen is then closed by means of a few sutures passing through the muscle-layers and the skin. The animals support the operation perfectly well, and the development of the ova of the opposite side is not in the least interfered with. When it is desired to study these the animal may be killed, or may be subjected to a second laparotomy if it be desired to preserve it for ulterior observations. During the fourth, fifth, and sixth days after copulation the ova of the rabbit are free in the uterine, cornua ; they are easily visible to the naked eye, and may be extracted by the same manipulations as those of the tubes. After the sixth day they are at rest in the uterus, but have not yet contracted adhesions with the mucosa, so that they can still be extracted whole. At this stage the parts of the cornua where the ova are lodged are easily distinguishable by their peculiar aspect, the ova forming eminences of the size of a pea. The cornua should be cut up transversely into as many segments as there are eminences, care being taken to have the ova in the centre of the segments. You then fix each segment by means of two pins on the bottom of a dissecting dish, with the meso- metrial surface downwards and the ovular eminence upwards. The dissecting dish is then filled up with serum or liquid of Miiller, or O'l per cent, solution of osrnic acid, or Kleinenberg's picro-sulphuric acid, or nitric acid, or acetate of uranium solution. With a small scalpel a longitudinal incision is made on the surface of the ovular eminence, not passing deeper than the muscular layer; the underlying* uterine mucosa is then gently dilacerated with two pairs of small forceps, and the ovum set free in the liquid. From the moment the ova have become adherent to the uterine mucosa they can no longer be extracted whole. The embryo being always situated on the mesometrial surface, the ovular eminence is opened by a crucial incision, and the strip of mucosa to which the embryo remains adherent is fixed with pins on the bottom of the dish. ED. v. BENEDEN (see Arch, de Biol., v, fasc. iii, 1885, p. 378) has been able by operating in this way in serum of Kronecker, and keeping the whole at blood temperature, to observe the circulation of the embryo for hours together. (If this be desired to be done, the crucial incision should not be too extended, so as to leave the terminal sinus intact.) EMBRYOLOGICAL METHODS. 337 RETTERER (C. E. Soc. de Biol., 1887, p. 99) advises that for ova of the seventh day the segment of uterus containing them be opened on the mesometrial surface, for at that date no adhesion has yet been contracted with that side. By running in liquid of Kleinenberg by means of a pipette between the ovum and the free surface of the uterus, the vum may be got away in the shape of a closed vesicle. 605. RABBIT ; Microscopic Preparations. In order to make permanent preparations of the different stages of fecundation and segmentation, v. BENEDEN (Arch, de Biol., i, 1, 1880, p. 149) recommends the following process : The living ovum is brought into a drop of 1 per cent, osmic acid on a slide, and thence into solution of Miiller (or bichromate of am- monia or solution of Kleinenberg). After an hour the liquid is changed, and the whole is put into a moist chamber, where it remains for two or three days. It is then treated with glycerin of gradually increasing strength, and at last mounted in pure glycerin acidified with formic acid. Ova may be -stained with Beale's carmine or picro-carmine, after removal from the osmic acid and careful washing. In order to bring out the outlines of blastoderm-cells the living ovum may be brought into one third per cent, solution of nitrate of silver. After remaining there for half a minute to two minutes, according to the age of the vesicle, it is brought into pure water and exposed to the light. The pre- parations thus obtained are instructive, but blacken rapidly, and cannot be permanently preserved. After the end of the third day the blastodermic vesicle can be opened with fine needles, and the blastoderm washed, stained, and mounted in glycerin or balsam ; v. Beneden has also obtained good preparations by means of chloride of gold. For embryonic areas and more advanced embryos KOLLIKER -recommends putting the ovum into 0*5 per cent, solution of osmic acid until it has taken on a somewhat dark tint, which happens in about an hour, and then treating it with successive alcohols for several hours. If the ovum be adherent to the uterine mucosa the portion of the membrane to which it is fixed should be left, stretched out with pins, in O'l per cent. solution of osmic acid for from four to six hours. The blasto- 22 CHAPTER XXVII. dermic vesicle can then easily be removed, and immersed for a few hours more in 0'5 per cent, solution of osmic acid, and finally be brought into alcohol. For sections Kolliker fixes with osmic acid. v. BENEDEN treats the ova for twenty-four hours with 1 per cent, solution of chromic acid, then washes well, and brings them through successive alcohols. Chromic acid has the advantage of hardening thoroughly the vesicle, and maintaining at the same time the epiblast-cells perfectly adherent to the zona pellucida. v. Beneden also recommends the liquid of Kleinenberg. HENNEGUY writes that he fre- quently employs it for embryonic areas and embryos of various ages, always with excellent results. Fol's modification of the liquid of Flemming, and Ranvier and VignaPs osmic acid and alcohol mixture ( 37), also give excellent results. For stain- j ing, Henneguy recommends borax-carmine or Delafield's ! hasmatoxylin for small embryos ; for large ones he found that his acetic acid alum-carmine was the only reagent that 3 would give a good stain in the mass. I think Carmalum is j now indicated. For sections, pure paraffin. Cut in series and mount in balsam. PIERSOL (Zeit.f. iviss. ZooL, xlvii, 2, 1888, p. 155) has been j lately using for fixation either Kleinenberg's solution or, for j young stages, Altmann's 3 per cent, nitric acid. Staining i and cutting as above. See also WEYSSE, Proc. Amer. Acad. Arts and Sci., 1894, 1 p. 285 (blastodermic vesicle of Sus scrofa) and SOBOTTA . j Arch. f. mik. Anat., xlv, 1895, p. 15 (fecundation and jj segmentation of the ovum of the mouse ; fixation in Flem- j ming's weak mixture, sections stained with Benda's iron | haematoxylin). Aves. 606. Superficial Examination. Excellent instructions on this _; head are given in FOSTER and BALFOUR'S Elements of Embryo- logy, to which, as it is certain to be in the student's hands, he may be referred. What follows here is given merely as being of more recent publication. If it be desired to observe a living embryo by transmitted light, the egg should be opened under salt solution, as de- EMBRTOLOGICAL METHODS. 339 scribed below. A little of the white is then removed through the window, the egg is lifted out of the liquid, and a ring of gummed paper is placed on the yolk so as to surround the embryonic area. As soon as the paper adheres to the vitel- line membrane, which will be in a few minutes, a circular incision is made in the blastoderm outside the paper ring. The egg is put back into the salt solution, and the paper ring removed, carrying with it the vitelline membrane and the blastoderm, which may then be brought into a watch- glass or on to a slide and examined under the microscope (DUVAL). 607. Gerlach's Window Method (Nature, 1886, p. 497; Journ. Boy. Mic. Soc., 1886, p. 359). Remove with scissors the shell from the small end of the egg ; take out a little white by means of a pipette ; the blasto- derm will become placed underneath the window thus made, and the white that has been taken out may be replaced on it. Paint the margins of the window with gum mucilage, and build up on the gum a little circular wall of cotton wool ; place on it a small watch-glass (or circular cover-glass), and ring it with gum. When the gum is dry, the cover is further fixed in its place by means of collodion and amber varnish, and the egg is put back in its normal position in the incubator. The progress of the development may be followed up to the fifth day through the window. A description of further developments of this method, with figures of special apparatus, will be found in Anat. Anz., ii, 1887, pp. 583, 609; see also Zeit.f. wiss. Mik., iv, 3, 1887, p. 369. 608. Preparation. During the first twenty-four hours of incubation it is extremely difficult to separate the blastoderm from the yolk, and they should be fixed and hardened together. In later stages, when the embryo is conspicuous, the blasto- derm can easily be separated from the yolk, which is very advantageous. To open the egg, lay it on its side and break the shell at the broad end by means of a sharp rap; then carefully remove the shell bit by bit by breaking it away with forceps, working away from the broad end until the blastoderm is exposed. The egg should be opened in salt solution, then lifted up a little, so as to have the blastoderm above the surface of the liquid ; the blastoderm is then treated with some fixing solution dropped on it from a pipette (1 per cent, solution of osmic acid, or Ranvier and Yignal's osmic acid and alcohol mixture, iodised serum, solution of Kleinen- berg, 10 per cent, nitric acid, &c.). By keeping the upper end of the pipette closed, and the lower end in contact with 340 CHAPTER XXVII. the liquid on the blastoderm, the blastoderm may be kept well immersed for a few minutes, and should then be found to be sufficiently fixed to be excised. (Of course, if you prefer it, you can open the egg in a bath of any fixing liquid [10 per cent, nitric acid being convenient for this purpose] of such a depth as to cover the yolk ; and having exposed the blasto- derm, leave it till fixed [fifteen to twenty minutes] ; but I think the procedure above described will generally be found more convenient.) The egg is put back into the salt solution, and a circular incision made round the embryonic area. The blastoderm may then be floated out and got into a watch-glass, in which it may be examined, or may be brought into a hardening liquid. Before putting it into the hardening fluid, the portion of vitelline membrane that covers the blastoderm should be removed with forceps and shaking. Fixation in 10 per cent, nitric acid has the advantage of greatly facilitating the separation of the blastoderm. The acid should be allowed to act for ten minutes, after which it is well to bring the preparation into 2 per cent, solution of alum (cf. HOFMANN, Ziet. f. wiss. Mik., x, 4, 1893, p. 485). In order to counteract the turning up of the edges of the blastoderm that generally happens during the process of hardening, it is well to get the blastoderm spread out on the convex surface of a watch-glass, and leave it so during the hardening. For hardening, FOSTER and BALFOUR recommend solution of Kleinenberg for five hours, followed by alcohol. Or chromic acid, a solution of 0*1 percent, for twenty-four hours, followed by a solution of 0*3 per cent, for twenty-four hours more, then by 70 per cent, alcohol for a day, 90 per cent, alcohol for two days, and lastly absolute alcohol. They also recommend a 0'5 per cent, solution of osmic acid, in which the embryo remains for two hours and a half in the dark, and after washing is brought into absolute alcohol. HENNEGUY prefers the osmic acid and alcohol mixture of Ranvier and Vignal, or Flemming's mixture followed by suc- cessive alcohols. Staining and imbedding may be performed by the usual methods. EMBRYOLOGICAL METHODS. 341 Up to about the fiftieth hour embryos may be mounted entire, in glycerin or balsam. 609. M. DUVAL'S Orientation Method (Ann. d. Sc. nat. ZooL, 1885). In the early stages of the development of the ova of Aves, before the appearance of the primitive streak, it is difficult to obtain a correct orientation of the hardened cica- tricula, so as to be able to make sections in any desired direc- tion. Duval, starting from the fact that during incubation the embryo is almost always found to be lying on the yolk in such a position that the big end of the egg is to the left, and the little end to the right of it, marks the position of the blastoderm in the following way. With a strip of paper 5 millimetres wide and 50 millimetres long you construct a sort of triangular bottomless box. You lay this on the yolk, enclosing the cicatricula in such a posi- tion that the base of the triangle corresponds to what will be the anterior region of the embryo, and its apex to the posterior region ; that is to say, if the big end of the egg is to your left, the apex of the triangle will point towards you. You now, by means of a pipette, fill the paper triangle with 0'3 solution of osmic acid. As soon as the preparation begins to darken you put the whole egg into weak chromic acid, remove the white, and put the rest into clean chromic acid solution for several days. After hardening you will find on the sur- face of the yolk a black triangular area, which encloses the cicatricula and marks its position ; you cut out this area with scissors and a scalpel, and complete the hardening with chromic acid and alcohol. See also the critique {ind modification of this method by KIONKA, Anat. Hefte, x, p. 391 ; Zeit. f. wiss. Mik., xi, 2, 1894, p. 250 (stain with borax carmine, adding to the acid alcohol used for washing out a few drops of aqueous solution of Orange Gr, which stains the vitellus yellow). See also the method of Hirota, Journ. Roy. Mic. Soc., 1895, p. 118. 610. VIALLETON'S Method (Anat. Anzeig., vii, 1892, pp. 624627; Journ. Roy. Mic. Soc., 1892, p. 889). Egg opened in salt solution, blasto- derm excised and removed to a glass plate, then treated with 1 per cent, nitrate of silver solution, washed with water, and put into 70 per cent, alcohol for six to twelve hours in the dark. Borax-carmine, alcohol, dammar. 342 CHAPTER XXVII. Reptilia. 611 General Directions. The methods described above for the embryology of birds are applicable to the embryology of reptiles. During the early stages the blastoderm should be hardened in situ on the yolk ; later the embryo can be iso- lated, and treated separately with Kleinenberg's solution and alcohol (STRAHL, Arch./. Anat. u. Phys., 1881, p. 123). 612. PEEENYI'S Method (Zool. Anzeig., 274, 1888, p. 139, and 276, p. 196). Fix in the following special mixture : 3 parts 20 per cent, nitric acid. 3 parts 1 per cent, chromic acid. 4 parts absolute alcohol. For embryos of Lacerta. Fix for twenty minutes. Wash out for an hour with 70 per cent, alcohol, and then with strong alcohol. Stain with Dela- field's hsematoxylin, and treat the stained material for three to five minutes with 1 per cent, chromic acid. 613. See also KUPFFEE'S Method (Arch.f. Anat. und Entwick., 1882, p. 4). SABASIN'S Method (SEMPEE'S Arbeiten, 1883, p. 159), and MITSU- CUEI (Journ. Roy. Mic. Soc., 1894, p. 750). Amphibia. 614. Preliminary. In order to prepare the ova of Amphibia for section cutting, it is essential to begin by removing their thick coats of albumen. This may be done by putting them for two or three days into 1 per cent, solution of chromic acid, and shaking well ; but ova thus treated are very brittle, and do not afford good sections. A better method is that described by WHITMAN (Amer. Natural., xxii, 1888, p. 857), and by BLOCHMANN (Zool. Anz., 1889, p. 269). Whitman puts the fixed eggs into a 10 per cent, solution of sodium hypo- chlorite diluted with 5 to 6 volumes of water, and leaves them there till they can be shaken free, which happens (for Necturus) in a few minutes. Blochmann takes eau de Javelle (potassium hypochlorite), and dilutes it with 3 to 4 volumes of water, and agitates the eggs previously fixed with solution of Flemming, for fifteen to thirty minutes in it. The ova are afterwards preserved in alcohol in the usual way. Some other means of attaining the same end are given in the following paragraphs. KMBRYOLOGICAL METHODS. 343 615. Axolotl. The ova are easier to prepare than those of the Anura, because the yoke is separated from the albuminous layer by a wide space filled with a liquid that is not coagulated by reagents. Put the eggs for a few hours into picro-sulphuric acid, then pierce the inner chorion with fine scissors or needles, and gently press out the ovum. Harden in alcohol. 616. Triton (Scorr and OSBORN, Quart. Journ. Mic. Soc., 1879, p. 449). The albumen is here present in the form of several concentric coats, which are very delicate. Incise each of them separately with fine scissors, turn out the ovum, and fix it. Solution of Kleinenberg is the reagent that gives the best results. 617. Triton (HERTWIG, Jen. Zeit.f.Naturw., 1881-2, p. 291). Put the eggs into a mixture of equal parts of 2 per cent, acetic acid and 0*5 per cent, chromic acid. After ten hours incise the membranes, opening one end of the inner chorion, and turn out the embryos and bring them into successive alcohols. 618. Salamandra (RABL, Morpliol. Jahrb., xii, 2, 1886, p. -~*'2). For his more recent methods see 602. 619. Eana (0. HERTWIG, Jen. Zeit. f. Naturiv., xvi, 1883, p. 249). The ova are thrown into nearly boiling water (90 to 96 C.) for five to ten minutes. The albuminous envelope of the ovum is then cut open, and the ovum extracted under water. The ova are then brought into O5 per cent, osmic acid, or into alcohol of 70, 80, and 90 per cent. Chromic acid makes ova brittle ; they ought not to remain in it for more than twelve hours. Chromic acid destroys or attacks the pigment of the ova, whilst alcohol preserves it, which is frequently important for the study of the germinal layers. MORGAN (Amer. Nat., xxv, 1891, p. 759; Journ. Roy. Mic. Soc., 1892, p. 284) has the following. During the periods in which it is difficult or impossible to remove the inner jelly- membrane the eggs can be freed as follows : Each egg is cut out with scissors from the general jelly-mass, and put for from one to twelve hours into saturated solution of picric acid in 35 per cent, alcohol containing "the same amount of sulphuric acid as in Kleinenberg's solution." Wash for 344 CHAPTER XXVII. several hours in several changes of alcohol, beginning with 35 per cent., and increasing the strength gradually up to 70 i per cent. About the second day in the 70 per cent, alcohol the inner membrane begins to swell, and on the third or fourth day may be pierced by a needle, and the egg removed and placed in 80 per cent, alcohol (see also WHITMAN, Meth. \ of Research, p. 156 ; and SCHULTZE, Zeit. f. wiss. Zool., v, 1887, p. 177). 620. Sulphate of Copper Hardening Liquid. I take from the j Lehrbuch of FOL (p. 106) the following formula, which was first published by EEMAK, then modified by GOETTE, and is said to be useful for hardening j the ova of Amphibia : 2 per cent, solution of sulphate of copper . . 50 c.c. Alcohol of 25 per cent. . . . . . 50 Eectified wood vinegar 35 drops. 621. Other Methods. Koux, Study of Cleavage Cells (Cytro- pism of the Ovum), see Arch. f. Entwickelungs-mechanik, i, p. 44 ; Amer. Natural., xxix, 1895, p. 511 ; Journ. Roy. Mic. Soc., 1895, p. 598. ] Production of Hemiembryos of the Prog, &c., Am at. Anz., ix, 8, 1894, p. 248, and 9, p. 265. BORN, Arch. f. mik. Anal., xliii, 1894, p. 1 ; EABL, Zeit. /. wiss. Mik., xi, 2, 1894, p. 165 (his fixing methods have been given). Pisces. 622. Teleostea in General. The ova of many of the bony fishes can be studied by transmitted light in the living state ; j but those of the Salmonidas must be hardened and removed from their envelopes for the study of the external forms of j the embryo. To this end the ova may be put for a few minutes into j water containing 1 to 2 per cent, of acetic acid, and thence : into 1 per cent, chromic acid. After three days the capsule of the ovum may be opened at the side opposite to the em- j bryo, and be removed with fine forceps. The ovum is put for twenty-four hours into distilled water, and then into succes- sive alcohols. Embryos thus prepared show no deformation, j and their histological elements are fairly well preserved. But the vitellus rapidly becomes excessively hard and brittle, so as greatly to interfere with section cutting. The following processes give good results as regards sec- tion cutting. EMBRYOLOGIOAL METHODS. 345 Put the ova for a few minutes into 1 per cent, osmic acid ; as soon as they have taken on a light brown colour bring them into Miiller's solution. Open them therein with fine scissors the vitellus, which immediately coagulates on con- tact with air, dissolves, on the contrary, in Miiller's solution and the germ and cortical layer can be extracted from the capsule of the ovum. They should be left in clean Miiller's solution for a few days, then washed with water for twenty- four hours, and brought through successive alcohols. Another method (HENNEGUY) is as follows : The ova are fixed in solution of Kleinenberg containing 10 per cent, of acetic acid. After ten minutes they are opened in water con- taining 10 per cent, of acetic acid, which dissolves the vitellus. The embryos are put for a few hours into pure solution of Kleinenberg, and are then brought through alcohol of gradually increasing strength. 623. KOLLMAXX'S Fixative (KOLLMAXX, Arch.f. Anat. u. Phys., 1885, p. 296). Bichromate of potash 5 per 100. Chromic acid . . . . . . . 2 ,, Concentrated nitric acid . . . . 2 For ova of Teleostea. Fix for twelve hours, wash with water for twelve hours, then remove the chorion, and put the ova into 70 per cent, alcohol. 624. RABL'S Method (see 602). 625. PERENYI'S Method (see 50). 626. KOWALEWSKY'S Method (see Zeit. f. wiss. ZooL, xliii, 1886, p. 434). 627. BOYER'S Methods. See Bull. Mus. Camp. ZooL, Harvard, xxiii, 1892, p. 93 ; Journ. Roy. Mic. Soc., 1892, p. 699. 628. RABL-RUCKHAICD'S Method (Arch.f. Anat. u. Entw., 1882, p. 67). Fix in 10 per cent, nitric acid for fifteen minutes. Remove the mem- branes to avoid deformation of the embryos, and put the ova back into the acid for an hour. Wash out in 1 to 2 per cent, solution of alum for an hour, and harden in alcohol. Modification of this method by GOROXOWITSCH (see fiforph. Jahrb., x, 1884, p. 381). 629. Pelagic Fish Ova (WHITMAN'S method ; Amer. Natural., xvii, 1883, pp. 1204-5 ; Journ. Roy. Mic. Soc. [N.S.], iii, 1883, p. 912, and Methods of Research, &c., p. 152). Fix by treatment first for five to ten minutes with a mixture of equal parts of sea water and per cent, osmic acid solution, and then for one or two days with a modified Merkel's solu- tion (due to Eisig), consisting of equal pails of 0'25 per cent, platinum 346 CHAPTER XXVII. chloride and 1 per cent, chromic acid. Prick the membrane before trans- ferring to alcohol. Whitman found that the usual Merkel's fluid caused maceration of the embryonic portion of the egg. Picro-sulphuric acid causes the embryonic cells to swell, and in many cases to become com- pletely disorganised. The osmic acid treatment is necessary in the case of segmenting ova because the Merkel's fluid does not kill rapidly enough, so that eggs placed in it may even pass through one or two stages of cleavage before dying. This fluid arrests the process of blackening by the osmium, or will even bleach the objects if blackening has set in. See also AGASSIZ and WHITMAN, in Proc. Amer. Acad. Arts and Sciences, xx, 1884. For later stages the authors recommend the method of Perenyi. And see the experiments detailed by COLLINGE, Ann. and Mag. Nat. Hist., x, 1892, p. 228 ; Joum. Roy. Mic. Soc., 1892, p. 883. Tunicata. 630. Distaplia. DAVIDOFF (Mitth. Zool. Stat. Neapel, ix, 1, 1889, p. 118) has some important observations on the fixation of the ova of D. magnilarva. The best reagent is a mixture of 3 parts of saturated solution of corrosive sublimate and 1 of glacial acetic acid. The ova to remain in it for from half an hour to an hour, and be then washed for a few minutes in water and brought through successive alcohols. Another reagent, almost as good, consists of 3 parts of saturated solu- tion of picric acid and 1 of glacial acetic acid, the objects to remain in it for three to four hours, and then be brought into 70 per cent, alcohol. 631. Amaroecium (MAURICE and SCHULGIN, Ann. Sci. Nat. Zool., xvii, 1884). Stain in borax-carmine, wash out, and stain for fifteen to twenty hours in very weak solution of Ueu de Lyon in 70 per cent, alcohol with a few drops of acetic acid. In sections the epiblast and hypoblast appear chiefly blue, the mesoblast cells, on the contrary, appearing almost entirely red. 632. Test-Cells of Ascidians (MOBGAN, Journ. of Morphol, iv, 1890, p. 195). Tease fresh ovaries in very weak osmic acid, wash in distilled water, treat for half an hour with 1 per cent, silver nitrate, wash for half an hour in 2 per cent, acetic acid, and reduce in sunlight. Imbed in paraffin. By this process the limits of the follicle cells are demonstrated. Mollusca. 633. Cephalopoda (Ussow, Arch, de BioL, ii, 1881, p. 582). Segmenting ova are placed, without removal of the mem- KMBRYOLOGICAL METHODS. 347 branes, in 2 per cent, solution of chromic acid for two minutes, and then in distilled water, to which a little acetic acid (one drop to a watch-glassful) has been added, for two minutes. If an incision be now made into the egg-membrane the yolk flows away and the blastoderm remains ; if any yolk still cling to it, it may be removed by pouring away the water and adding more. WATASE (Journ. of AIorphoL, iv, 1891, p. 249 ; Journ. Roy. J/V'-. Soc., 1892, p. 152) kills the ova in the macerating mix- ture of the Hertwigs ( 553), and as soon as the blastoderm turns white and opaque removes it under dilute glycerin. Treatment with liquid of Perenyi is recommended for surface views of cleavage. 634. Gastropoda (HEXNEGUY). Ova of Helix may be fixed for from four to six hours in Mayer's picro-nitric solution ( 76). The carbonate of lime that encrusts the external membrane is thus dissolved, and the albuminous coat of the egg is coagulated. The egg is opened with needles, the albumen comes away in bits, and the embryo can be removed. Treat with successive alcohols, and imbed in paraffin. 635. Limax (early stages) (MARK, Bull. Mus. Comp. ZooL, Harvard Coll., vi, 1881). The ova are treated with acetic acid of 1 to 2 per cent, for four or more hours. The two external membranes are incised with fine scissors, and the egg squeezed out in its albumen membrane. This is dis- sected off on a slide, the egg is separated from the albumen, stained, and mounted in glycerin. For later stages, or for making sections, osmic acid is used instead of acetic acid, and the egg is hardened with its albuminous coats. Miss A. HENCHMAN (Bull. Mus. Comp. Zool., Harvard, xx, 1890, p. 171; Journ. Roy. Mic. Soc., 1891, p. 274; Zeit. /. wiss. Mik., viii, 2, 1891, p. 216) finds that the best way of obtaining embryos is to keep some twenty-five or thirty adults (of L. maximus) in a large tin pail with a cover perforated with small holes. They should be fed on cabbage, and the vessel kept very clean. Eggs are generally found in the morning in bunches of thirty to forty. As they are more abundant in the early stages of confinement it is better to obtain a few slugs often than many at once. In a moderately warm room hatching occurs between the twenty-second and twenty-seventh day. The eggs must be carefully protected from desiccation. 348 CHAPTER XXVII. Kill with 0'33 per cent, chromic acid, or with liquid of Perenyi. It is best to remove only the outer envelope before putting into the chromic acid, the inner membrane being removed after two or three minutes therein. Where Perenyi is used the membranes must be removed first, as the albumen will else coagulate in such a way as to prevent the removal of the embryos. See also SCHMIDT, Studien zur EntwicJcelungsgesch. d. Pul- monaten, Dorpat, 1891 ; and WASHBURN, Amer. Natural., xxviii, 1894, p. 528; or Journ. Roy. Mic. Soc., 1894, p. 531. The paper of KOFOID in Bull. Mus. Comp. ZooL, Harvard Coll., xxvii, 2, p. 35, contains a detailed account of the treatment appropriate to the ova of Agriolimax agrestis, which are smaller than those of Limax maximus, studied by Miss Henchman. See also the abstract of these methods in Journ. Roy. Mic. Soc., 1895, p. 701. 636. CHITON, see METCALF, Stud. Biol. Lab. Johns Hopkins Univ., v, 1893, p. 251, or Journ. Boy. Mic. Soc., 1894, p. 531. Arthropoda. 637. Fixation of Ova. In most cases the ova of Arthropods are fixed by heat in a more satisfactory way than by any other means. This may be followed either by alcohol or some watery hardening agent. If it be desired to avoid heating, picro-sulphuric acid or liquid of Perenyi may be tried. 638. Removal of Membranes. This is frequently very difficult, and it may often be advisable not to attempt to remove them, but to soften them with eau de Javelle or eau de Labarraque (see the methods of Looss and LIST) . MORGAN (Amer. Natural., xxii, 1888, p. 357; Zeit. f. wiss. Mik., vi, 1, 1889, p. 69) recommends (for the ova of Peri- planeta] eau de Labarraque diluted with five to eight volumes of water, and slightly warmed. Thus used, it will soften the chitin membranes sufficiently in thirty to sixty minutes if employed before fixing. Fixed ova take longer. The fluid must, of course, not be allowed to penetrate into the interior of the ovum. 639. HENKING'S Methods. For the whole subject of the technique of the embryology of Insecta see an elaborate KMBRYOLOGICAL METHODS. 349 paper by this specialist in Zvit. f. wiss. Mik., viii, 2, 1891, p. 15t>. Henking agrees with other workers at this subject, that in the majority of cases heat is the only available fixing agent that will give fair results. He kills ova by plunging them into hot water, or by pouring hot water on to them in a watch-glass, and then removing into 70 per cent, alcohol. But, as might be expected, he finds that the preservation of structures by this method is far from being perfectly satisfac- tory, cell-contours being not at all sharply brought out by it, and achromatic cell-structures being but imperfectly pre- served. He finds that in some cases ova may be fixed with liquid of Flemming, which, as may be supposed, gives incom- parably better results in these respects. Suitable ova may be put into liquid of Flemming (Henking does not say which formula) for half an hour, then for two hours into the same diluted with three volumes of water, then treated with alcohol as usual. Boveri's picro-ace^ic acid was found not to pene- trate the membranes. Henking thinks that eau de Javelle for softening mem- branes is best avoided. Membranes should either be dissected away or left in situ, and cut with the rest of the egg, accord- ing to the nature of the case. The great obstacle to section cutting is the brittleness of the yolk. This difficulty may be overcome as follows : After fixing and treating with alcohol, prick the chorion and stain with borax-carmine. Put the stained ova for twelve hours into a mixture containing 20 c.c. of 70 per cent, alcohol, one drop of concentrated hydrochloric acid, and a knife-pointful of pepsin (it is not necessary that all the pepsin should be dissolved). The ova may then be treated with alcohol, oil of bergamot, and paraffin, and (with some exceptions, amongst which is Bombyx mori) will be found to cut without crumbling. The contents of fresh ova may conveniently be studied by means of the following fluid : Distilled water . . . .80 c.c. Glycerin . . . . . 16 Formic acid . . . . 3 1 per cent, osmic acid . . . 1 Dahlia 0*04 grin. The eggs are simply teased in a drop of the liquid, and a 350 CHAPTER XXVII. cover-glass put on. If it be desired to preserve the prepara- tion, nothing more is necessary than to lute the cover-glass. 640. Lepidoptera (BOBRETZKY, Zeit. f. wiss. ZooL, 1879, p. 198). Ova (of Pieris cratsegi and Porthesia chrysorrJicea) are slightly warmed in water and put for sixteen to twenty hours into 0*5 per cent, chromic acid. The membranes can then be removed, and the ova brought for a few hours into absolute alcohol, stained with carmine, and cut. 641. Blattida (PATTEN, Quart. Journ. Hie. Sci., 1884, p. 549). The ova or larvae are placed in cold water, which is gra- dually raised to 80 C. You leave off heating as soon as the ova have become hard and white. Pass very gradually through successive alcohols, beginning with 20 per cent.; stain with Kleinenberg's ha3matoxylin or Mayer's cochineal (only alcoholic stains will traverse the chorion). The ova may remain in the hasmatoxylin for five or six days, and be washed out in alcohol containing one drop of HC1 per 20 grms., in which they should remain for several days, and then be soaked in pure alcohol until they have regained their violet colour. Penetrate with benzol and imbed in paraffin. WHEELEE (Journ. of Morph., iii, 1889, p. 292 ; Journ. Roy. Mic. Soc., 1890, p. 250) dissects out ovarian ova in salt solu- tion and fixes in liquid of Perenyi (fifteen minutes), then treats with alcohol, and stains with borax-carmine. Laid eggs may be killed by Patten's method. After heating, the two lips of the crista of the capsule may be separated with fine forceps and pieces of the walls torn away, and the eggs pushed out of the compartments formed by their choria and hardened as desired. Good results are also obtained by heating to 80 C. for ten minutes in liquid of Kleinenberg, and preserving in 70 per cent, alcohol. This causes the en- velopes to dilate and stand off from the surface of the egg, so that they can easily be dissected away. CHOLODEOWSKY (Mem. Acad. Imp. St. Petersburg, xxxviii, 1891 ; Zeit. f. wiss. Mik., ix, 1, 1892, p. 80) recommends cutting off the ends of the cocoons and fixing for twelve hours in liquid of Perenyi, or for a few minutes in a solution of' 1 part iodine, 1 part iodine of potassium, and 300 parts water, heated to boiling-point. EMBRYOLOGICAL METHODS. 351 HEYMONS (Zeit.f. wiss. ZooL, liii, 1892, p. 434; Zeit. f. wiss. ),iik., ix, 3, 1893, p. 343) finds that Cholodkowsky's methods are good for the study of general relations of parts, but not satisfactory for the preservation of delicate detail. For young embryos it is better to incise the cocoon at the end by which it inheres in the body of the mother, bring it for two minutes into water heated to 90 C., and open in Flemming, in which the embryo should be dissected out. 642. Diptera (HEXKING, Zeit.f. wiss. ZooL, xlvi, 1888, p. 289; Zeit.f. wiss. Mik., 1889, p. 59). Ova still contained within the fly may be fixed by plunging the animal for some time into boiling water, then dissecting out and bringing them into 70 per cent, alcohol. Laid eggs may have boiling water poured over them, or be put into solution of Flemming in a test-tube which is plunged into boiling water until the eggs begin to darken (about a minute) . Cold solution of Flemming easily causes a certain vacuolisation of the contents of the ova. Open the ova at the larger end, stain with borax- carmine for fifteen to thirty hours, and cut in paraffin. See also (for Chironomus) RITTEE, Zeit. f. wiss. ZooL, i, 1890, p. 408; Zeit.f. wiss. Mik., viii, 1, 1891, p. 87 (strings of ova fixed with hot 30 per cent, alcohol containing a little sublimate, and stained in the mass by immersion for several days in picro-carmine) . 643. Phalangida (BALBIAXI). The ova of Phalangium opilio are enclosed in a chorion covered with yellow corpuscles which renders them quite opaque. They may be cleared by treating them with water contain- ing a little solution of caustic potash and raised to boiling point. The ova are then laid on blotting-paper, and the chorion is removed by rubbing them gently with a small brush. The vitelline membrane remains intact and transparent, and the embryo may be studied through it. 644. Phalangida (HESKLN-Q, Zeit. f. wiss. Mik., iii, 4, 1886, pp. 470 et. seq.). Fix with boiling water or " Flemming." Preserve the ova in 90 per cent, alcohol. To open the chorion, bring them back into 70 per cent. alcohol, which causes them to swell up so that the chorion can easily be pierced with needles, and the ovum turned out. 645. Araneina. See BALFOUE (Quart. Journ. Mic. Sci., 1880, p. 167) ; KISHLNOFYE (Joum. Coll. Sci. Imp. Univ. Japan, iv, 1891, p. 55 ; Zeit.f. wiss. Mik., ix, 2, 1892, p. 215) ; LOCY (Bull. Mus. Comp. ZooL, Harvard, xii, 3, 1886 ; Zeit. f. wiss. Mik., iii, 2, 1886, p. 242). 646. Aphides (WiLL, Semper's Arbeiten, 1883, p. 223). Sections to be 352 CHAPTER XXVII. made through the entire animals containing the ova and embryos. The animals are killed in water of 70 C., and brought into alcohol. 647. Astacus (REICHENBACH, from Zeit.f. wiss. Mik., 1886, p. 400). Fix in water gradually warmed to 60 or 70 C. (if the chorion should burst, that is no evil), harden for twenty -four hours in 1 to 2 per cent, bichromate of potash or O5 per cent, chromic acid, wash out for the same time in running water, and bring into alcohol. Remove the chorion, remove the embryo from the yoke by means of a sharp knife, and stain with picro- carmine and mount in balsam. 648. Amphipoda (Orchestia) ULIANIN, Zeit.f. wiss. Zool., xxxv, 1881, p. 441). Ova in the earliest stages of development were treated for two hours with picro-sulphuric acid (Kleinenberg's formula). This causes the ohorion to swell and burst. Ova in later stages, in which the embryo is surrounded by a cuticular membrane, which encloses an albuminous liquid, must have this membrane torn with needles and the albuminous liquid allowed to ooze out before placing in the picro-sulphuric acid. Venues. 649. Tsenia (v. BENEDEN, Arch, de BioL, ii, 1881, p. 187). Ova in which a chitinous membrane has formed around the embryo are impervious to reagents. They may be put on a slide with a drop of some liquid and covered. Then, by with- drawing the liquid by means of blotting-paper, the cover may be made to gradually press on them so as to burst the mem- branes, and the embryo may then be treated with the usual reagents. 650. Planaria (!IJIMA, Zeit. f. wiss. Zool., xl, 1884, p. 359). The capsule containing the ova (of fresh-water Planaria) is opened with needles on a slide, in a drop of 2 per cent, nitric acid. The ova are extracted and covered (the cover being supported by paper, or by wax feet). After half an hour they are treated with successive alcohols under the cover, and finally mounted in glycerin. For sections, the whole of the contents of a capsule is hardened in the mass in 1 per cent, chromic acid and cut together. 651. Lumbricus (KLEINENBERG, Quart. Journ. Mic. ScL, 1879, p. 207). Fix with Kleinenberg's picro-sulphuric acid, or, which is not quite so good, with vapours of osmium, pass through successive alcohols, stain with Kleinenberg's haema- toxylin, and cut in paraffin. WILSON (Journ. of Morph., iii, 1889, p. 445; Journ. Roy. EMBRYOLOGICAL METHODS. 353 Mic. Soc., 1890, p. 402) finds that liquid of Perenyi is by far the best fixing reagent, being in most respects superior even to Flemming's. Fix for fifteen to sixty minutes ; wash out in 70 per cent, alcohol. For Nereis, see VON WISTINGHAUSEN, Mitth. Zuol. Stat. Neapel, x, 1891, p. 41 ; or Zeit.f. iviss. Mik., x, 4, 1893, p. 479. 652. Ascaris. Seethe chapter on " Cytological Methods." Echinodermata, Ccelenterata, and Porifera. See the paragraphs treating of these groups in the chapter on " Zoological Methods." For the maturation and fecundation of the ova of the Echi- nodermata, see also the chapter on " Cytological Methods." 23 CHAPTER XXVIII. CYTOLOGIOAL METHODS. 653. Subjects for Study. One of the best objects for this purpose is the tail of young larvae of Amphibia, both Anura and Urodela. In the living animal the epithelial cells and nuclei (in the state of repose) are so transparent as to be invisible in the natural state. They may, however, be brought out by cura- rising the larva; or, still better, by placing the curarised larva for half an hour in 1 per cent, chloride of sodium solu- tion. Normal larvae may be used for the study of the active state of the nucleus, but much time is saved by using curare. Curare. Dissolve 1 part of curare in 100 parts water, and add 100 parts of glycerin. Of this mixture add from 5 to 10- drops (according to the size of the larva), or even more for large larvae, to a watch-glassful of water. From half to one hour of immersion is necessary for curarisation. The larvae need not be left in the solution until they become quite motionless; as soon as their movements have become slow they may be taken out and placed on a slide with blotting- paper. If they be replaced in water they return to the normal state in eight or ten hours, and may be re-curarised several times. Etlierisation. Three per cent, alcohol, or 3 per cent.j ether maybe used in a similar way. These reagents cause no obstruction to the processes of cell-division, and are useful, but their action as anaesthetics is inconstant. Indifferent Media. One per cent, salt solution, iodised serum, syrup, cold water (+ 1 C.), and warm water (35 40 C.). The tail may be excised from the living animal and studied for a long time in these media (PEREMESCHKO, Arch. \ f. mik. Anat., xvi, 1879, p. 437). Perhaps (FLEMMING, ibid. } pp. 304 et seq.) the very best subject for these studies is Salamandra. The adult offers for study the thin transparent bladder; in the larva the gills and caudal "fin." mav be studied in the living state. The- CYTOLOGICAL METHODS. 355 gills are difficult to fix in position for observation, and are obscured by pigment. In the fin there is always a spot, near to the hind limbs, that is free from pigment; and on lightly coloured larvae other such spots may be found on the ventral half of the fin and on the lateral line. On a flat-finned larva it is possible to study these spots with high-power glasses. The larva may be fixed in a suitable cell, or wrapped in moist blotting-paper, or may be curarised ; or the tail may be excised. (It is preferable to cut through the larva close in front of the hind limbs.) Objects for Preparation. A favorable object for prepara- tion is found in the gill-plates, delicate laminae that are to be found attached to the gill-cartilages on the mouth side. The lungs, parietal peritoneum, and mesentery of the larvae are also very favorable objects for preparations (see FLEMMING, Arch. f. mik. Anat., xxxv, 1890, p. 275 ; and xxxvii, 1891, pp. 249 and 685). To prepare the lungs the larva, which should be of not more than 4 cm. length, should be killed by immersion in chromo-aceto-osmic acid, the body- cavity cut into, and the viscera gently drawn out and exposed to the action of the liquid, care being taken not to let the lungs get into folds. After fixation they should be carefully got on to a slide, and a small strip removed from their mar- gins on either side by means of a scalpel, after which the two walls may be separated from each other, and utilised as thin, flat preparations. Another excellent object is the intestine of the adult, of which sections may be made by the paraffin method, as re- commended in the important paper of M. HEIDENHAIN, " Uber Kern und Protoplasma," in Festschr. z. 50 jdhr. Jubil. d. H. Prof. Geheimr. v. Kolliker (also in separate reprint), Engel- mann, Leipzig, 1892, p. 111. This organ offers for study, besides the large epithelium cells of the intestinal crypts, numberless examples of leucocytes, an extremely favorable object. Larvae may be bred from adults kept in confinement, and supplied with a vessel of water, in which they will place the larvae of their own accord. In May gravid females may be killed and the larvae extracted. The larvae must be kept in frequently changed water and fed every day or two. Aquatic worms may be used for feeding them, e. g. Tubifex rivulorum. 356 CHAPTER XXVIII. It is extremely important that they should be fed regularly and abundantly, for, if not, cell-divisions in the tissues become rare, and may even cease altogether. Adults may easily be kept in a vivarium. The bottom should be covered to a depth of about four inches with mould ; a shallow dish (a photographic developing dish is as good as anything) filled with water should be sunk in the mould, and the mould should be covered with moss. The animals should be fed with worms, which they are sure to take during the night (HENNEGUY, Leq.ons sur la Cellule, p. 68). LONNEBEEG (see Zeit. f. wiss. Mik., x, 3, 1893, p. 377) recom- mends the intestinal epithelium of hibernating snails as an object that furnishes an abundance of karyokinetic figures. The snails should be kept for some days in a warm room, their epiphragm should then be removed, and they should be fed for three days with cabbage leaves before being dissected. Other classical subjects of study will be found mentioned in the following paragraphs. 654. Stains for living Cells. It is sometimes of the very greatest importance to be able to stain a cell in the living state, even though it be but feebly and imperfectly. See 213. 655. Study of Fresh and lightly fixed Cells. It has been rightly pointed out by Flemming that so-called " indifferent " liquids must not be believed to be without action on nuclei. Iodised serum, salt solution, serum, aqueous humour, lymph, better deserve the name "of weak hardening agents. Between these, and such energetic hardening agents as Flemming's mixture, come such light fixing agents as picric acid or very dilute acetic acid. These it is whose employment is indicated for the study of fresh isolated cells. A typical example of this kind of work is as follows : Tease out a piece of living tissue in a drop of acidulated solu- tion of methyl green (O75 per cent, of acetic acid). This is a delicate fixing agent, killing cells instantly without change of form. Complete the fixation by exposing the preparation for a quarter of an hour to vapour of osmium, and add a drop of solution of Ripart and Petit and a cover. Or you may fix the preparation, after teasing, with vapour of CYTOLOGICAL METHODS. 357 osmium for halt' a minute to two minutes, then add a drop of methyl green, and after five minutes wash out with 1 per cent, acetic acid, and add solution of Ripart and Petit and cover. Or you may kill and fix the cells by teasing in solution of Ripart and Petit (to which you may add a trace of osmium if you like), and afterwards stain with methyl green. I have found Pictet's chloride of manganese ( 386) very useful as an examination medium. A little solution of dahlia may be added to it. HENKIXG'S mixture, which has been given above ( 639), may also be found useful. For FLEMMING'S methods for the study of the division of the ova of Echinodermata, see Arch.f. mik.Anat., xx, 1881, p. 3, or previous editions of this work. Other fixing agents, such as picric acid or weak sublimate solution, may of course be used, and in some cases doubtless should be preferred. Other stains, too, such as Bismarck brown, may be used as occasion dictates ; and of course other examination media than solution of Ripart may be employed. But, for general purposes, the methyl-green-osmium-and- Ripart's-medium method gives such good results, and is so very convenient, that it may well be called the classical method for the study of fresh cells. I think great credit is due to CARNOY for his frequent insistence on the excellence and handiness of this method. 656. Some Microchemical Reactions. Methyl green is a test for chromatin, in so far as it colours nothing but the chro- matin in the nucleus. It is, however, not a perfect test, for the intensity of the coloration it produces varies greatly in different nuclei, and may in certain nuclei be extremely weak, or (apparently) even altogether wanting. In these cases other tests must be applied in order to establish with certainty the presence or absence of that element. The following sug- gestions are taken from CARNOY, who is, I believe, the only writer on the zoological side, at all events who has insisted on the necessity of applying microchemical methods in a systematic manner to the study of cells. Chromatin is distinguished from the lecithins and from albuminoids by not being soluble, as these are, in water and in weak mineral acids, such as 0*1 per cent, hydrochloric 358 CHAPTER XXVIII. acid. It is easily soluble in concentrated mineral acids, in alkalies, even when very dilute, and in some alkaline salts, such as carbonate of potash and biphosphate of soda. In the presence of 10 per cent, solution of sodium chloride it swells up into a gelatinous mass, or even, as frequently happens, dissolves entirely (Biol. Cell., pp. 208-9). It is only partially digestible (when in situ in the nucleus) in the usual laboratory digestion fluids. The solvents of chromatin that are the most useful in practice are 1 per cent, caustic potash, fuming hydrochloric acid, or cyanide of potassium, or carbonate of potash. These last generally give better results than dilute alkalies. They may be employed in solutions of 40 to 50 per cent, strength. If it be desired to remove all the chromatin from a nucleus the reaction must be prolonged, sometimes to as much as two or three days, especially if the operation be con- ducted on a slide and under a cover-glass, which is the safer plan. It must be remembered that these operations must be per- formed on fresh cells, for hardening agents bring about very considerable modifications in the nature of chromatin, render- ing it almost insoluble in ammonia, potash, or sodic phos- phate, &c. Hydrochloric acid, however, still swells and dissolves it, though with difficulty. Partial digestion may render service in the study of the chromatic elements of nuclei. Chromatin resists the action of digestive fluids much longer than the albumins do ; so that a moderate digestion serves to free the chromosomes from any caryoplasmic granulations that may obscure them, whilst at the same time it clears up the cytoplasm. The term " chromatin " has been used in the above paragraphs in a sense in which the term " nuclein " is employed by many writers. It is now known that there exists a whole series of nucleins, differing chiefly in respect of their richness in phosphorus and proteids. At one end of the chain is nucleic acid, with 9 to 11 per cent, of phosphorus, and without any proteids (this compound occurs in nature in the heads of spermatozoa) ; in the middle are what are generally termed the nucleins, consisting of proteid with vary- ing amounts of nucleic acid ; and at the other extreme are nucleins which are nearly all proteid, containing only 0'5 to 1 per cent, of phosphorus, and are in fact the same substances which have received the name of " nucleo- albumin ;" they may also be termed the artificial plastins. These substances have both been isolated from the most diverse tissues of CYTOLOGICAL METHODS. 359 the animal body, and have been prepared artificially. A corresponding series of nucleins exists within the nucleus itself. There are those that contain most nucleic acid; these are readily soluble in alkalies, and precipi- table with difficulty by acid : chromatin is one of these. There are others more insoluble in alkalies and poorer in nucleic acid ; these are the plastins, the pyrenin of nucleoli being one of them. And there are others even poorer in nucleic acid ; these are the nucleo-albumins (which exist also in the cytoplasm) : the paralinin, or "nuclear sap," appears to be in part com- posed of these, in part of phosphorus-free compounds. There appeal's to be some doubt whether chromatin is or is not nucleic acid itself. The principal reactions in which it resembles nucleic acid are given by HALLIBUBTON (Goulstonian Lectures on the Chemical Physiology of the 'Animal Cell, 1893, p. 574 of the Report in the British Medical Journal, No. 1681, March 18th, 1893, from which place also I have con- densed the above remarks on the chemistry of the nucleins) as follows : " 1. It does not give Millon's nor the xantho-proteic reactions. 2. It is easily soluble in alkalies, soluble with difficulty in acids. 3. It is soluble in an acetic acid solution of potassium ferrocyanide. 4. After treatment with concentrated copper sulphate solution for twenty-four hours it loses its affinity for stains. It is not, however, dissolved by the copper sulphate as Schwarz stated. 5. It has a great affinity for anilin dyes, especially for basic dyes like methyl green. If a mixture of methyl green and acid fuchsin is employed, nucleic acid is stained green. The nucleins next richest in phosphorus are stained a blue-violet tint, whereas the phosphorus-poorest are coloured red. Now in the dividing nucleus, when the amount of chro- matin is at its maximum, the nucleus stains green ; whereas in the resting nucleus, where there is more pyrenin, a blue colour is observed." It results from experiments of the nature of that mentioned under No. 5 (which, by the way, does not appear to me to be quite correctly stated), that chromatin is a basophilous body in EHELICH'S sense (explained in 268), and that the albumins, and consequently cytoplasm in general are acido- philous. These considerations appear to justify the employment of the term " chromatin" for the element of the nucleus that stains with methyl green, the term " nuclein " having obtained a wider extension. See also KOSSEL, in BEHBENS, KOSSEL, und SCHIEFFEEDECKEK'S -Das Mikroskop, &c., ii, p. 47 ; the same, in Verh. d. physiol. Ges., Berlin, Oct. 21st, 1892 ; MALFATTI, Ber. d. naturw. med. Vereines in Innsbruck, 1891-2 ; SCHWABZ, Cohn's Beitr. z. Biol. d. Pflanzen, v, 1, 1887, p. l,.or Zeit.f. wiss. Mik., iv, 4, 1887, p. 530 ; ZACHAEIAS, Ber. d. deutscheti botan. Ges. x, 1893, p. 188, and xi, 1893, p. 293, or Zeit. f. wiss. Mik. t x, 1, 1893, p. 80, and x, 3, 1893, p. 373 ; LILIENFELD, Verh. d. phys. Ges. Berlin, 1892-3, No. 11, or Zeit.f. wiss. Mik., x, 1, 1893, p. 80; ZIMMEBMANX, Zeit.f. wiss. Mik., xii, 4, 1896, p. 458. For microchemical methods for the study of the distribution of assimilated iron compounds in cells, see the masterly paper of MACALLUM, in Quart. Journ. Mic. Sci., No. 150, 1895, p. 175. For the microchemical detection of phosphorus in tissue-elements see the paper of LILIENFELD and MONTI, in Zeit. f. physiol. Chemie, xvii, 1892, 360 . CHAPTER XXVIIT. p. 410 (Report in Zeit.f. wiss. Mik., ix, 3, 1893, p. 332) ; also a short notice in HALLIBUETON'S Goulstonian Lectures, 1893 (see Brit. Med. Journ., March llth, 1893, p. 505). 657. Cytological Fixing Agents. The following is in great part taken from the numerous papers of FLEMMING in the Arch. f. mik. Anat. from the year 1879 onwards, and from his Zellsubstanz Kern- und Zelltheilung. Osmic acid (y 1 ^ to 2 per cent.) preserves the form of the entire cell, but swells the nuclei and rounds off nucleoli. It renders the nuclear "reticulum" undiscernible. Picric acid, either concentrated or dilute, and chromic acid, O'l to O5 per cent., are to be preferred to alcohol and other agents for the study of the cells of Vertebrates. Shrinking and distortion of the nuclear figures (and, with picric acid, swellings of them) are to be expected, but other agents have the same defect to a much greater degree; alcohol especially causes entanglement of the threads. Acetic acid does the same, and causes swelling besides. Stronger chromic acid solutions cause shrinking. Neither of these reagents is harmless as regards the nuclei of red blood-corpuscles. The salts of picric acid (potash-, soda-, and baryta-salts) are most harmful. Weak (i. e. not more than 1 per cent.) acetic, hydrochloric, or nitric acid, combined with clearing in glycerin and staining, may be useful for bringing out chroniatin and nucleoli. Chloride of gold preserves the form well, but generally leaves the nuclear structures unstained. Nitrate of silver is hopelessly uncon- trollable in its action. Alcohol has much the effect of chromic acid, but often causes a much greater shrinking of the nuclei. Bichromate of potash and chromate of ammonia bring out very sharply the appearance of a reticulum, but these appearances cannot be accepted as true (1. c., p. 334, et seq.). " Those who seek to study cell-division by means of bichromate of potash or other chromic salts are hopelessly in the wrong road." And this because of the injurious action of the bichromate, not on the body of the cell, which it preserves wellj but on the chromatin structures. Chromic salts are excellent reagents for general histological work, but not for nuclear structures. They dissolve nucleoli, destroy nuclear "networks," and swell up and distort karyokinetic figures to such a degree that the appearances obtained from them are merely unnatural caricatures of the true structure. CYTOLOGICAL METHODS. 361 Altmann's nitric acid method is excellent for the purpose of hunting for cell-divisions in tissues; but the minute struc- ture of the figures is not so well preserved as it is by means of chromic or picric acid. The same must be said of Kleinen- berg's picro-sulphuric acid method. (I am not alone in hold- ing that this is a most untrustworthy cytological reagent ; see, for instance, HOLL [Sitzb. k. Akad. Wiss. Wien, xcix, 1890, p. 311 ; Zeit.f. wiss. Hik.,ix, 1, 1892, p. 89], who found that it frequently reduced chromosomes to the state of mere lumps, "Kriimeln.") There are two fixing mixtures which may be said to be classical for cytological studies, FLEMMING'S ehromo-aceto-osmic acid mixture, 46, 47, and HERMANN'S platino-aceto-osmic acid mixture, 65. As to the former of these, Flemming has the following explanations: Attempts to omit the chromic acid from the formula did not give good results. The omission of acetic acid (as in Max Flesch's formula, 45) causes the figures to be far less sharply brought out. The presence of acetic or formic acid in all osmium solutions is favorable to the precision of subsequent staining with haematoxylin, picro- carmine, or gentian violet. But mixtures of osmic and acetic acid without chromic acid (Eimer) do not give such good results as the chromo-aceto-osmic acid mixture. Mixtures of picric acid with osmic acid or with osmic and acetic acid (proportions of the latter as in the chromo-aceto-osmic mix- ture [ 46], but of picric acid about 50 per cent.) fix quite as well as the chromic mixtures, but precise staining is even more difficult than with pure osmic acid preparations. Flem- ming concludes that the beneficial effects of the osmium in all these mixtures are to be ascribed to the instantaneous rapidity with which it kills, the function of the other acids of the mixture being to render the structures distinctly visible. Mixtures containing osmic acid should therefore be em- ployed whenever it is desired to fix the chromatic figures as faithfully as possible; whilst pure chromic acid should be taken whenever very sharp staining is the more important point. For the study of achromatic figures he recommends the chroino-acetic acid mixture ( 43), followed by staining in haematoxylin (anilins, he states, do not give so good results for this purpose). 362 CHAPTER XXVIII. For the study of polar corpuscles lie recommends the osmium mixtures, or pure chromic acid followed by staining with gentian violet. The above account stands nearly as it stood in the first edition. The state of things at present is as follows : It is admitted by all competent observers that the chromo-aceto- osmic mixture is, with at most one or two possible exceptions, by far the best fixing agent for nuclei. But some observers have stated that it does not always preserve the cell-body well. This is a question that has been already discussed in 46 and 47. I will only add here that after considerable experience I see no reason to distrust Flemming's mixture as a preservative of any kind of protoplasm, provided it be used in the proper way. It must be taken of the proper strength, it must be used with very small objects, so that it may act on all parts of them with its full strength, and not be filtered and diluted through thick walls of tissue before coming into con- tact with the object of study; and it must be allowed to act for the proper time. This brings us to another point. There are two Chromo- aceto-osmic mixtures the old weaker one, and the new stronger one. Flemming recommended the strong one primarily as affording a means of differentiating kinetic chromatin from resting chromatin. He did not recommend it as a reagent for general work. Whether of these two solutions should be used for general work ? According to my experience, the strong solution does preserve both nuclear structures and caryoplasmic structures quite as faithfully at least as the old formula, and some structures most decidedly much better. Of course the one and the other should be taken according to the nature of the object you are dealing with ; but I think it may safely be stated as a general rule that if you take the strong mixture, and fix thoroughly in it, you are not likely to go far wrong. And what is meant by a thorough fixation? Half an hour may be taken to be generally enough ; but for very delicate things, such as the Nebenkern and the achro- matic figure, eighteen hours or more may be desirable. As to the platino-aceto-osmic mixture of HERMANN, it has already been explained in 65 that the point of superiority over Flemming's mixture that is claimed for it lies in a more faithful preservation of cytoplasm and achromatic structures. CYTOLOGICAL METHODS. 363 That the alleged superiority really exists appears to be the general opinion of those who have worked with this reagent. Flemming (Arch.f. mik. Anat., xxxvii, 1891, p. 685; Zeit. /. ?//>>-. Mik., viii, 3, p. 343) agrees that it gives a peculiarly sharp demonstration of spindle fibres, centrosomes, and polar corpuscles ; but thinks that the chromo-aceto-osmic mixtures give a somewhat more faithful preservation of the chromatic elements. According to my experience there are slight differences in the behaviour of these two reagents which may be taken advantage of (see 65). XIESSING (Arch. f. mik. Anat., xlvi, 1895, p. 147) has the following two modifications of Hermann's mixture : (1) Platinic chloride, 10 per cent, solution . 25 Osmic acid, 2 per cent. . . . .20 Glacial acetic acid . . . . .5 Distilled water ...... 50 (2) The same with saturated aqueous solution of corrosive sublimate instead of the water. 0. VOM RATH'S picro-platinic mixtures have been given (80) . Liquid of MEEKEL, or the modification of it by BRASS, which will be given in the chapter on Protozoa, may be found useful. For LINDSAY JOHNSON'S platinic mixture see 97. Some observers have had good results with liquid of PEEENYI, especially for achromatic structures. Two or three of the fixing agents proposed by other writers may also rank as first-class reagents for this kind of work. There is RABL'S chromo-formic acid ( 44). Fix in this for twelve to twenty-four hours, wash out well with water, and pass into alcohol. And there is the same observer's platinum chloride solution ( 64). In Rabl's paper in Anat. Anz., iv, 1889, p. 21, he recommends that Salamandra larvae be fixed (for twenty-four hours) in a solution of from one tenth to one eighth per cent, strength. In his earlier work he used solu- tions of 1 : 300 strength. Platinum chloride has the pecu- liarity of causing a slight shrinkage of the chromatiu, which helps to bring into evidence the granules of Pfitzner and the longitudinal division of the chromosomes. There remain to be mentioned several fixing agents with which very important work has been done. These, however, are, I think, not quite first- class reagents for the purpose, the brilliant results that have been obtained with them having been obtained rather in spite of their defects than on 364 CHAPTER XXVIII. account of their good qualities. For instance, acetic alcohol is a reagent with which some of the most important work in recent cytology has been done namely, much of that on the maturation and fecundation of the ovum of Ascaris. It is evident that for such an extraordinarily impenetrable object as the ovum of Ascaris, the employment of some such highly pene- trating fluid as acetic acid is imperatively indicated, notwithstanding the serious defects that it may have. CABNOY (La Cellule, iii, 1, 1886, p. 6) used at first a mixture of three parts .of absolute alcohol with 1 of glacial acetic acid ; later (ibid., iii, 2, 1887) the chloroform mixture ( 69). From five to fifteen minutes is enough for even the most resistent ova. VAN BENEDEN and NEYT (Nouvelles Recli. sur la Fee. et la Division mitosique, 1887) employed a mixture of equal parts of absolute alcohol and glacial acetic acid, or even pure acetic acid. Acetic alcohol may be washed out with either pure alcohol, or with dilute glycerin (Calberla's formula would be a good one in many cases). For further details see ante, 69. M. HEIDENHAIN (Ueb. Kern u. Protoplasma, 1892, p. 113) has been using corrosive sublimate on account of its convenience, and, above all, on account of the great facility it affords for the employment of certain stains. The beautiful figures of " attraction spheres " and other cytoplasniic struc- tures given in this paper show that the most brilliant results may be obtained by this means. But I would remark that the figures of nuclear structures appear to me less convincing. The author figures and describes, under the name of " Lanthanin," an acidophilous caryoplasmic substance exhibiting a minutely reticular arrangement. The figures remind me of appearances which I frequently obtained in certain nuclei when working with sublimate some years ago, and which I regarded as artefacts, and in consequence was led to the abandonment of sublimate as a cytological fixa- tive. I do not find in Heidenhain's paper that he has instituted control experiments to show that the reticular arrangement of his " lanthanin " is preformed in the nucleus, and would point out the need of such experiments before either the existence of the lanthanin reticulum or the fidelity of the reagent can be deemed established. It should further be noted that with sublimate material it is impossible to obtain with safranin, gentian violet, and some other stains, the delicate differentiations that they give with chrom- osmium material. ALTMANN (Arch.f. Anat. u. Entwickel., 1892, p. 223 ; Zeit.f.wiss.Mik., ix, 3, 1893, p. 331) has a new fixative for resting nuclei, viz. a 2'5 per cent, solution of ammonium molybdate to which is added about 0'25 per cent, of chromic acid. He also mentions (Die Elementarorganismen, 1890; cf. Zeit.f.wiss. Mik., vii, 2, 1890, p. 201) a fixation with nitrate or picrate of mercury, for the demonstration of his " bioblasts." I have no personal knowledge or other information concerning either of these methods. Lemon juice (fresh, filtered) has been warmly recommended as a fixative for nuclei by VAN GEHUCHTEN (Anat. Anzeig., iv, 1889, p. 52). Fix for five minutes, wash well with water and stain with methyl green, and exa- mine in liquid of Ripart and Petit. CYTOLOGICAL METHODS. 365 Heat has been recommended, but I believe it to be altogether objection- able. HEXKING (Zeit. f. wiss. Zool, xcix, 3, 1890, p. 503 ; Zeit.f. wise. Mil:., vii, 2, 1890, p. 211) has found that it totally destroys achromatic structures. 658. Chromatin Stains. Stains appropriate for fresh or lightly fixed tissues have been mentioned in 655. For sections of hardened tissues we have the choice bet the finer hsematein stains (or carmalum) , and those o by means of safranin, gentian violet, and some other used according to the regressive method. Of the haematein stains, hasmalum gives very good resu with sublimate material; but for chrom-osmium material I decidedly prefer in general an iron lake, M. HEIDENHAIN'S or BENDA'S. Thionin has been warmly recommended by M. HEIDENHAIN as giving a stain even superior to that of safranin. After trial, I think the recommendation is justified. Methyl green is, in my experience, inferior for preparations destined to be mounted in balsam. Carmalum is a very precise stain, and useful for sublimate material, but is decidedly too weak for chrom-osmium material. BABES'S supersaturated safranin stain (Arch.f. mik. Anat., xxii, 1883, p. 361) may also occasionally be useful. It is as follows : A supersaturated solution of safranin in water is warmed to 60 C. and filtered warm. On cooling, it becomes turbid through the formation of small crystals. Sections are placed in a watch-glass with some of this turbid solution, and the whole is warmed for a few seconds (till the liquid becomes clear) over a spirit lamp. Allow the whole to remain for one minute, and wash out with water, and treat with alcohol and turpentine in the usual way. Tissues which do not take on the stain at once must be warmed over and over again. Clove oil must be avoided for clearing. For some remarks of BATAILLOX and KOEHLEE on the stain of borax- methylen-blue, see Comptes Rendus, cxvii, 1893, p. 521, or Journ. Roy. Mic.'Soc., 1894, p. 41. 659. Plasma Stains. I have been unable to discover a single thoroughly satisfactory one. All of those known to me are of an imperfect electivity, in so far as it is difficult if not impossible to limit their action with the desired precision and certitude to the element that it is desired to bring into promi- nence by staining. Almost all of them colour too readily the enchylema or hyaloplasm afc the same time as the plasmatic reticulum. And, on the other hand, there are many im- 366 , CHAPTER XXVIII. portant elements of the cell which refuse to stain sufficiently. Spindles, for instance, can be made to stain, but only weakly at the best : it is impossible to get them to stain vigorously and at the same time distinctively. The best, or least defective plasma stain for hardened tissues that I know of is KEENSCHWARZ ; see 334, and also my paper " Sur le Nebenkern et sur la formation du Fuseau," in La Cellule, xi, 2, 1896, p. 257. After Kernschwarz, perhaps the best results I have had have been obtained by means of FLEMMING'S orange method. It is a very good stain for normal cytoplasmic structures, but a very poor one for the Nebenkern (" Spheres " of some authors) and other spindle relics. BENDA'S Safranin and Lightgrun or Sdureviolett gives sometimes splendid results, but is capricious. It is very goad for the Nebenkern, less so for equatorial spindle relics. For Sdurefuchsin and Orange G, see 305 and 300. EHRLICH-BIONDI mixture is a celebrated plasma stain. It will work with chrom-osmium material. It is of no use whatever for polar corpuscles or spindle relics. For KA.WITZ' Inversion Stain, see 308. I have obtained some vigorous images of polar corpuscles by this process, but I certainly do not think that it is a recommendable one. The Osmic Acid and Pyrogallol Process, 377, gives a very fair and frequently useful plasma stain ; but I do not con- sider it to be a method of quite the first class. The Iron-Hsematein Lakes of BENDA and M. HEIDENHAIN give good plasma stains, according to the degree of extrac- tion. The iron lake of M. HEIDENHAIN is said to be a specific stain for " centrosomes." I am not concerned to dispute the alleged fact, but must be allowed to hold that the status of the corpuscles stained by this process has not been placed on a perfectly satisfactory footing. It is said by HEIDENHAIN, and by other observers who have repeated his observations, that the reaction in question is obtained in a sharper form by combining the hsematein stain with a foregoing stain with Bordeaux R. It is held by HEIDENHAIN that the foregoing stain with Bordeaux, which is a general stain taking effect on both chromatin and plasma, but having no affinity for cen- trosomes, so satisfies the chemical affinities of the first-named elements that they no longer hold the iron lake with the CYTOLOGICAL METHODS. 367 usual tenacity, but give it up freely in the extraction process, whilst the centrosomes, not having taken up so much Bor- deaux, are free to bold the haematein strongly; which, it is stated, they do. This hypothesis is put forward as a theory under the name of Subtractive Staining by HEIDENHAIN, and under the name of Tinctorial Preoccupation by UNNA (ZtiLf. wiss. Mik., xii, 4, 1396, p. 454). The instructions given by HEIDENHAIN for the employment of the Bordeaux are not so detailed as might be desired. He states (Arcli.f. mik. Anat., xlii, 1894, p. 665) that the sec- tions (sublimate sections were used by him) are to be stained for twenty-four hours or more in " a weak " solution of Bor- deaux, until they have attained such an intensity of colour as that " they would just be fit for microscopic examination with high powers 5 ' (1. c., p. 440, note), and that they be then brought into the ferric alum. After mordanting and staining, the haematein is to be extracted in the iron alum until the chromatin has become entirely or almost entirely colourless. The Bordeaux is not supposed in this process to act as a plasma stain ; it goes away in the subsequent processes. Instead of Bordeaux, " anilin blue " may be used in the same way. I have repeatedly tried the Bordeaux process, and with my objects (I have not tried Heidenhain's) have observed not the slightest difference in the preparations obtained in this way, and those obtained by the iron hasruatein without the Bordeaux. The solution of Bordeaux used by me was of 1 per cent, strength. I have obtained some good plasma stains both with EHRLICH'S triacid and with his acidophilous mixture, and for some purposes should prefer these to the Ehrlich-Biondi mixture. HERMANN (Arch. f. mik. Anat., xxxvii, 4, 1891, p. 583) recommends a modification of the hsematoxylin impregnation method of PAL : Testes of Proteus put for twelve to eighteen hours into hsematoxylin, 1 grm. ; absolute alcohol, 70 c.c. ; water, 30 c.c., in the dark, and washed out for the same time in 70 per cent, alcohol, also in the dark. Sections having been made are treated with dilute pale rose-coloured solution of permanganate of potash till they turn ochre-coloured, rinsed in water, and washed out for removal of the brown peroxide of manganese in Pal's oxalic acid mixture diluted with 5 to 10 volumes of water. They are finally stained for three to five minutes in safranin. See also on this subject the paper of HERMANN, " Methoden zum Studium 368 CHAPTER XXVIII. des Archoplasmas und der Centrosomen tierischer und pflanzlicher Zellen," in Ergebnisse der Anatomie, Band ii, 1892 (1893), p. 23. For HEIDENHAIN'S Vanadium lisBmatoxylin, see COHN in Anat. Hefie, 1895, p. 302, or Zeit. f. wiss. Mik., xii, 3, 1896, p. 359. The achromatic structures of the ovum of Ascaris are best demonstrated, according to VAN BENEDEN et NETT (Nouv. Rech.) by fixing with acetic alcohol (ante, 657), and bringing the ova into one-third glycerin in which is dissolved a little malachite green. The " spheres attractives " stain green. See also BOVERI'S Zellen-Studien (in Jen. Zeitschr. f. Naturw., xxi, 1887, p. 423, or sold separately). HEELA (Arch, de Biol., xiii, 1893, p. 423) advises a mixture of vesuvin 0'25, mala- chite green 0'25, distilled water 100, and glycerin 10 ; the stain to be washed out with dilute glycerin. 660. Nucleus of BALBIANI ("Noyau Vitellin," " Cellule Embryo- gene") (Zool. Anz., 1883, p. 659). This may be observed in the fresh state, without the addition of any reagent, in the ova of some animals, amongst others a great number of Arachnida and Myriapoda. It may be brought out more distinctly by treating the ova with a mixture of equal parts of acetic acid and 1 per cent, osmic acid, to which is added a little sodium chloride. This mixture does not render ova so granular as pure dilute acetic acid. For sections, stain by HENNEGUY'S permanganate safranin process. 661. Cell-granules. These for the most part undoubtedly metabolic products are best studied in gland-cells and blood- and lymph- corpuscles, and in certain elements belonging to the group of connective tissues, and the reader is therefore referred to the sections on " Connective Tissues " for the appropriate methods. It will suffice here to state that the most generally employed stains for cell-granules are the mix- tures of EHRLICH. For ALTMANN'S " Bioblasts," see, besides that author's Studien iiber die Zelle, 1886, his Die Elementarorganismen, Leipzig, 1890, and a paper in Arch.f. Anat. u. Entwickel., 1892, p. 223; also Zeit. f. wiss. Mik., vii, 2, 1890, p. 199 ; ix, 3, 1893, p. 331 ; and L. and K. ZOJA, in Mem. R. 1st. Lombardo di Sci. e Lettere, xvi, 3, vii, p. 237. Omitting minutise and variations, it may be said that these granules may be demonstrated by fixing for twenty-four hours in a mixture of equal parts of 5 per cent, bichromate of potash and 2 per cent osmic acid, imbedding in paraffin, staining sections for a minute on the slide held over a flame with a solution of 20 grms. of acid fuchsin in 100 c.c. of anilin water (p. 190), and washing out with CYTOLOGICAL METHODS. 369 saturated alcoholic solution of picric acid diluted with 2 volumes of water, heat being used as before to aid the differentiation, and finally clearing with xylol and mounting in balsam. FISCHER (Anat. Anz., ix, 1894, No. 22, p. 678) has given reasons for believing that these granules may consist, in part at all events, of precipitates of albuminates (principally peptones) thrown down by the fixing agents (see also 657). 662. More Special Methods. For accounts of the methods that have been employed in the study of tissue-cells, and more especially of sexual products, now suppressed for want of space, see previous editions, or the Traite des Methodes techniques de VAnat. microscopiqne, LEE et HENNEGUY. 24 CHAPTER XXIX. TEGUMENTAEY OEGANS. 663. Epithelium. One of the chief methods of obtaining preparations giving instructive surface views of epithelia is the nitrate of silver method. For this see ante, 358, et seq.,. in the chapter on " Impregnation Methods." The reader may also consult with advantage the admirable instructions given by RANVIER in his Traite technique, p. 246, et seq., and the memoir of TOURNEUX and HERMANN in the Journ. de I' Anat. > 1876, p. 200. The perchloride of iron and pyrogallic acid method of the HOGGANS, 379, and the osmic acid and pyrogallic acid pro- cess, 377, may also be found useful here. But in many cases impregnation with methylen blue will doubtless be found preferable. Sections are easily made by the usual methods. The best hardening agent for skin appears to be Mailer's solution. This was the conclusion of F. E. SCHULTZE in 1867 (Arch.f. mik. Anat., p. 145) ; and it is that of TIZZONI, the author of important researches on this organ (Bull, delle Sc. med. di Bologna, 1884, p. 259), and of BEEN (Arch. f. mik. Anat.,' xxxix, 1892, p. 581). Simple bichromate of potash solution will do about as well. For glandular epithelium it is frequently better to employ a chromic acid liquid, or osmic acid (see, for example, RANVIER, loc. cit., p. 258, et seq.), or absolute alcohol (BLAUE, Arch. f. Anat. u. Phys., 1884, p. 231) ; ' ' Kleinenberg " is not so good. Macerating Media. For soft epithelia, mild macerating agents, such as iodised serum, one-third alcohol, saliva, or Schultze's mixture of saliva and solution of Miiller, or a mixture of saliva with three to four volumes of physiological salt s-olution (BIZZOZERO, Intern. Monatsschr. f. Anat., 1885, p. 278); for hard epithelia, energetic dissociating agents, such as 40 per cent, solution of caustic potasli. TEGUMENTABY ORGANS. 371 Mi NOT (Amer. Natural., xx, 1886, p. 575; cf. Journ. Eoy. Mi'-. Soc., 1886, p. 872) recommends maceration for several dnvs in 0'6 pet* cent, solution of sodium chloride containing O'l per cent, of thymol, which allows the isolation of the epidermis of embryos, and is useful for the study of the development of hairs. Another method, given by MITROPHANOW (see Zeit. f. wiss. Mik., v, 4, 1888, p. 513), is as follows : 'An embryo of axo- lotl is fixed for a quarter of an hour in 3 per cent, nitric acid, and then brought into one-third alcohol. After an hour the epidermis begins to come away in places ; and if the embryo be put for twenty-four hours into stronger spirit, it will come away almost entirely. For the purpose of separating the epidermis from the corium, LOEWY (Arch. f. mik. Anat., xxxvii, 1891, p. 159 ; Zeit. f. iviss. Mik., viii, 2, 1891, p. 222) recommends macerat- ing for twenty-four to forty-eight hours, at a temperature of about 40 C., in 6 per cent, pyroligneous acid. Acetic acid of 4 per cent. (PHILIPPSON) is also good. For ciliated epithelium, see the methods of ENGLEMANN under " Mollusca." 664. Prickle-Cells and Intercellular Canals. Besides macera- tion, which is one of the most important of the methods for the study of these objects, impregnation may be useful. MITROPHANOW (Zeit. f. wiss. Zool., 1884, p. 302, and Arch. f. Anat. u. Phys., 1884, p. 191) recommends the following pro- cess : Wash with distilled water the tail of an axolotl larva; put it for an hour into 0*25 per cent, solution of gold chloride with one drop of hydrochloric acid to a watch-glassful of the solution ; wash, and reduce in a mixture of one part of formic acid with six parts of water. On this subject see the important memoirs of IDE, in La Cellule, iv, 2, 1888, p. 409, and v, 2, 1889, p. 321. 665. Plasma -fibrils of Epithelium. KROMAYER'S process for demonstrating his iiitra- and intercellular fibrils of epithelia (Arch. f. mik. Anat., xxxix, 1892, p. 141 ; Zeit. f. wiss. Mik., ix, 1, 1892, p. 84, and ix. 3, 1893, p. 355) is as follows: Sections are stained for fiva minutes in a mixture of equal volumes of anilin water (p. 190) and concentrated aqueous 372 CHAPTER XXIX. solution of methyl violet 6 B. They are well washed in water and treated with solution of iodine in iodide of potassium until they become blue-black (one to thirty seconds). They are again washed with water, dried with blotting-paper, and treated with a mixture of 1 vol. of anilin to 2 vols. of xylol until sufficiently differentiated, when they are brought into pure xylol, Very thin sections will require more xylol in propor- tion to the anilin, viz. 1 : 3 or 1 : 4 ; thicker ones may require more anilin, viz. 3:5 or 3:3. Gentian or Kry stall violett will do instead of methyl violet, but not quite so well. For a discussion of the points of difference between the fibrils of Kromayer and the fibres of HEEXHEIMEE, see EHEMANN, and JADASSOHN, Arch. f. Dermatol. u. Syphilis, 1892, 1, p. 303; Zeit. f. ivissMik., ix, 1893, p. 356. For the same object UNNA (Monatsh.f. prakt. Dermatol., xix, 1894, p. 1 and p. 277, et seq. ; Zeit.f. iviss. Mile., xii, 1, 1895, pp. 61, 63) has given a whole series of minutely detailed methods, from which the following are some extracts : 1. WASSEEBLAU-ORCEIN. Stain sections for ten minutes in a neutral aqueous 1 per cent, solution of Wasserblau, rinse with water and stain 'for five or ten minutes in a neutral alcoholic 1 per cent, solution of Griibler's orcein. Dehydrate, clear, and mount in balsam. This method may be varied as follows : (a) Ten minutes in the Wasserblau, and thirty minutes or more in the orcein. (b) Take for the second stain an acid solution of orcein. (c) Stain for only one minute in the Wasserblau, but for thirty or more in the neutral orcein. 2. Stain for half an hour or more in a strong solution of haBmalum, rinse with water, stain for half a minute in a satu- rated aqueous solution of picric acid, and dehydrate for thirty seconds in alcohol containing 0*5 per cent, of picric acid. 3. Haemalum for two hours, neutral orceiu as above for ten to twenty minutes. Besides these methods, UNNA gives half a dozen others which can hardly be usefully detached from the theoretical part of his papers. 666. Horn, Hair, and Nails. The elements of hairs and nails may be isolated by prolonged maceration in 40 per cent, potash TEGUMENTAEt ORGANS. 373 solution, or by heating with concentrated sulphuric acid. See also vox XATHUSIUS, Zool. Anz.,*v, 1892, p. 395. Horny tissues stain well in safranin or gentian violet (REIXKE, Arclt.f. mik. J.?ia./xxx, 1887, p. 183; Zeit. f. wiss. Mik., iv, 3, 1887, p. 383). 667. Tactile Hairs. RANVIER (Traite, p. 914) recommends for the study of the nerve-endings the boiled formic acid and gold-chloride method, 371. A tactile hair having been isolated with its bulb, and its capsule incised, is put for about an hour into the formic acid and gold-chloride mixture, the gold is reduced in slightly acidulated water, hardening is completed in alcohol, and longitudinal and transverse sections are made. For DRASCH'S method see Zeit. f. wiss. Mik., iv, 4, 1887, p. 492. 668. Skin Nerves. WOLTERS (Arch.f. Dermatol. u. Syphilis, 1892 ; Zeit. f. wiss. Mik., ix, 3, 1893, p. 360) employs the chloride of vanadium and haematoxylin method, which will be given in the chapter on " Nerve-cell and Cell-process 669. Intra-epidermic Nerve-fibres. May be studied by the gold method. RAXVIER (Traite, p. 900) recommends the boiled formic acid and gold-chloride method, 371. He also (p. 910) recommends this method for the study of the tactile menisci of the pig's or mole's snout. Pieces of skin are impregnated as directed 371, and after reduction are brought into alcohol, which completes the hardening, and stays the further reduction of the gold. Sections are then made. For the study of the tactile menisci of the snout, Kanvier also recommends the lemon juice and gold-chloride method, 372. The methylen blue impregnation method should also be employed in the study of these nerve-endings. 670. Tactile Corpuscles (FISCHER, Arch. f. mik. Anat., 1875, p. 366). Fischer employed the gold method of Lowit see 370. Ranvier (Traite', p. 918) also recommends this method, 374 CHAPTER XXIX. as well as his two gold methods, 371, 372. Pieces of skin are first impregnated whole, then hardened by alcohol and sectioned. He finds (as do other authors) that osmic acid and picro-carmine are invaluable aids to the study of these structures, and to that of the corpuscles of Pacini. Purpurin and hasmatoxylin may also be used for after-staining. See BANVIEE, Traite, p. 919; and aee also LANGEEHANS, Arch. f. mik. Anat., 1873, p. 730; KULTSCHIZKY, ibid., 1884, p. 358; and SMIENOW, Intern. Monatschr. f. Anat., &c., x, 1893, 6, p. 241; Zeit.f. wiss. Mik., x, 2, 1893, p, 254 (this observer recommends, besides the gold method of Lowit, the rapid bichromate of silver method of GOLGI). 671. Corpuscles of Herbst and Corpuscles of Grandry. DOGIEL (Arch. f. Anat. u. Entwickel, 1891, p. 182; Zeit. f. iviss. Mik., viii, 4, 1892, p. 520) prefers the methylen blue method (Chap. XVII). Four per cent, solution of methylen blue, warmed to 40 C., is injected into blood-vessels of the heads of ducks or geese ; pieces of skin are removed from the beaks, sectioned in pith, and the sections brought 011 to slides and moistened with aqueous or vitreous humour from the animal, and left for a few minutes exposed to the air (it is well to add to the aqueous or vitreous humour a few drops of y 1 -^ per cent, methyl blue solution). After about ten to thirty minutes the nerve-endings are seen to be stained, and the sections are then brought into picrate of ammonia, and treated as described in the chapter on " Methylen Blue/' GEBEEG (ibid., x, 2, 1893, p. 244) has also employed this method. He has also made use of simple osmic acid, and of the gold method of AENSTEIN, which is as follows : Pieces of skin are macerated for twenty-four hours in lime water, after which the horny layer may easily be removed. This being done, the skin is cut up into small pieces which are put for five minutes into a 0*25 per cent, solution of chloride of gold. Reduction sets in very rapidly, and the preparations become brown in a few minutes. The reduction is completed by putting them for twenty- four hours into dis- tilled water. During this time there forms a granular pre- cipitate which is removed by putting the pieces into a 0'25 per cent, solution of cyanide of potassium and brushing them vigorously with a camel's-hair brush; after which thej are TEGt'MENTARt ORGANS. 375 mounted in damar. GEBERG used the gold chloride of 0'5 per cent, strength, and allowed it to act for thirty minutes. See also the method of CARRIERE, Arcli. f. mik. Anat., 1882, p. 146, or previous editions of this work. 672. Corpuscles of Meissner and of Krause (Cornea and Con- junctiva Bulbi and Palpebrarum) (DooiEL, Arch. f. mik. Anat., xxxvii, 1891, p. 602, and xliv, i, 1894, p. 15). A fresh bulb should be enucleated in toto, and incised along a line running parallel to the equator and 5 to 8 mm. behind the corneal margin. The anterior segment thus obtained is freed from the ciliary body, lens, &c., the conjunctiva being left in situ on the cornea. It is then cut into pieces which are stained for an hour to an hour and a half in methylen blue + aqueous humour, and further treated for examination and preservation as described in the chapter on " Methylen Blue." See also LONGWORTH'S methods, Arch. f. mik. Anat., 1875, p. 655. 6Y3. Similar Objects. Papillae Foliatae of the Rabbit (HERMANN >. See Zeit.f. v:iss. Mik., v, 4, 1888, p. 524. Olfactive Organs of Verte- brates (DoGIEL, Arch.f. mik. Anat., 1887, p. 74). Organs of a " Sixth Sense " in Amphibia (MITBOPHANOW). See Zeit. f. wiss. Mik., v, 4, 1888, p. 513. (This paper contains some details as to staining with " Wasserblau," for which see also Biol. Centralb., vii, 1887, p. 175.) Nerve-endings in Tongue of Frog (FAJERSTAIX [FEUEBSTEIX], Arch, de Zool exper. et gen., vii, 1889, p. 705; Zeit.f. u-iss. Mil:, vii, 3, 1889, p. 357). (Amongst other methods for the study of the terminal discs, the methylen hlue method is recommended ; see previous editions). Tongue of Rabbit, VON LENHOSSEK, Zeit.f. wiss. Mik., xi. 3, 1894, p. 377 (Ramon y Cajal's double Golgi-method). 674. Cornea. There are three chief methods for the study of the corneal tissue the methylen blue method, the silver method, and the gold method. For the methylen blue method see Chap. XVII, particularly 295 and 296. Negative images of the corneal cells are easily obtained by the dry silver method (KLEIN). The conjunctival epithelium should be removed by brushing from a living cornea, and the corneal surface well rubbed with a piece of lunar caustic. After half an hour the cornea may be detached and examined in distilled water. 376 CHAPTER XXIX. In order to obtain positive images of the fixed cells tlie simplest plan (RANVIER) is to macerate a cornea that has been prepared as above for two or three days in distilled water. There takes place a secondary impregnation,, by which the cells are brought out with admirable precision. The same result may be obtained by cauterising the cornea of a living animal as above, but allowing it to remain on the living animal for two or three days before dissecting it out, or by treating a negatively impregnated cornea with weak salt solution or weak solution of hydrochloric acid (His). But the best positive images are those furnished by gold chloride. RANVIER prefers his lemon-juice method to all others for this purpose (see 372). It is important that the cornea should not remain too long in the gold solution, or the nerves alone will be well impregnated. Ranvier also recommends this method as being the best for the study of the nerves. ROLLETT (Strieker's Handbuch, p. 1115) recommends a double impregnation with silver followed by gold for obtaining gold-stained negative images. A cornea having been treated for a short time only with 0'5 per cent, silver nitrate solution, and the silver reduced, is treated with O5 per cent, gold chloride solution. The brown stain of the silver disappears immediately the preparation is placed in the gold solution ; after a few minutes the preparation is exposed to the light in acidulated water. Reduction of the gold rapidly takes place, and in the place of the former brown stain of the silver the ground-substance shows the well-known blue of reduced gold. The cells are, however, visible, being recognisable by their granular appearance and pale yellow tint. RENACT (Oomptes Rend. } 1880, l er sem,, p. 137) gives the following process for corneal corpuscles : Cornea of frog : formic acid, 20 per cent., ten minutes ; gold chloride, 1 per cent., twenty-four hours; formic acid, 33'3 per cent., twenty- four hours. HOYER'S method has been given, 374. 675. Cornea, other Methods (ROLLETT, Strieker's ffandb., p. 1102). Rollett strongly recommends the following plan : A fresh cornea is placed (in humor aqueus) in a moist chamber, and exposed to the action of iodine vapour. As soon as it has TEGUMKXTARY ORGANS. 377 become brown the epithelium may easily be peeled off. If the reaction is not complete the cornea may be put back into the iodine chamber. AVhen sufficient iodine has been absorbed the preparation may be examined, and it will be found that the network of corneal cells is brought out with an evidence hardly inferior to that of gold preparations. The method never fails, which is not the case with the gold method. It is admirable as a fixing method. For dissociation of the fibres Rollett recommends macera- tion in a solution of permanganate of potash or a mixture of this solution with alum. As soon as the tissue has become brown it is shaken in a test-tube with water, and breaks up into fibres and bundles of fibres. TARTUFEEI (Anat Anz., v, 1890, p. 524; Zeit.f. wiss. Mik., vii, 3, 1890, p. 365) has the following method for demonstrating corneal cells and the ramifications of their processes : A cornea is placed for three or more days in a solution of 15 grammes of hyposulphite of soda to 100 c.c. of distilled water, kept at a temperature of about 26, then removed for two days into water containing very finely powdered chloride of silver (if left longer the corneal cells will not be stained, but innumerable elastic fibres will be demonstrated). The preparations are said to be permanent. Some further details are mentioned in Zeit.f. wiss. Mik., xi, 3, 1894, p. 346. 676. Crystalline (Hardening of) (LowE, Arch. f. mik. Anat., 1878, p. 557). A fresh bulb is placed in a vessel containing several litres of 1 per cent, bichromate of potash solution, which is frequently changed for stronger solutions until the strength of a cold saturated solution is attained. The bulb must remain in this for at least a year and a half, in order that the crystalline may attain the right degree of hardness. For Maceration, use Max Schultze's sulphuric acid solution, supra, 557. Formaldehyde may perhaps be found useful for hardening. CHAPTER XXX. MUSCLE AND TENDON (NERVE-ENDINGS). Striated Muscle. 677. Muscle-cells. For the study of these and allied sub- jects see, inter alia, BEHRENS, KOSSEL, und SCHIEFFERDECKER, Das Mikroskop, &c., vol. ii, pp. 154- 161; also, for the ap- plication of the gold method to the study of muscle- cells, SCBAFER, Proc. Roy. Soc. y xlix, 1891, p. 280 ; or Journ. Roy. Mic. 8oc., 1891, p. 683* 678. Sarcolemma. Besides the places quoted in last section, see SOLGER, Zeit.f.wiss. Mik., vi, 2, 1889, p. 189 (small pieces of fresh muscle teased and examined in cold saturated solu- tion of carbonate of ammonia) . 679. Sections (ROLLETT, Denkschr. math, naturw. Kl. k. Akad. Wiss. Wien, 1885; Zeit. f. wiss. Nik., 1886, p. 92). Besides the usual section methods, the following methods of Rollett should be noted : (1) The method mentioned ( 183) of freez- ing living tissue in white of egg. (2) The same method applied to recently fixed muscle. 680. Dissociation. See Chap. XXV. LANGERHANS' methods for Amphioxus (Arch. f. mik. Anat., 1875, p. 291). For isolation of the muscle-plates macerate the fresh animal in 20 per cent, nitric acid. For isolation of the nervous system macerate an animal for three days in 20 per cent, nitric acid, then place it for twenty-four hours in water, and shake forcibly. The whole of the nervous system may thus be separated, almost down to the finest peripheral terminations of nerves. 681. Nerve -endings. For the study of nerve-endings in muscle, both motor and sensory, the four chief methods are the methylen-blue method, the gold method, the silver method, and the bichromate of silver method of Golgi. MUSCLE AND TENDON NERVE-ENDINGS. 379 682. Nerve-endings the Methylen-blue Method. The prin- ciple* of the impregnation of nerve-tissue with methylen blue have been explained in Chap. XVII. BIEDERMANN'S procedure for the muscles of Astacus has been indicated in 291 (see also Zeit. f. wlss. Mik., vi, 1, 1889, p. 65). After impregnating as there directed the carapace should be opened, and the muscles exposed to the air in a roomy moist chamber for from two to six hours, in order that the stain may differentiate. The abdominal and caudal muscles are those which give the best results. For Hydropliilus piceus, Biedermann proceeded by inject- ing 0'5 c.c. of methylen-blue solution between the ultimate and penultimate abdominal rings, in the ventral furrow, and keeping the animals alive in water for three to four hours. After this time the thorax should be opened by two lateral incisions, and the muscles of the first pair of legs (which are the most suitable) removed and exposed to the air for three or four hours in a moist chamber, and finally examined in salt solution. GERLACH (Sitzb. It. math.-phys. Cl. k. layer. Akad. Wiss. Milnclien, 1889, ii, p. 125; Zeit. f. wiss. Mik., vii, 2, 1890, p. 220) injected frogs, either through the abdominal vein or through the aorta, with 4 to 5 c.c. of a 1 : 400 solution in 1 per cent, salt solution, and examined pieces of muscle (pre- ferably the head and eye muscles) in serum of the animal, afterwards fixing the preparations with picrate of ammonia and mounting in glycerin jelly. The procedure of DOGIEL has been given in 291. 683. Nerve-endings the Gold Method. FISCHER (Arch. f. mik. Anat., 1876, p. 365) used the gold method proposed by LOWIT (Wien. Sitzgsber., Bd. Ixxi, Abth. 3, 1875, p. 1), and employed by himself in his researches on the tactile corpuscles (Arch.f. mik. Anat., xii, p. 366). See ante, 370. BIEDERMAXX, in the paper quoted in the last section, recom- mends for Astacus a similar procedure, the preliminary treat- ment with formic acid being omitted, and the muscles being put for a couple of days into glycerin after reduction in the acid. The procedure of TRINCHESE (Mem. R. Accad. 1st. Bologna, 5, ii, p. 279; Zeit. f. iv'tss. Mik., ix, 2, 1892, p. 238) is also practically identical. 380 CHAPTER XXX. RANVIEE (Traite, p. 813) finds that for the study of the motor terminations of Batrachia the best method is his lemon- juice and gold-chloride process ( 372). The delicate ele- ments of the arborescence of Kiihne are better preserved by this method than by the simple method of Lowit. For the study of the motor plates of reptiles, fishes, birds, and mammals, he finds (ibid., p. 826) that his formic acid and gold-chloride method ( 371) gives preparations infinitely superior to those obtainable by the method of Lowit ; but the lemon-juice method is still better, especially for lizards and mammals. The branches of the terminal arborescence are more regular than in preparations obtained by the formic acid process. 684. Nerve-endings the Silver Method. RANVIER finds that the silver nitrate method of Cohnheim is also useful. He employs it as follows (ibid., p. 810) : Portions of muscle (gastrocnemius of frog) having been very carefully teased out in fresh serum are treated for ten to twenty seconds with nitrate of silver solution of 2 to 3 per 1000, and exposed to bright light (direct sunlight is best) in distilled water. As soon as they have become black or brown they are brought into 1 per cent, acetic acid, where they remain until they have swelled up to their normal dimensions (the swelling induced by the acid serving to make up for the shrinkage caused by the nitrate of silver). They are then examined in a mixture of equal parts of glycerin and water. This process gives negative images, the muscular substance is stained brown, except in the parts where it is protected by the nervous arborescence, which itself remains unstained. The gold process gives positive images, the nervous structures being stained dark violet. 685. Nerve-endings the Bichromate of Silver Method. The osmium bichromate and silver method of Golgi has been suc- cessfully applied by RAMON Y CAJAL to the study of the ter- minations of nerves and of tracheas in the muscles of insects, and is doubtless susceptible of still wider applications. The process used by him is the rapid one. In Zeit. f. u-iss. Mik., vii, 3, 1890, p. 332, he gives it as follows : Fresh muscle of insects put into a mixture of 20 MVM-LK AND TENDON (XEKVE-EXDINGs). 381 parts 3 per cent, bichromate solution and 5 parts 1 per cent, usmium solution for twelve to twenty-four hours, then into <-7"> per cent, nitrate of silver for one day, then alcohol of 40 d threes (presumably Bauine, i. e. about 90 per cent.), then clove oil followed by resinified turpentine and (if I understand rightly) balsam. 686. Nerve -endings other Methods. For the following see pre- vious editions : BREMEE (Arch. f. mik. Anat., 1882, p. 195) ; CIACCIO (Journ. de Jficrographie, 1883, p. 38); WOLFF (Arch.f. mik. Anat., 1881, p. 355) ; GAEL SACHS (ibid.) ; KBAUSE (Intern. Monatschr.f. Anat. u. Hist. ; Zeit.f. wiss. Mik., 1885, p. 547) ; XEGEO (Zeit.f. wiss. Mik., v, 2, 1888, p. 240); SIHLEE, Verhand. d. Physiol. Ges. Berlin, 1894-5 ; Zeit.f. wiss. Mik., xii, 3, 1896, p. 389 (these two are hsematoxylin stains) ; BOCCAEDI (Zeit.f. wiss. Mik., iv, 4, 1887, p. 492) ; KUHKE (Zeit.f. Biol, xxiii, v, 1887, p. 1 ; Zeit.f. wiss. Mik.j iv, 4, 1887, p. 495 this paper contains a critical review of the different go Id methods) ; GOLGI (Mem. delle R. Accad. di Sci. di Torino, ii, 32) ; MARSHALL (Quart. Journ. Mic. Sci., 1890, p. 73 ; Journ. Boy. Mic. Soc., 1890, p. 404; : MAYS (Zeit.f. Biol., 1884, p. 449; Zeit.f. iciss. Mik., 1885, p. 242). Tendon. 687. Corpuscles of Golgi (KAXVIER, Traite, p. 929). Take the tendon of the anterior and superior insertion of the gemini muscles of the rabbit. Free it as far as possible from adherent muscle-fibres. Treat it according to the formic acid and gold method ( 371), and after reduction of the gold scrape the tendon with a fine scalpel, in order to remove the muscle-fibres that mask the " musculo-tendinous organs/' 688. Corpuscles of Golgi (in the tendons of the motores bulbi oculi) (VON MARCHI'S methods, Archivio per le Scienze Mediche, vol. v, No. 15). The enucleated eyes, together with their muscles, were put for not less than three days into 2 per cent, bichromate of potash. The muscles and tendons were then carefully dissected out, stained with gold chloride and osmic acid (Golgi's method, supra, 686), and by the methods of MANFREDI, given in 374. Mount all these preparations in glycerin (balsam clears too greatly). The methods only succeed completely during fine sunny weather. 689. Corpuscles of Golgi (CATTANEO, Arch. Hal. de Biol., x, 1888, p. 337 j. The method here recommended is the arsenic acid gold method of Golgi quoted above, 686. 382 CHAPTER XXX. See also RTJFFIXI (Atti R. Ace. Lincei Roma, 1892, p. 442 ; Zeit. f. wise. \ Mik., ix, 2, 1892, p. 237), who recommends the method of Fischer. 690. Corpuscles of Golgi (CiACCio, Mem. R. Ace. Sci. Bologna j [4], t. x, 1890, p. 301 ; Zeit. f. wiss. Mik., vii, 4, 1891, p. 507). For Amphibia the usual gold methods are not satisfactory, because the ground-substance of the tendon "takes the stain at the same time as the nerve- endings. Pieces of tendon, should be put into O'l per cent, hydrochloric acid or 0*2 per cent, acetic acid until quite transparent. They should then be put for five minutes into a mixture of O'l per cent, gold chloride and 0*1 per cent, potassium chloride. After that they are put back into the acetic acid, and remain there for | a day in the dark, and for two or three hours more in the sunlight. When they have become somewhat violet they are put for a day into O'l per cent, osmic acid, and finally mounted in Price's glycerin acidulated with 0'5 per cent, of acetic or formic acid. 691. The Methylen-blue Method. I find no mention of the application of this method to the study of the relations of nerve and tendon, for which it would seem a priori to be peculiarly suitable. Smooth Muscle. 692. Test for Smooth Muscle (RETTERER, Comptes Rend. Soc, BioL, iv, 1887, p. 645 ; Journ. Roy. Hie. Soc., 1888, p. 843). If a specimen of tissue be fixed in a mixture of ten volumes of 90 per cent, alcohol and one volume of formic acid, well washed, and stained for twenty-four to thirty-six hours with alum-carmine, the cytoplasm of smooth muscle will be found to be stained red, whilst connective-tissue ceils remain un- stained, and are swollen. 693. Smooth Muscle Isolation of Fibres (SCHWALBE, Arch.f. mik. Anat., 1868, p. 394). Maceration in weak chromic acid solution (0'02 per cent, proved a generally useful strength). This is a better reagent than osmic acid, 1 per cent, acetic acid (Moleschott), weak sulphuric acid, pyroligneous acid (Meissner), 20 per cent, nitric acid (Reichert), 32 to 35 per MUSCLE AND TEXDON (XHIJVE-EXDIXGS). 383 cent, potash solution (Moleschott), as it preserves better than any of these the finer struct tire of the cells. GAGE'S methods. See Journ. Roy. Hie. Soc., 1887, p. 327 ; and 539, 551, and 555, ante. MOBIUS, liquid for maceration of the muscle of Cardium (see above, 550). BALLOWITZ, muscle of Cephalopoda, see Arch. f. mik. Anat., xxxix, 1892, p. 291 ; Zeit.f. iviss. Mik., ix, 3, 1893, p. 344. 694. Smooth Muscle, Specific Stain for (UNNA, Monatsh. f. prakt. DermatoL, xix, 1894, p. 533; Zeit. f. iviss. Mik., xii, 2, 1895, p. 243). Sections stained for ten minutes in poly- chromatic methylen blue solution, rinsed in water, and brought for ten minutes into 1 per cent, solution of red prussiate of potash. This fixes the colour, so that the sections -will now bear differentiating with acid alcohol. They are treated ac- cordingly with alcohol acidified with 1 per cent, of hydro- chloric acid for about ten minutes (until the collagen ground comes out white). Absolute alcohol, essence, balsam. In the same place see also another stain with acid orcein, haematein, Saurefuchsin, and picric acid. 695. Iris (DoaiEL, Arch. f. mik. Anat., 1886, p. 403). An enucleated eye is divided into halves, and the anterior one with the iris brought for some days into a mixture of two parts one-third alcohol and one part 0*5 per cent, acetic acid. The iris can then be isolated, and split from the edge into an anterior and posterior plate, and these stained according{to the usual methods. 696. Iris (KOGANEI, Arch. f. mik. Anat., 1885, p. 1). The pigmented epithelium can be removed by brushing with a small brush after prolonged maceration in solution of Miiller. The pigment may also be bleached by chlorine water, which, however, should only be allowed to act for a few hours, until the pigment has become of a light brown; complete de- coloration may be obtained by prolonging the reaction for twenty-four hours, but then the tissues suffer. (See Journ. Roy. Mic. Soc. f 1886, p. 874.) See also CANFIELD, in Arch.f. mik. Anat., 1886, p. 121 ; and DOSTOIEWSKY, ibid., p. 91 (sections stained with hserna- 384 CHAPTER XXX. toxylin and eosin, or [LIST, Zeit. f. wiss. Mik., iii, 4, 1886, p. 514] with Renaut's hgematoxylic glycerin). 697. Stomach of Triton (see STILLING and PFITZNER, in Arch, mik. Anal., 1886, p. 396). 698. Bladder of Frog, Innervation of (WOLFF, Arch. /. mik. Anat.j 1881, p. 362). A frog* is killed and a solution of gold chloride of 1 : 20,000 injected into the bladder through the anus. (If the injection flows out on removal of the syringe, tie the frog's thighs together.) Now open the frog, dissect away the attachments of the bladder, ligature the intestine above the bladder, and cut away the abdomen of the frog so as to have in one piece bladder, rectum, and hind legs. (All this time the bladder must be kept moist with weak gold solution.) The bladder and the rest are now put into gold solution of 1 : 2000 for four hours ; the bladder is then ex- cised, slit open, and pinned (with hedgehog spines) on to a cork (outside downwards). Place it under running water until 1 all the epithelium is washed away. Use a pencil if necessary. Put for twenty-four hours into gold solution of 1 : 6000. Wash in pnre water, and put away in the dark ' ' for some time" in acidulated water, and finally reduce in fresh water in common daylight. The muscles should be pale blue- red ; medullated nerves dark blue-red sympathetic nerves and ganglia carmine-red. RANVIER (Traite, p. 854) recom- mends one or the other of his two gold processes. The bladder of frogs should be carefully distended by injection of the lemon juice or gold chloride and formic acid through the cloaca. See also the method followed by BERNHEIM, Arch. f. Anat. u. PhysioL, Physiol. Abth., 1892, supp., p. 29; Zeit. f. wiss. Mik., x. 4, 1893, p. 484 ; likewise a gold method. The methylen-blue method would appear to be very much indicated for this object, but has not been used for it so far as I am aware. CHAPTER XXXI. NEUROLOGICAL METHODS INTRODUCTION AND SECTION METHODS. 699. Introduction. The technique of the microscopic ana- tomy of the nervous system is highly special. The ordinary methods of microscopic anatomy are not sufficient to elucidate either the delicate and complicated structure of nervous elements, or their anatomical relations. These problems can only be successfully attacked by means of special methods of hardening, and the employment of highly specific methods of coloration. Histological research into the structure of the nervous system pursues two ends. Either it is desired to elucidate the minute structure of the nervous elements or neurons (neurites FISH), that is to say, the internal organisation of nerve-cells and nerve- fibres: the processes employed to this end forming a group of cytological methods. Or it is desired to study the form of nerve-cells, the exact distribution of the divers groups of nerve -cells in the grey matter, the connec- tions that are formed by means of nerve-fibres between these groups of nerve-cells or " nuclei," and to follow out the intri- cate course of the tracts of fibres that enter into the constitu- tion of the white matter of the cerebro-spinal axis. The pro- cesses employed in all these researches form a group of the anatomical methods of neurology. It is more especially in this group that we find highly special methods of selective coloration. This group may be divided as follows. A. Nerve-fibres. (a) Myelin stains ; comprising the methods of WEIGERT, and similar methods. (b) Axis-cylinder stains, and axis-cylinder and myelin stains. 25 386 CHAPTER XXXI. B. Nerve-cells. (c) Axis-cylinder and protoplasm stains, comprising the methylen-bliie method and some rather old-fashioned general stains. (d) Axis-cylinder and protoplasm impregnations, con- sisting chiefly of the methods of GOLGI (the sublimate method and the three bichromate of silver methods), and the gold methods that have been given in previous chapters. Nature acknowledges no absolute distinction between a central and a peripheral nervous system ; and as, moreover, the chief neurological methods are applicable both to the study of central and of peripheral nerve-tissue, it does not seem advisable to make a formal distinction of that sort here. The three following chapters wear the aspect of being devoted chiefly to the " Central Nervous System " simply because a large proportion of the methods used in the study of nerve- tissue in peripheral organs have already been extensively treated of in the chapters on "Methylen Blue/' on "Im- pregnation Methods," on " Tegumentary Organs," and on "Muscle and Tendon. " The reader will kindly bear in mind that a considerable part of the subject properly compre- hended under the term " Neurological Methods " is contained in those chapters, which should be referred to in order to complete the account given in the following pages. The remainder of this chapter will be devoted to the special section methods employed for the central nervous system, a knowledge of which is a necessary preliminary to further study, and to the Cytological Methods of Neurology. Group A of the Anatomical Methods will be given in Chap. XXXII, and Group B in Chap. XXXIII. For more minute details concerning the dissection and hardening of the voluminous encephala of Man and the larger Vertebrates than can be given here see MEECIEE, Les Coupes du Systems Nerveux Central (1894, Paris, Rueff ) ; DEJEBINE, Anatomie des Centres Nerveux, 1895 ; BEVAN LEWIS, The Human Brain; Histological and Coarse Methods of Research, London, Churchill ; and OBEESTEINEE, Anleitung beim Studium des Baues d. nervosen Centraloryane im gesunden u. kranken Zustande t Leipzig, Toeplitz. These very welcome additions to the literature of the' subject relieve me from the obligation of treating the matter with all the minuteness that might be desired by specialists ; the more so as they show : that so to treat it would require a volume, not a chapter. XKUROLOGICAL METHODS. 387 SECTION METHODS. 700. Fixation by Injection. Fixation, in the proper sense of the word, is of course out of the question in the case of the human subject. But in the case of the lower animals it is possible to introduce fixing liquids into the living nerve- centres by means of injection, thus ensuring a much more rapid penetration of the reagents than can be obtained by simple immersion. This method was, I believe, first suggested by GOLGI (Arch. Ital. de Biologie, t. vii, p. 30). He injected 2'5 per cent, solution of bichromate of potash, through the carotid if he wished to limit the hardening to the encephalon, or through the aorta if he desired to fix the spinal cord. DE QUEEVAIN (Virchow's Archiv, cxxxiii, 1893, p. 489 ; Zeit.f. wiss.Mik., x, 4, 1893, p. 507) proceeds as follows with dogs or cats. The subject, lightly chloroformed, is fixed securely on the operating table. A carotid is laid bare and cut through, and a canula fitted with a stopcock is tied both into the central and the distal portion. The cock of the central portion is opened first, and blood is allowed to flow out till no more comes away. Then liquid of Muller, at the temperature of the body, is injected under a moderate pressure through the peripheral canula (therefore towards the brain). This kills the subject at once and begins the fixation. Then more liquid of Muller is in- jected alternately through the central canula and the peri- pheral one, until a quantity has been injected equal to the quantity of blood that has been abstracted. For dogs 300 c.c. to 600 c.c. will be required, for cats from one third to one half of that quantity. As soon as the injection has been made, ligatures are placed on the cut ends of the vessel; the encephalon is removed, and is put for some weeks into liquid of Muller kept at 37 C. MANN (Zeit.f. iviss. Mik. 3 xi, 4, 1894, p. 482) proceeds in a similar manner. He injects through the aorta. Before throw- ing in the fixing liquid, he injects for about twenty seconds physiological salt solution warmed to 39 C. This washes out the capillaries, and prevents the blood from coagulating there. The fixing solution employed by him consists of saturated solution of corrosive sublimate, warmed to 39 C. After five minutes of injection the brain ought to be fixed throughout. It is removed and put for twelve hours into the same sub- 388 CHAPTER XXXI. limate solution, after which it is either put for permanent preservation into O'l per cent, solution of sublimate, or is at once passed through alcohol for imbedding in paraffin. STRONG (New York Acad. of Sci., January 13th, 1896 ; Anat. Anz.y xi, 21, 1896, p. 655) advises injecting formalin diluted with an equal volume of water into the cephalic vessels until it runs from the cut jugulars. After a few minutes the same quantity is again injected, and once or twice again after a lapse of fifteen to twenty minutes. When only the Golgi method is to be used, an equal volume of 10 per cent, solution of potassium bichromate is to be taken instead of the water. I must say this treatment seems" to me very heroic (see 86). Hardening. 701. Hardening by the Freezing Method. This is in many cases a very good method, and in particular may be of service for the histological study of the cortex. If it be desired to freeze an organ that has been already hardened by reagents, the freezing may be done by means of a freezing mixture of ice and salt ; but in this case the preparation should first be penetrated by a mucilaginous or gelatinous freezing mass ( 177, et seq.), in order to avoid the formation of ice crystals in the tissues. But in the case of fresh tissue the ether freezing method is to be preferred. This method allows of rapidly producing any desired degrees of hardness, and maintaining them or allow- ing them to diminish as occasion may require, and is perhaps the only method by which satisfactory sections of unhardened nerve -tissue can be obtained. The sections should be floated on to water, treated for a minute on the slide with O25 per cent, osmic acid solution, and stained or otherwise treated as desired. For a detailed description of these manipulations see BEVAN LEWIS'S The Human Brain. 702. Generalities on Hardening by Reagents. If large pieces of nerve-tissue are to be hardened, it is necessary to take special precautions in order to prevent them from becoming deformed by their own weight during the process. Spinal cord or small specimens of any region of the encephalon may be cut into slices of a few millimetres thickness, laid out on cotton wool, and brought on the wool into a vessel in which they may have the hardening liquid poured over them. The wool performs two functions : it forms an elastic cushion on NEUROLOGICAL METHODS. 389 which the preparations may lie without being distorted by their own weight ; and it allows the reagent to penetrate by the lower surfaces of the preparations as well as by their ex- posed surfaces. A further precaution, which is useful, is to hang up the preparations, lying on or in the cotton wool, in a glass cylinder or other tall vessel ; by hanging them near the top of the liquid the processes of diffusion and the penetra- tion of the reagent are greatly facilitated. If the preparations are placed on the bottom of the vessel, they should never be placed one on another. If it be desired to harden voluminous organs without divid- ing them into portions, they should at least be incised as deeply as possible in the less important regions. It is perhaps better in general not to remove the membranes at first (except the dura mater), as they serve to give support to the tissues. The pia mater and arachnoid may be removed partially or entirely later on, when the hardening has already made some pro- gress. The spinal cord, the medulla oblongata, and the pons Varolii may be hardened in toto. The dura mater should be removed at once, and the preparation hung up in a cylinder- glass, with a weight attached to its lower end. The weight has the double function of preventing any part of the preparation from floating above the level of the hardening liquid (a thing that easily happens where somewhat dense liquids, such as Mailer's solution, are used), and of preventing the torsions of the tissues that may otherwise be brought about by the elastic fibres of the pia mater and arachnoid. The cerebrum should be very delicately laid out on a layer of cotton wool, or, if possible, hung up in it. Plugs of the wool should be put into the fissure of Sylvius, and as far as possible between the convolutions. Unless there are special reasons to the contrary, the brain should be divided into two symmetrical halves by a sagittal cut passing through the median plane of the corpus callosum. Betz recommends that after a few hours in the hardening liquid the pia mater should be removed wherever it is accessible, and the choroid plexuses also. I have found this by no means easy, and think it is an operation that can only be recommended for experienced hands. The cerebellum should be treated after the same manner. 390 CHAPTER XXXI. The temperature at which the preparations are kept in the hardening solution is an important point. The hardening action of most solutions is greatly enhanced by heat. Thus WEIGERT (Centralb. f. d. med. Wiss., 1882, p. 819 ; Zeit. f. wiss. Mik., 1884, p. 388) finds that at a temperature of from 30 to 40 C. preparations may be sufficiently hardened in solution of Miiller in eight or ten days, and in solution of Erlicki in four days ; whilst at the normal temperature two or three times as long would be required. But it is not certain that this rapid hardening always gives the best definite results. SAHLI, who has made a detailed study of the hardening action of chrome salts, is of opinion that it does not, and thinks it ought for this reason to be abandoned (see Zeit. f. wiss. Mik., 1885, p. 3). On the other hand, the slowness of the action of chromic salts at the normal temperature is such that decomposition may easily be set up in the tissues before the hardening and preserving fluid has had time to do its work. For this reason voluminous preparations that are to be hardened in the slow way should be put away in a very cool place best of all in an ice-safe. 703. The Reagents to be employed. The hardening agents most used are the chromic salts. Chromic acid was much used at one time, but most workers now agree that its action, though much more rapid than that of the salts, is much more uneven, and frequently causes a disastrous friability of the tissues. Osmic acid is excellent according to BEVAN LEWIS it is the best of hardening agents ; but its employment is un- fortunately very restricted, as it can hardly be used for objects of more than a cubic centimetre in size. It has already been noted that the liquid of Erlicki has a more rapid action than the other solutions of chromic salts ; for this reason it is one of the most commonly employed solutions. SAHLI, however (1. c.), after having studied the action of the usual solutions, concludes that the best harden- ing agent for fresh tissues is pure bichromate of potash, in 3 or 4 per cent, solution, the hardening being done in a cold place. And he does not approve of the addition of sodium sulphate (Miiller), and rejects the liquid of Erlicki on account of the precipitates it so frequently gives rise to (see 96). NEUROLOGICAL METHODS. 391 OBERSTEINER is of the same opinion, recommending pure bichromate for general hardening purposes ; whilst for the study of the most delicate structural relations he recommends fixing in FoPs modification of Flemming's liquid (p. 32) for twenty-four hours, followed by washing with water and hardening in 80 per cent, alcohol. In view of the slowness of penetration of chromic salts it is often advisable to treat preparations for twenty-four hours or more with alcohol of 80 to 90 per cent, before putting them into the hardening liquid, in or der to avoid maceration of the deeper layers of tissue. Two recent observers, FISH (The Wilder Quarter- Century Book, 1893, p. 335) and DONALDSON (Journ. of Morphol., ix, 1894, p. 123; Journ. Roy. Mic. Soc., 1894, p. 642), have made numerous determinations of weight and volume with the object of ascertaining what changes are produced by reagents in encephala of sheep. They have found that bichromate of potash produces a slight increase both of weight and volume, whereas all the other reagents tried produce a diminution of both these factors. Formaldehyde is a reagent that has been too recently introduced for it to be desirable to enunciate a definitive opinion concerning its fixative and hardening properties. I have quoted in 86 and 109 some authors who have found it advantageous, especially in the form of mixtures with bichro- mate or other reagents, in the hardening of nervous tissue, and some other formulae of authors will be given further on. I have already stated ( 86) that I find that used pure it does not faithfully preserve the minute structure of cells. There is no doubt that it is excellent for the preservation of specimens intended only for macroscopical study, but that is another thing. See further 711 and 765 below. For the question as to how far certain so-called pathological alterations of ganglion-cells should be attributed to putrefractive changes or to the influence of reagents see Neurologisches Centralb. for the years 1884 and 1885. As to the so-called " pigment spots " produced by the liquid of Erlicki see supra, 96. 704. Strengths of the Reagents. All hardening reagents (except osmic acid) should afc first be taken as weak as is con- sistent with the preservation of the tissue, and be changed by degrees for stronger. Osmic acid may be taken of 1 per cent, strength, and will harden small pieces of tissue sufficiently in five to ten days (EXXER). Bichromate of potasli should be taken at first of not more than 2 per cent, strength; this is then gradually raised to 3 392 CHAPTER XXXI. or 4 per cent, for the cord and cerebrum, and as much as 5 per cent, for the cerebellum. Obersteiner begins with 1 per cent., and proceeds gradually during six to eight weeks to 2 or 3 per cent. (This is at the normal temperature : at a tem- perature of 35 to 45 C. the hardening can be got through in one or two weeks.) Bichromate of ammonia should be taken of half the strength recommended for bichromate of potash, or even weaker at first ; it may be raised to as much as 5 per cent, for cerebellum towards the end of the hardening. Chromic acid is not much used alone (see 88). It forms part of some of the mixtures mentioned below. A very little chromic acid (say one to two drops of 1 per cent, solution for each ounce) added to bichromate solu- tion will do no harm, and will quicken the hardening. Nitric acid has been and still is employed in strengths of 10 to 12 per cent., and gives particularly tough preparations. Neutral acetate of lead in 10 per cent, solution affords an excellent pre- servation of ganglion-cells, according to ANNA KOTLAKEWSKI (see Zeit.f. wiss. Mik., iv, 3, 1887, p. 387). TEZEBINSKI (Virchow's Arch., 1887, p. 1 ; Zeit.f. wiss. Mik., iv, 4, 1887, p. 497) finds that, as regards the faithful preservation of ganglion-cells (of the spinal cord of the rabbit and dog), the best results are obtained by hardening for eight days in 7 per cent, solution of corrosive sublimate, followed by hardening in alcohol containing 0'5 per cent, of iodine. DIOMIDOFF (ibid., p. 499) also obtained very excellent results by harden- ing small pieces of brain (as suggested by GAULE, OGATA, and BECHTEREFF) for from five to nine days (not more in any case) in 7 per cent, sublimate solution, and then putting the tissues for twenty-four hours into 50 per cent, alcohol, and for the same time into 70 per cent, and 96 per cent, alcohol successively. (This process produces artificial " pigment spots," similar to those produced by solution of Erlicki ; they may be dissolved out by prolonged treatment with warm water, or in five minutes by strong solution of LUGOL.) The tissues are of a good consistence for cutting. Chloride of zinc has been recommended for some purposes (see below, 710, 711). The next following paragraphs give in detail some methods of hardening recommended by some of the most competent workers. 705. BETZ'S Methods (Arch. f. mik. Anat., 1873, p. 101). The spinal cord, medulla oblongata, and pons Varolii are treated as follows : The dura mater is removed, and they are hung up in a cylinder containing 75 to 80 per cent, alcohol, to which is added enough iodine to produce a light brown coloration. After from one to three days the preparation will be found to be somewhat surface-hardened; it is taken down, and the pia NEUROLOGICAL METHODS. 393 mater and arachnoid are removed. If the pia mater does not come away completely enough the preparation is put back for some days into the alcoholic iodine. The membranes having been removed, the preparation is put back into the original fluid, which is found to have become colourless owing to ab- sorption of the iodine by the tissues. Fresh quantities of a strong solution of iodine in alcohol are from time to time added to the liquid in order to keep it at its original strength of iodine (as shown by the colour). If the membranes have been carefully removed, it will be found that after about six days the preparation ceases to take up further quantities of iodine. The preliminary hardening may now be considered complete. The preparation is now brought into a 3 per cent, solution of bichromate of potash. (A small weight is attached to it to prevent any portion of it from floating above the surface of the liquid. After a day or two it will have lost much of its alcohol, and will sink to the bottom of the vessel, which is equally undesirable ; this must be watched for, and the preparation hung up or otherwise supported.) The vessel is put away in a cool place. As soon as a brown turbidity is seen in the liquid, together with a brown deposit on the pre- paration, the hardening may be considered to be complete. The preparation must be at once washed with water, and put away until wanted in a 4 to 1 per cent, solution of bichromate. Cerebellum. Must be quite fresh, and before placing in the iodine the membranes and vessels must as far as possible he very carefully removed. (If the pia mater does not come away freely the organ must be macerated for a few hours in iodine solution in which other preparations have been kept, and which is diluted before using for this purpose.) The membranes having been removed, the cerebellum is placed (supported on cotton wool, with which the different organs are so propped up as to preserve their natural position) in solution of iodine for two or three days, and fresh iodine -olution frequently added. The pia mater is now removed from the rest of the pre- paration, which is put back for seven to fourteen days into the iodine solution. If at the expiration of this time it be found that the cerebellum can be supported on the finger 394 CHAPTER XXXI. by the vermiculus alone without bending, the preliminary hardening is complete, and it is brought into a 5 per cent, solution of bichromate, where it remains until fit for cutting. Cerebrum. The cerebrum is divided into two halves along the median line of the corpus callosum, and put into the iodine solution. After a few hours the pia mater is removed from the fissure of Sylvius and from the corpus callosum, and if possible the choroid plexus is removed likewise. The preparation is now put away in the iodine solution in a cool place (in summer in a cool cellar), and fresh iodine added as soon as the liquid is seen to lose colour (which must be watched for) . After twenty-four to forty-eight hours the remaining pia mater is carefully removed by means of scissors and forceps from the fissures and convolutions, and one half- volume of fresh iodine solution is added to the liquid. (To facilitate the penetration of the liquid, wads of cotton wool are stuffed into the fissure of Sylvius and between the " operculum" and the central lobe [" CcntraUappen"'], in the direction of the descending cornu, and between the convolutions.) After twenty-four to seventy-two hours the brain is brought into fresh solution of iodine in 70 per cent, alcohol, where it re- mains until the hemispheres are hard enough to be supported on two fingers without bending. (This will not be before ten to fourteen days.) It is then put into 4 per cent, solution of bichromate and left to acquire its definitive hardness. If an excessive brown deposit make its appearance, and the brain be found notwithstanding to be not hard enough for cutting, it must be rinsed with water and the bichromate solution changed. When ripe for cutting the brain ought to show an almost equal intensity of yellow-brown stain over the whole surface of a cut made through the total thickness of a hemisphere. Brains that are not fresh require for hardening longer time and stronger alcohol. The methods of Betz are particularly adapted to the harden- ing of voluminous specimens, and of tissues that are in a state of post-mortem softening. 706. Cerebrum (BEVAN LEWIS, The Human Brain, p. 102). Methylated spirit, twenty-four hours in a cool place. Miiller's solution, three days in a cool place. Then change the liquid; NEUROLOGICAL METHODS. 395 and after three days more change it again, or, preferably, substitute a 2 per cent, solution of potassium bichromate. At the end of the second week a solution of double the strength may be added ; and if at the termination of the third week the mass is still pliable, and of the consistence of ordinary rubber, it as yet unfit for section cutting, and the reagent should be replaced by a solution of chromic acid. 707. Brain (HAMILTON, Journ. of Anat. and Physiol., 1878, p. 254). Take a fresh brain and make a series of incisions into different parts, still keeping everything in situ ; or slice it into any number of segments about one inch thick, but of the whole length or breadth of the organ, as may be desired. Do not remove the membranes ; they form a protection for the superficial layers, and do not interfere with the hardening process. The large segments are placed flat in a large vessel | padded with cotton ; do not put them one above the other. I Cover them with the following fluid : Miiller's fluid ... 3 parts. Methylated spirit . . 1 part. | (Heat is evolved on mixing these liquids, and the mixture I must be allowed to cool before pouring it over the brain I tissue.) Pufc the preparations away in an ice-safe. Turn the segments over next day. Change the solution in a fortnight or three weeks ; or if on examining a section of one of the pieces it is found that the hardening reagent has penetrated I to the interior, they may be at once removed to the following ' mixture : Bichromate of ammonia . . 1 grm., Water . . . .400 c.c., in which they remain for one week. Then change the solu- tion to one of 1 per cent, for one "week ; and let this be followed by a solution of 2 per cent, for another week, or longer if required. The pieces will now be sufficiently hard for cutting; they may be kept permanently in solution of chloral hydrate, twelve grains to the ounce. Probably the chloral hydrate serves to attenuate the y.ellow coloration produced by the chromic liquids. This is a process particularly adapted to the preparation of large segments of brain. The consistence is very tough and firm. 396 CHAPTER XXXI. 708. Entire Encephalon (DEECKE, Journ. Roy. Hie. $oc.,1883, p. 449). To harden the entire brain so that the inside and the outside shall be hardened equally and properly, Dr. Deecke finally adopted bichromate of ammonia in 4 to 1 per cent, solution, according to the consistence of the brain. If it happens to be soft he adds say -^ to -^ per cent, of chromic acid to the solution, and always -i- to J of the whole volume of alcohol. It is then placed in a refrigerator and the fluid changed frequently. After a month add a little more alcohol from week to week until the alcohol is 90 per cent. This is changed as often as it is discoloured. The treatment requires from twelve to eighteen months. 709. Encephalon (M. DUVAL, Eosm's Journal de VAnatomie, 1876, p. 497). First Method. Place the fresh tissues in solution of bichromate of potash 25, water 1000; change the liquid after the first twenty-four hours, and again after three or four days, After two or three weeks place the prepara- tions in chromic acid of 3 per 1000, change the liquid every day for the first week, and after that every eight days until the middle of the second month, after which time it is no longer needful to change the liquid. The preparations must remain at least two months in the chromic acid ; the longer they remain in it the better. A few fragments of camphor should be added to the liquid in order to prevent the growth of mould. 710. Encephalon (FiSH, from an interesting paper on " Brain Preservation " in The Wilder Quarter -Century Book, 1893, p. 393). The following is said to be not ideal in its effects, but to answer the requirements of economy, fixation of the struc- j tural elements, differentiation of tissue, firmness of texture, and rapidity of action : Water 400 c.c. 95 per cent, alcohol . . 400 Glycerin . . . . 250 Zinc chloride .... 20 grms. Sodium chloride ... 20 Immerse in this, filling the cavities of the brain with it, and if practicable also injecting the blood-vessels with it, for XKUROLOGICAL METHODS. 397 about three days, then transfer for a week or more to a mix- ture of equal parts of the fluid and 70 per cent, alcohol, and finally store in 90 per cent, alcohol. 711. Formaldehyde. The liquids employed by STRONG and DORIG are given below in 765. See also 109. WEIGERT (Beitr. zur Kenntn. d. normalen menschlichen Neuroglia, 1895, quoted from Neurol. Centralb., 1895, p. 1146) puts portions of material of not more than half a centimetre in thickness for four days into a 4 per cent, solution of f ormol (by which is presumably meant commercial formol diluted with 9 volumes of water). MARCUS (quoted from FISH, see below) recommends harden- ing the spinal cord for two or four weeks in a ^ per cent, solution of formalin, then small pieces one half-centimetre thick are cut out and placed in Miiller's fluid for a week in an oven at 37 C. VAN GIESON (Anat. Anz., x, 1895, p. 494) states that he obtained good results by using " solutions of formalin of 4, 6, and 10 per cent./' followed by 95 per cent, alcohol. Myelin was found to be well preserved and to give, the characteristic blue reaction with Weigert's haematoxylin (the 1885 method), just as if a chrome salt were present. LACHI (cf. Zeit.f. wiss. Mik.,xii, 1895, p. 32) states that he has had good results with " 20 per cent, solutions of formol." FISH (Proc. Amer. Mic. Soc., xvii, 1895, p. 319) recommends the following formula : Water .... 2000 c.c. Commercial formalin . . 50 Sodium chloride . . .100 grms. Zinc chloride . . 15 Brains should be left in this mixture for a week or ten days or more, then transferred to a 2*5 per cent, solution of formalin (water 2000 c.c., formalin 50 c.c.), in which they may remain indefinitely if the jar be kept tightly covered. After an immersion of two weeks in these solutions a human brain lost only 6*8 per cent, of its weight ; but after an immersion for a similar period of eight days in 50 per cent, alcohol and eight days in 70 per cent, alcohol it was found to have lost 22 per cent, of its first weight. PARKER and FLOYD (Anat. Anzeiger, Bd. xi, 1895, No. 5, 398 CHAPTER XXXI. p. 156) find that a " 2 per cent, solution of formol," by which is meant a mixture of two volumes of forrnol with 98 of water, will harden a sheep's brain in a week or ten days in a \ satisfactory manner as regards consistency, bat with a marked increase of volume, which may amount to as much as 40 per cent. To obviate this they advise a mixture of Alcohol 95 per cent. . . .6 volumes, Forrnol 2 per cent, (the above mixture) 4 ,, which has the same excellent and rapid hardening qualities and gives only a hardly perceptible increase of volume. Brains may be kept for months in the mixture (ibid., 1896,; p. 568). 712. BEKKLEY, cortex of cerebellum of the dog (cf. Zeit. f\ wiss. Mik.j x, 3, 1893, p. 388). Liquid of Flemming, twenty- four to thirty-six hours ; absolute alcohol; celloidin. 713. Nervous Centres of Reptiles and Amphibia (MASON, Central Nervous System of Certain Reptiles, &c. ; WHITMAN'S Methods, p. 196). I Iodised alcohol, six to twelve hours ; 3 per cent, bichromate, with a piece of camphor in the bottle, and to be changed once a fortnight until the harden- ing is sufficient (six to ten weeks). BURCKHARDT (Das Centrdlnervensystem von Protopterus, Berlin, 1892 ; Zeit.f. wiss. Milt., ix, 3, 1893, p. 347) recommends a liquid composed of 300 parts of 1 per cent, chromic acid, 10 parts of 2 per cent, osmic acid, and 10 parts of concentrated nitric acid, in which brains of Protopterus are hardened in twenty-four to forty-eight hours. FISH (Journ. of MorphoL, x, 1, 1895, p. 234) employed for the encephalon of Desmognathus fusca a mixture of 100 c.c. of 50 per cent, alcohol, 5 c.c. of glacial acetic acid, 5 grins, of corrosive sublimate, and 1 grm. of picric acid, fixing for twelve to twenty-four hours, and passing through the usual alcohols. Imbedding and Cutting. 714. The Methods of Imbedding. The paraffin infiltration method can only be used for the smaller objects of this class. Human spinal cord (which is quite at the upper limit as re- gards size) can be properly penetrated with paraffin by taking the precaution of first cutting it up into slices of not more than a few millimetres preferably not more than one in thickness. The largest objects of this class, such as entire hemispheres of man, cannot be properly penetrated by any known imbedding mass ; and the anatomist must be content with simple superficial imbedding a proceeding which is ^ITY NEUROLOGICAL METHODS. 399 here of the greatest service. For intermediate objects those whose size varies between that of a small nut and a walnut it appears to me that no hesitation as to the proper course is possible; such objects should be treated by the collodion method, which is at once the safest, the most convenient, and the most advantageous as regards the ulterior treatment of sections. Imbedding is not a necessary process. Sections can be obtained from any part of the central nervous system without imbedding. The material should be very well hardened, and a suitable piece should be glued on to a piece of wood or cork by means of a rather thick solution of gum arabic. As soon as it begins to stick to the support the whole is thrown into 80 per cent, alcohol to harden the joint, after which it may be fixed in the object-holder of the microtome and cut. If the collodion method has been taken a difficulty may arise. It may be found that, notwithstanding every pre- caution, the collodion has not thoroughly penetrated the tissues. Good sections may, however, still be obtained by Duval's method of collodionising the sections. The cut sur- face of the tissue is dried by blowing on it, and is covered with a thin layer of collodion laid on it with a brush. As soon as this layer has somewhat dried, which happens very rapidly, a section is cut, and the cut surface collodionised as before, and so on for each section. This process gives very good results, and may be advantageously employed even with material that has been successfully imbedded, as it gives a better consistency to the tissue, and enables thinner sections to be obtained (VAN G-EHUCHTEN, in litt.). BEVAN LEWIS recommends the ether free/ing method for fresh brain. For hardened brain he recommends some of the old-fashioned wax-and-oil and other fatty mixtures, a doctrine at which I am surprised. HAMILTON recommends freezing in the gum and syrup mass given above, 178. He also recommends a method of penetrating with collodion, which is hardened in the usual way, the hardened mass being cut with an ice-and- salt freezing microtome (see Journ. of Anat. andPhysiol., 1887, p. 444; or Journ. Roy. Mic. Soc., 1888, p. 1051). GOODALL'S Rapid Method for preparing Spinal Cord (Brit. Med. Journ., May, 1893, p. 947 ; Journ. Roy. Mic. Soc., 1893, p. 405). Cut sections of fresh tissue with a freezing microtome ; float them on to water, and as soon as possible drain them and float them on to pyridin. After a quarter of an hour wash in water ; stain with 0'2o per cent, aqueous solution of anilin blue- 400 CHAPTER XXXI. black, followed by picro-carmine ; dehydrate and clear in pyridin ; mount in balsam thinned with pyridin. See also ante, 107. BENDA (Centralb.f. Allgem. Path, u. path. Anat., vi, 1895, p. 803) finds there is great advantage in using formaldehyde for removing the alcohol from hardened tissues before freezing. Small pieces of material should be left for from a quarter of an hour to several hours in 1 per cent, formal- dehyde solution (2J per cent, of commercial formalin), and washed in dis- tilled water and frozen therein. The tissues after thawing are not found to be brittle, as is the case with those that have not been treated with formaldehyde, but have a peculiar tough soapy consistency. For sections of entire human brain, DEECKE (1. c., 708) proceeds as fol- lows : The brain to be cut is placed upon the piston of the microtome (a Ranvier model) and held in situ by several pieces of soft cork. It is then imbedded in a cast of .paraffin, olive oil, and tallow, which, after it has become hard, is held in position by a number of small curved rods attached to, and projecting upwards from, the piston to the height of about an inch. Before cutting, and as it proceeds, the cast is carefully removed from around the specimen to the depth of about half an inch (which is easily done by the use of a good-sized carpenter's chisel), so that the knife never comes in con- tact with the cast. Cutting is done under alcohol, the entire microtome being immersed in a copper basin. The sections are floated, with the aid of a fine camel-hair brush, on to sheets of glazed writing-paper. They are removed thereon successively into staining, washing, and clearing fluids. After clearing, they are brought on the paper on to a slide, and the paper is gently pulled away from them; they are then mounted in chloroform- or benzol -balsam. It should be noted that the membranes should not be removed from the brain ; they present no obstacle to cutting if this is done with a slight sawing movement, or with a series of short cuts, instead of one sweep of the knife. By this plan the sections are much more perfect and uniform in thickness, and the loss in a series of from four to five hundred to the inch through the entire cerebrum of man may not amount to more than 2 or 3 per cent. OSBOEN (Proc. Acad. Nat. Sci. Philadelphia, 1883, p. 178, and 1884, p. 262: WHITMAN'S Methods, p. 195) found advantage in employing Euge's egg-mass (for the brain of Urodela). He recommends that the mass be injected into the ventricles. The DEJEEINES (Anat. des Centres Nerveux, t. i, 1895, p. 29) have found advantage in the employment of both collodion and paraifin at the same time, for cutting entire hemispheres. The preparation is thoroughly impregnated with the collodion, which after hardening should form a wall of a couple of centimetres in thickness all around it. On the lower surface of this wall of collodion, incisions of about one centimetre in depth are made, running in various directions. The whole is then plunged into the cylinder of a Grudden microtome containing melted paraffin, and oriented according to the direction in which it is desired to make the sections. It is not neces- sary that the preparation should be entirely covered by the paraffin ; it is sufficient that it should plunge therein for about one third of its depth. NEUROLOGICAL METHODS. 401 The paraffin penetrates into the cuts that have been made in the collodion, and after it has solidified is found to hold the preparation firmly. A very considerable quantity of paraffin is required for this operation, and as in drying it undergoes great shrinkage, it is well to wait for twenty-four hours before making the sections. FEIST (Zeit. f. wiss. Mile., viii, 4, 1892, p. 492) gives a useful hint for marking the right and left sides of spinal cord. He imbeds with each segment of the cord a small cylinder (of about 1 square millimetre in section) of hardened liver, stuck vertically in the imbedding mass (either celloidin or paraffin) against the side of the cord that it is desired to mark. CYTOLOGICAL METHODS. 715. General Cytological Methods. For the general prin- ciples of cytological research as applicable to nervous ele- ments the reader is referred to the chapter on " Cytological Methods/' The following paragraphs are of a more special nature. (a) Nerve-cells. 716. NISSL'S Fuchsin Method (Neurol. Centralb., quoted from MERCIER'S Coupes du Systeme Nerveux Central, 1894, p. 183). Perfectly fresh material is to be cut into pieces of not more than one cubic centimetre in size. These are put for two days into a " chromic solution in 70 per cent, alcohol " (strength not stated). They are then transferred for five days to absolute alcohol, and may then be sectioned. The sections are brought into a saturated solution of fuchsin contained in a deep watch-glass, only one section being treated at a time. The watch-glass is then warmed over a flame, care being taken not to overheat the preparation, until vapours begin to be given off from the liquid. Then the section is removed and plunged for one to two minutes into absolute alcohol. It is then got on to a slide and covered abundantly with clove oil, in which it remains until no more colour is given off. It is then drained, washed once with fresh clove oil, drained and mopped up, and mounted in balsam. Nuclei and the chief protoplasmic processes are demon- strated, as also neuroglia cells, but not myelin. 26 402 CHAPTER XXXI. 717. NISSL'S Methylen-blue Method (Neurol. Centralb.,1894, p. 508). Fresh, material is hardened in 96 per cent, alcohol, and sectioned. The sections are brought into a watch-glass with the following stain : Methylen blue (Methylenblau pat.) . 3' 75 pa.rts. Venice soap . . . 1*75 Distilled water . . . lOOO'O The watch-glass is warmed over a flame to about 65 to 70 C., till bubbles are given off which burst at the surface of the liquid. The sections are then brought into a mixture of ten parts of anilin oil with 90 parts of 96 per cent, alcohol, and are differentiated therein until colour is no longer given off from them. They are got on to a slide, dried with filter- paper, cleared with oil of cajeput, dried again with filter- paper, treated with a few drops of benzin, and mounted in benzin colophonium. Professor VAN G-EHUCHTEN, to whom I am indebted for this process, obtains the same results in a more commodious manner. Sections are cut by the paraffin method, and fixed on a slide by the distilled- water method (see 186). The paraffin is removed by means of xylol, the slide is passed rapidly first through absolute alcohol, then through 80 per cent, alcohol, and is put into a dish with Nissl's staining mixture. The whole is put for five or six hours into a stove kept at about 35 to 40 C. Differentiation is performed as in Nissl's process, and the preparation is mounted in xylol- damar. In this way a considerable series of sections can be treated at one time. 718. ROSIN'S Method (Neurol Centralbl, xii, 1893, p. 1 ; Zeit.f. wiss. Mile., xii, 1, 1895, p. 77) consists in staining with the Ehiiich-Biondi mixture. 719. The methods of REHM (Milnchener med. Wochenschr., 1892, No. 13 ; Zeit.f. wiss. Mik., ix, 3, 1893, p. 390) are as follows : Sections are stained for a few minutes in concentrated aqueous solution of Congo, washed in alcohol, and treated for ten minutes, until they become blue, with alcohol acidulated with hydrochloric or nitric acid, then cleared with origanum oil and mounted. A sharp axis-cylinder stain with considerable richness of other detail. But REHM prefers the following simple process: Sections of alcohol- hardened material are placed for one to two days in an aqueous solution of j hsematoxylin of 0'5 per cent, strength, then washed out in aqueous solution of carbonate of lithia (strength not given) until no more colour comes away NEUROLOGICAL METHODS. 403 from them, dehydrated and mounted. Axis-cylinders, cells, and processes, grey -black. The sections may be after-stained for a few minutes with Ol per cent, aqueous solution of Bismarck brown. (b) Nerve Fibres. 720. Structure of Medullated Nerve. In order to demonstrate the axis-cylinder and the sheath of Schwann, the myelin may be removed. This may be done by boiling in caustic soda, and then neutralising ; by boiling in a mixture of absolute alcohol and ether, and adding caustic soda; by boiling in glacial acetic acid; by boiling in fuming nitric acid, and adding caustic potash ; or by treating with eau de Javelle ; or (VAN GEHUCHTEN, in Hit.) the myelin may be extracted in the cold by leaving the nerves for some time in a mixture of alcohol and ether. 721. KUPFFEE'S Method (Sitzb. math. phys. Kl. ~k. Bayr. Akad. Wise., 1884, p. 446 ; Zeit.f. wiss. MiJt., 1885, p. 106). A nerve is stretched on a cork and treated for twenty-four hours with 0*5 per cent, osmic acid. It is then washed in water for two hours and stained for twenty-four to twenty- eight hours in saturated aqueous solution of Saurefuchsin ; after which it is washed out for from six to twelve hours (not more in any case) in absolute alcohol, cleared in clove oil, imbedded in paraffin, and cut. Sections are said to show the axis-cylinder as a bundle of fibrils (stained red) floating in an albuminous liquid. 722. Neuroceratin Structures (GALLI, Zeit.f. wiss. Mik.,ui,l, 1886, p. 467). An ischiatic nerve is excised, and fixed and hardened for eighteen to twenty minutes in solution of Miiller. Small portions of the nerve are then further treated for one or two days with solution of Miiller diluted with 2 parts of water, then for a quarter of an hour with glycerin contain- ing 1 or 2 drops of glacial acetic acid for each cubic centimetre, and finally (without previous washing with water) are stained for fifteen to twenty minutes in aqueous solution of China blue (the best China blue for this pur- pose is that supplied by the Badische Anilin- und Soda-Fabrik, at Stuttgart). They are washed out in alcohol, cleared in essence of turpentine, and mounted in dammar. For the results see the paper and the plate, 1. c. PLAINER (Zeit.f. wiss. Mik., vi, 2, 1889, p. 186) obtains a specific stain of the neuroceratin framework of medullated nerve in the following way: Small nerves are fixed and hardened for several days in a mixture of 1 part of Liq. Ferri Perchlor. (Ph. G., ed. 2) and 3 to 4 parts of water or alcohol. They are then to be washed out in water or alcohol till no traces of iron remain in them (the reaction of the washings with rhodanide of potassium being a good test of this), and are stained for several days or weeks in a con- centrated solution of " Echtgriin " (dinitrosoresorcin, not " dinitroresorcin," 404 CHAPTER XXXI. as erroneously in Platner's paper) in 75 per cent, alcohol ; after which they are dehydrated, imbedded, and sectioned. See also the papers of GEDOELST in La Cellule, iii, 1887, p. 117, and v,| 1889, p. 126 (good details of digestion methods) ; also the report in Zeit. f. wiss. Mik., vii, 1, 1890, p. 57. 723. Other Methods. KANVIER, Traite, p. 718, et. seq. ; KEZZONICO, Arch, per le Sci. Med., 1879, p. 237 ; TIZZONI, ibid., 1878, p. 4 (a process of boiling in chloroform for an hour or two, then staining and mounting in glycerin) ; BOVEEI, Zeit. f. wiss. Mik., iv, 1, 1887, p. 91 ; JAKIMOVITCH, Journ. de VAnat., xxiii, 1888, p. 142, or Zeit. f. wiss. Mik., v, 4, 1888, p. 526 (instructions for impregnating the axis-cylinder with silver, followed by reduction in formic acid and amyl alcohol) ; SCHIEFFERDECXER, in BEHRENS, KOSSEL, u. SCHIEFFERDECKER, Das Mikroskop, Bd. ii, p. 227 ; HUBER, Zeit. f. wiss. Mik., x, 3, 1893, p. 394 (stains with BENDA'S saf ranin and Lichtgriin) ; RABL, ibid., xi. 1, 1894, p. 42 (the lines of Frommana are artefacts due to the silver nitrate) ; FISCHEL, ibid., p. 48 (similar con- clusion) ; TIRELLI, ibid., xi, 3, 1894, p. 391; SEGALL, Journ. de I'Anat. xxix, 1893, p. 586. CHAPTER XXXII. NEUROLOGICAL METHODS NERVE-FIBRE STAINS (WEIGERT AND OTHERS). a. Myelin Stains. 724. Introduction. The most important of the methods for the study of tracts of medullated nerve-fibres are the haema- toxylin methods of WEIGERT. There have been in all three methods of WEIGERT ; the 1884 method, the 1885 method, and the 1891 method. The ordi- nary methods of staining with haematoxylin depend on the employment'of an aluminium lake of hsematoxylin. Weigert's method depends on the formation of another lake, a chromium or copper lake. In consequence of the formation of these lakes haematoxylin acquires the property of staining the myelin of nerves in a quite specific way. In Weigert's process the formation of these lakes takes place in the tissue itself. The details of the process have been considerably modified, both by other workers and by Weigert himself. The 1884 method (Fortschr. d. Med., 1884, f pp. 113, 190; Zeit.f. wiss. Mik., 1884, pp. 290, 564), which depends on the formation of a chrome lake, may be considered to be superseded. Not so the two others, which depend on the formation of a copper lake. 725. WEIGERT'S 1885 Method (Fortschr. d. Med., 1885, p. 136; Zeit.f. wiss. Mik., 1885, pp. 399, 484). The tissues are to be hardened in bichromate of potash (the solutions of Miiller or Erlicki will do as well, so far as I know). The hardening need only be carried to the point at which the tissues have acquired a brown, not a green coloration (but green tissues may be used, provided they have once passed through the brown stage). The preparation is then (but this is not necessary) imbedded by infiltration with celloidin, and the celloidin block fastened on cork and hardened in 406 CHAPTER XXXII. the usual way. The hardened block is put for one or two days into saturated solution of neutral acetate of copper diluted with one volume of water, the whole being kept at the temperature of an incubating stove. By this treatment the tissues become green, and the celloidin bluish green. The mordantage of the tissues is now terminated, and the pre- paration may be kept, till wanted for sectioning, in 80 per cent, alcohol. Sections are made with a knife wetted with alcohol, and are brought into a stain composed of Hsematoxylin . . . . O f 75 to 1 part. Alcohol . . . . . .10 parts. Water 90 Saturated solution of lithium car- bonate ...... 1 part. (A trace of any other alkali may be added in the place of lithium carbon- i ate. The object of adding a little of some base is to " ripen " the htema- : toxylin solution.) The sections remain in the stain for a length of time that varies according to the nature of the tissues : Spinal cord, two hours ; medullary layers of brain, two hours ; cortical layers, twenty-four hours. They are then rinsed with water, and brought into a de-j colourising solution composed of Borax ...... 2*0 parts. Ferricyanide of potassium . . 2*5 ,, Water 200" They remain in the solution until they are decoloured to the right degree that is, until complete differentiation of the nerves (half an hour to several hours) and are then rinsed with water, dehydrated with alcohol, and mounted in balsam. They may be previously stained, if desired, with alum-carmine for the demonstration of nuclei. For very difficult objects, such as pathological nerves, the decolouring solution should be diluted with water, and the immersion in it prolonged. GELPKE (Zeit.f. wiss. MiJc., 1885, p. 489) states that for transverse sections of atrophied nerves the solution should be diluted with fifty volumes of water, and the immersion be prolonged to twelve hours at the least ; for] longitudinal sections it should be diluted with ten volumes of water. The process is applicable to tissues that have been hardened in alcohol or in any other way, provided that they be put into a solution of a chromic NKUROLOGICAL METHODS. 407 salt until they become brown, before mordanting them in the copper solu- tion. As above stated, it is not necessary that the mordantage be done in bulk with tissues imbedded in celloidin. MAX FLESCH (Zeit.f. wiss. Mik., iii, 1, 1886, p. 50) finds that this practice is unfavorable to subsequent staining with other reagents than haematoxylin, and prefers (following LICHTHEIM) to make the sections first, bring them on closet-paper into the mordant, and after mordanting bring them on a spatula into 70 per cent, alcohol, and thence into the stain. In the process given above, a copper lake is formed in the tissues. In the earlier form of the process the mordantage with the copper salt was omitted, and the stain depended on the formation in the tissues of a chromic lake. The results were not quite so good, and the process may be taken to be superseded by the copper process. If very many large sections have to be prepared, and if the staining solu- tion be thrown away after using, the process may be found somewhat expensive. The following method for regenerating the staining solution is given by FANNY BERLINEEBLAU (Zeit.f. wiss. Mik., 1886, p. 50) : About 2'5 to 5 per cent, of baryta water is added to the used solution ; it is well shaken and allowed to stand for twenty-four hours ; carbonic acid (obtained from the action of crude hydrochloric acid on marble) is led through it, it is allowed to stand for twenty-four hours more, and then filtered. PANETH (ibid., 1887, p. 213) makes the stain with extract of logwood instead of pure haematoxylin. One part of commercial extract of logwood is dissolved in 90 parts of water and 10 of alcohol. To the filtered solution is added 8 drops of concentrated solution of lithium carbonate for each 100 c.c. Sections require from eighteen to twenty-four hours in the stain at the normal temperature. BREGLIA (ibid., vii, 2, 1890, p. 236 ; see also Journ. Roy. Mic. Soc., 1890, p. 817) stains with liquid extract of logwood or Pernambuco wood, prepared by extracting 7 to 10 grms. of the wood for five or six days with 100 c.c. of alcohol of 90 to 95 per cent. The results obtained by Weigert's method are most splendid. The blue-black nerves stand out with admirable boldness on a golden ground. The method is applicable to the study of peripheral nerves as well as to nerve-centres, and is likely to be of great utility in Vertebrate embryology. Nerve-tissue is not the only tissue stained by the process, which can be usefully applied to lymphatic glands and to skin (see SCHIEFFEKDECKER, in Anat. Anz., ii, 1887, p. 680). 726. WEIGERT'S 1891 Method (Deutsche med. Wochenschr., 42, 1891, p. 1184; Zeit. f. wi**. Mik., viii, 3, 1891, p. 392).- The material is to be hardened in bichromate and imbedded in celloidin in the usual way. The hardened blocks of cel- loidin are brought into a mixture of equal parts of a cold 0*+ + 408 CHAPTER XXXII. saturated solution of neutral acetate of copper and 10 per cent, aqueous solution of potassio-tartrate of sodium (C 4 H 4 O 6 KNa + 4H 3 0, salt of Seignette). They are left in the mixture for twenty-four hours in an incubator. (Large specimens [Pons] will require forty-eight hours, the mixture being changed for fresh at the end of twenty-four hours.) They are then brought for twenty-four hours into aqueous solution of neutral acetate of copper, either saturated or diluted with 1 volume of water, being kept as before in the incubator. They are then rinsed with water and brought into 80 per cent, alcohol, in which they may either remain till wanted or be cut after half an hour. The sections are stained for from four to twenty-four hours at the temperature of the room in a freshly prepared mixture of 9 vols. of (A) a mixture of 7 c.c. of saturated aqueous solu- tion of carbonate of lithium with 93 c.c. of water, and 1 vol. of (B) a solution of 1 grm. of hsematoxylin in 10 c.c. of alcohol (A and B maybe kept in stock, but A must not be too old). The sections must be loose ones, not such as have been seriated in celloidin, and must not be thicker than 0'025 mm. The stain is poured off and the sections are washed in several changes of water poured on to them. They are then treated with 90 per cent, alcohol followed by carbolic-acid-and-xylol mixture (for a short time only), or by a mixture of 2 parts of anilin oil with 1 of xylol, then pure xylol and xylol balsam (not chloroform balsam, which injures the stain). Medullated fibres dark blue on a light, sometimes rosy ground. If it be wished to have the ground particularly colourless, take instead of the second wash-water a mixture of -J- to J volume of common (not glacial) acetic acid with 100 volumes of water. Thick sections or series in celloidin require a special differentiation. They may be differentiated either with the above-mentioned acetic acid mixture, or in the usual borax-ferricyanide mixture diluted with water. In the latte7^aseTh^^r^uli3rwlirbe yellow. If the impregnation with the copper be imperfect (as, for instance, may happen if the treatment with the copper salt be performed at the normal temperature instead of in an in- cubator) some instructive differentiations of ganglion-cells may be obtained, the processes of the cells of Purkinje in the cerebellum, for instance, being very sharply brought out; NEUROLOGICAL METHODS. 409 but such preparations have a tendency to after-blackening, which does not happen with those that have been thoroughly impregnated with the copper. The advantages of the improved method are that differen- tiation after staining is not necessary; that the annoying precipitates formed on the surface of the preparations by the copper in the old method do not appear ; that the divers manipulations are simpler and easier ; the preparations are equal in beauty to those of Pal, and can be obtained with greater certainty. Since the first publication of this method, it has been dis- covered (WEIGEKT, Ergebnisse der Anat., iii, 1894, p. 21) that preparations made as above, without differentiation in the ferricyanide liquid, do not keep well. Weigert therefore now advises that they be mordanted as above with salt of Seignette, which has the advantage of preventing the formation of pre- cipitates on the surface of the preparations, but that they be also differentiated in the ferricyanide, as in the 1885 method. Modifications of Weigert' s Method. 727. PAL'S Method (Wien. med. Jahrb., 1886; Zeit. f. wiss. Mik., iv, 1, 1887, p. 92; Med. Jahrl., 1887, p. 589; Zeit. f. iL-iss. Mik., 1888, p. 88). This is a chrome-lake process. You proceed at first as in Weigert's process, but omitting the copper bath, and you stain as in Weigert's process. After staining in the haematoxylin solution the sections are washed in water (if they are not stained of a deep blue a trace of lithium carbonate must be added to the water). They are then brought for twenty to thirty seconds into 0'25 per cent, solution of permanganate of potash, rinsed in water, and brought into a decolouring solution composed of Acid. Oxalic, pur. . . . 1*0 Potassium Sulphite* (Kalium Sul- furosum [SO 3 K 2 ]) . . 1-0 Aq. Dest. . . . . . 20OO In a few seconds the grey substance of the sections is de- colourised, the white matter remaining blue. The sections should now be well washed out, and may be double-stained * Xot "sulphide," as erroneously given in MERCIEE'S Les Coupes du Systime Nerveux Central, p. 190. 410 CHAPTER XXXII. with Magdala red or eosin, or (better) with picro-carmine or acetic-acid-carraiiie. For further details as to the somewhat elaborate minutiae of the process see the papers quoted, or BEHRENS, KOSSEL, and SCHIEFFERDECKER'S Das Mikroskop, i, p. 199. PAL'S process gives more brilliant results than that of Weigert, the ground of the preparations being totally colour- less. But it has a defect ; it is less certain, or, to put it in another way, less easy to control. The differentiation is more energetic and rapid than is desirable. The whole pro- cess of differentiation only lasts some seconds; evidently, then, an error of judgment of only a few seconds may entirely vitiate the result. 728. KAISER'S Modification of Weigert (Neurol. Centrall;., xii, 1893, No. 11, pp. 364, 368; Zeit. f. iviss. Mih, xi, 2, 1894, p. 249; anterior methods suppressed). Put the material into liquid of Miiller. After two or three days divide it into slices of two to four millimetres in thickness, and put them back into the liquid of Muller for five or six days more. Then put them for eight days into liquid of Marchi ( 733). Wash, pass through alcohol, and imbed in celloidin. Make sections, and mordant them for five minutes in the following mixture : Liquor Ferri Sesquichlorati . 1 part. Aq. Dest. . . . . 1 Spirit. Rect. . . . .3 parts. Wash them in Weigert's haBinatoxylin, then warm them in a fresh quantity of the same (not to boiling-point) for a few minutes. Wash with water and differentiate in Pal's liquid. Neutralise the oxalic acid by washing in water containing a little ammonia. 729. KULTSCHITZKY (Anat. Anz., 1889, p. 223, and 1890, p. 519 ; Zeit.f. wiss. Mik., vi, 2, 1889, p. 196, and vii, 3, 1890, p. 367) has given two forms of his well-known process, of which the following is the later : Specimens are hardened for one or two months in solution of Erlicki, imbedded in celloidin or photoxylin, and cut. Sections are stained for from one to three hours, or as much as twenty-four, in a stain made by adding 1 grm. of hgematoxylin dissolved in a little alcohol to 100 c.c. of 2 per cent, acetic acid. They are washed out in saturated solution of carbonate of lithium or sodium. NEUROLOGICAL METHODS. 411 By adding to the carbonate of lithium solution 10 per cent, of a 1 per cent, solution of red prussiate of potash, and de- colourising therein for two or three hours or more, a finer differentiation is obtained. After this the sections are well washed in water and mounted in balsam. 730. WOLTEBS (Zeit. f. iviss. Mil: , vii, 4, 1891, p. 466) proceeds as Kult- schitzky, except that he stains in a solution kept warm by placing it on the top of a stove kept at 45 C. for twenty-four hours, after which time the sections are dipped in solution of Miiller, and differentiated by the method of Pal. KAES (ibid., viii, 3, 1891, p. 388 ; Neurol. Centralb., 1891, No. 15) modi- fies this by staining for as much as two or three days, and performing the differentiation several times over. It appears doubtful whether either of these modifications is an improvement. 731. BERKLEY'S Rapid Method (Neurol. Centralb., xi, 9. 1892, p. 270 ; Zeit.f.wiss. Mik., x, 3, 1893, p. 370). Slices of tissue of not more than two and a half millimetres in thickness are hardened for twenty-four to thirty hours in mixture of FLEMMING, at a temperature of 25 C. Without washing out, they are brought into absolute alcohol, which is changed twice during the first twenty-four hours. After sufficient hardening they are imbedded in celloidin and cut. After washing in water the sections are put overnight into a satu- rated solution of acetate of copper (or they may be simply warmed therein to 35 to 40 C. for half an hour). They are then washed, and stained for fifteen to twenty minutes in the fluid given below, warmed to 40 C., allowed to cool, and differentiated for one to three minutes in WEIGERT'S ferri- cyanide liquid, which may be diluted if desired with one third of water. "Water, alcohol, bergamot oil, xylol -balsam. The stain is made as follows : Two cubic centimetres of saturated solution of carbonate of lithia are added to 50 c.c. of boiling water, and the solution boiled for two minutes more, when \\ to 2 c.c. of 10 per cent, solution of haematoxylin in absolute alcohol are added. This method is most suited to fresh material, and does not give good results with tissues that have suffered post-mortem changes. 732. Other Modifications or Similar Methods. FLECHSIO (Arch. f. Anat. u. Phys., Phys. Abth., 1889, p. 537; Zeit.f. wise. Mile., vii, 1890, p. 71 ; Journ. Roy. Mic. Soc., 1890, p. 538 ; BBEGLIA, Zeit., vii, 2, 1890, 412 CHAPTER XXXII. p. 36 ; Eossi, ibid., vi, 2, 1889, p. 182 ; MERCIEE, ibid., vii, 4, 1891, p. 480 ; HAUG, ibid., vii, 2, 1890, p. 153 ; WALSEM, ibid., xi, 2, 1894, p. 236. Other Myelin Stains. 733. MARCHI'S Method for Degenerate Nerves (Rivista sperim. di Fren. e di Med. legate, 1887, p. 208 ; taken from the report by Schiefferdecker of a paper by EIJKMAN, in Zeit.f. wiss. Mik. } ix, 3, 1893, p. 350). Nerves are first hardened for a week in solution of Muller, and then put for a few days into a mixture of 2 parts solution of Miiller and 1 part 1 per cent, osmic acid solution. The treatment with the chrome salt deprives the medullary sheath of normal fibres of the faculty of im- pregnating with osmium, whilst the degeneration products in diseased sheaths retain that faculty. In consequence the sheaths in normal nerves acquire a yellow coloration, those of degenerated tracts a black one. For the study of degenerate nerve-tracts the method of MAECHI has an advantage over that of WEIGERT, in that it gives positive images of the degenerated elements, Weigert's process only giving negative ones. 734. AZOULAY'S Osmic Acid Method (Anat. Anz., x, 1, 1894, p. 25). An application of the osmic acid stain given 377. (A) Material that has been for several months in liquid of Miiller is washed for a couple of days in water, imbedded in celloidin, and sectioned. The sections washed in water are put for five to fifteen minutes into solution of osmic acid of 1 : 500 or 1 : 1000 strength. Rinse with water and put them for two to five minutes into a 5 or 10 per cent, solution of tannin, warming them therein over a flame till vapours are given off, or in a stove at 50 to 55 C. Wash for five minutes in water, double-stain if desired with carmine or eosin, and mount in balsam. Thin sections are necessary to ensure good results. If they should be too thick it will be necessary after staining to differentiate by PAL'S process, or by eau de Javelle diluted with 50 vols. of water. (B) Material that has been in an osmic mixture (liquid of Flem- ming, of Marchi, or of Golgi). Sections as before, then the tannin bath, warming for three to ten minutes, and the rest as before. 735. Osmic Acid (ExNER, Sitzb. It. Ahad. Wiss. Wien, 1881, Ixxxiii, 3 Abth. ; BEVAN LEWIS, The Human Brain, p. 105). A small portion of NEUROLOGICAL METHODS. 413 brain, not exceeding a cubic centimetre in size, is placed in ten times its volume of 1 per cent, osmic acid. The solution should be replaced by fresh after two days, a proceeding which may advantageously be repeated at the end of the fourth day. In from five to ten days the piece is usually hardened throughout, and may be washed with water, treated with alcohol, and imbedded. The sections may be treated by a drop of caustic ammonia, which clears up the general mass of the brain substance, leaving medullated fibres black. B. Lewis says that this method exhibits a wealthjof structure which no other method displays. The sections may be mounted in soluble glass. The chief value of this method is for tracing the course of medul- lated fibres. BELLOXCI (Arch. Hal. de Biol., vi, p. 44)5) employed this method in his researches on the optic nerve of mammalia. He used an osmic acid solution of 0'5 to 1 per cent., hardened for only fourteen to twenty hours, made sections, and treated them for three or four hours with 80 per cent, alcohol, and then with ammonia. 736. The methods of PALADINO (next section) and ZIEHEN ( 780) are also more or less specific myelin stains. b. Nyelin-and-axis-cylinder Stains. 737. PALADINO'S Iodide of Palladium Method (Bendic. R. Accad. Scienze Fis. e Mat., Xapoli, iv, 1890, p. 14, and 1891 [1892], p. 227; Zeit.f. wiss.Mik., vii, 2, 1890, p. 237, and ix, 2, 1892, p. 238 ; Journ Roy. Hie. Soc., 1890, p. 817, and 1892, p. 439). Pieces of material hardened in bichromate, chromic acid, or corrosive sublimate, and not more than 5 to 8 mm. in thickness, are put for two days into a large quantity (at least 150 to 200 c.c. for each piece) of 0*1 per cent, solution of chloride of palladium (which may be made as directed in 66). They are next put for twenty-four hours into a 1 per cent, solution of iodide of potassium (the later paper quoted says the iodide solution should be of 4 : 100 strength, and that a relatively small volume of it should be taken ; other- wise the iodide of palladium, which is rapidly formed in the tissues, may be again extracted by the liquid : small pieces of tissue should not remain in it for more than one or two hours). Dehydrate; imbed, if necessary, in paraffin by the chloroform method ; mount in balsam. A brown stain, being both a myelin stain and an axis- cylinder and cell-process stain, and applicable both to central and to peripheral nervous structures. It is very well spoken of. 738. SAHLI (Zeit.f. wiss. Mil:, 1885, p. 1) gives the following method : Sections of tissue hardened in bichromate to the degree required for 414 CHAPTER XXXII. Weigert's haematoxylin process are washed for not more than five or ten minutes in water, and stained for several hours, until they are of a dark blue colour, in concentrated aqueous solution of methylen blue. They are then rinsed with water, and stained for five minutes in saturated aqueous solution of Saurefuchsin. If now they be rinsed with alcohol and brought into a liberal quantity of water, the stain becomes differentiated, axis-cylinders being shown coloured red and the myelin sheaths blue. If, instead of rins- ing with pure alcohol, alcohol containing from O'l to 1 per cent, of caustic potash be taken, the stain differentiated in water, and the sections cleared with cedar oil and mounted in balsam dissolved in cedar oil, still finer images are obtained. Axis-cylinders are red as before, but the myelin sheaths are blue in some places, red in others. Sahli thinks that this reaction points to some difference of kind in the nerve-tubes that exhibit it. The same author (1. c., p. 50) also gives a method for obtaining a specific stain of nerve-tubes by means of methylen blue alone. Sections of material hardened as before are stained for a few minutes or hours in the following liquid : Water 40 parts. Saturated aqueous solution of methylen blue . 24 5 per cent, solution of borax . . . . 16 ,, (Mix, let stand a day, and filter.) The sections are then washed either in water or alcohol until the grey matter can be clearly distinguished from the white, are cleared with cedar oil, and mounted in balsam. Nerve-tubes are stained blue, ganglion-cells greenish, nuclei of neuroglia blue. Micrococci are stained, if any be present in the tissues. The preparations are not perfectly permanent. 739. Safranin followed by methylen blue gives a very special stain of spinal cord. The method is due to ADAMKIEWICS (8itzb. k. Akad. Wiss. Wien. Math. Naturw. Kl, 1884, p. 245 ; Zeit.f. wiss. Mik., 1884, p. 587). Sections (of material hardened in liquid of Miiller for not less than one month and not more than three) are washed first with water, then in water acidified with a little nitric acid, and stained in concentrated solution of safranin. They are then treated with alcohol and clove oil till no more colour comes away, and are brought back again into water, washed in water acidified with acetic acid, stained in methylen blue, and cleared as before. Myelin (" erythrophilous substance" of Adamkiewics) is red, nuclei of nerves, of neuroglia, and of vessels violet. The erythrophilous substance of pathological nerve-tubes does not take the stain, so that the method is valuable for the study of degenerative changes. 740. NIKIFOEOW (Zeit.f. wiss. Mik., v, 3, 1888, p. 338) has a modifica- tion of the foregoing method, which consists in impregnating with gold chloride or other metallic salt after the safranin stain. 741. Similar to the methods of ADAMKIEWICS are those of CIAGLINSKI (Zeit.f. wiss. Mik., viii, 1, 1891, p. 19) and of STEOEBE (ibid., x, 3, 1893, . p. 336), both of them employing safranin followed by anilin blue. For NISSL'S Congo-red method, see Miinchener med. Wochenschr., 1886, p. 528, or Zeit.f. iviss. Mik., iii, 3, 1886, p. 398. CHAPTER XXXIII. NEUROLOGICAL METHODS, AXIS-CYLINDER AND PROTOPLASM STAINS (GOLGI AND OTHERS). 742. Introduction. There are three chief methods for the study of axis-cylinders and protoplasmic nerve-cell processes, viz. the methylen blue method, the sublimate method of GOLGI, and the bichromate-of-silver method of GOLGI. The methylen blue method may be considered to be a staining method in the proper sense of the word, whereas the methods of Golgi are true impregnation methods. The methylen blue method having been given in Chap. XVII, it remains to group together here some other subordinate but useful methods that are also stains proper; after which will be given the methods of GOLGI and some other impregnation methods. c. Stains Proper. 743. Ammonia- carmine is old-fashioned, but may be used for general views. Beale's formula is a good one, especially where prolonged staining is required. The secret of success lies in staining very slowly in extremely dilute solutions. Bichromate material ought to be brought direct into the stain without passing through alcohol (see 94). Picro -car mine has much the same action as ammonia-carmine, but gives a better demonstration of non-nervous elements. Chromic objects stain very slowly in both these media. Sections may, however, be stained with them in a few minutes if they be put into a watch- glass with the stain, and the whole be kept on a wire net over a water-bath heated to boiling-point (OBEBSTEINEB). HENLE (Handb. d. Nervenlehre, 1871) gives the following, after MEEKEL. In order to do away with the slowness of staining of tissues hardened in chromates, sections should he placed in solution of chloride of palladium (1 in 300 to 1 in 600) till they are of a straw-colour (one or two minutes), rinsed in water, and stained in strong ammonia-carmine. Myelin, yellow ; axis-cylinders, nerve-cells, and neuroglia, deep red. Borax-carmine is chiefly useful when employed for double-staining with indigo-carmine or an anilin blue to follow. I have obtained some superb stains with Seller's borax-carmine and indigo-carmine process ( 339). MERKEL'S mixture of borax-carmine and indigo-carmine ( 340) has been 416 CHAPTER XXXIII. strongly recommended by MAX FLESCH (Zeit. f. wiss. Mik., 1884, p. 566, and 1885, p. 349), who says that it gives extremely rich and instructive images. Alum-carmine (Grenadier's or Csokor's) may be used as a nuclear stain (OBERSTEINER). The stain principally takes effect on non-nervous nuclei. See also SCHMAUS (Munch, med. Wochenschr., 1891, No. 8; see Zeit.f. wiss. Mik., viii, 2, 1891, p. 230, and Journ. Roy. Mic. Soc., 1892, p. 439) ; UPSON (Neurolog. Centralb., 1888, p. 319; Zeit.f. wiss. Mik., v, 4, 1888, p. 525) ; FEEEBOEN (Amer. Mon. Mic. Journ., 1888, p. 231 ; Journ. Roy. Mic. Soc., 1889, p. 305). 744. Anilin blue-black was first recommended by SANKEY (Quart. Journ. Mic. Sci., 1876, p. 69). He stained in a 0'5 per cent, solution, and, in order to obtain a differential stain, washed out for twenty to thirty minutes in solution of chloral hydrate. BEVAN LEWIS (Human Brain, p. 125) considers this to be one of the most valuable stains for nervous centres. He stains sections for an hour in O25 per cent, aqueous solution, and clears and mounts (in the case of brain or cord sections) ; for the cortex of the cerebellum he washes out for twenty to thirty minutes in 2 per cent, chloral solution. SANKEY and STIRLING have also used anilin blue-black in a much weaker solution, which Bevan Lewis does not recommend. YEJAS, however (Arch. f. Psychiatrie, xvi, p. 200), obtained good results by staining from eighteen to twenty-four hours in a solution of 1 in 3000. G-IERKE (Zeit. f. wiss. Mik., 1884, p. 379) was not able to obtain good results with anilin black procured in Germany, and finds that the treatment with chloral is injurious to the preservation of the tissues. MARTINOTTI (ibid., p. 478) comes to the same conclusion. LUYS (Gaz. med. de Paris, 1876, p. 346) greatly recommends the anilin colour known as Noir Colin. He stains for three to four minutes in a O'l per cent, solution. JELGERSMA (Zeit.f. wiss. Mik., 1886, p. 39) finds that anilin blue-black gives excellent results provided that the English preparation of the colour be alone employed. He makes solu- tions of 1 : 100, 1 : 800, and 1 : 2000, of which the first stains sections in a quarter of an hour, the second in five hours, the third in twelve hours. The stain takes effect on ganglion- cells and their processes, and on axis-cylinders, but does not demonstrate neuroglia or connective tissue. SCHMAUS (Munch, med. Wochenschr., No. 8, 1891, p. 147; NEUROLOGICAL METHODS. 417 frit./, wiss. Mik., viii, 2, 1891, p. 230) recommends English blue-black in 0*25 per cent, solution in 50 per cent, alcohol, with the addition of a little picric acid ; sections to be stained for an hour. The addition of picric acid has the advantage of leaving celloidin almost colourless, whilst pure aqueous solutions of blue-black stain it strongly. 745. MAKTINOTTI (1. c., 1884, p. 478) finds that picro- nigrosin gives very good results, especially for pathological objects. He stains for two or three hours or days in a saturated solution of nigrosin in saturated solution of picric acid in alcohol, and washes out in a mixture of 1 part of formic acid with 2 parts of alcohol until the grey matter appears clearly differentiated from the white to the naked eye. 746. ROSIN (Neurol. Centralb., xii, 1893, p. 1; Zeit.f. wiss. Mik., xii, 1, 1895, p. 77) recommends Ehrlich-Biondi mixture. LiNDSAY-JoHXSOX (in Hit.} adds to it about one third of a 20 per cent, (saturated) solution of nigrosin. 747. KAISEE (Zeit.f. wiss. Mik. t vi, 4, 1889, p. 471) advises, for celloidin sections of spinal cord, naphthylamin brown (obtainable from Griibler)- Sections are stained for a few hours in a solution containing 1 part of naphthylamin brown, 200 parts of water, and 100 parts of alcohol, washed with alcohol, cleared with origanum oil, and mounted. Chromophilous ganglion-cells, dark brown ; chromophobous cells, light on a dark ground. 748. REHM (Munch, med. Wochenschr., 1892, No. 13; Zeit.f. iciss. Mik., [ ix, 3, 1893, p. 389) gives the following: Sections (of alcohol-hardened material) stained for five minutes in 1 per cent, ammonia-carmine, and washed out in 70 per cent, alcohol acidified with 1 per cent, of nitric acid ; the acid removed by pure alcohol ; the sections stained for half a minute [ in 0*1 per cent, solution of methylen blue, differentiated in alcohol, cleared [ in origanum oil, and mounted in colophonium. Nuclei and axis-cylinders, [ red. This stain is said to prove that the nuclei of the ganglion-cells of the rabbit contain only one true nucleolus, instead of two or more, as has-been hitherto believed. 749. For a plasma-stain, REHM '(1. c.) gives a method modified from XISSL. Sections of alcohol-hardened material are stained for half a minute to a minute in a hot O'l per cent, solution of methylen blue, washed in 96 I per cent, alcohol till no more colour comes away, cleared with origanum oil, 1 and mounted in balsam or benzin-colophonium. Nerve-cells, dark blue ; I connective-tissue cells lighter, and greenish. 750. To obtain a sharper distinction between nerve-cells and connective- tissue cells, REHM (1. c.) stains as before in the hot methylen-blue solution 27 418 CHAPTER XXXIII. for not more than half a minute, and washes out as before with 96 per cent, alcohol. The sections are then stained for fifteen to thirty minutes in a O'l per cent, solution of f uchsin in 96 per cent, alcohol, washed out for a minute, until no more red colour comes away, in alcohol, cleared in clove oil, and mounted. Nerve-cells blue-red, their nuclei being unstained ; nuclei of connective tissue and of vessels, brilliant red. This distinction is not obtained in embryonic tissues. Nuclei of connective tissue and vessels may also be brought out by stain- ing for a few minutes in 1 per cent, aqueous solution of eosin, followed by a few minutes in warm O'l per cent, aqueous solution of dahlia, dehydration, and mounting (the nuclei blue, all else red). Or 1 per cent, aqueous solu- tion of nigrosin may be taken instead of the eosin, and O'l per cent, alcoholic solution of fuchsin (half an hour) instead of the dahlia (nuclei red, all else grey-blue). 751. For MALLOEY'S Phospho-molybdic Acid Haematoxylin Stain, see Anat. Anz., 1891, p. 375 ; Zeit.f. wiss. Mik., viii, 3, 1891, p. 341 ; MEECIEE, Les Coupes du Systeme nerveux Central, p. 228. SCHIEFFEEDECKEE and VOBIS find that celloidin sections of material hardened in Miiller stain well in it. 752. WOLTERS'S Chloride of Vanadium process for axis- cylinder and cell-staining is as follows (Zeit.f. wiss. Mik., vii, 4, 1891, p. 471): The material (either central or peripheral nervous tissue) is hardened in liquid O/KULTSCHITZKY, 53, followed by alcohol, as there described. It is imbedded either in celloidin or paraffin, and cut. The sections are mordanted for twenty- four hours in a mixture of 2 parts of 10 per cent, solution of chloride of vanadium and 3 parts of 3 per cent, solution of acetate of aluminium, washed for ten minutes in water, and stained for twenty-four hours in a solution of 2 grammes of haematoxylin (dissolved in a little alcohol) in 100 c.c. of 2 per cent, acetic acid. They are washed out until they are of a light blue-red colour (it is not possible to specify exactly the time required) in 80 per cent, alcohol acidulated with 0'5 per cent, of hydrochloric acid. Kemove the acid thoroughly by washing with pure alcohol, dehydrate, clear with origanum oil, and mount. A sharp axis-cylinder stain, myelin being coloured only if the differentiation in the acid alcohol is insufficient. 753. ALT (Munch, med. Wochenschr., 1892, No. 4; Zeit.f. wiss. Mik., ix, 1, 1892, p. 81) stains for a couple of hours in solution of Congo in abso- lute alcohol, and washes out with pure alcohol. The results are said to be specially adapted to the study of peripheral axis-cylinders. SCHIEFFEE- NEUROLOGICAL METHODS. 419 DECKEB, reporting on the method, does not recommend it. SQUIRE (using u '2 per cent, aqueous solution) says it is one of the best stains he has tried. d. Impregnations. 754. The Methods of GOLGI. There are two methods of GOLGT, viz. the Corrosive Sublimate Method, and the Bichromate and Nitrate of Silver Method. The corrosive sublimate method will be given later on. The bichromate and nitrate of silver method has been worked out by GOLGI in three forms. These are known as the slow process, the rapid process, and the mixed process.* The rapid process is the one that is the most in use at the present time for researches into the distribution and relations of axis-cylinders and protoplasmic processes ; it may be taken to be the classical method of inquiry into the finer relations of the neurons in hardened tissue. It may be said that it is to the study of hardened tissue that which the methylen blue method is to the study of fresh tissue. Before proceeding to describe these processes it is desir- able to indicate briefly the general characters of the impreg- nations obtained by them. The preparations have not in the least the appearance of stains, and are even very different in aspect from the impregnations obtained on fresh tissue by the ordinary methods of impregnating with nitrate of silver or chloride of gold. The impregnation is a partial one, by which is meant that of all the elements, whether nervous or not, that are present in a preparation, only a limited number are coloured. That is the peculiar quality not by any means the defect, but rather the advantage of the method. For if all the elements present were coloured equally with the great intensity with which they take the colour in this method, you would not be able to see the wood for the trees, in fact you would hardly be able to distinguish any detail at" all in the preparations. But Golgi's method selects from * In a recent text-book, the Leitfaden of RAWITZ, the sublimate method is called " the slow method of GOLGI," and the bichromate and silver nitrate method is given under the form of the slow process, and called " the rapid method of GOLGI." That is a very " nice derangement of epitaphs" indeed. RAWITZ further attributes the rapid method to RAMON T CAJAL, which is equally erroneous. Similar confusions are made by MEECIEB in his Coupes dn Xijstcme Nervevx Central. 420 CHAPTER XXXIII. among the elements present a small number which it stains with a great intensity and very completely, that is to say, throughout a great length, so that they are at the same time very clearly separated from those elements that have remained uncoloured, such as supporting cells and the like, and can be followed out for a great distance. There is no other method which will allow cell-processes to be followed out for such great distances. But the method does not demonstrate at the same time the histological detail of other tissues that may be present in the preparations, and all cytological detail is lost. It is par excellence a special method. Nervous tissue is not the only thing that is impregnated in these preparations ; indeed, the method has been applied with success to the study of such things as bile-capillaries, gland-ducts, and the like. Both on account of this character, and on account of the capriciousness with which the impreg- nation takes hold of only certain elements of the preparations, care must be exercised in the interpretation of the images obtained. It has been constantly taught in the chapter de- voted to gold impregnations in this book (see 366) that " the very best gold preparations give images that are only worthy of credence as to what they show, and furnish absolutely no evidence whatever as to the non-existence of anything that they do not show; for you can never be sure that the imbibi- tion of the salt has not capriciously failed, or its reduction capriciously stopped, at any point. " This warning applies with at least equal force to Golgi's impregnations. And in their case a further source of error is found in the fact that the method frequently gives precipitation-forms of chromate of silver that simulate dendrites and other structures (see the paper of FRIEDLAENDER in Zeit. f. wiss. Mik., xii, 2, 1895, p. 168, and the plate in the following number). A corre- spondent writes me that he has " Golgified a potato, and obtained beautiful nerve-fibres/' and FRIEDLAENDER'S paper describes similar results obtained with white of egg, &c. Clearly, then, caution is necessary in the interpretation of the images. It has been said that the method does not give good results with the tissues of invertebrates. It has, however, been applied with success by RAMON Y CAJAL to the study of the NEUROLOGICAL METHODS. 421 muscles of insects ; by VON LENHOSSEK to the nerves of Lumbricus ; by RETZIUS to the retina of Cephalopods ; and other anatomists have been successful with various objects. The details of the method have been considerably modified at the hands of various workers, the most important modifica- tion being that of the ft double " or " intensified " impreg- nation of RAMON Y CAJAL. The method has been described at length by GOLGI in the Archives Italiennes de Biologie, t. iv, p. 32, et seq. The follow- ing account is from the paper by GOLGI himself in the Archives de Biologie, t. vii, p. 15, et seq. The account given in former editions of this work represents an earlier form of the method, and should not be followed. 755. GOLGI'S Bichromate and Nitrate of Silver Method, SLOW Process (1. c., p. 17). (a) The hardening. This must be done in a bichromate solution. Either pure bichromate of potash may be employed or liquid of Miiller (the reaction can be obtained with liquid of Erlicki, but it is not to be recommended). The normal practice is to take bichromate of potash, beginning with a strength of 2 per cent., and changing this frequently for fresh solution of gradually increased strength, 2, 3, 4, and 5 per cent. The tissue to be operated on should be as fresh as possible ; though satisfactory results may be obtained from material taken twenty-four to forty-eight hours after death. It should be in pieces of not more than 1 c.cm. or 1J c.cm. in size. The most difficult point of the method consists in hitting off the exact degree of hardening in the bichromate that should be allowed before passing to the next stage of the process, the silver bath. The degree of hardening arrived at in any given time depends on so many factors (state of the tissues, temperature, &c.) that it is impossible to formulate rules in this respect. In summer good results may be obtained after fifteen to twenty days, and the material may continue in a favorable state for impregnation up to thirty, forty, or fifty days. In cold weather good results can seldom be obtained under a month ; when obtained, the material may continue to give good results up to two, three, and even four months of hardening. The only way to make sure is to pass trial portions of the tissue at intervals into the silver bath, in 422 CHAPTER XXXIII. summer frequently, in winter every eight or ten days, and observe whether the reaction is obtained. Good results are obtained by injecting the organs with the hardening fluid (2'5 per cent, bichromate). Stoving at a temperature of 20 to 25 C. is useful for abridging the hardening, but there is risk of over-hardening ; and G-OLGI thinks the results are never quite so delicate as after hardening in the cold. (6) Impregnation. As soon as the pieces of tissue have attained the proper degree of hardening in the bichromate, they are brought into a bath of nitrate of silver. The usual strength of this bath is 0*75 per cent., bat it is not necessary to hold rigorously to this strength : O50 per cent, may be taken, and even seems to be better for material that has not been quite enough hardened, and solutions of 1 per cent, may also be used and even seem to be better adapted for material that has been slightly over-hardened. A relatively large quantity of solution should be taken for the bath. For two or three pieces of tissue of 1 c.cm. each, GOLGI says that about "half a glassful of solu- tion" should be taken (that distinguished anatomist having evidently forgotten for the moment that glasses vary in capa- city from a liqueur-glass to a soda-water tumbler) . The moment the pieces of tissue are put into the silver bath, an abundant yellow precipitate of chromate of silver is formed. This of course weakens the bath pro tanto. It is therefore well, before putting the pieces into the final silver bath, to first wash them well in a weaker silver solution, until on being put into a fresh quantity of it no further precipitate is formed. Used solutions will do for this purpose. The final silver bath in general needs no further attention, unless it be that sometimes, in the case of tissues that have taken up a great deal of bichromate of potash, the solution may after six to ten hours become somewhat yellow, in which case it should be changed for fresh. It is not necessary to keep the preparations in the dark during the impregnation-bath; in winter it is well to keep them in a warm place. The time necessary for impregnation by the silver salt is from twenty-four to forty-eight hours. The normal time is from twenty-four to thirty hours, forty-eight being quite ex- NEUROLOGICAL METHODS. 423 ceptional. By this is meant that the reaction is not obtained in less time, but tissues may remain in the bath without hurt for days, weeks, or months. (c) Preservation. As soon as a trial has shown that a suffi- ciently satisfactory impregnation has been obtained, the pieces are brought into alcohol. The alcohol is changed two or three times, or even more, until it remains transparent even after the preparations have been two or three days in it. For in view of good preservation it is necessary that the ex- cess of nitrate of silver should be washed out from them thoroughly. Sections are now made. They are to be washed very thoroughly in three or four changes of absolute alcohol. They are then cleared, first in creosote, in which they should remain only a few minutes, then in oil of turpentine, in which they should remain for ten to fifteen minutes (they may re- main there for days without hurt). They are then mounted in Damar (rather than in balsam), and without a cover. Pre- parations mounted under covers in the usual way always go bad sooner or later, whilst those that are mounted without a cover keep very well, especially if they le kept in the dark. GOLGI states that he has a large number that have kept with- out change for nine years. The order in which the elements of tissues impregnate is first, axis cylinders, then ganglion cells, and, lastly, neu- roglia cells. 756. GOLGI'S Bichromate and Nitrate of Silver Method, RAPID Process (op. cit.,ip. 33). Small pieces of very fresh tissue are thrown into the following mixture : Bichromate solution of 2 to 2*5 per cent, strength, 8 parts. Osmic acid of 1 per cent, strength . . . 2 The hardening being much more rapid than with the slow process, the tissues will begin to be in a fit state for taking the silver impregnation from the second or third day; in the next following days they will be in a still more favor- able state, but the favorable moment does not last long, the faculty of impregnation soon declines, and is generally quite lost by the tenth or twelfth day. The silver impregnation is conducted exactly as in the slow 424 CHAPTER XXXIII. process, and sections are prepared and mounted in the same manner. There is this difference, that the impregnated material can- not be preserved for any length of time in alcohol, but must not remain for more than two days in it. But it may be kept until wanted for sectioning in the silver solution. This process has the advantage of great rapidity, and of sureness and delicacy of result, and is the one that has found the most favour with other workers. But for methodical study of any given part of the nervous system, GOLGI himself prefers the following. 757. G-OLQI'S Bichromate and Nitrate of Silver Method, MIXED Process (op. cit., p. 34) . Fresh pieces of tissue are put for periods varying from two to twenty-five or thirty days into the usual bichromate solution. Every two or three or four days some of them are passed on into the osmio-bichromate mixture of the rapid process, hardened therein for from three or four to eight or ten days, and finally impregnated with silver, and subsequently treated exactly as in the rapid process. The reasons for which Golgi prefers this process are the certainty of obtaining samples of the reaction in many stages of intensity, if a sufficient number of pieces of tissue have been operated on ; the advantage of having at one's disposition a notable time some twenty-five days during which the tissues are in a fit state for taking the silver, and the possi- bility of greatly hastening the process whenever desired by simply bringing the pieces over at once into the osinic mix- ture; lastly, a still greater delicacy of result, especially remarkable in the demonstration of the " functional" or nervous process of nerve-cells. 758. Critique of GOLGI'S Method. The above-described methods have been found extremely valuable in the most various departments of nervous anatomy. They have given brilliant results in the study of peripheral nerves and their origins or terminations, and in the study of the relations of fibres and cells in the central nervous system. It has been found, at the same time, that they have the defect of con- siderable uncertainty in the production of the desired reac- NEUROLOGICAL METHODS. 425 tion, and in the preservation of the stain. These defects have given rise to a most elaborate discussion, which unhappily has not as yet led to very satisfactory results. Golgi's method is apparently (but this is by no means certain) a double decomposition method, based on the com- bination of the bichrofnate of potassium in the tissues with the silver nitrate, and formation of a brown precipitate of bi- chromate of silver (K 2 Cr 2 7 + 2AgN0 3 =Ag,Cr 2 7 +2KNO ; the precipitate is brown by reflected light, but appears black by transmitted light) . The problem is to preserve this pre- cipitate in the tissues free from chemical or molecular change. And the problem is not an easy one ; without special precau- tions the preparations will not resist the processes necessary for imbedding, will not always resist those necessary for merely mounting in balsam, and even then may easily " go bad " after they have been mounted for a short time. Modifications concerning the Impregnation of the Tissues. 759. RAMON Y CAJAL, who has done a great deal of im- portant work by Golgi's method, has always used the rapid process. For the times and strengths used by him in his researches on the cerebral cortex of mammals, see his paper in La Cellule, vii, 1891, p. 125, or Zeit. /. iviss. Mik., ix, 2, 1892, p. 239; also Journ. Roy. Hie. Soc., 1892, p. 154. He found it useful to adopt Sehrwald's gelatin process (infra, section 766) for avoidance of peripheral precipitates. He prefers not to adopt Greppin's treatment with hydrobromic acid, nor Obregia's treatment with gold chloride, finding that although they serve to render the preparations per- manent, they obscure the finer relations of fibres. iFor embryos of the fowl he employs the same process ; see his paper in Anat. Anz., v, 1890, p. 85, or Zeit. f. viiss. Mik., vii, 2, 1890, p. 235. 760. RAMON Y CAJAI/S Double-Impregnation Process. In a paper on the structure and relations of the sympathetic gan- glia (which I have not seen, and quote from Zeit. f. wiss. Mik., 1. c.) RAMON Y CAJAL describes a process of " intensified " or " double " impregnation. After hardening for three days (embryos of fowl) in the osmium-bichromate mixture, the preparations are put for thirty-six hours into nitrate of silver 426 CHAPTER XXXIII. solution (O5 to O75 per cent.) They are then brought back into the same osmium-bichromate mixture, or into a weaker one containing only 2 parts of osmic acid solution to 20 of the bichromate. After treatment with this they are washed quickly with distilled water, and put for a second time into the silver solution for thirty-six to forty-eight hours. It is important to hit off the proper duration of the first impreg- nation in the bichromate. If it has been too long (four days) or too short (one day), the second impregnation will not succeed. In this case a third impregnation must be resorted to, the objects being again treated with the weak osmium- bichromate mixture, and afterwards again with the silver solution. This modification of the original process is, perhaps, the most important that has hitherto been made. 761. KALLIUS (Anat. Hefte, x, 1894, p. 527; Zeit.f. wiss. Mile., xi, 2, 1894, p. 154) states that he has often found it advantageous to employ bichromate of ammonia or of soda instead of the bichromate of potash, and to perform all the reactions in the dark. Preparations made by the ammonia or soda salt rarely require a double impregnation. 762. SMIBNOW (Intern. Monattsschr., x, 2, 1893, p. 241; Zeit.f. wiss. Mik., x, 2, 1893, p. 254), for the study of nervee ndings in the human skin, performs the hardening in ALTM ANN'S mixture (equal parts of 5 per cent, bichromate of potash and 2 per cent, osmic acid), leaving the pieces therein for 5 to 10 days, and impregnating with 1 per cent, solution of nitrate of silver. For nerve endings in the skin of the earthworm, he hardens (Anat. Anz., ix, 9, 1894, p. 571) in a mixture of equal parts of 5 per cent, bichromate and 1 per cent, osmic acid, for from 5 to 28 days. 763. Reviving Over-Hardened Tissues. Tissues that have been too long (three to four weeks) in the osmium-bichromate mixture will no longer take on the silver impregnation, as has been explained above. They can, however, be re- vivified, and made to impregnate, in the following manner, due to GOLGI, and published by SACEEDOTTI (Intern. Monatsschr., xi, 1894, 6, p. 326; Zeit. f. iviss. Milt., xi, 3, 1894, p. 389). They are washed in a half-saturated solution of acetate of copper until they no longer give a precipitate, and are then put back again for five or six days into the osmium-bichromate mixture. Sections of the impregnated material give remarkably fine images, and will bear mounting in thickened oil of cedar under a cover. NEUROLOGICAL MKTHODS. 427 764. BOEHM, and afterwards OPPEL (Anal. Anz., v, 1890, p. 143, and vi, 1891, p. 165; Zeit.f. wiss. Mik., vii, 2, 1890, p. 222, and viii, 2, 1891, ]>. -'2 1) have modified the hardening part of the process by taking instead of bichromate of potash (slow process) the one, an 0'5 per cent, solution of chromic acid (forty-eight hours), the other, an 05 per cent, solution of neutral chromate of potash (twenty-four hours, the pieces having been pre- viously hardened in alcohol). This is for liver. 765. Formaldehyde Mixtures. STRONG (Anat. Anz., x, 15, 1895, p. 494) states that formaldehyde can with advantage be substituted for the osmic acid in the osmio-bichromic mixture of GOLGI'S impregnation process. He adds from 2*5 to 5 per cent, of " formaline " to the 3'5 to 5 per cent, bichromate solution. The advantage of using formaldehyde instead of osmic acid is stated by STRONG to be that the stage of hardening favor- able for impregnation lasts longer; in other words, the formaldehyde-bichromate does not over-harden. DURIG (ibid., p. 659) makes the same recommendation. He obtained the best results by means of 3 per cent, bichromate solutions containing 4 to 6 per cent, of formaldehyde (if I understand rightly). Durig hardens therein for three days, then performs double impregnation by Ramon y Cajal's process. FISH (Proc. Amer. Mic. Soc., xvii, 1895, p. 319) has also obtained good results with the following mixtures for the Golgi process : Formalin ...... 2 c.c. 3 per cent, bichromate . . . 100 leaving the tissues three days in this liquid and three days in the silver nitrate (f per cent.). Or, with advantage : Liquid of Miiller . . . .100 c.c. 10 per cent, formalin . . . 2 1 per cent, osmic acid . . . 1 ,, The formalin and bichromate mixtures should be kept in the dark. It is well only to make them up at the instant of using them. KOPSCB (Anat. Anz., xi, 1896, p. 727) states that he has obtained good impregnations with a mixture of 4 parts of 3'5 per cent, bichromate solution, and one of commercial 428 CHAPTER XXXIII. formaldehyde solution. He considers the results more certain than with the osmic acid mixture. VAN GEHUCHTEN (in litt.) has tried the substitution of formaldehyde for the osmic acid in the Golgi process, and has not obtained good results. 766. SEHRWALD'S Gelatin Process (Zeit. f. wiss. Mik., vi, 4, 1889, p. 456). One of the annoyances of Golgi's process is that it fre- quently gives rise to the formation at the surface of the preparations of voluminous precipitates that are destructive of the clearness of the images. SEHEWALD finds that this evil can be avoided by putting the tissues into gelatin solution before bringing them into the silver-bath. A 10 per cent, solution of gelatin in water may be made. The tissues are imbedded in this in a paper imbedding box with the aid of a little heat (the gelatin melting at a sufficiently low tem- perature), and are brought therein into the silver-bath. After the silvering the gelatin is removed by warm water saturated with chromate of silver. MARTINOTTI wraps the tissues simply in blotting-paper. Modifications concerning the Preservation of tlie Preparations. 767. SEHRWALD, in an elaborate paper (Zeit. f. wiss. Mik., vi, 4, 1889, p. 443), develops the theory that the deterioration of the preparations is due to solution of the precipitate in the reagents employed in the ulterior processes of preparation in water, in chlorides (which may be present in alcohol), in the xylol, in the paraffin, in the very balsam itself. He made elaborate attempts (fruitless, but instructive) to turn the diffi- culty by transforming the soluble chromic salt of silver into some insoluble compound. He tried to replace the acid of the salt; he tried to replace the metal of the salt ; he tried to make a sulphide of it ; he tried to reduce it to the metallic state with photographic developing solutions (and had some success with hydroquinone, but does not recommend the pro- cess on account of its giving rise to deceptive precipitates that simulate organised elements). None of these experi- ments gave the desired result ; and the upshot of the paper is that it is better not to attempt to modify the state of the pre- NEUROLOGICAL METHODS. 429 cipitate, but rather so to modify the preparation liquids as to reduce their solvent action on the precipitate. This may be done by supersaturating them with bichromate of silver before using. The powdered salt should be added to each of them in excess, including the clearing media, the medium used for fixing the sections to the slide, the paraffin itself, the solvent used for removing it, and the balsam, and made to dissolve by the aid of heat. By this means thin sections may be pre- pared and mounted in balsam without injury to the stain. Sehrwald does not say how long they will keep. 768. SAMASSA (op. cit., vii, 1, 1890, p. 26) points out that bichromate of silver is not soluble either in absolute alcohol, toluol, xylol, paraffin, or Canada balsam, nor otherwise chemi- cally affected by them ; and that therefore Sehrwald's ex- planation is untenable. He thinks that the true explanation of the deterioration of the preparations is that the precipitate forming the impregnation is little by little floated away from the tissues by the mechanical force of the diffusion currents set up on the passage of the preparations through the dif- ferent reagents, and particularly those long-continued ones caused by the slow drying of balsam under a cover-glass. He recommends, therefore, simply that the preparations be preserved without a cover, as directed by GOLGI. 769. FICK (Zeit. f. iviss. Nik., viii, 2, 1891, p. 168) does not admit Samassa's theory of the deterioration of sections through diffusion currents (supra). He points out that bichromate of silver is soluble in water, especially with the aid of heat ; and after an elaborate series of experiments, concludes that the water of the reagents or damp confined by the cover-glass is the cause of the ruin of the preparations. Watery fluids should be avoided, and sections should be mounted without a cover, or on a cover raised free of contact with the slide by means of wax feet, or the like. Or sections mounted without a cover may be later on provided with one if the balsam be first rendered perfectly anhydrous by careful heating. This last method is also recommended by HUBER (Anat. Anz., vii, 1892, p. 587; Journ. Roy. NIC. Soc., 1892, p. 707; Zeit.f. wiss. Nik., ix, 4, 1893, p. 479). The reader will of course understand that the balsam is to be heated on the 430 CHAPTER XXXIII. slide, with the section in it, until it immediately sets hard on cooling. 770. GBEPPIN (Arch. f. Anat. u. Entw.,A.nat. Abth., 1889, Supp., p. 55 ; Zeit.f. wiss. MiTc., vii, 1, 1890, p. 66) finds that by means of hydrobromic acid, suggested by NEUMANN, preparations made by the slow method may be rendered sufficiently resistent to bear mounting under a cover. After silvering, sections are made with a freezing microtome and treated for thirty to forty seconds with 10 per cent, solution of hydrobromic acid, and may then be well washed in several changes of water and mounted in the usual way. If they be cleared in clove oil and exposed therein to sunlight for ten or fifteen minutes, they will take on a violet tone, and details will be more strongly brought out. It is sometimes well to treat them, after the 10 per cent, acid, with 40 per cent, acid for twenty to thirty seconds. They may also be treated by Pal's modification of Weigert's hsematoxylin process. 771. OBKEGIA ( Virckow's Archiv, cxxii, 1890, p. 387 ; Zeit. f. wiss. Mik., viii, 1, 1891, p. 97 ; Journ. Roy. Hie. Soc., 1891, pp. 536, 830; Amer. Mon. Micr. Journ. , 1891, p. 210) gives the following. Sections of silvered material are made, either without imbedding or after imbedding either in paraffin or celloidin, care being taken in either case not to use alcohol of a lower grade than 94 or 95 per cent. They are brought from absolute alcohol into a mixture of eight to ten drops of 1 per cent, solution of gold chloride with 10 c.c. of absolute alcohol, which should be prepared half an hour beforehand and exposed to diffused light until the sections are placed in it, when it should be put into the dark. After fifteen to thirty minutes therein, according to their thickness, the sections are quickly washed in 50 per cent, alcohol, then in water, then treated for five or ten minutes with 10 per cent, solution of hyposulphite of soda. They are lastly washed well with water, and may then be mounted at once in balsam under a cover, or if desired may be previously stained with carmine or hsematoxylin, or Pal's modification of Weigert's process, or the like. Obregia thinks that the reason why Sehrwald did not succeed in substituting gold for the silver in his preparations (vide supra) is that he took the gold salt in aqueous solution. This method is also applicable to material treated by Grolgi's sublimate process, 774. 772. SALA (Zeit. f. wiss. ZooL, lii, 1, 1891, p. 18; Zeit. f. NEUROLOGICAL METHODS. 431 wiss. Mik.j viii, 3, p. 389), in a paper written in Golgi's labora- tory, finds Greppin's hydrobromic acid variation not merely useless, but hurtful. And he thinks that Sehrwald's process for imbedding the material in paraffin with the object of get- ting very thin sections is a mistake. The chief quality of Golgi's process is that it admits of the following of nerve-cell processes for a very great distance. Evidently this cannot be done with very thin sections. It is better simply to wash the preparations taken from the silver-bath with water, fix: them to a cork with gum, put the whole into alcohol for a few hours to harden the gum, and cut with a microtome without imbedding. 773. KALLIUS (Anai. Hefte, ii, 1892, p. 269 ; Zeit.f. wiss. Mik., ix, 4, 1893, p. 477) has worked out the following process. Take 20 c.c. commercial hydroquinone developing solution and 230 c.c. distilled water (the hydro- quinone solution may be made up with 5 grms. hydroquinone, 40 grms. sodium sulphite, 75 grms. carbonate of potassium, and 250 grms. distilled water). At the instant of using, further dilute the solution with one third to one half its volume of absolute alcohol, and put the sections into it for several minutes ; they become dark grey to black. In order to ascertain whether reduction is complete, throw a section into a solution of hypo- sulphite of soda (about 10 parts to 50 of water) : chromate of silver will quickly dissolve, whilst metallic silver will not be attacked. As soon as re- duction is complete the sections are put for ten to fifteen minutes into 70 per cent, alcohol, then brought for five minutes into the above-given solu- tion of hyposulphite of soda, and thence into a large quantity of distilled water, where they should remain for twenty-four hours or more. Lastly, dehydrate in the usual way, and mount under a cover. All the details which in the undeveloped preparations were brown are now black on a light ground. After-staining with carmine, &c., may be employed. Other developers were tried, and gave satisfactory reductions, but they caused a red or brownish discoloration of the preparations. 774. GOLGI'S Bichromate and Sublimate Method (Archivio per le Scienze Mediche, 1878, p. 3; Archives italiennes de Biologie y t. iv, p. 32; Arch, de BioL, t. vii, p. 35). This method, which may be said to be in principle identical with the bi- chromate of potash and silver nitrate method of the author, consists, like the latter, of two processes : 1, hardening in bichromate ; 2, treatment with bichloride of mercury. For hardening, use either a solution progressively raised in concentration from 1 per cent, to 2^ per cent., or Miiller's solution. It is best to take small pieces of tissue (not more than 1 to 2 c.c.), large quantities of liquid, and change the 432 CHAPTER XXXIII. latter frequently, so as to have it always clear. But the re- action can be obtained with much larger pieces, even entire hemispheres. In this case the brain should at first be treated by repeated injections of the liquid, so as to ensure as rapid a permeation of the interior as possible. Fifteen to twenty days' immersion will suffice, or even six to eight, but twenty to thirty should be preferred, and an immersion of several months is not injurious. The tissues when hardened are passed direct from the bi- chromate into the bichloride of mercury. The solutions of the latter, first employed by Golgi, varied from O25 per cent, to O50 or 1 per cent.; he now always takes 0'50 per cent. The immersion in the bichloride must be much longer than the immersion in the nitrate of silver bath of that process : for the latter, twenty-four to forty-eight hours suffice ; but in the bichloride an immersion of eight to ten days is necessary in order to obtain a complete reaction through the whole thickness of the tissues, or for entire hemispheres two months or more. During the bath the bichromate will diffuse out from the tissues into the bichloride, which must at first be changed every day, and later on as often as it becomes yellow. At the end of the reaction the preparations will be found decolourised, and offering the aspect of fresh tissue. They may be left in the bichloride for any time. In Rendiconti R. 1st. Lombardo di Sci. Milano, 2, xxiv, 1891, pp. 594, 656 (see Zeit. /. wiss. Nik., viii, 3, 1891, p. 388) Golgi says that for the study of the diffuse nervous reticulum of the central nervous system the best results are obtained by keeping the preparations in 1 per cent, sub- limate for a very long time, two years being not too much in some cases. The reaction may be said to have begun by the time the tissues are nearly decolourised. From that time onwards sections may be made day by day and examined, and those which it is desired to preserve may be mounted. Before mounting, the sections that have been cut must be repeatedly washed with water (if it be wished to mount them permanently), otherwise they will be spoilt by the formation of a black precipitate. In the last place quoted Golgi says that after washing they may be toned by putting them for a few minutes into a photographic fixing-and-toning bath, after which it is well to wash them again, and stain them with NEUROLOGICAL METHODS. 433 some acid carmine solution. Mount in balsam or glycerin ; the latter seems the better preservative medium. The result of this process is not a true stain, but an " appa- rently black reaction," the tissues appearing black by trans- mitted light, white by reflected light. Golgi thinks that there is formed in the tissue elements a precipitate of some substance that renders them opaque. The elements acted on are (1) the ganglion- cells, with all their processes and rami- fications of the processes. These are made more evident than by any other process except the bichromate and silver-nitrate process. An advantage of the mercury process is that it demonstrates nuclei, which is not the case with the silver process. (2) Connective-tissue corpuscles in their charac- teristic radiate form. But the reaction in this case is far less precise and complete than that obtained by the silver process. (3) The blood-vessels, and particularly their muscular fibre cells. The method gives good results only with the cortex of the cerebral convolutions, hardly any results at all with the spinal cord, and very scanty results with the cerebellum. And, on the whole, the method shows nothing more than can be demonstrated by the silver-nitrate method, but it is superior to it as regards two points : the reaction can always be obtained with perfect certainty in a certain time, and the preparations can be perfectly preserved by the usual methods. But Golgi holds (Arch, de BioL, t. vii, p. 41), that the method deserves an honorable place amongst neurological methods, by the side of the silver-nitrate methods. See also FLATATT, in Arch.f. mik. Anat., xlv, 1895, p. 158 ; Zeit. f. wiss. Mik., xii, 2, 1895, p. 257. The method is recommended by BLOCHMAXN (BioL Centralbl., xv, 1895, p. 14; Zeit.f. wiss. Mik., xii, 2, 1895, p. 226) for the nervous system of Cestodes. Modifications of Golgi's Bichromate and Sublimate Method. 774a. TAL (Gazz. degli Ospitali, 1886, No. 68) finds that if sections made by this process be treated with solution of sodium sulphide, a much darker stain is obtained. Sections may then advantageously be double-stained with Magdala red. Golgi's method may be combined with Weigert's nerve stain (see PAL, Wien. med. Jahrb., 1886; Zeit.f. wiss. Mik., v, 1, 1887, p. 93). 28 434 CHAPTER XXXIII. 775. Cox (Arch. f. mik. Anat., xxxvii, 1891, p. 16 ; Journ. Roy. Hie. Soc., 1891, p. 420) finds the sublimate and bichromate may be used together, and give a uniform impregnation. He used a fluid consisting of 20 parts 5 per cent, bichromate, 20 parts 5 per cent, sublimate, 16 parts 5 per cent, simple chromate of potash, and 30 to 40 parts of water. The mixture should be as little acid as possible. The duration of the impregnation is from two to three months. There is considerable difficulty in preserving sections, which must be made with a freezing microtome, alcohol being avoided, treated with solution of sodium carbonate, and mounted without a cover. 776. MAGINI (Boll. Accad. Med. di Roma, 1886 ; Zeit.f. wiss. Mik., 1888, p. 87) recommends a development of Golgi's process in which zinc chloride is used in place of sublimate. Portions of tissue of 2 to 3 cm. cube are hardened for at least two or three months in Miiller's solution. They are well washed with distilled water, and brought into a O'l to 1 per cent, solu- tion of chloride of zinc. This is changed for fresh every day for seven to ten days (until it does not become yellower than bichromate solution). Sec- tions are then made, washed quickly with alcohol, imperfectly cleared with kreasote, and mounted in dammar. This process is said to demonstrate better than Golgi's the finer structure of ganglion-cells and their processes. 777. FLECHSIG'S modifications, see Arch. f. Anat. u. Phijs., Physiol. Abth., 1889, p. 537 ; Zeit.f. wiss. MiJc., vii, 1, 1890, p. 71. 778. MONTI (Atti d. R. Accad. del Lincei Roma, Kendic., v, 1889, 1 sem., p. 705 ; Zeit. f. wiss. Mik., vii, 1, 1890, p. 72) describes a brown impregna- tion brought about by the combined action of bichromate of potash and sul- phate of copper. The method does not appear to have been yet brought to a practical form. 779. ZIEHEN'S gold and sublimate method (Neurol. Centralb., x, 1891, No. 3, p. 65 ; Zeit.f. wiss. Mik., viii, 3, 1891, p. 385). Ziehen gives the following derivative of Grolgi's sublimate method as an improvement on it : Small pieces of fresh material are thrown into a large quantity of a mixture of 1 per cent, sublimate solution and 1 per cent, chloride of gold solu- tion in equal parts. They remain therein for at least three weeks, preferably for several months (up to five), by which time they will have become of a metallic red-brown colour. They are gummed on cork and sectioned without imbedding. The sections are treated either with Lugol's solution ( 73) diluted with four volumes of water, or with dilute tincture of iodine, until duly differentiated, which will require more or less time according to the thickness of the sections. They are then washed and mounted in balsam. The result is a bluish-grey impregnation; both medullated and non-medul- NEUROLOGICAL METHODS. 435 lated nerve-fibres are stained, also nerve- and glia-cells and their processes. Other Methods. 780. WEIGERT'S Specific Neuroglia Stain (WEIGERT, Beitr. zur Kenntniss der normalen menschlichen Neuroglia, Frankfurt- a-M., 1895; quoted from Neurol. Centralbl., 1895, xxiii, p. 1146). Pieces of tissue of not more than half a centimetre in thickness are put for at least four days into " 10 per cent, solution of forinol." They are then mordanted for four or five days in an incubating stove (or for at least eight days at the temperature of the laboratory) in a solution containing five per cent, of neutral acetate of copper, five per cent, of acetic acid, and two and a half per cent, of chrome alum, in water. (Add the alum to the water, raise to boiling point, and add the acetic acid and the acetate, powdered.) After the mordanting, the tissues are washed with water, dehydrated, imbedded in celloidin and sectioned. The sec- tions are treated for ten minutes with a one third per cent, solution of permanganate of potash, and well washed in water. They are then treated for two to four hours with a solution of " Chromogen." {C Chromogen " is a naphthalin compound prepared by the Hoechst dye manufactory. The solution of " Chromogen " to be used is prepared as follows : five per cent, of " Chromogen'' and five per cent, of formic acid are dissolved in water and the solution carefully filtered. To 90 c.c. of the filtrate are added 10 c.c. of ten per cent, solu- tion of sodium sulphite. After this bath, the sections are put till next day into a saturated (5 per cent.) solution of Chromogen. They are next carefully washed and submitted to the stain. The stain is a modification of WEIGERT'S fibrin stain. Instead of saturated aqueous solution of methyl violet, you take a warm-saturated solution in 70 per cent, or 80 per cent, alcohol, decant it after cooling, and add to it 5 per cent, of aqueous solution of oxalic acid. And instead of treating with pure anilin, you take a mixture of equal parts of anilin and xylol. This is afterwards removed from the sections by means of pure xylol, and they are mounted in balsam. 436 CHAPTER XXXIII. WEIGEET has concluded from the reactions obtained by this process that neuroglia is not a network of nerve-cell pro- cesses, but an intercellular substance of the nature of con- nective tissue substance. 781. See also ABONSON, a gallein stain, in MERCIER'S Coupes du systeme nerveux Central, p. 255 ; KULTSCHIZKY, Saurerubin for neuroglia, Anat. Anzeig., viii, 1893, p. 357, or Zeit.f. wiss. Mik., x, 2, 1893, p. 256. 782. UPSON'S Methods (MERCIER, in Zeit. f. wiss. Mik., vii,4, 1891, p. 474; or, in his "Coupes du systeme nerveux Central, p. 234; I pass over the older methods given in Neurol. Cen- tralb., 1888, p. 319; and in Zeit.f. wiss. Mik., 1888, p. 525). A. The Gold and Iron Method. Material must be very carefully hardened for from four to six months in bichromate of potash (not solution of Miiller, which does not allow of the same precision of stain). A 1 per cent, solution should be taken at first, and should at first be frequently changed. Only after some weeks should the strength be gradually increased up to 2 or 2'5 per cent. All the hardening should be done in the dark. Over-hardened material is not available. The surface of section of a pro- perly hardened piece of material should show macroscopically no marked difference of colour between the white and the grey matter. If the white substance appear very dark, almost black, and the grey substance whitish grey, good results will probably not be obtained ; a medullary stain will probably result, whereas the method ought to give an axis-cylinder and cell- process stain. After hardening, the specimens are washed with water, and put for two or three days into 50 per cent, alcohol, changed as often as necesssary, and then into 95 per cent, alcohol, in which they should remain until they show a de- cidedly green coloration (two to four weeks), the alcohol being changed as often as precipitates occur. Sections are made either under water or by the celloidin process (in the former case they should not be allowed to remain in contact with the water longer than is absolutely necessary, and should be put back into alcohol for two or three days before passing to the next stage). The next stage consists in a bath of from one to two hours' duration in 1 per cent, solution of chloride of gold acidified with 2 per cent, of hydrochloric acid. After this the sections are rinsed with water, and treated for half a minute with a freshly prepared solution of ferricyanide of potassium in 10 per cent, potash solution (a lump of ferricyanide not half so big as a pea to 5 c.c. of the potash solution). They are then washed for half a minute more in pure 10 per cent, potash solution, and after that for some time in distilled water. The following reducing mixture (which must be freshly made up at the very instant of using it for each section) is now to be prepared : Acidum Sulfurosum ...... 5 c.c. Tinctura lodi (3 per cent, strength) . . . 10 15 drops. Mix, and add Liquor Ferri Chloridi 1 drop. NEUROLOGICAL METHODS. 437 You must mix quickly in a graduated glass. The section should now be brought on a piece of filter-paper into a watch-glass, and the reducing mix- ture should be poured over it evenly at one pour, just as developing solution is poured on to a photographic negative. The section should remain in this bath until it is of a fine rosy j-ed tint, not a second longer, or it will become reddish black and useless. As soon as the rose-red colour appears the section is removed into distilled water. The water is changed once, the section is placed on a slide and brought into absolute alcohol, and after five to fifteen minutes therein into clove oil and mounted in balsam. The sections must be kept in the dark. (The treatment with ferricyanide of potassium sometimes gives rise to the formation later on of precipitates of Berlin blue in the tissues ; and as this treatment is not essential it may be omitted, the sections being simply treated with pure potash solution as directed.) 783. UPSON'S Methods. B. The Gold and Vanadium Method Sections (made as before) are placed in the following solution : 1 per cent, chloride of gold solution . . .5 c.c. Saturated solution of vanadate of ammonium . . 10 drops. Hydrochloric acid . . . . . . . 3 ,, After two hours therein they are washed with distilled water, and put for half to one minute into a mixture of 10 per cent, solution of caustic potash . . .5 c.c. 10 per cent, solution of permanganate of potash . 10 drops. Vanadate of ammonium . . . . .a trace. They are rinsed in distilled water, and treated until they become red, as in method a, with the following freshly prepared reducing fluid : a. 3 per cent, tincture of iodine, to which has been added chloride of tin until of a white or yellowish tint (may be kept in stock) . . . .15 drops. 6. Distilled water 3 c.c. c. Saturated solution of phosphate of iron in distilled water (the usual pharmaceutical solution) . . 3 o drops. d. Sulphurous acid 3 c.c. Mix a, 6, and c, and add d, which will cause a voluminous precipitate, at which instant the mixture is to be poured over the section, or the section brought into it, as in method a, the remaining treatment being exactly as there described. The principle of the two methods is the decoloration of the impregnated elements (especially glia-cells) up to a certain point by means of the potash, or potash and ferricyanide, or the vanadate and permanganate. It is im- portant to take care that this decoloration be not overdone, as if carried too far the axis-cylinders and ganglion-cells will be decolourised. 784. GIACOMINI'S "Dry" Process for Preserving Brains (Arch. per le Scienze Mediche, 1878, p. 11). Although this is in intention a macro- scopic method, it appears worth while, both on account of its thorough success and on account of its suggestiveness, to give a description of it here. The object is to make " dry" preparations of the encephalon, by which 438 .. CHAPTER XXXIII. is meant preparations that can be permanently preserved in the air. The methods hitherto employed were not successful because they consisted in making preparations that were " dry" in the literal sense of the word that is, deprived of their natural water ; and since brain-substance contains 88 per cent, of water, such preparations could not of course be obtained without so great an amount of shrinkage as to most seriously diminish the scientific value of the result. The principle of Giacomini's method is, on the contrary, to retain the natural water of the tissues, or an equivalent for it, by means of impregnation with a hygroscopic substance glycerin. The process consists of two divisions: 1, hardening; 2, impregnation with glycerin. 1. For hardening may be used zinc chloride, bichromate of potash, chromic acid, nitric acid, or alcohol. Chloride of zinc gives the best results. Perfectly fresh brain is put into a saturated aqueous solution of the salt (if there be reason to fear that the tissues are somewhat softened through having been left too long after the death of the subject, it is well first to inject 600 grins, of the solution through the internal carotid arteries). After forty-eight hours' immersion (during which time the floating brain must be turned over three or four times, so that all parts of it may duly come into contact with the liquid) the surface of the brain will have attained a consistency that will allow of the removal of the arachnoid and pia mater. The meninges having been removed the encephalon is put back into the solution for two or three days more, during which time it will be seen that, increasing in specific gravity, it tends towards the bottom of the vessel containing it. When this is seen to happen it must be removed into commercial alcohol, as if allowed to remain longer in the chloride of zinc solution it would take up too much water. In the alcohol it may remain for an indefinite time, or it may be removed if desired after ten or twelve days. (During the alcohol-bath it must be frequently turned over in order that no malformation may arise from con tinuance of pressure on the same part.) It is then removed into glycerin (either pure or with 1 per cent, of carbolic acid). It floats at first, but gradually sinks as the alcohol evaporates. As soon as it has sunk just below the surface it may be removed and exposed to the air. It is set aside to " evaporate" in a convenient place for a few days. As soon as the surface has become dry, it is varnished with India rubber or (better) with marine glue varnish diluted with a little alcohol. This com- pletes the process. If it be desired to make dissected preparations, the necessary dissection should be made on removing the encephalon from the alcohol before putting into glycerin. Bichromate of potash may be used for hardening in solutions gradually increasing in concentration from 2 to 4 per cent. The liquid must be fre- quently changed ; the immersion must be of not less than a month's dura- tion. Six to eight days will suffice for the alcohol-bath, or this may be altogether omitted. Nitric acid is used in solutions of from 10 to 12 per cent, for twelve to fifteen days. (Encephala float in this liquid, and must therefore be fre- NEUROLOGICAL METHODS. 439 quently turned over. It is this reagent that gives the toughest prepara- tions.) Concerning the value of the process, Golgi (from whose abstract I take the foregoing account) states that after a series of experiments he is able to affirm that for preservation of the volume, the colour, the finer relations of the parts, and the physiognomy proper to the organ, the process is far superior to any hitherto known. I am able to add that I saw specimens of Giacomini's preparations at the Milan International Exhibition of 1881, and think it would be hard to over-praise their beauty of aspect. It should be added that histological detail is preserved to a remarkable extent by this process, and that excellent sections may be cut at any time from the hardened brains. And as the preparations take up as little room as possible, there seems no reason why the process should not be generally adopted in medical schools, lunatic asylums, and similar institutions. The method may also be applied, with most perfect success, to the pre- servation of small animals entire, such as Batrachia, Reptilia. It is well to inject them with the zinc solution. Another " dry " method has been given by MAX FLESCH (Mt. Naturf. Ges. Bern., 1887, p. xiii). He hardens in alcohol, and then brings the brains through Calberla's mixture ( 425) into glycerin containing (1 part to 3000 of) sublimate. He does not appear to varnish his preparations. See Journ. Roy. Mic. Soc., 1888, p. 507. Retina. 785. Fixation and Hardening. For section cutting, the retina of small eyes is best prepared by fixing the entire un- opened bulb with osmium vapour. According to RANVIER (Traite, p. 954) you may fix the eye of a triton (without having previously opened the bulb) by exposing it for ten minutes to vapour of osmium. The sclerotic being very thin in this animal, such a duration of exposure is generally suffi- cient. Then divide it by an equatorial incision, and put the posterior pole for a few hours into one-third alcohol. Somewhat larger eyes, such as those of the sheep and calf, may be fixed in solutions without being opened. But it is generally the better practice to make an equatorial incision, and free the posterior hemisphere before putting it into the liquid. The older practice was to use strong solutions of pure osmic acid ; but most of the best recent work has been done with chromic mixtures. LINDSAY JOHNSON (in litt.) gets the best results by fixing the globe over the steam of a 2 per cent, osmic acid solution 440 CHAPTER XXXIII. raised nearly, but not quite, to boiling point, for five minutes ; or, if the eye be that of an adult, by removing the cornea and lens and injecting three to six drops of a mixture of equal parts of acetic acid and 2 per cent, platinic chloride into the vitreous cavity of the eye. After that the eye is put for twelve hours into the mixture, 97; it is then washed in running water, and suspended in a large volume of 2*5 per cent, bichromate of potash for two days, then passed gradu- ally through successive alcohols, beginning with 20 per cent., and ending with absolute, taking five days from first to last. This is the treatment recommended for adult human eyes. The eyes of children, small animals, e.g. rabbits, monkeys, and cats, and especially foetal eyes, will require a much gentler treatment or the retina will shrink into folds and the bulb collapse owing to the small amount of supporting and con- nective tissue present, and the thinness of the sclerotic. These eyes should not be opened at all during the fixing stage. After steaming for two or three minutes over osmic vapour, the bulb should be put entire into mixture of Flemming or Her- mann, or the mixture No. 97, for three to six hours, and then into bichromate solution, and only after two or three days should the bulb be opened. He also finds that eyes are preserved perfectly in a mixture of formaldehyde and platinic chloride, the best proportions being : Commercial formol or formalin, 4 parts, and 1 per cent, solution of platinic chloride, 30 parts. This mixture hardens slowly, but the retina remains nicely adherent, and the fovea well preserved, provided that the eye has previously been fixed by steaming over a 2 per cent, osmic acid solution raised to nearly boiling point, or solid osmic acid fumes pro- duced by heating the crystals in a test tube. Johnson finds as the result of repeated trials that the preliminary rapid fixing of the bulb over osmic vapour is absolutely essential to prevent the swelling and folding over of the retina at the macula, and to a less extent of the retina generally, nor has he been able to find any reliable substitute for this prelimi- nary treatment. As regards the after-hardening, a number of processes may be employed with good results, Flemming's mixture, Miiller's fluid, bichromate solution, formol and platinic chloride, and his mixture (97) being especially recommended. NEUROLOGICAL METHODS. 441 LEBEE (Milnch. med. Wochenschr., xli, 30, 1894; cf. Zeit.f.wiss. Mile., xii, '2. 1 ^.5, p. 256) confirms Hermann's observation concerning eyes ( 109). He a-lvises a -1 per cent, solution (formol 1, water 10). After a few days' hard- ening in this, the eyes may be cut through, it is said, without derangement of the parts. The retina lies flat, and is at least as well preserved as with solution of Miiller. The eyes may be passed without hurt direct into suc- cessive alcohols; the vitreous will shrink a little, but less rather than more than after solution of Miiller. I must be allowed to doubt the correct cyto- logical preservation of the elements by this process. 786. Staining. KAMON Y CAJAL employs the rapid chromate of silver method of Golgi, double impregnation process, as given above, 760. KUHNT (Jen. Zeit.f. Naturw., Bd. xxiv, H. 1, 1889, p. 177; Zeit. f. wiss. Mik., vii, 1, 1890, p. 65) employs Pal's modifica- tion of AVeigert's haeruatoxylin process. DOGIEL employs the methylen-blue method, as given in 291, 293. SCHAFFER (Sitzb. k. Akad. wiss. Wien, xcix, 1890,3, p. 110; Zeit. f. wi#s. Mik., viii, 2, 1891, p. 227) recommends mordant- ing sections in 1 per cent, chromic acid for some hours, washing for a short time only with water, staining for twenty hours in Kultschitzky's acetic acid hasmatoxylin ( 729), and differentiating for twelve hours in Weigert's ferricyanide solution. KBAUSE (1. c., 788) obtains instructive preparations by treating fresh retina with perchloride of iron or of vanadium in 1 per cent, solution, and then with a 2 per cent, solution of tannic or pyrogallic acid. These reagents only stain the granule layers and the nuclei of the ganglion-cells. The ele- ments of the other layers may then be stained with Saurefuchsin, or some other anilin. LENNOX (Arch. f. Ophthalm., xxxii, 1; Zeit.f. wiss. Mile., iii, 3, 1886, p. 408; and Journ. Roy. Mic. Soc., 1887, p. 339) has been applying Weigert's hseniatoxylin method to the retina, with some remarkable results. CUCCATI stains with concentrated aqueous solution of Saurefuchsin, and mounts in balsam. See also BERNHEIMER, Sb. k. Akad. wiss. Wien, 1884 ; or Journ. Roy. Mic. Soc., 1886, p. 167 ; and KAMON Y CAJAL, Rev. trim, de Hist. Norm, y Path., i, 1888, p. 1 ; Anat. Anz., 1889, p. Ill ; Zeit. f. wise. Mik., v, 3, 1888, p. 373, and vi, 2, 1889, p. 204. COLUCCI (Zeit.f. iviss. Mik., xii, 1, 1895, p. 87) recommends above every- thing Paladino's iodide of palladium impregnation, 737. 787. Sections. Some workers recommend celloidin ; but I see no reason whatever for not employing paraffin. Sections may be mounted in dammar or (FLEMMIXG) in glycerin. 442 , CHAPTER XXXIII. 788. Dissociation Methods. For maceration preparations you may use weak solutions (O2 to O5 per cent.) of osmic acid for fixation, and then macerate in 0'02 per cent, chromic acid (M. SCHULTZE), or in iodised serum (M. SCHULTZE), or in dilute alcohol (LANDOLT), or in Miiller's solution, or (RANVIER, Traite, p. 957) in pure water, for two or three days. THIN (Journ. of Anat., 1879, p. 139) obtained very good results by fixing for thirty-six to forty-eight hours in one-third alcohol, or in 25 per cent, alcohol, and then staining and teasing. SCHIEFFERDECKER macerates fresh retina for several days in the methyl mixture, 559. KRAUSE (Intern. Monatsch. f. Anat. u. Hist., 1884, p. 225 ; Zeit.f. wiss. Mik., 1885, pp. 140,396) recommends treatment for several days with 10 per cent, chloral hydrate solution. Barrett finds that this process preserves the rods and cones admirably. Inner Ear. 789. SCHWALBE (Beitr. z. Phys., 1887 ; Zeit. f. wiss. Mik., iv, 1, 1887, p. 90; Journ. Roy. Mic. Soc., 1887, p. 840). Fix (cochlea of guinea-pig) for eight to ten hours in " Flemming," wash in water, decalcify (twenty-four hours is enough) in 1 per cent, hydrochloric acid, wash the acid out, dehydrate, and imbed in paraffin. PRENANT (Intern. Monatsschr. f. Anat. u. Physiol., ix, 1, p. 6; Zeit. f. wiss. Mik., ix, 3, 1893, p. 379). For sections, open the cochlea in solution of Flemming or of Hermann, and fix therein for four to five hours. Avoid decalcification. as far as possible, as it is inimical to good preservation of elements; but if necessary, take 1 per cent, palladium chloride. Make paraffin sections and stain with safranin, or with methyl violet B, or with anilin green and orange, or with Renaut's eosin-haematoxylin. Isolation preparations of the stria vascularis may be made by putting a cochlea for a day into 1 per cent, solution of osmic acid, then for four to five days into 0*1 per cent, solu- tion ; the stria may then be got away whole. 790. Other Methods. WALDEYEE, Strieker's Handb., p. 958 (decalci- fication either in O'OOl per cent, palladium chloride containing 10 per cent, of HC1, or in chromic acid of 0'25 to 1 per cent.). UBBAN PEITCHAED (Journ. Roy. Mic. Soc., 1876, p. 211). Decalcification in 1 per cent, nitric acid. LAVDOWSKY (Arch.f. mik. Anat., 1876, p. 497). Fresh tissues (from the NEUROLOGICAL METHODS. 443 cochlea) are treated with 1 per cent, solution of silver nitrate, then washed for ten minutes in water containing a few drops of Oo or 1 per cent, osmic acid solution, and mounted in glycerin. MAX FLESCH (Arch. f. mik. Anat., 1878, p. 300). TAFAXI (Arch. Ital. de Biol., vi, p. 207). POLITZER, '" Die anatomische u. histologische Zergliederung d. menschlichen Gehororganes," Stuttgart (Enke), 1889 (see Zeit. f. wis9. Mik., vii, 3, 1890, p. 33.4}.-- The reviewer (HAUO) liere calls attention to the admirable qualities of phloroglucin as a decalcification agent for this object (see 587). EICHLER (Abh. d. math-phys. Cl. d. k. Sdchsischen Ges. d. Wiss., Bd. xviii, 1892, p. 311 ; Zeit. f. iciss. Mik., ix, 3, 1893, p. 380). Detailed account of manipulations for injection of blood-vessels of the labyrinth. See also SIEBEXMANX, Die Blutgefdsse im Labyrinthe des menschlichen Ohres, Wiesbaden, Bergmann, 1894; cf. Zeit. f. . Mik. f xi, 3, 1894, p. 386. 790a. Methylen Blue for Central Nervous System (SEMI MEYER, Arch. f. mik. Anat., xlvi, 1895, p. 282) .Methylen blue has not hitherto given good results with the central nervous system of the higher animals, either by the method of intra- venous injection or by that of staining by immersion. MEYER has obtained better results (for the central nervous system, not for the peripheral) by means of subcutaneous injection. The following points should be observed : Large quantities of solution must be injected, but they may be divided into fractional doses. A young rat will require at least 5 c.c. of 1 per cent, solution; a rabbit of a few weeks about 40 c.c.; a cat of the same size about three times as much. Mice are not good subjects. The total dose should be given in several portions, at intervals of one to several hours. If 20 c.c. of 1 per cent, solution be injected into a young rabbit, and again the same quantity after the lapse of two hours, the subject will generally be found to be dead or dying at the end of two hours more. It is not necessary to wait till death by intoxica- tion has taken place, and after a suitable interval the subject may be killed. It is not necessary to expose the organs to the air for the sake of " oxydising" the stain. They should be thrown direct into the liquid of BETHE, 294. The liquid ought to be well cooled before use. The preparations should remain in it till the next day. CHAPTER XXXIV. SOME OTHER HISTOLOGICAL METHODS. Connective Tissues. 791. Connective Tissue. S. MAYER (Sitzb. k. Akad. Wiss., ixxxv, 1882, p. 69) recommends, for staining fresh tissue, a solution of 1 gramme of " Violet B " (Bindschedler and Busch, Bale) in 300 c.c. of 0*5 per cent, salt solution. In this liquid connective-tissue cells stain rapidly and energeti- cally. Elastic fibres and smooth muscle also stain, but of different tints. For KANVIER'S method of artificial oedemata for the study of areolar tissue, see his Traite, p. 329. For FLEMMING'S observations on the development of connective-tissue fibrils, see Festschr. E. Virchow gewidmet, &c., 1, 1891, p. 215; Zeit.f. wiss. Mile., ix, 2, 1892, p. 225. For PIANESE'S very complicated double stain with carmine and picro- nigrosin, see Journ. Roy. Mic. Soc., 1892, p. 292. FEEEBOEX (Amer. Mon. Mic. Journ., 1888, p. 231 ; Journ. Roy. Mic. Soc., 1889, p. 305) recommends (for sections) picro-nigrosin, made by mix- ing 5 c.c. of 1 per cent, aqueous solution of nigrosin with 45 c.c. of aqueous solution of picric acid. Stain for three to five minutes, wash with water, and mount in balsam. Connective-tissue fibres bright blue, nuclei blackish, all the rest greenish yellow. 792. BENECKE'S Stain for Fibrils. BENECKE (Verh. d. anat. Ges., vii Vers., 1893, p. 165; Zeit. f. wiss. Mik., xi, 1, 1894, p. 79) finds that by a slight modification of WEIGERT'S Fibrin Stain (post, 813) good stains of connective-tissue fibrils and some other elements may be obtained. The modification consists in taking for the decoloration a mixture of anilin with xylol instead of pure anilin, as in WEIGERT'S form of the process. BENECKE takes a mixture of two parts of anilin with three of xylol. The process is therefore essentially similar to that of KROMAYER given above, 665. Connective-tissue fibrils are stained of a deep blue, elastic tissue red or violet, glia-fibres of the same tone as the connective tissue. 793. UNXA'S Specific Stains for Collagen (Monatsh. f. prakt. DermatoL, xviii, 1894, p. 509 ; Zeit. /. wiss. Mik., xi, 4, 1894, SOME OTHER HISTOLOGICAL METHODS. 445 p. 518). Unna finds that the method of BENECKE (last ) is unsurpassed for the demonstration of collagen fibrils alone, but prefers the following whenever it is desirable to obtain at the same time good images of the ground substance and of other elements of preparations. 1. The Orcein Method. Sections of alcohol material are stained for five minutes in Griibler's strong solution of poly- chromatic methylen blue. They are then brought for fifteen minutes into a neutral 1 per cent, solution of orcein in abso- lute alcohol, rinsed in pure alcohol, cleared in bergamot oil, and mounted. Nuclei blue, collagenous ground-substance dark red, granules of Mastzellen carmine red, protoplasm of Plasmazellen blue. 2. The Method of Sulphosalts. (a) Stain for five or ten minutes in an aqueous 2 per cent, solution of Saurefuchsin, rinse, treat for one or two minutes with saturated aqueous solution of picric acid, dehydrate (two minutes) in absolute alcohol saturated with picric acid, rinse with pure alcohol, clear and mount, (b) Stain for twenty seconds in aqueous 1 per cent, solution of Wasserblau, rinse, treat for five minutes with neutral aqueous 1 per cent, solution of safranin. Rinse and put into absolute alcohol until the blue colour reappears, clear with bergamot oil and mount. Collagen light blue, nuclei red. 794. Fat. DEKHUYSEX (see FLEMMING, in Zeit. f. wiss. Mik., 1889, pp. 39, 178) has discovered that fat that has been stained black bj treat- ment with chromo-aceto-osmic acid (not with pure osmic acid) is dissolved in the course of a few hours in turpentine. It is dissolved also in xylol, ether, and kreasote. FLEMMING finds that very good demonstration pre- parations may be made by treating fatty tissue with chromo-aceto-osmic acid, staining with safranin or gentian, and then treating for a few hours with turpentine until all the fat is dissolved. The optical hindrance caused by the high refraction of the fat being thus eliminated, nuclei and cytoplasm may be studied to far greater advantage than in the usual preparations. 705. Granule-cells ("Plasmazellen" and "Mastzellen "). In 1874 there were described by WALDEYEE (Arch.f. mil'. Anat., Bd. xi) cer- tain special cells existing between the bundles of connective tissue, besides the flat cells, and lymphatic and fat cells. They are large round cells con- taining large granules ; Waldeyer called them plasma cells (" Plasma- zellen"). Later on, EHELICH (ibid., xiii, 1877) distinguished in the same tissue and in other places certain cells containing large granules, which have a superficial resemblance to Waldeyer's plasma cells, but which differ 446 CHAPTER XXXIV. f rom them .in staining reaction (Verhandl. Berl. Physiol. (res., January 17th, 1879 ; Eeichert. u. Du Bois Reymond's Arch., 1879, p. 166). Ehrlich named these food cells (" Mastzellen "), intending to express thereby the opinion that these cells are derived from fixed connective-tissue cells by a transformation brought about by exalted nutrition. If (KORYBUTT-DASZKIEWICZ, Arch. f. mik. Anat., xv, 1878, p. 7) frogs be kept for two months (in summer) without food, then placed in a reservoir of running water and well fed for four weeks, " plasma-cells " will be found in abundance, .especially in the neighbourhood of the nerves. It is a pretty general character of these elements that they take the stain of anilin colours and retain it on treatment with alcohol with greater energy than other tissue cells. And further (at least so far as regards the true Mastzellen of Ehrlich) that in successful preparations they show the nucleus unstained, the general mass of cytoplasm unstained or but slightly coloured, and, in the cytoplasm, the characteristic granules very intensely stained. The staining reactions of the granules are very similar to those of bacteria. In order to distinguish them from these it may be observed that the stain of anilins is removed from the granules, but not from bacteria, by treatment with a weak solution of carbonate of potassium (SQUIRE, Methods and Formulae, p. 46). 796. EHRLICH'S Classification of Granules. Cell-granules in general have been classified by Ehrlich, according to their respective degrees of affinity for one or the other of the three groups of dyes defined in 268, into (1) a granulations, or eosinophilous granulations, being such as in a mixture of dyes select the most acid stain there present; (2) the /3 granula- tions, or amphophilous granules, '.being such as in a mixture of basic and acid dyes take up both ; (3) the y or basophilous granules, being such as take up basic dyes only (such are the granules of Mastzellen ; (4) the S granulations, also baso- philous ; and (5) the c granulations, or neutrophilous granules, being such as take up neutral anilin dyes, such as methyl blue and ' ' acid " f uchsin [acid f uchsin is, however, a weakly acid dye]. I do not propose to enter minutely into the subject of these reactions, as it is one that cannot profitably be treated apart from the histology of the elements in question, and will confine myself to mentioning a few well-known methods. SOME OTHER HISTOLOGICAL METHODS. 447 Some of these have already been given; viz., the EHRLICH- BIONDI mixture, 306; EHRLICH'S C mixture, or acidophi- lous mixture, 323, for eosinophilous granulations ; and his triacid mixture, 307, which stains the a, /3, and e granula- tions. 797. EHRLICH'S " Mastzellen " (Arch. f. mik. Anat., 1876, p. 263). The tissues must be first well hardened in strong alcohol (chromic acid and its salts must be avoided). They are then placed for at least twelve hours in a staining fluid composed of Absolute alcohol .... 50 c.c. Aqua . . . . . 100 c.c. Acid. acet. glacial .... 12 c.c. to which has been added enough dahlia to give an almost saturated solution. After staining, the preparations are transferred to alcohol, which washes out the stain from all but the plasma-cells, and may then be mounted in resin- turpentine solution. Mucus-cells and fat- cells are also sometimes stained by these solutions. Other Hedia. In a similar way other soluble anilins may be employed (in the form of a fluid containing 7 per cent, of acetic acid), primula, iodine violet, methyl violet, pur- purin, safranin, fuchsin ; of these, methyl violet gives the best results. 798. " Mastzellen." SCHIEFFEEDECKEE (SCHIEFFERDECKER AND KOSSEL'S Gewebelehre, p. 329) recommends the following, after ORTH. A piece of mesentery of a rat is brought into a solution of gentian violet in anilin water (p. 190), which is carefully heated over a flame until vapour begins to be given off and then allowed to remain for a couple of hours (or the heating may be omitted, and the preparation allowed to stand for twenty-four hours). It is then rinsed in water, washed out until almost colourless in hydrochloric acid alcohol, rinsed in water, counterstained (if desired) in carmine, and mounted in balsam. Nuclei red, granules blue. 799. Plasma-cells (XORDMAXX, JBeitr. z. Kenntniss d. Mast- zellen, Inauguraldiss., Helmstedt, 1884). Nordrnann finds it useful to employ a solution of vesuvin containing 4 to 5 per 448 CHAPTER XXXIV. cent, of hydrochloric acid. Sections should remain for a few- minutes in the solution, and then be dehydrated with absolute alcohol. The paper quoted contains a detailed discussion of the microchemical reactions of granule-cells. 800. Plasma Cells and Mastzellen. UNNA, in his paper, Zeit. f. wiss. Mifc., vii, 4, 1892, p. 475, gives the following: A. For Plasma Cells. Methylen blue . . . . 1*0 Caustic potash . . . . O05 Distilled water . . . lOO'O Add a few drops of this to ten, fifty, or one hundred vols. of anilin water (p. 190) in a watch-glass, and stain (alcohol material, or at most sublimate and alcohol material, not chromic material) for half an hour, several hours, or over night. Dehydrate rapidly in absolute alcohol, differentiate in creosol (details not given), rinse in xylol, and mount in balsam. B. General Stain, also bringing out Plasma Cells. Methylen blue . . . . 1-0 Carbonate of potash . . . TO Distilled water. . . . 10OO Alcohol .... 20-0 Heat on a water-bath until reduced to 10OO. Use for staining undiluted, or diluted with one vol. of anilin water. Differentiate (details not given) with glycol, styron, or creosol. Mastzellen are not differentiated. c. Stain giving Red Mastzellen with Blue Plasma Cells. Methylen blue . . . . I/O Kali Carbon, (natron carbon, ammon. carbon) . . . . 1-Q Aq. dest. (Aq. carbolisata, chloroforma) 10OO Dilute about 100-fold, stain slowly, treat with 70 to 80 per cent, alcohol, differentiate in styron, and bring through ber- gamot oil or xylol into balsam. In this process the granules SOME OTHER HISTOLOGICAL METHODS. 449 of the Mastzellen stain red in consequence of the formation of methylen red in the staining bath. In a paper " Ueber Plasinazellen, insbesondere bei Lupus " (for which see Monatsh.f. prakt. DermatoL, xii, 1891, p. 296, or Zeit. f. wiss. Mik., ix, 1, 1892, p. 92), UNNA gives some directions concerning the process of differentiation with creosol. Creosol only differentiates the stain, it does not dehydrate the sections. Sections should, therefore, first be dried with blotting-paper, and should then be treated with absolute alcohol for a few seconds, or with anilm oil before applying the creosol. The time required for differentiation in the creosol is from a few minutes to a few hours. When the proper stage of differentiation is attained, it should be fixed with xylol and the sections mounted. The method shows Mastzellen of a cherry-red tone. In a paper, 1. c., xiii, 1891, p. 364 (see Zeit. f. wiss. Mik., xi, 1, 1892, p. 89), VAN DEB SPEK and UXXA describe some further experiments. Main- taining the method of staining given above under A, they find that besides the differentiating agents given under B, a lengthy series of other reagents are available. In all of them Mastzellen are distinguishable from plasma cells by the reddish coloration of their granules. See also UNXA, Monatsh.f. prakt. DermatoL, xix, 1894, p. 225 ; Zeit. /. wiss. Mik., xii, 2, 1895. p. 58. 801. Plasma Cells and Mastzellen. BERGONZINI (Anat. Anz., vi, 1891, pp. 595 600; Zeit. f. wiss. Mik., ix, 1, 1892, p. 95) gives the following : Mix 1 volume of 0*2 per cent, solution of Saurefuchsin with 2 volumes of a like solution of methyl green, and 2 volumes of a like solution of gold-orange, and filter through cotton wool. Stain alcohol or sublimate sections (after washing in water) for three to four minutes, wash for one or two minutes in water, bring into absolute alcohol for two minutes, clear in bergamot oil or pure creo- sote, wash in turpentine, and mount in balsam. One sort of gold-orange precipitates methyl green, and therefore cannot be used in this mixture. Orange G may be used instead (thus giving something very like Ehrlich-Biondi mixture), but in this case the acidophilous granules will stain greyish instead of red or orange. Basophilous granules ought to be green, weakly acidophilous granules red, strongly acidophilous ones orange or brown, nuclei always green. Other details 1. c. 29 450 OH AFTER XXXIV. 802. Plasma Cells. JADASSOHN (Arch. f. Dermatol. u. Syphilis, Erganzungsheft 1, 1892, p. 58; Zeit.f. wiss. Mik., ix, 2, 1892, p. 226) re- commends staining for not too long in a 1 : 2000 strongly alkaline or borax solution of Thionin, and washing out with acidulated water. See also VON MAESCHALKO, Arch. f. Dermatol. u. Syphil., xxx, 1895, p. 3 ; Zeit.f. wiss. Mik., xii, 1, 1895, p. 64. 803. Clasmatocytes. RANVIEE (C. E. Acad. des Sci., 1890) lias described under the name of " clasmatocytes" certain cells that occur in the thin connective membranes of verte- brates, and that show a close affinity to Mastzellen, possessing processes that break up and form islands of granules. He demonstrates them as follows : A piece of suitable membrane (epiploon of mammalia, mesentery of batrachia) is stretched secundum artem on a slide, and a few drops of 1 per cent, solution of osmic acid are allowed to fall on to it. After one or two minutes it is washed with water and stained with concentrated aqueous solution of methyl violet OB diluted with ten parts of distilled water. When sufficiently stained the preparation is covered with a thin glass cover and examined. Glycerin may be added to make the preparation permanent, but does not succeed very well, as it causes the stain to diffuse. Brim's glucose medium ( 420) would probably be preferable. 804. Elastic Tissue. Two of the most salient characters of elastic fibres are that they have a great affinity for osmium, staining with much more rapidity than most other tissue- elements, and that they are not changed by caustic soda or potash. A further character is that they have a great affinity for certain anilin dyes, especially Victoria blue. For a review of the older methods of BALZER, UNNA, LUST- GARTEN, and HERXHEIMER, see the paper by MARTINOTTI in Zeit. f. wiss. Mik., iv, 1, 1887, p. 31. The method of LUSTGAETEN has been given in 281. The colour used by him was called " Victoriablau 4B./' and this is probably an important detail. The method of MARTINOTTI (I.e.) is as follows : Fix in a chromic liquid, wash, stain for forty- eight hours in strong (5 per cent. Pfitzner's) solution of safranin, wash, dehydrate, clear, and mount in balsam. Elastic fibres are stained an intense black, the rest of the preparation showing the usual characters of a safranin stain. SOME OTHER HISTOLOGICAL METHODS. 451 The staining will be performed quicker if it be done at the temperature of an incubating stove (GRIESBACH, ibid., iv, 1887, p. 442). And FERRIA (ibid., v, 3, 1888, p. 342) says that clearer preparations will be obtained if the sections be left for a long time, say twenty-four hours, in the alcohol, or be treated for a short time with very dilute alcoholic solution of caustic potash. This decolourises more completely the ground of the preparations. Another safranin method, which seems to have the fault of requiring a too minute attention to details, is that of MIBELLI, see Mon. zool. italiatio, 1, p. 17, or Zeit.f. wiss. Mik., vii, 2, 1890, p. 225 (the report in Journ. Boy. Mic. Soc., 1890, p. 803, is vitiated by a misprint). 805. UNNA'S Modified Orcein Method (Monatsh. f. prakt. V- I'matoL, xix, 1894, p. 397; Zeit.f. wiss. Mik., xii, 2, 1895, p. 240). The following solution is made : G rubier' s orcein, 1 part ; hydrochloric acid, 1 part ; absolute alcohol, 100 parts. The sections are put into a porcelain capsule with just enough of the stain to cover them, and the whole is warmed in a stove or over a naked flame to abo*at 30 C. After ten to fifteen minutes the stain becomes quite thick, owing to the evaporation of the alcohol. The sections are then well rinsed in alcohol, cleared and mounted. Elastin dark brown, collagen light brown. For UNNA'S earlier orcein method, see last edition, or Monatsh. f. prakt. Dtrmatol, xii, 1891, p. 394 (Zeit.f. wiss. Mik., ix, 1, 1892, p. 94). See also ZENTHOEFER, in Unna's Dermatol. Studien, 1892, or Zeit. f. u'iss. Mik., ix, 4, 1893, p. 509 ; KOPPEX, Zeit. f. wiss. Mik., vi, 4, 1889, p. 473 ; and vi, 1, 1890, p. 22, or last edition ; BURCI, Journ. Roy. Mic. Soc., 1891, p. 831, and 1892, p. 292, or last edition ; HAXSEX, Yirchow's Archiv, cxxxvii, 1894, p. 25 ; Zeit.f. wiss. Mik., xi, 3, 1894, p. 383 ; KULTSCHIZKY, ib '].. xiii, 1, 1896, p. 74, or the original, Arch.f. mik. Anat., xlvi, 1895, p. 806. Bone, Non-decalcified (RAXVIER, Traite, p. 297). ivier points out certain precautions that it is necessary to ike in the preparation of sections of dry bone. In general, the bones furnished by " naturalists," or procured in ana- tomical theatres, contain spots of fatty substance that prevent good preparations from being made. Such spots are formed * For a minutely detailed review (40 pages, with references to 80 memoirs) of the whole subject of the technique of bone, see the paper of FFER, Die Methodik der histoloyischen Uniei'suchung. des Knochen- gewebes, in Zeit.f. wiss. Mik., x, 2, 1893, p. 167. 452 CHAPTER XXXIV. when bones are allowed to dry before being put into water for maceration ; when a bone is left to dry the fat of the medullary canals infiltrates its substance as fast as its water evaporates. Bones should be plunged into water as soon as the sur- rounding soft parts have been removed, and should be divided into lengths with a saw whilst wet. The medulla should then be driven out from the central canal by means of a jet of water; spongy bones should be submitted to hydrotomy. This may be done as follows : An epiphysis having been removed, together with a small portion of the diaphysis, a piece of caoutchouc tubing is fixed by ligature on to the cut end of the diaphysis, and the free end of the piece of tubing adapted to a tap through which water flows under pressure. As soon as the bones, whether compact or spongy, have been freed from their medullary substance they are put to macerate. The maceration should be continued for several months, the liquid being changed from time to time. As soon as all the soft parts are perfectly destroyed, the bones may be left to dry. When dry, they should be. of an ivory whiteness, and their surfaces exposed by cutting of a uniform dulness. Thin sections may then be cut with a saw and prepared by rubbing down with pumice-stone. Compact pumice-stone should be taken and cut in the direction of its fibres. The surface should be moistened with water and the section of bone rubbed down on it with the fingers. When both sides of the sections have been rubbed smooth in this way, another pumice-stone may be taken, the section placed between the two, and the rubbing continued. As soon as the section is thin enough to be almost transparent it is polished by rubbing with water (with the fingers) on a Turkey hone or lithographic stone. Spongy bone should be soaked in gum and dried before rubbing down (but see VON KOCH'S copal process, ante, 174, and EHRENBAUM'S colophoniuni process, 175). The process of WEIL (Zeit. f. wiss. Mik., v, 2, 1888, p. 200), which is intended for bone and teeth, has been given 176. KOSE (Anat. Anz., vii, 1892, pp. 512519; Zeit. f. wiss. Mik., ix, 4, 1893, p. 506) points out some precautions that it is well to take. He penetrates first with a mixture of cedar oil and xylol, then with pure xylol, SOME OTHER H1STOLOGICAL METHODS. 453 and imbeds in solution of dauiruar in chloroform or xylol. The method can 1-e combined with Golgi's impregnation. XEALEY (Amer. Un. Mic. Journ., 1884, p. 142 ; Journ. Roy. Mic. Soc., 1885, p. 348) says that perfectly fresh portions of bone or teeth may be ground with emery on a dentist's lathe, and good sections, with the soft parts in situ, obtained in half an hour. WHITE (Journ. Roy. Mic. Soc., 1891, p. 307) recommends the following : Sections of osseous or dental tissue should be cut or ground down moderately thin, and soaked in ether for twenty-four hours or more. They should then be put for two or three days into a thin solution of collodion stained with fuchsin (made by dissolving the dye in methylated spirit, adding the requisite quantity of ether, then the pyroxylin). The sections are then put into spirit to harden the collodion. After this they are ground down to the requisite thinness between two plates of old ground glass, with water and pumice powder, and mounted, surface dry, in stiff balsam or styrax, care being taken to use as little heat as possible. Lacunas, canaliculi, and dentinal tubuli are found infiltrated by the coloured collodion. For the method of MATSCHINSKY (Arch. f. mik. Anat., xxxix, 1892, p. 151 ; Zeit. f. wiss. Mik., ix, 3, 1893, p. 353), see last edition. For a similar infiltration and grinding method of RUPRECHT, see Zeit. f. ids*. Mik., xiii, 1, 1896, p. 21, wherein see also quoted (p. 23) a method of ZIMMERMAXN. HOPEWELL- SMITH (Journ. Brit. Dent. Ass., xi, 1890, p. 310; Journ. Roy. Mic. Soc., 1890, p. 529) says that for preparing sections of teeth showing odontoblasts in situihs best plan is to take embryonic tissues. A lower jaw of an embryonic kitten or pup may be taken, and hardened in solution of Miiller followed by alcohol, then cut with a freezing micro- tome. The knife cuts quite easily the thin cap of semi- calcified dentine and bone. 807. VIVAXTE (Intern. Monatssch.f. Anat. u. Phys., ix, 1892, p. 394; Zeit.f. wiss. Mik., ix, 3, 1893, p. 351) has made out that thin specimens of bone can be successfully treated by Golgi's bichromate of silver process. He places portions of frontal bone of four to six months calves, which are not more than 3 to 4 mm. thick, for eight days in solution of Miiller, then in the osmium bichromate mixture, and then in the silver solution. After impreg- 454 CHAPTER XXXIV. nation the specimens should be decalcified, which may be done by putting j them for twenty days into von Ebner's mixture ( 577), after which they ' should be well washed with water and brought into solution of carbonate of j soda, and finally imbedded in paraffin. Another method recommended by Vivante is as follows : Fix for five or j six 'days in solution of Flemming, wash and decalcify with von Ebner's mixture, wash, treat with carbonate of soda, imbed in paraffin, cut, and stain the sections for an hour in O2 per cent, solution of quinolem blue ( 321). : Wash out in equal parts of alcohol and water followed by pure water, dry j the sections in a stove at 40 C. (both alcohol and glycerin must be avoided), clear with bergamot oil, and mount in dammar. Nuclei violet, protoplasm and ground substance different shades of blue. For UNDEEWOOD'S gold process for teeth, see p. 257 ; and for that of LEPKOWSKY, see Anat. Anz., vii, 1892, p. 274; Zeit. f. iviss. Mik., ix, 3, i 1893, p. 355 ; or the last edition of this work. 808. Bone, Decalcified (FLEMMING, Zeit. f. iviss. Mik., 1886, p. 47). Sections of decalcified bone are made with the free hand. They are soaked in water, and brought in a drop of water on to a glass plate, where they are spread out flat. The excess of water is removed with blotting-paper, and the sections are covered with another glass plate, to prevent them from rolling. The whole is brought into a plate and covered with alcohol. After the lapse of half an hour the sections have become fixed in the flat position, and may be brought into absolute alcohol without risk of their rolling. To mount them, wash them with fresh alcohol (which may be followed by ether) ; lay them again flat on glass, and cover them with a double layer of blotting-paper and a somewhat heavy glass plate, and let them dry for a day in the air or in a stove. When they are dry, put a drop of melted balsam on a slide, and let it spread out flat and cool. Prepare a thin glass cover in the same way, put the section on the prepared slide, cover it with the prepared cover, put on a clip, and warm. By this process sections can be very expeditiously pre- pared, which show the lacunar system injected with air in quite as instructive a manner as non-decalcified sections. KOLLIKER (Zeit. f. wiss. Zool., xliv, 1886, p. 662) recom- mends the following process for the demonstration of the fibres of Sharpey in decalcified bone. Sections are treated with concentrated acetic acid until they become transparent, and are then put for one quarter to one minute into a con- centrated solution of indigo-carmine, then washed in water and mounted in glycerin or balsam. In successful prepara- SOME OTHER HISTOLOGICAL METHODS. 455 tions the fibres of Sharpey appear stained of a pale or dark red, the remaining bone-substance blue. ZACHABIADES (Zeit.f. wiss. Mik., x, 4, 1893, p. 447) has the following : Bone is decalcified by means of picric acid, is repeatedly washed until all the acid is removed, then put into alcohol and sectioned. The sections are placed on a slide and treated for a few seconds with 1 per cent, solution of osmic acid. They are then stained, either for twenty-four hours in a weak aqueous solution of quinolein blue, or for a few minutes in a saturated aqueous solution of safranin. They are then treated on a slide with a drop of 40 per cent, solution of caustic potash, the slide being warmed over a flame until the sections spread out flat. The excess of potash is then removed and the sections are carefully washed with water, covered and examined. The safranin preparations may be permanently preserved in glycerin containing a small proportion of safranin. It is said that prepara- tions thus made demonstrate that there exist in fresh bone, at all ages of the subject, ramified cells possessing a membrane, and easily isolated by the action of potash. Their ramifications anastomose with one another and with the ramifications of other cells, the whole forming a protoplasmic network enveloped in a membrane. 809. Stains for Cartilage (and Decalcified Bone). For an excellent discussion (especially as regards staining) of the methods that have been recently recommended for these objects, see the exhaustive paper of SCHAFFER in Zeit.f. wiss. Mik., v, 1, 1888, which gives in sufficient detail all the methods in question. The following appear to be the best : SCHAFFER'S safranin method. This method is due in its principle to BOUMA (Centralb.f. d. med. Wiss., 1883, p. 866). I give it in the form to which it has been brought by the careful study of Schaffer (Zeit.f. wiss. Mik., v, 1, 1888, p. 17). Sections of bone decalcified with nitric acid (chromic acid may be used, but the stain will be less brilliantly contrasted) are stained for half an hour to one hour in 0'05 per cent, aqueous solution of safranin, washed with water, put for two or three hours inO'l per cent, solution of corrosive sublimate, and examined in glycerin. In order to make permanent preparations, the sections on removal from the sublimate are rinsed with alcohol, pressed on to a slide with filter- paper, cleared for a long time in bergamot oil or clove oil, and mounted in xylol balsam. This is a double stain ; cartilage, orange ; bone, uncoloured (or green in chromic objects) ; marrow, red. BAYERL'S method for ossifying cartilage (Arch. f. mik.Anat. y 456 CHAPTER XXXIV. 1885, p. 35), is as follows : Portions of ossified cartilage are decalcified as directed 578, cut in paraffin, stained in Merkel's borax-carmine and indigo-carmine mixture. 340, and mounted in balsam. MAX FLESCH (Zeit. f. wiss. Mik., 1885, p. 351) particularly recommends this process for the study of the development of dental tissue. Aqueous solution of benzoazurin has been commended as a stain for ossifying cartilage by ZSCHOKKE, see Zeit. f. wiss. Mik.,x, 3, 1893, p. 381. A process recommended by BAUMGARTEN has been given, 342, MOEENEE (Skandinavisches Arch. f. Physiol., i, 1889, p. 216 ; Zeit. f. wiss. MiJc., vi, 4, 1889, p. 508) gives several stains for tracheal cartilage, chiefly as microchemical tests, for which see the last edition. See also a critique of these methods by WOLTERS in Arch.f. mik. Anat., xxxvii, 1891, p. 492; Zeit.f. wiss. Mik., viii, 3, 1891, p. 383; and on the whole subject of cartilage, see SCHIEFFEEDECKER'S Gewebelehre, p. 331. Blood. 810. It might be supposed that for the study of blood it would suffice to prick a finger, place a drop of blood on a slide, cover, and examine it. But this is by no means the case. " Blut ist ein ganz besonderer 8 aft,"*' and will not yield up its secrets to such simple wooing. The technique of blood is most elaborate ; see, for instance the voluminous work of HAYEM, Du sang et de ses alterations anatomiques , pp. 1035, with 126 figures, Paris, Masson, 1889 (a report of over twenty pages on this important work is contained in Zeit. f. wiss. Mik., vi, 3, 1889, p. 330, et seq.) ; LOWIT, Sitzb. k. Akad. wiss. Wien, 3, Ixxxviii, 1883 ; xcii, 1885; xcv, 1887; Zieg- ler's Beitr. z. path. Anat., x, 1891, p. 214; Zeit. f. wiss. MiJc., vi, 1889, pp. 74, 76; viii, 3, 1891, p. 371 ; Arch.f. mik. Anat., xxxviii, 1891, p. 524; Zeit.f. Wiss. Mik., ix, 2, 1892, p. 233; Studien zur Physiol. u. Path. d. Blutes u. d. Lymphe, Jena, Fischer, 1892 ; 'EHRLICH, Zeit. f. klin. Medicin, i, 1880, 3, p. 558; Zeit.f. wiss. Mik., i, 1884, p. 382, and other papers; MULLER, Sitzb. k. Acad. Wiss. Wien, xcviii, 3, p. 219; Zeit. f. iviss. Mik., ix, 3, 1893, p. 365; GRIESBACH, Zeit. f. iriss. Mik., vii, 3, 1890, p. 326; and many other investigators. * GOETHE'S Faust, i, 4, line 1387. SOME OTHER HISTOLOGICAL METHODS. 457 It is out of the question for me to attempt to abstract these memoirs in the space at my disposal, so I must confine myself to giving a few methods that may be useful to the general student, referring the specialist to the original papers. 811. Fixing and Preserving Methods. The time-honoured process of drying drops of blood over a flame gives rise to great deformation of the elements, and should be abandoned as far as possible. It is better to mix the blood at once with some fixing and preserving medium, and study it as a fluid mount. Some examination liquids and stains for fresh blood in the fluid state are given in the next . Most recent authors (BIONDI, Mosso, MAX FLESCH) are agreed that by far the most faithful fixing agent for blood- corpuscles is osmic acid. A drop or two of blood (Biondi re- commends two drops exactly) is mixed with 5 c.c. of osmic acid solution, and allowed to remain in it for from one to twenty-four hours. The exact degree of concentration of the osmium solution is a somewhat important point, and must be made out by experiment for each form. Ae a rule it should be strong, 1 to 2 per cent. According to Biondi, 2 per cent, is best. Fixed specimens may be preserved for use in acetate of potash solution (MAX FLESCH, Zeit.f. wiss. Mik., v, 1, 1888, p. 83). GKIESBACH also (op. cit., p. 328) prefers osmic acid, not only as being a first-rate fixing agent, but because it can be combined with certain stains without decomposing them. He mentions methyl green, methyl violet, crystal violet, saf- ranin, eosin, Saurefuchsin, rhodamin, and iodine in potassic iodide. Rossi (Zeit.f. wiss. Mik. y vi, 4, 1889, p. 475) advises a mix- ture of equal parts of 1 per cent, osmic acid, water, and strong solution of methyl green, permanent mounts being made by means of glycerin cautiously added. The mercurial liquids of PACIXI ( 400) used to be con- sidered good. HAYEM (op. cit. ; see also Zeit. f. wiss. Mlk., vi, 3, 1889, p. 335) has a similar formula, viz. sublimate 0'5, salt 1, sulphate of soda, 5, and water 200. This should be mixed with blood in the proportion of about 1 : 100. Eosin may be added to it. L6 WIT'S formula (Sitzb. Jc. Akad. Wiss. Wien, xcv, 3, p. 129; Zeit. f. wiss. Mik., vi, 1, 1889, p. 75) 458 CHAPTER XXXIV. consists of 5 c.c. cold saturated sublimate solution, 5 grins, sulphate of soda, 2 gums, salt, and 300 c.c. water. Mosso finds, however, that both of these are too weak in sublimate. Of course other well-tried fixing fluids, such as Flemming's solution, or Hermann's, may also be used for blood. LAVDOWSKY (in a long paper in Zeit.f.wiss. Mik., x, 1, 1893, p. 4) describes some remarkable results obtained by fixing with 2 per cent, iodic acid, and staining with Neu-Victoriagrun, methyl violet 6 B, or gentian violet, a process which is said to reveal the presence of nuclei in elements generally considered to be apyrenematous. 812. Stains for Blood. Blood prepared as above can be satisfactorily stained with many of the usual reagents. Eosin stains rose-red all parts of blood-corpuscles that contain haemoglobin (see WISSOWSKY, Arch. f. mik. Anat., 1876, p. 479) ; parts that do not contain hemoglobin, such as the nucleus, remaining unstained. This suggests double- staining with eosin and haematoxylin. WISSOWSKY (1. c.) stains in a solution of equal parts of eosin and alum in 200 parts of alcohol, and then with haema- toxylin. MOORE (The Microscope, 1882, p. 73 ; Journ. Roy. Mic. Soc., 1882, p. 714) stains for three minutes in a similar solution without the alum, washes, and stains for two minutes in a 1 per cent, aqueous solution of methyl green. Eed corpuscles, red nuclei and white corpuscles, bluish green. The liquid of CHENZINSKY has been given ( 315). It stains nuclei and eosinophilous granules. MERKEI/S carmine and indigo- car mine stain has been dis- cussed above ( 340). Fresh (unfixed) blood is perhaps best treated as follows (BizzozERO and TORRE, Archivio per le Scienze mediclie, vol. iv, No. 18, 1880, p. 390) : -Dilute a drop of blood with 0'75 per cent, salt solution in which has been dissolved a little methyl violet. This liquid in no wise affects the form of the elements, stains intensely the nucleus of the red corpuscles, and, in the white, stains the nucleus intensely, and the protoplasm less intensely. May be used for the study of bone-marrow and spleen. For the staining of the blood-plates of BIZZOZERO, this observer (Arch. f. path. Anat. u. Phys. ; Zeit. f. wiss. Mik., SOME OTHER HISTOLOGICAL METHODS. 459 1884, p. 389) employs a 0.02 per cent, solution of methyl violet in salt solution, or a 1 : 3000 solution of gentian violet. TOISON (Journ. Sci. med. de Lille, fev., 1885 ; Zeit. f. wiss. Mik., 1885, p. 398) recommends that blood be mixed with the following fluid : Distilled water . . . .160 c.c. Glycerin (neutral, 30 Baume) . 30 Pure sulphate of sodium . . 8 grammes. Pure chloride of sodium . . 1 gramme. Methyl violet 5 B . . 0*25 (The methyl violet is to be dissolved in the glycerin with one half of the water added to it ; the two salts are to be dissolved in the other half of the water, and the two solu- tions are to be mixed and filtered.) White blood-cor- puscles stain in this medium in five or ten minutes; the maximum of coloration is attained in from twenty to thirty minutes. White blood- corpuscles, violet; red blood-cor- puscles, greenish. FERRIER'S liquid is said to have a sp. gr. similar to that of liquor sanguinis. Fuchsin, 1 grm. ; water, 150 c.c. ; rectified spirit, 50 c.c. ; dissolve, and add glycerin, 200 c.c. (from Squire's Methods and Formulae, p. 39). LECLEEQ'S fuchsin followed by malachite green, or Congo followed by gentian and eosin, see Bull. Soc. Beige de Mic., xvi, 1890, p. 61: or Journ. Roy. Mic. /Soc., 1890, p. 675. DEKHUYSEX'S methylen blue and acid fuchsin mixture, see Verhandl. Anat. Gesellsch., 1892, p. 90; or Journ. Roy. Mic. Soc., 1893, p. 116. It goes without saying that the EHRLICH-BIONDI mixture, ( 306), and Ehrlich's triacid and acidophilous mixtures, ( 307 and 323) will be found most valuable reagents in many haematological researches. LOWIT (Ziegler's Beitr. z. path. Anat., &c., x, 1891, p. 214; Zeit. f. wiss. Milt., viii, 3, 1891, p. 371) obtained instructive results by staining sublimate preparations for one to two minutes in a concentrated Ehrlich- Biondi solution, and examining in water or glycerin. For details as to the reactions of the granules of leucocytes and of Lo'wit's " pyrenogenous " corpuscles, see the original paper; also EHBLICH'S ' Methodologische Beitr. z. Physiol., &c., der Leucocyten," in Zeit.f. klin. Med., \, 1880, 3, p. 558 ; cf. Zeit. /. wiss. Mik., i, 1884, p. 382, and later papers of Lowitz in Anat. Anz., vi, 1891, p. 344, and Arch.f. mik. Anat., xxxviii, 1891, p. 524 (Zeit.f. wiss. Mik., ix, 2, 1892, p. 233). Also, LOVELL 460 CHAPTER XXXIV. GULLAND (Journ. of PhysioL, xix, 1896, p. 385). For some details con- cerning the carrying out of EHKLICH'S cover-glass preparation method, see EEINBACH, in Arcli.f. Tclin. Chirurg., xlvi, 1893, p. 486 (Zeit.f. wiss. Mik., xi, 2, 1894, p. 258). For the process employed by Foi (Festschr. R. VircJiow (jewidm., &c., 1891, i, p. 481; Zeit.f. wiss. jflik., ix, 2, 1892, p. 227), see 352. The elaborate paper of MULLER mentioned in 810 is too rich in detail to bear abstracting here. A novelty in it is the impregnation of cover-glass preparations with gold chloride by Ranvier's formic acid process. 813, WEIGEET'S Fibrin Stain (Fortschr. d. Med., v, 1887, No. 8, p. 228 ; Zeit.f. wiss. Mik., iv, 4, 1887, p. 512). Sections (alcohol material) are stained in a saturated solution of gen- tian or methyl violet in anilin water ( 278). They are brought on to a slide and mopped up with blotting-paper, and solution of LUGOL is poured on to them. After this has been allowed to act for a sufficient time they are differentiated and cleared in anilin oil without previous dehydration with alcohol. They are simply mopped up with blotting-paper, and a drop of anilin is poured on to them. The anilin soon becomes dark, and is then changed for fresh once or twice. The sections are by this means differentiated and cleared at the same time. When this has been done, the anilin is thoroughly removed by means of xylol, and a drop of balsam and a cover are added. This stain may be applied to celloidin sections with- out previous removal of the celloidin. Fibrin is sharply stained in blue, bacteria and fungi are also stained of a very dark blue. See also the modifications of this method by KROMAYER ( 665) and BENECKE ( 792). 814. Demonstration of Blood-plates of Bizzozero (KEMP, Studies fr. the Biol. Lab. Johns Hopkins Univ., May, 1886, iii, No. 6; Nature, 1886, p. 132). The mere demonstration of the blood- plates of Bizzozero is easy enough. A somewhat large drop of blood is placed on a slide, and quickly washed with a small stream of normal salt solution. The blood-plates are not washed away, because they have the property of adhering to glass ; and on bringing the slide under the microscope they will be seen in large numbers. If it be desired to make permanent preparations of them, they should first be fixed. This is done by putting a drop of osmic acid solution on the finger before pricking it. SOME OTHER HISTOLOGICAL METHODS. 461 For BIZZOZERO'S recent methods for the numeration of these elements and for the study of their regeneration, see his paper in Festschr. R. Vir- chow gewidm., &c., 1, 1891, p. 459 ; or the report of the methods in Zeit. f. wise. Mik., ix, 2, 1892, p. 229. For the application of some digestion methods to the study of blood-plates, see LILIENFELD, Arch.f.Anat.u. PhysioL, Physiol. Abth., 1892, p. 115; or Zeit.f. iviss. Mik.. ix, 3, 1893, p. 363. For methods for obtaining large quantities of blood-plates, see DEUEBIN, Die Herstellung wagbarer Mengen von Blutplattchen, Jurjew, 1893 ; Zeit. f. wise. Mile., x, 4, 1893, p. 493. 815. BIONDI'S Section Method for Blood (Arch.f. mile. Anat., xxxi, 1888, p. 103). A process of imbedding in agar-agar. See last edition, or Journ. Roy. Mic. Soc., 1888, pp. 313, 659. SCHIEFFEBDECKEE says that celloidin may be employed (see his Gewebelehre, p. 389). Glands. 816. Mucin. HOYER, who has made a special study of the staining reactions of mucin in tissues (Arch. f. mik. Anat., xxxvi, 1890, p. 310; see also Zeit.f. wiss. Mik., viii, 1, 1891, p. 67), has the following conclusions : The mucin of mucus cells and goblet cells, both of Verte- brates and Invertebrates, stains with basic tar colours, but not with acid tar colours (see above, 268). More or less specific stains of mucin are obtained, for instance, with hydrochlorate or nitrate of rosanilin, commercial fuchsin, Griibler's " neutral " fuchsin (" n. Unna"), magenta, Magdala red, iodine green, methyl green, dahlia, methyl violet, gen- tian, iodine violet, crystal violet, Victoria blue. A similar reaction is obtained with alum-haematoxylin solutions, whilst carmine behaves like the acid coal-tar dyes, and affords no stain. HOYER obtained his best results by means of thionin (violet of Lauth), which gives a double stain, the tissues blue, the mucin elements ruddy violet. The dye called amethyst, prepared by Geigy and Co., of Bale, is a good succedaneum, and so are toluidin blue (obtainable from Griibler) or the phenylen blue of Oehler in Offenbach, and the p-phenylen blue of the Hochst manufactory. Like thionin, all these give metachromatic stains. Results less brilliant than those given by the above- mentioned stains, but nevertheless excellent, are obtained by means of methylen blue or Bismarck brown. Methylen green and safranin also give good reactions, but are somewhat 462 CHAPTER XXXIV. inconstant in their effects. Methylen blue is particularly useful from its power of bringing out the merest traces of mucin. All of these colours may be used in the same way. Speci- mens should be fixed for two to eight hours in 5 per cent, sublimate solution, imbedded in paraffin, cut, and the sections stained for five to fifteen minutes in a very dilute aqueous solution of the dye (two drops of saturated solution to 5 c.c. of water). It is theoretically interesting to observe that hyaline carti- lage, the jelly of Wharton, and the Mastzellen of Ehrlich give the same reactions with basic dyes as mucin does, even their inetachromatic reactions being identical. These conclusions had already been in part formulated by SUSSDORF (Deutsche Zeit. f. Thiermed., xiv, pp. 345, 349 ; see Zeit.f. wiss.Mik., vi, 2, 1889, p. 205). See also the important series of papers by BIZZOZERO, " Sulle ghiandole tubulari deltubo gastro-enterico," &c., in the Atti R. Accad. di Sci. di Torino, 1889 to 1892 ; reports in Zeit. f. wiss. Mik., vii, 1, 1890, p. 61 ; andix, 2, 1892, p. 219. As regards the safranin reaction, it is well to note that it is not obtained with all brands of the dye ; that of Bindschedler and Busch, in Bale, gives it, whilst safranin of Griibler does not. For the distinctive reactions of old and young mucin see the original, or the last-quoted report of the Zeit. f. iviss. Mik. The subject has recently been carefully investigated by PAUL MAYER ("Ueber Schleimfarbung," Mitth. Zool Stat. Neapel, xii, 2, 1896, p. 303). As regards the haematein reac- tion, he establishes the following propositions. 1. If the staining solution contain free acid, as in Mayer's acid haemalum or Ehrlich's haematoxylin, then as a general rule the secretion of mucus gland cells does not stain in it. 2. If it contain a relatively large proportion of alum, say 5 per cent., as in hsemalum, or more, then also as a general rule the secretion of mucus gland cells does not stain in it. 3. If it contain a relatively small proportion of alum, but a large proportion of haematein, then it stains many sorts of mucus, and at the same time stains chro matin strongly. As the result of his investigations Mayer gives the follow- ing formulae for specific stains of the secretion of mucus cells SOME OTHER HISTOLOG1CAL METHODS. 463 (the distinction between mucin and mucigen is not taken in this paper). Mucicarmine. One gramme of carmine is rubbed up in a capsule with 0*5 gramme of aluminium chloride (must be dry, not damp and yellow), and 2 c.c. of distilled water. The capsule is heated over a small flame for two minutes, until the originally light-red mixture has become quite dark. Stir thoroughly. The liquid having become thick, add a little 50 per cent, alcohol, in which the warm mass ought to dissolve easily, and rinse the whole with more alcohol into a bottle. Make up to 100 c.c. with 50 per cent, alcohol, let it stand for at least twenty-four hours, and filter. This gives a stock solution, which is as a rule to be diluted for use tenfold with distilled or tap water. Exceptionally it may be diluted instead five or ten fold with alcohol of 50 per cent, or 70 per cent. The stock solution may be obtained from Griibler and Co. Mucicarmine stains in sections or thin membranes mucus only. Muchaematein. Hasmatein 0'2 g., aluminium chloride 0*1 g., glycerin 40 c.c., water 60 c.c. Rub up the haematein in a mortar with a few drops of the glycerin, then add the other ingredients. If it be desired to avoid employing a watery liquid, an alcoholic solution may be made in the same way by dissolving the hasmatein and aluminium chloride in 100 c.c. of 70 per cent, alcohol, with or without the addition of two drops of nitric acid. Either of these solutions is a nearly pure mucus stain for sections or thin membranes. Mayer's paper contains such other matter not abstracted here, which will well repay careful study. A fairly detailed abstract may be found in Zeit. f. wiss. Mik. y xiii, 1, 1896, p. 38. For the methods of UNNA for obtaining a specific stain of mucin by means of polychromatic methylen blue, see ibid., p. 42, or the original, Monatsh. f.prakt. DermatoL, xx, 1895, p. 365. 817. Goblet Cells. So far as these contain mucin they give the reactions above described (see FLEMMING, Zeit. f. wiss. Mik.j 1885, p. 519; and PAULSEN, ibid., p. 520). But the reactions appear to be different for different animals. Thus PANETH (Arch. f. mik. Anat., xxxi, 1888, p. 113, et. seq.) 464 CHAPTEE XXXIV. found that in the small intestine of the mouse the contents of the goblet cells did not stain with Bohmer's ha3matoxylin. And the goblet cells of the small intestine of man did not stain with safranin. KANVIEB, in a paper too long to be abstracted bere (Comptes rend., 1887, 3, p. 145; see also Zeit.f. wiss. Mik., v, 2, 1888, p. 233), describes a specific reation of perrutbenic acid (Ru0 4 ) on goblet cells. By treating the pharyn- geal mucosa of the frog first for ten to twelve hours with vapour of osmium, and then for three minutes with vapours of perruthenic acid, the goblet cells are brought out with remarkable distinctness. The contained mucigen is stained black, but the vacuoles are unstained. Since perruthenic acid is very rapidly reduced by organic matter, Ranvier regards this reaction as a proof that the vacuoles do not contain any organic substance, but probably only water and inorganic salts. For detailed instructions for the study of goblet cells, see LIST, in Arch.f. mik. Anat., xxvii, 1886, p. 481. 818. Salivary Glands. SOLGEE (TJnters. z. Naturlehre d. Henschen, xv, 5 and 6, pp. 2 15; Zeit.f. wiss. Mik., xii, 3, 1896, p. 374) demonstrates the granules in serous cells and half-moons of the submaxillary gland by means of formal- dehyde. The gland is hardened for two days or more in a 10 per cent, solution of formol, and may then either be sectioned and examined in the wet way or imbedded in paraffin, and the sections stained with hgernatoxylin of DE LA- FIELD or of EHRLICH, the granules taking the stain. See also 348. 819. Liver. See hereon the important papers of BANVIER, " Les membranes muqueuses et le syst. glandulaire" in the Journ. deMicrogr., ix, x, 1885-6; IGACUSCHI, in Arch.f. path. Anat., xcvii, p. 142, or Zeit. f. iviss. Mik., 1885, p. 243 (gold process for study of fibrous networks) ; KUPFFER, Sitzb. Ges. f. Morph., &c., Miinchen, Juli, 1889, or Zeit. f. wiss. Mik., vi, 4, 1889, p. 506 (hesmatoxylin stain for demonstration of ulti- mate bile-ducts, and application of Golgi's silver bichromate method to the same object and to the study of fibrous net- works) ; OPPEL, Anat. Anz., v, 1890, p. 143 ; vi, 1891, p. 165 ; and Zeit. f. wiss. Mik., vii, 2, 1890, p. 222 ; viii, 2, 1891, p. 224 (also concerning the application of G-olgi's process to the above objects). SOME OTHKR HISTOLOGICAL METHODS. 465 820. Other Methods for Glandular Structures. Amongst numerous important papers that cannot be quoted here, see RANVIER, " Le mecanisme de la secretion/' in Journ. de Microgr., x, 1886-7, and the valuable papers of HEIDENHAIN in Pfliiger's Archiv. The peculiar applicability of the EHRLICH-BIONDI stain to this kind of work hardly needs pointing out. 30 CHAPTER XXXV. SOME ZOOLOGICAL METHODS. 821. Introduction. It is the purpose of this chapter to describe such variations of the usual histologies! processes as are indicated for the study of organisms which offer special difficulties. The methods described are all of them such as give results applicable to histological study, and no account has been taken of such methods as are merely useful for the preparation of organisms for museum specimens or for coarse dissection. But of course in many cases the methods recom- mended for histological work will be found to give admirable results for the preparation of show specimens, and may be used for that purpose if desired. A further word here as to the employment of formaldehyde. The introduction of this reagent has singularly facilitated museum work. I consider it to be established that as a pre- servative agent for delicate organisms, it is in many cases greatly superior to alcohol. For as there is not even a partial dehydration, all risk of shrinkage on that head is done away with. But it is only, I think, as a preservative agent that it oifers signal advantages. As a fixing agent, even for mere museum specimens, it will only, I think, be found to give good results in a very few cases. In the vast majority of cases, organisms should first be fixed secundum artem, in one of the tried and appropriate fixing agents, and then, after proper washing if necessary, be transferred to solution of formaldehyde for preservation. A valuable paper giving an account of a number of the processes employed in the Naples Zoological Station for the preservation of marine animals has been published by SALVATOBE Lo BIANCO in Mitth. Zool. Stat. Neapel, ix, 1890, p. 435. Eeferences to the work of S. Lo BIANCO in the remainder of this chapter are to that paper. An abstract of it is contained in Amer. Natural, xxiv, 1890, p. 856, and Jowrn. Roy. Hie. Soc., 1891, p. 133, and a very full account in Zeii.f. wiss. Mik., viii, 1, 1891, p. 54. SOME ZOOLOGICAL METHODS. 467 Tunicata. 822. Fixation of Tunicata. A method of SALVATORE Lo BIANCO for killing simple Ascidians in an extended state has been given above, 22. In the paper quoted above this plan is recommended for Oiona, Ascidia, and Rhopalea. But many other forms, such as Clavellina, Perophora, Phallusia, Molgula, Cynthia, &c., should first be narcotised by treatment for from three to twelve hours with chloral hydrate (1 : 1000 in sea water), then killed in a mixture containing chromic acid of 1 per cent. 10 parts, acetic acid 100 parts, and finally hardened in 1 per cent, chromic acid. The small proportion of chromic acid in the above mixture is stated to be sufficient to neu- tralise the swelling action. of the acetic acid. The compound Ascidians with contractile zooids are difficult to manage if one does not go the right way to work. The best process known to me is the following (due to VAN BEN- EDEN, kindly communicated to me by Dr. C. Maurice). Place the corms in clean sea water, and leave them alone for a few hours, in order that the zooids may become fully extended. Seize the corms with your fingers, and plunge them suddenly into glacial acetic acid. Leave them there for two, four, or six minutes, according to the size of the corms (which, of course, you will have taken care to select of as small a size as possible). Take them out of the acid with your fingers (or in some manner that may dispense with the employment of steel instruments, which would blacken the tissues) and bring them into 50 per cent, alcohol. Wash them thoroughly in that, and then bring them in the usual way through successively stronger alcohols. I most strongly recommend this process, which gives admirably preserved preparations quite free from any opacity either in the tissues or the tunic. The acid will not hurt the fingers if they be washed immediately. S. Lo BIANCO recommends for this group the chloral hydrate process, followed by fixation with sublimate or chromo-acetic acid. Small pelagic Tunicates are very easily fixed with osmic acid or acid sublimate solution, with the exception of Anchinia. The not very numerous preparations I have made of this ex- ceedingly delicate form have all been unsatisfactory. And 468 CHAPTER XXXV. some other similar forms may be found difficult. I have had a striking failure with Salpa virgula, which I fixed with "Flemming," and got a very poor preparation. The very similar S. pinnata is fixed perfectly in this medium. Molluscoida. 823. Bryozoa. For some methods of killing and fixing see 11, 18, and 19. S. Lo BIANCO employs for Pedicellina and Loxosoma the chloral hydrate method, fixing with sub- limate. For Flustra, Cellepora, Bugula, Zoobothrium, he employs the alcohol method of EISIG, 16. For Crist at ella } see 17. Mollusca. 824. Fixation of Mollusca. Two groups at least amongst the Mollusca offer considerable difficulties in the way of fix- ation Lamellibranchiata and Gastropoda. If it be attempted to take living and normal Lamellibran- chiata from the water they are contained in, in order to throw them into a fixing solution, they invariably withdraw their siphon and foot, shut their valves, and die in a state of con- traction. And if it be attempted to open the shell by force after death, the mantle is generally injured, and it is im- possible to get the foot and siphon into the extended state. DE CASTELLARNAU (La Estacion Zoolog. de Napoles, Madrid, 1885) advises that they be killed by the method of EISIG and ANDRES described for Actiniae in 16. Before dying, the animals protrude largely their feet, siphons, branchiae, and tentacles, and die with their shells open. They may be fixed as soon as insensibility has supervened, by bringing them into picro-sulphuric acid, or , some other rapidly killing fixing agent. The same methods recommended for Lamellibranchiata sometimes give good results with Gastropoda. The asphyxia- tion method has been described in 23. S. Lo BIANCO advises that Lamellibranchiata, Prosobran- chiata, and, amongst the Heteropoda, Atlantidas, be narcotised with 70 per cent, alcohol, 16. Opisthobranchiata ought not to give much trouble, and I recommend sudden killing with liquid of Perenyi, or the acetic acid method, 822. Aplysia SOME ZOOLOGICAL METHODS. 469 may first be narcotised by subcutaneous injection of about 1 c.c. of a 5 to 10 per cent, solution of hydro chlorate of cocain (ROBERT, Bull Sclent, de la France, &c., 1890, p. 449 ; Zeit.f. wiss. Mik., ix, 2, 1892, p. 216). For Ancylusj ANDRE (Rev. Suisse de Zool. y 1, 1893, p. 429) recommends boiling water. For Pteropoda in general, liquid of Perenyi. Creseis is a difficult form. S. Lo BIANCO advises the alcohol method, 16. Xote the hydroxylainin method of HOFER, 20. For preservation it may be noted that for Heteropoda and Pteropoda, formaldehyde (preceded by due fixation in a chromic or sublimate solution) is an admirable medium, so far at least as macroscopic appearances are con- cerned, and for this purpose superior to alcohol. 825. Terrestrial Gastropods. The asphyxiation method has been described in 23. The quantity of mucus that exists in the integument of Gastropoda is often a serious obstacle in the way of preparation. MAKCHI (Arch.f. mik. Anai., 1867, p. 204) finds that if a living Limax be thrown into moderately concentrated salt solution it will throw off enormous quantities of mucus, and die in a few hours. The epidermis will be found well preserved. If the animal be thrown into osmic acid or Miiller's solution, if I understand the writer justly, no secretion of mucus will occur. 826. Eyes of Gastropoda (FLEMMING,X!>T/Z,./. mik. Anat., 1870, p. 441). The first difficulty here is to obtain the excision of an exserted eye. It is impossible to sever the exserted peduncle in a living animal without its retracting at least partially before the cut is completed. Xever mind that ; make a rapid cut at the base, and throw the organ into very dilute chromic acid, or 4 per cent, bichromate ; after a short time it will evaginate, and remain as completely erect as if alive. Harden in 1 per cent, osmium, in alcohol, or in bichromate. CARRIERS (Zool. Anz., 1886, p. 221) gives the following in- structions : Remove the eye, together with a portion of the tentacle, and fix it by exposing it for some minutes to vapour of osmium. Make sections according to the usual methods and fix them on a slide with Schallibauin's collodion. Stain them with picro-carmine ; or first depigment them by very careful treatment with very dilute eau de Javelle, and then stain with picro-carmine. Mount in dammar. Successful 470 CHAPTER XXXV. preparations show the tissues perfectly preserved; but Car- riere has only been able to make the depigmentation process succeed with Helix pom atia; with Prosobranchiata he failed. 827. Eyes of Cephalopoda and Heteropoda (G-RENACHER, Abh. naturf. Ges. Halle-a.-S., Bd. xvi; Zeit. f. wiss. Mik., 1885, p. 244). Fix iu picro-sulphuric acid, or in a saturated solu- tion of corrosive sublimate in picro-sulphuric acid (this mix- ture is especially useful for Octopus, Eledone, and Sepia, but does not succeed with the pelagic forms, such as Loligo, Ommatostrephes, and Rossia). Depigment the specimens with hydrochloric acid (in preference to the nitric acid used by Grenadier in former researches) . The mixture 599 may also be used. The operation of depigmentation may be combined with that of staining ; if you stain with borax-carmine and wash out in the last-mentioned mixture the pigment will be found to be removed quicker than the stain is washed out. But this process is delicate, and requires a practised hand. The operation of depigmentation may be carried out on sec- tions, but it is better to use portions of retina of 2 to 5 mm. in thickness. Grenadier mounted his preparations in castor oil, see 447. Similar methods are recommended by the same author for the eyes of Heteropoda (see Abh. naturf. Ges. Halle-a.-S., 1886; Zeit.f. wiss. Mik., 1886, p. 243). 828. Eyes of Chitonidae (MOSELEY, Quart. Journ. Mic. Sci., 1885, p. 40). Moseley worked by decalcifying the shell with nitric acid of 3 to 4 per cent, and making sections. 829. Eyes of Pecten and other Forms, see PATTEN, in Mitth. Zool. Stat. Neapel, vi, 4, 1886, p. 733. 830. Shell. Sections of non-decalcified shell are easily obtained by the usual methods of grinding, or, which is often a better plan, by the methods of v. Koch or Ehrenbauin, 174, 175. For sections of decalcified shell, MOSELEY, who has had great experience of this kind of work, particularly recommends the method of decalcification given above, 828. SOME ZOOLOGICAL METHODS. 471 831. Injection of Acephala (FLEMMIXG, Arch. f. mik. Anat., J878, p. 252). To kill the animals freeze them in a salt-and- ice mixture, and throw them for half an hour into lukewarm water. They will be' found dead, and in a fit state for in- jection. Chloroform and ether are useless (but see 20). The injection-pipe may be tied in the heart ; but when this has been accomplished there remains the problem of occlud- ing cut vessels that it is impossible to tie. To this end, after the pipe has been tied, the entire animal is filled and covered up with plaster of Paris. As soon as the plaster has hardened the injection may be proceeded with. 832. Maceration Methods for Epithelium of Mollusca. For the study of ciliated epithelium the following methods are recom- mended by ENGELMANN (Pfliiger's Arch., xxiii, 1880, p. 505) : Cyclas Cornea (intestine), maceration in osmic acid of 0*2 per cent, (after having warmed the animal for a short time to 45 to 50 C.). Also, concentrated boracic acid solution. The Intra-cellular Processes of the Cilia. The entire intra- cellular fibre apparatus may be isolated by teasing fresh epi- the Humfrom the intestine of a Lamellibranch (e. g. Anodonta) in either bichromate of potash of 4 per cent., or salt solution of 10 per cent. To get good views of the apparatus in situ in the body of the cell, macerate for not more than an hour in concentrated solution of boracic or salicylic acid. Very dilute osmic acid (e.g. O'l per cent.) gives also good results. The " lateral cells " of the gills are best treated with strong boracic acid solution (5 parts cold saturated aqueous solution to 1 part water). BELA HALLER'S Mixture, see 554. BEOCK'S Medium, 549. MOBIUS'S Media, 550 ; the second of these is much recom- mended by DROOST (MorphoL Jahrb., xii, 2, 1866, p. 163) for Cardium and My a. See also the media recommended by PATTEN (Mitth. Zool. Stat. Neapel, vi, 4, 1886, p. 736). Sulphuric acid, 40 drops to 50 grammes of water, is here recommended as a most valuable macerating and preservative agent. Entire molluscs, without the shell, may be kept in it for months. 472 CHAPTER XXXV. Arthropoda. 833. General Methods for Arthropoda. It may safely be stated that, as general methods for the study of chitinous structures, the methods worked out by Paul Mayer (see 7 and 9, and also 75, 240, and 241) are superior to all others. It is absolutely necessary that all processes of fixation, wash- ing, and staining should be done with fluids possessing great penetrating power. Hence picric acid combinations should in general be used for fixing, and alcoholic fluids for washing and staining. Concentrated picro- sulphuric acid is the most generally useful fixative, 70 per cent, alcohol is the most useful strength for washing out, and tincture of cochineal in alcohol of 70 per cent. ( 241) is a very generally useful stain- ing fluid. Mayer's hsemacalcium ( 257) may sometimes be preferable, and alcoholic carmine and borax-carmine will occasionally give more satisfactory results. Alcoholic picro-sulphuric acid may be indicated for fixing in some cases. Some forms are very satisfactorily fixed with sublimate. Such are the Copepoda and the larvse of Decapoda. It is sometimes indicated to use the sublimate in alcoholic solution. Some Copepoda, however (Copilia, Sapphirina), are better preserved by means of weak osmic acid, and so are the Ostra- coda. In many cases the osmic acid will produce a sufficient differentiation of the tissues, so that further staining may be dispensed with ; Copilia and Phyllosoma are examples of forms that may be prepared in this simple manner. The pyrogallic process ( 377) may often prove helpful in the study of such forms. For Ostracoda, MULLER (.Fauna u. Flora d. Golfes von Neapel, xxi (Ostracoda) 1894, p. 8) recommends fixing in a mixture of 5 parts of ether and 1 of absolute alcohol, followed by 70 per cent, alcohol. 834. Methods for Clearing and Softening Chitin. The employ- ment of eau de Javelle or eau de Labarraque, as suggested by Looss, for making chitin transparent and permeable to reagents has been described above, 570. LIST (Zeit. f. wiss. Mik., 1886, p. 212) has obtained good results with Coccidae by treating them (after hardening) fo x SOME ZOOLOGICAL METHODS. 473 eighteen to twenty-four hours with eau de Javelle, diluted with four volumes of water. After washing out with water, the objects may be dehydrated with alcohol and imbedded in paraffin, the chitin being sufficiently softened to allow of their being penetrated and good sections being obtained. You may stain before imbedding, with alum-carmine or picro-carmine (five to six days). The same methods are applicable to the preparation of the ova of Insecta for instance, Periplaneta (see MORGAN, Am. Man. Hie. Journ., ix, 1888, p. 234). 835. Other Depigmentation Methods. Besides the depig- nientatiou processes discussed in Chap. XXVI, the following methods are available. SAZEPIN'S Method for Antennae of Chilognatha (Mem. Acad. Imp. St. Petersb., xxxii, 9, 1884, pp. 11, 12). Sazepin treated antennae that have been dehydrated with alcohol by steeping them in chloroform. The reaction is slow, the chitin becomes gradually less opaque, but the pigment does not entirely disappear. In order to remove the last trace of it, it will be sufficient if a drop of fuming nitric acid be now added to the chloroform. The mixture must be occa- sionally agitated, in order to prevent the acid from floating on the chloroform. The reaction is complete in twenty-four hours. Employed in this manner, nitric acid has no injurious action on tissues. 836. Eyes of Arthropods. LANKESTER and BOURNE (Quart. Journ. Mic. Sci., 1883, p. 180) prepared the eyes of Limulus as follows : Alcohol, turpentine, paraffin ; sections made and carefully depigmented under the microscope with nitric acid of 5 to 10 per cent., then mounted in balsam, some after staining with borax-carmine, others unstained. Non-depig- mented sections also mounted in the same manner. HICKSOX (ibid., 1885, p. 243) prepared the eye of a fly as follows : Remove the posterior wall of the head, and expose the rest, with the eyes in situ, for twenty minutes to vapour of osmium. Wash for a few minutes in 60 per cent, alcohol. Harden in absolute alcohol. Make sections. To depigment them, mount them on a slide with Mayer's albumen, remove 474 CHAPTER XXXV. the paraffin with turpentine, treat them with absolute alcohol, and invert the slide over a capsule containing* 90 per cent, alcohol to which a few drops of strong nitric acid have been added. Nitrous vapours are freely given off, and the pigment dissolves. The reaction may be stopped at any moment by washing with pure alcohol. For dissociation preparations, put the eye or the optic nerve for twenty-four hours into 5 per cent, solution of chloral hydrate, tease, and mount in glycerin. If the elements of the teased tissues be fixed to. the slide by means of Mayer's albumen, they may be washed with alcohol and stained in situ, or they may be depigmented before staining. The methods of PAEKEE for eyes of scorpions have been given, 593. For the eye of Homarus see Bull. Mus. Comp. Zool., Gam- bridge, U.S.A., xx, 1890, p. 1 (Zeit.f. u-iss. Mik., viii, 1, p. 82). In a later paper (Mitth. Zool. Stat. Neapel,x.\\, 1895, p. 1 : Zeit.f. uiss. Mik., xii, 4, 1896, p. 496) PAEKEE describes the application of the methylen-blue method to the study of the retina and optic ganglia in Decapods, especially in Astacus. He injected 0*1 c.c. of a 0'2 per cent, solution into the ventral sinus. After twelve to fifteen hours the animals were killed, the ganglia quickly dissected out and brought into aqueous solution of corrosive sublimate, which fixes the stain (see 294) . After fixation the preparations were either dehydrated by the methylal method, described in 294, or in a modification of that process without the methylal. A stock of 8 per cent, solution of corrosive sublimate in absolute alcohol is prepared, and from this are prepared weaker grades of sublimate- alcohol, by dilution with saturated aqueous sublimate solution. For instance, to make 30 per oent. sublimate-alcohol you take 30 c.c. of the absolute alcohol solution and 70 c.c. of the aqueous solution, and so on. The preparations are dehydrated by passing them through a succession of grades of sublimate- alcohol thus prepared, and of the strengths of 30 per cent., 50 per cent., 70 per cent., and 95 per cent., remaining in each for a quarter of an hour. They then come into the 8 per cent, solution in absolute alcohol for an hour, then into a mixture of one part of this solution with one of xylol, remain- ing there also for an hour, and finally into pure xylol, in which they may remain till imbedded. SOME ZOOLOGICAL METHODS. 475 For the methods of PURCELL for the eyes of Phalangida, see Zeit. f. wiss. Zool., Iviii, 1894, p. 1 ; Zeit. f. wiss. Mik., xii, 1, 1895, p. 44. He has the following stain. The objects are brought for twenty minutes into 50 per cent, alcohol warmed to 45 or 50 C., and saturated with picric acid. The pigment dissolves in this solution and stains the nuclei and some other parts of the rhabdoms, so that no further stain is required. See also the methods of VIALLANES (Ann. d. Sci. Nat., xiii, 1892, p. 354; Journ. Roy. 3//c. Soc., 1893, p. 260). 837. Nerve and Muscle of Arctiscoida (DoYEBE, Arch. f. mik. Anat., 1865, p. 105). A score or so of Milnesium tardigradum are collected (it is well to have a large number, as the process by no means succeeds with all individuals) and put into a test-tube with water that has been deprived of its air by boiling. A drop of oil is run on to the surface of the water, so as thoroughly to exclude the air. After twenty-four to forty-eight hours the animals will be found, not dead, but fixed and extended in a cataleptic state ; the circulation of the perivisceral fluid has ceased, the pigment of the cuticle has disappeared or collected into patches that are no hindrance to observation, the entire animal has gained in transparency, and the nervous and muscular systems stand boldly out. The animals are examined in boiled water, unless it be wished to study the phenomena of resuscitation, in which case spring water should be used. 838. Phalangida (RossLEB ; see previous editions, or Zeit.f. wiss. Zool., xxxvi, 1882, p. 672). 839. Macrotoma plumbea (SomiER ; see previous editions, or Inaug. Diss., 1884, p. 4; Zeit.f. n-iss. Mik., 1885, p. 234). 840. BETHE'S Stain for Chitin (Zool. Jahrb., Abth. f. Anat., viii, 1895, p. 544; Zeit. f. wiss. Mik., xii, 4, 1896, p. 498). Desirous of staining the chitinous hairs and plates of the otocyst of My sis } Bethe found advantage in employing a process, borrowed from industrial dyeing, in which anilin black is produced on the tissue itself. Anilin black is a pro- duct of the oxidation of anilin hydrochloride. Bethe proceeds as follows : Series of sections mounted on a slide are put for three or four minutes into a freshly prepared 10 per cent, solution of anilin hydrochloride, to which has been added one drop of hydrochloric acid for every 10 c.c. They are then rinsed in water, and the slide is put with the sec- tions downwards into 10 per cent, solution of bichromate of OF THF 476 CHAPTER XXXV. potash. The stain quickly begins to show itself, but is at first in general not sufficiently intense. The process is then repeated until the desired intensity of stain is obtained, care being taken to rinse the sections well with water after each of the operations, in order to avoid the formation of precipitates. The stain is at first green, but becomes blue in tap water or alcohol containing ammonia. The same paper contains a hint concerning the preparation of telsons for section cutting. They are put for eight to fourteen days into 40 per cent, alcohol, to which nitric acid is gradually added, so that by the end of that time they have been brought into alcohol containing 20 per cent, of the acid. This softens the chitin, and somewhat breaks down the structure of the otolith, so that good sections through it are occasionally obtained. Vermes. 841. Cestodes. This group must of course be chiefly studied by the usual section methods. As pointed out by VOGT and YUNG (Traite d'Anat. comp. prat., p. 204), the observation of the living animal may be of service, especially in the study of the excretory system. And, as shown by PINTNER, taeniae may be preserved alive for several days in common water to which a little white of egg has been added. LONNBERG (Centralb. f. Bakteriol. u. Parasitenk., xi, 1892, p. 89; Journ. Roy. Mic. Soc., 1892, p. 281) has kept Triseno- phorus nodulosns, a parasite of the pike, alive for a month in a slightly acid pepsin-peptone solution containing from 3 to 4 per cent, of nutritive matter, and less than 1 per cent, of Nad. ZEENECZE (Zool. Jdhrl., Abth. f. Anat., ix, 1895, p. 92; Zeit.f. wiss. Mile., xii, 4, 1896, p. 494) has employed with success the bichromate of silver im- pregnation of GOLGI. He kills Ligula in the osmio-bichromic mixture (4 : 1), impregnates as usual, makes sections in liver, and treats them by the hydroquinon process of KALLIUS. Besides the peripheral and central nervous system, muscle- fibres, parenchyma cells, and the excretory vascular system are impregnated. He has also obtained good results by the methylen-blue method. 842. Trematodes (FISCHER, Zeit. f. wiss. Zool., 1884, p. 1). Opisthotrema cochleare may be mounted entire in balsam. For sectioning, Fischer recommends imbedding in a mass SOME ZOOLOGICAL METHODS. 477 made by dissolving 15 parts of soap in 17'5 parts of 96 per cent, alcohol. This mass melts at about 60 C., penetrates very rapidly, and solidifies very quickly. The sections should be studied in glycerin. WEIGHT and MACALLUM (Journ. of Morph., i, 1887, p. 1) find that Sphyranura is for most purposes best fixed in liquid of Flemniing, and stained with alum-cochineal. Cercarise. SCHWAEZE (Zeit.f.wiss. Zool., xliii, 1886, p. 45) found that the only fixing agent that would preserve the his- tological detail of these forms was cold saturated sublimate solution warmed to 35 40 C. 843. Turbellaria. Methylen blue will in some cases be found useful for the study of living specimens. Fixing is difficult, and generally unsatisfactory. For Rhabdoccela, BBAUN (Zeit.f.wiss. Mik., iii, 1886, p. 398) proceeds as follows : For preparing entire animals, the specimens are got on to a slide, lightly flattened out with a cover, and killed by running under the cover a mixture of three parts of liquid of Lang with one of 1 per cent, osmic acid solution. Other fixing media than that described were not satisfactory. (BoHMiG, however, commenting on this, says that for some of the tissues, such as muscle and body paren- chyma, nitric acid and picro-sulphuric acid are very useful.) Sections may be made by the usual paraffin method. DELAGE (Arch, de Zool. exp. et gen., iv, 2, 1886; Zeit.f. wiss. Mik., iii, 2, 1886, p. 239 ; Journ. Roy. Mic. Soc., 1886, p. 1073) strongly recommends fixation (of Rhabdoccela Accela) by the osmium-carmine mixture, 237. Concentrated solution of sulphate of iron is also an excellent fixing medium. The animals (Convoluta) die in it fully extended. Liquid of Lang was not successful. For staining, he recommends either the osmium-carmine stain or impreg- nation with gold (i formic acid, two minutes ; 1 per cent, gold chloride, ten minutes ; 2 per cent, formic acid, two or three days in the dark. It is well to allow an excessive reduction to take place, and then lighten the stain by means of 1 per cent, solution of cyanide of potassium). BOHMIG, commenting on the above, says that he has obtained very in- structive images with Plagiostomidse fixed with sublimate and stained with osmium-carmine. VON GRAFF (Die Organisation d. Turbellaria Acoela, Leipzig, 1891 ; see Zeit. f. u-iss. Mik., ix, 1, 1892, p. 76) has the 478 CHAPTER XXXV. following remarks : Chromo-aceto-osmic acid, followed by haematoxylin, is good for the skin ; but even this method will not afford a satisfactory preservation of the Rhabdites, which in Accela and Alloiocoela seem to be destroyed by swelling, whilst in terrestrial and fresh- wal^r Planaria, Polyclada, and most Rhabdoccela they are better preserved. The same method is also good for the parenchyma of Amphichoerus cinereus, Convoluta paradoxa, and C. sordida. Sublimate is not good for these forms, but it is good for Convoluta Eoscoffensis. For some forms it is important to avoid picro- carmine, which destroys the central parenchyma. The ner- vous system may be investigated by the methods of DELAGE. For Dendroccela sublimate solutions, sometimes hot, appear indicated for fixing. CHICHKOFF (Arch, de BioL, xii, 1892, p. 438; Journ. Roy. Mic. Soc., 1893, p. 262) recommends the following for fresh-water Dendroccela : 2 per cent, sublimate solution, 6 parts; 15 per cent, acetic acid, 4 parts; pure nitric acid, 2 parts; 14 per cent, chloride of sodium, 8 parts; and 2 per cent, alum, 1 part. The animals are said to die in it without contraction. Note also the mixtures of LANG, 58. Mayer's tincture of cochineal, 241, may be found useful for the study of glands, for which purpose the Ehrlich- Biondi stain may also be employed. 844. Nemertina. After considerable experience of this difficult group I have to say that I know of no method of fixation that will certainly give good results. My best results have always been obtained with cold saturated sublimate solution, acidified with acetic acid. I have tried most of the energetically hardening fixing agents, such as the osmic and chromic mixtures, and do not recommend them for this group, for they seem (the chromic mixtures and perchloride of iron in particular) to act as irritants, and provoke such violent muscular contractions that the whole of the tissues are crushed out of shape by them. And, besides, they do not kill as quickly as sublimate. I have found it a good plan to decapitate the animals (in the larger forms), cut them up quickly into lengths (not too long), and throw these sharply into the sublimate, the mus- cular contractions being less energetic in segments that are no longer in connection with the cerebral ganglia. SOME ZOOLOGICAL METHODS. 479 Perhaps a better method than this will be found in the simple process, suggested to me by Prof. DU PLESSIS, of fixing with hot (almost boiling) water. On the few occasions on which I have tried it the animals have died in extension, without vomiting their proboscis ; and I think it is certainly worth trial, especially for the larger forms. I have tried FOETTINGER'S chloral hydrate method ( 18). My specimens died fairly extended, but vomited their pro- hoscides. According to S. Lo BIANCO narcotisation with a solution of 0*1 to 0*2 per cent, in sea water is found successful at Naples. DE CASTELLARNAU (Estacion Zool. de Napoles, p. 137) says that Xemerteans can be successfully narcotised by Eisig's alcohol method, described 16, and I think the process may be a good one for some of the larger forms. DENDY (see Journ. Roy. Mic. Soc., 1893, p. 116) has suc- ceeded with Geonemertes by exposing it for half a minute to the vapour of chloroform. Intra vitam staining with methylen blue may be found useful in some cases. For the application of the methylen- blue method to the study of the nervous system see 290. For staining fixed specimens in toto I hold that it is well- nigh necessary to employ alcoholic stains, for even the most delicate species are not satisfactorily penetrated by watery stains in any reasonable lapse of time. Borax-carmine or Mayer's alcoholic carmine may be recommended ; not so cochineal or haematoxylin stains, on account of the energy with which they are held by the mucin which in general exists in such great abundance in the skin of these animals. Sections by the paraffin method, after penetration with oil of cedar (chloroform will fail to penetrate sometimes after the lapse of weeks). 845. Nematodes. The extremely impermeable cuticle of these animals is a great obstacle to preparation. According to Looss (Zool.Anz., 1885, p. 318) this difficulty may be over- come by treating the animals (or their ova, which are in the same case) with eau de Javelle or eau de Labarraque, in the manner described in 570. For fixing, most recent authors recommend sublimate solu- 480 CHAPTER XXXV. tions ; chromic solutions seem to have a tendency to make the worms brittle. But according to ZUR STEASSEN (Zeit. f. wiss. ZooL, liv, p. 655) } Bradynema rigidum, a parasite of Aphodius fimetarius, ought to be fixed for at least twelve hours in mixture of Flemming. AUGSTEIN (Arch. f. Naturg., Jahrg. Ix, 1, 1894, p. 255 ; Zeit. f. wiss. Mik., xii, 2, 1895, p. 227) found that for Stron- gylus filoria the best fixing agent was MAYER'S picro-nitric acid. Staining is frequently difficult, and sometimes alcoholic carmine, 240, is the only thing that will give fair results. BBAUN (see Journ. Boy. Mic. Soc., 1885, p. 897) recommends that small unstained Nematodes be mounted in a mixture of 20 parts gelatin, 100 parts glycerin, 120 parts water, and 2 parts carbolic acid, which is melted at the moment of using. Canada balsam, curiously enough, is said to some- times make Nematodes opaque. 846. Demonstration of Living Trichinae (Barnes, Amer. Mon. Mic. Journ., xiv, 1893, p. 104 ; Journ. Roy. Mic. Soc., 1893, p. 406). A piece of trichinised muscle of the size of a pea should be placed in a bottle in a mixture of 3 gr. of pepsin, 2 dr. of water, and 2 minims of hydrochloric acid. The whole should be kept at body temperature for about three hours with occasional shaking. The flesh and cysts being dissolved, the fluid is poured into a conical glass, and allowed to settle ; the trichinae are drawn off from the bottom with a pipette, got on to a slide with water, and exa- mined on a hot stage. 847. Acanthocephali. It is very difficult to kill Echino- rhynci so as to have the animals duly extended and the tissues well preserved. Neither corrosive sublimate nor strong osmic acid will, as a rule, attain this end, even after preliminary intoxication with tobacco smoke or chloroform, the animal thus treated dying contracted. HAMANN, however (Jen. Zeit. f. Naturu\, xxv, 1890, p. 113; Zeit.f. wiss. Mik., viii, 2, 1891, p. 209), has succeeded with sublimate, and also with alcohol containing a little platinum chloride. SAEFFTIGEN (Morphol. Jahrb., x, 1884, 120; Journ. Roy. Mic. Soc. [N.S.], v, 1885, p. 147) obtained the best results by killing gradually with O'l per cent, osmic acid; the animals placed in this contract during the first hours, but stretch out again and die fully extended. Another method of killing is treatment with O'l per cent. SOME ZOOLOGICAL METHODS. 481 chromic acid ; Echinorhynci live for days in it, but eventually die fully extended. KEISER (Biblioth. ZooL, H. vii, 1 Halfte, 1891 ; Zeit. f. wiss. Mik., viii, 3, 1891, p. 363), found that a saturated aqueous solution of cyanide of mercury, warmed to 45 to 50 C., and allowed to act for from fifteen to sixty minutes, and then washed out with 70 per cent, alcohol, was the best of all fixing media for Acanthocephali. He also found the following mixture excellent : Picric acid ... 1 gramme. Cone, sulphuric acid . . 10 grammes. Chromic acid ... 1 gramme. Water .... 1000 grammes. To be warmed to 55 C., allowed to act for fifteen to twenty minutes, washed out for five to ten minutes with hot water, and afterwards for some days in 60 per cent, alcohol. 848. Gephyrea. YOGT and YUNG (Anat. comp. prat., p. 373) direct that Siphunculus nudus be kept for some days in per- fectly clean basins of sea water, in order that the intestine of the animals may be got free from sand, which would be an obstacle to section cutting, and then anaesthetised with chloro- form, under which treatment they die extended, and may be fixed as desired. WAED (Bull. Hus. Comp. Zool., Cambridge, Harvard Coll., xxi, 3, p. 144) found the best plan was to put the animals into a shallow dish with sea water and pour 5 per cent, alcohol in a thin film on to the surface of the water. After four to eight hours, if the animals make no contractions on being stimu- lated, they may be removed to 50 per cent, alcohol. S. Lo BIANCO says killing with 0'5 per cent, chromic acid or with O'l per cent, chloral hydrate in sea water may be tried, but either method is uncertain. Phascolosoma and Phoronis should be treated by the alcohol method. APEL (Zeit. /. wiss. ZooL, xlii, 1885, p. 461) says that Priapulus and Halicryptus can only be satisfactorily killed by heat. The animals may either be put into a vessel with sea water and be heated on a water-bath to 40 C. ; or they may be thrown as rapidly as possibly into boiling water, which paralyses them so that they can be quickly cut open and 31 482 CHAPTER XXXV. thrown into one third per cent, chromic acid, or picro- sulphuric acid. 849. Rotatoria. By far the most important method for the study of this group consists in the observation of the living animals. Great difficulty exists in the way of getting them to keep sufficiently quiet. VOGT and YUNG (Anat. com. prat., p. 420) say that a drop of solution of any of the soluble salts of strychnin run under the cover sometimes renders service. WEBEE (Arch, de BioL, viii, 4, 1888, p. 713) finds that strychnin, prussic acid, and curare act too strongly; of all the reagents he tried, 2 per cent, solution of hydrochlorate of cocain gave the best results. Warm water gave him good results for large species, such as those of Hydatina and Brachionus. HARDY (Journ. Roy. Mic. Soc., 1889, p. 475) recommends thick syrup added drop by drop to the water. HUDSON (ibid., p. 476) mentions weak solution of salicylic acid. HOFER'S hydroxylamin method has been given, 20, and TULLBERG'S chloride of magnesium method, 21 ; the pro- cesses of EISMOND and JENSEN, 872, may be tried. Methylen blue, 288, may be found useful. Permanent preservation of Rotifers has, until lately, been considered by those who have tried it to be well-nigh impos- sible. Now, however, thanks to the patient experimentation of ROUSSELET, this difficult problem may be considered to be fairly conquered. Rousselet now proceeds as follows (Journ. Quekett Mic. Club, v, March, 1895, p. 1) : The animals are got together in a watch-glass. They are narcotised by adding to the water at intervals a few drops of the following mixture Hydrochlorate of cocain, 2 per cent, solution . 3 parts. Methylated spirit . . . .1 part. Water . . . 6 parts. They are watched under a dissecting microscope, and at the moment when the cilia have ceased to beat/ or are seen to be on the point of ceasing to beat, they are fixed by adding a drop of liquid of Flemming or of i per cent, osmic acid. The fixing agent is allowed to act for half a minute or less, after which the animals are taken out with a pipette, and thoroughly SOME ZOOLOGICAL METHODS. 483 washed by passing them through two or three watch-glasses of distilled water. They are then definitely mounted in 2J per cent, solution of formaldehyde (forinol 2J parts, distilled water 37 J parts). For some details concerning variations of this method adapted to the preservation of the different forms, see the paper quoted. Mr. Rousselet has been good enough to show me a large series of preparations made by this method, and I can testify that they are most beautiful. Annelida. 850. Cleansing Intestine of Lumbricus (KUKENTHAL, Journ. Hoy. Mic. Soc., 1888, p. 1044). Put the animals into a tall glass vessel which has been filled up with bits of moistened blotting-paper. They gradually evacuate the earthy particles from the gut, and fill it instead with paper. YOGT and YUNG (Traite d'Anat. Comp. Prat., v) recommend coffee-grounds instead of paper; paper becomes rather hard when imbedded, whereas coffee-grounds cut fairly well. 851. Chaetopoda. Lumbricus maj be anaesthetised by putting the animals into water with a few drops of chloroform. PERKIER has pointed out that it is better not to let the chloroform act directly in solution on the animals, but to put them into water in a shallow dish, set up a watch-glass with chloroform in the corner of it, and cover the whole. In half an hour the worms will be more or less narcotised, and if allowed to remain will die in a state of extension. CERFONTAINE (Arch, de Biol., x, 1890, p. 327; Zeit. f. wiss. -J///.\, viii, 2, 1891, p. 210) much recommends curare, ad- ministered by interstitial injection of a dose of about 2 c.c. of a 1 : 500 solution. The animal should afterwards be put into water, and after a quarter of an hour will be found dead. In order to kill Criodrilus lacuum, COLLIN (Zeit. f. wiss. Zool., xlvi, 1888, p. 474) puts the animals into a closed vessel with a little water, and hangs up in it a strip of blotting- paper soaked in chloroform. KUKENTHAL (Die mik. Technik, 1885 ; Zeit.f. wiss. Mik., 1886, p. 61) puts Annelids into a glass cylinder filled with water to the height of 10 centimetres, and then pours 70 per cent, alcohol to a depth of 1 to 2 centi- metres on to the water. The animals will be found sum- 484 CHAPTER XXXV. ciently narcotised for fixation in from four to eight hours. For Opheliadds he also employs 0*1 per cent, of chloral hydrate in sea water. Many marine Chsetopoda may be successfully narcotised (S. Lo BIANCO) in sea water containing 5 per cent, of alcohol, or by means of the mixture, 16. The Polycliteta sedentaria offer the difficulty of a complex and very contractile branchial apparatus. They may some- times be satisfactorily fixed by bringing them rapidly into corrosive sublimate. Cold, not hot solutions should be taken, as heat frequently shrivels up the branchiaa. The species of Polychseta errantia that offer a contractile branchial appara- tus, as Eunice and Onuphis, may be treated in the same way. S. Lo BIANCO advises killing Chastopteridaa, Sternaspidae, Spirogr aphis } Protula, by putting them for half an hour into 1 per cent, chromic acid. I have satisfied myself that good show specimens can be obtained in this way ; but I doubt the histological preservation of the parts being so good as with sublimate specimens. Some of the sedentaria may be got protruded from their tubes by leaving them for some hours in O'l per cent, chloral hydrate in sea water (S. Lo BIANCO). See also the methods 18 to 23. Note also the liquid of EHLERS, 43. Staining. For the staining of small Annelids entire, I find carmalum gives very good results, I think better than borax- carmine or paracarmine. 852. Blood-vessels of Annelids (KUKENTHAL, Zeit. f. wiss* Mik., 1886, p. 61). The animals should be laid open and put for two or three hours into aqua regia (4 parts of nitric acid to 2 of hydrochloric acid). The ramifications of the vessels will then be found to be stained black, the rest of the preparation yellow. 853. Nerves of Annelids. The methylen-blue method and the bichromate of silver method of Golgi (the rapid method) . For the latter see v. LENHOSSEK (Arch. f. mik. Anat., xxxix, p. 102 ; Zeit. f. wiss. Mik., ix, 3, 1893, p. 432). 854. Hirudinea. For the methods of killing see those given for Lumbricus in 851, also 18 to 23. WHITMAN (Meth. in mic. Anat., p. 27) recommends that they SOME ZOOLOGICAL METHODS. 485 be killed with sublimate. This reagent is said to kill leeches with such rapidity that they die in general without having time to change the attitude in which they were found at the moment when the liquid came into contact with them. I have obtained better results myself by narcotising with carbonic acid ( 24), and fixing with liquid of Flemming. Small specimens of Nephelis are narcotised in a few minutes ; large ones will require several hours. I have also found that lemon juice kills them in a state of very fair extension. Carmalum I find excellent for staining entire animals; curiously enough, I have found it to have a better penetration than borax-carmine or paracarmine. Ehrlich-Bioudi mix- ture sometimes gives fine results with, sections. GRAF (Jen. Zeit., 1893, p. 165) states that he has obtained good results by narcotising with decoction of tobacco. Injection. WHITMAN (Amer. Natural., 1886, p. 318) states that very perfect natural injections may often be obtained from leeches that have been hardened in weak chromic acid or other chromic liquid. He considers that these injections are the best for the purpose of the study of the circulatory system by means of sections. Of course Hirudinea (or any other Annelids) on which it is desired to make artificial injections must be killed by some procedure that leaves the tissues in a state that will allow the injection to run freely. JACQUET (Mitth. Zool Stat. Neapel, 1885, p. 298) advises that leeches be put into water with a very small quantity of chloroform ; they soon fall to the bottom of the vessel and remain motionless. They should be allowed to remain a day or two in the water before injecting them. Echinodermata . 855. Holothurioidea. These animals are difficult to fix on account of their contracting with such violence under the in- fluence of irritating reagents as to expel their viscera through the oral or cloacal aperture. 8. Lo BIANCO puts Holothurids into pure sea water until they have expanded their tentacles, then seizes them with forceps behind the tentacles, so as to mechanically render impossible their withdrawal, and immerses the anterior part 486 CHAPTER XXXV. of the body in acetic acid, whilst at the same time an assistant injects 90 per cent, alcohol through the anus. VOGT and YUNG (Anat. Comp. Prat., p. 641) say that Cucu- maria Planci (C. doliolum, Marenzeller) is free from the vice of expelling its intestines under irritation ; but they recom- mend that it be killed with fresh water, or by slow intoxi- cation with alcohol, chromic acid, or sublimate added to the sea water in which it is contained. Synapta may be allowed to die in a mixture of equal parts of sea water and ether or chloroform (S. Lo BIANCO). Holothurids, Dr. WEBER informs me, are admirably pre- served in formaldehyde ; a weak solution is sufficient. 856. Asteroidea. HAMANN (Beitr. z. Hist. d. Echinodermen, ii, 1885, p. 2) finds it best to inject the living animal with a fixing liquid. The cannula should be introduced under the integument at the extremity of a ray, and the liquid injected into the body-cavity. The ambulacral feet and the branchiae are soon distended by the fluid, and as soon as it seems to have penetrated sufficiently the animal is thrown into a quan- tity of the same reagent. The study of the eyes presents points of special difficulty. In order to study them in sections, with the pigment preserved in situ, the eye should be removed by dissection, should be hardened in a mixture of equal parts of 1 per cent, osmic acid and 1 per cent, acetic acid, and imbedded in a glycerin gum mass, or some other mass that does not necessitate treatment with alcohol (which dissolves out the pigment, leaving the pigmented cells perfectly hyaline). For maceration use one- third alcohol, the aceto-osmic mixture failing to preserve the rods of the pigmented cells. Formaldehyde is not to be recommended for the preser- vation of Asteroidea (WEBER) . 857. Ophiuridea. Should be killed in fresh water if it be desired to avoid rupture of the rays (DE CASTELLARNAU, La Est. Zool. de Napoles, p. 135). 858. Echinoidea. I advise that they be killed by injection of some fixing liquid. For preservation, formaldehyde has proved admirable in all respects, and greatly superior to alcohol (WEBER). SOME ZOOLOGICAL METHODS. 487 859. Larvae of Echinodennata. I am greatly obliged to my able friend Dr. BARROIS for kindly writing down for me (for the Traite des Meth. techn., from which they are translated) the following, instructions, which are the outcome of a pro- longed and minute study of the metamorphoses of the Echino- dermata. Pluteus. In order to a fruitful study of the metamorphoses of the Echinoidea and Ophiuridea it is necessary to obtain preparations that offer the advantages presented by the study of the living larvas; and especially such as give distinct images of the different organs, and show the calcareous skeleton preserved intact (a point of considerable importance, since this skeleton frequently affords landmarks of the greatest value). These preparations should further possess the fol- lowing points : They should give clear views of the region of formation of the young Echinoderm (which is generally opaque in the living larva). And they should possess suffi- cient stiffness to allow of the larva being turned about in any desired way, and placed in any position under the micro- scope. It is not very easy to obtain preparations fulfilling these conditions, on account of the difficulty of obtaining a selective stain whilst preserving the integrity of the calcareous skeleton. The following method is recommended : Pluteus larvae are fixed in a cold saturated solution of corrosive sublimate, in which they remain not more than two or three minutes. They are then washed with water, and brought into dilute Mayer's cochineal ( 241). This should be so dilute as to possess a barely perceptible tinge of colour. The objects should remain in the stain for from twelve to twenty-four hours, being care- fully watched the while, and removed from the stain at the right moment and mounted in balsam, or, which is frequently better, in oil of cloves or cedar-wood. This method is per- f ectly satisfactory for the study of the chief phases of meta- morphosis. Auricularia and Bipinnaria. The method described above is equally applicable to these forms, and seems to be altogether the best method for the study of the metamorphosis of Bipin- naria. The earlier stages of the metamorphosis of Auricularia are better studied by fixing with osmic acid, staining with Beale's carmine, and mounting in glycerin. 488 CHAPTER XXXV. Larvss of Comatula. The best method for the study of the embryonal development of Comatula consists in fixing with liquid of Lang, and staining with dilute borax-carmine. It is important (for preparations that are not destined to be sec- tioned) to use only dilute borax-carmine, as the strong solution produces an over-stain that cannot easily be reduced. Narcotisation by chloral hydrate before fixing is useful, especially for the study of Pentacrinus larvae and of the young Synaptse formed from Auricularia. Without this precaution you generally get preparations of larvae either shut up (Pentacrinus), or entirely deformed by contraction (young 8ynaptss) . See also MA.CBBIDE on the development of Amphiura squamata, Quart. Journ. Mic. Sci., xxxiv, 1892, p. 131 ; Journ. Roy. Mic. Soc., 1893, p. 117 (osmic acid followed by liquid of Miiller and alcohol ; decalcification with nitric acid in alcohol ; staining with Mayer's paracarmine or hseinaluin) ; and SEELIGER on the development of Antedon, Zool. Jahrb., Abth. f. Anat., vi, 1892, p. 161 ; Zeit.f. wiss. Mik., x, 2, 1893, p. 229. Coelenterata. 860. Actinida. Narcotisation. For suitable narcotisation methods see 13 to 23. Fixation. In Le Attinie, Fauna u. Flora d. Golfes v. Neapel } ANDRES gives the following hints : Hot corrosive sublimate often gives good results. In the case of the larger forms the solution should be injected into the gastric cavity, and a further quantity of the liquid be poured over the animals. Freezing sometimes gives good results. A vessel contain- ing Actiniae is put into a recipient containing an ice-and-salt freezing mixture and surrounded by cotton wool. After freezing, the block of ice containing the animals is thawed in alcohol or some other fixing liquid. Maceration. For the HERTWIGS' well-known method (Jen. Zeit., 1879, p. 457) see 553. The tissues should be left to macerate in the acetic acid for at least a day, and may then be teased in glycerin. LIST (Zeit. f. wiss. Mik., iv, 2, 1887, p. 211) recommends dilute liquid of Flemming. Tentacles of Anthea cereus and Sagartia parasitica treated for ten minutes with a mixture of 100 c.c. of sea water with 30 c.c. of Flemming's liquid (the strong solution, 47), then washed out for two or three hours SOME ZOOLOGICAL METHODS. 489 iii 0'2 per cent, acetic acid, and teased in dilute glycerin, give fine dissociations of the connective, sensory, and urticant cells of the ectoderm, and after removal of the epidermis allow of the demonstration of ganglion- cells and the supporting lamella. Picro-carmine may be used for staining. 861. Zoantharia with Calcareous Skeletons are difficult to deal with on account of the great contractility of the polyps. Sublimate solution, which ought very often to be taken boil- ing, sometimes gives good results. DE CASTELLARNAU (La Est. Zoul. de Napoles, p. 132) says that this process succeeds well with Dendrophyllia, Antipathes, Astroides, Cladocora and Caryophyllia. Sections. For preparing sections, besides the usual methods for sectioning decalcified specimens, we have the valuable methods of von Koch and Ehrenbaum, 174 and 175, which, being applicable to undecalcified specimens and furnishing preparations showing at one and the same time soft parts and hard parts in situ, render most inestimable services. 862. The Alcyonaria have also extremely contractile polyps. In a former edition I suggested for their fixation either hot sublimate solution or glacial acetic acid ( 68). S. Lo BIANCO has since recommended essentially similar processes. GAEBIXI (Mann. ale, p. 151) says that the polyps may be fixed in the state of extension by drenching them with ether, and then bringing them into strong alcohol. WILSOX minutes, the liquid is drawn off and the preparation is washed for half an hour with alcohol of medium strength. The objects are then stained for ten to twenty minutes in picro-carmine, washed with water till the picric acid is removed, and mounted in a mixture of equal parts of glycerin and water. 878. Other General Methods. Du PLESSIS (Voox et YUNG, Trait. Atw.t. Comp. Prat., p. 92) recommends fixation with 0'2 per cent, solution of corrosive sublimate. Let the preparation dry up, and if the organisms have preserved their shape, stain and mount in balsam. This seemingly barbarous mode of procedure is said to give very fine preparations when successful. FOL (Lehrb., p. 102) fixes delicate marine Infusoria (Tintinnodea] with the perchloride of iron solution ( 67), added to the water containing them, and stains with gallic acid as directed ( 379), and states that this is the only method that has given him good results, especially as regards the pre- servation of cilia. See also the methods of FABRE-DOMERGUE, Ann. de Microgr., ii, 1889, p. 545 ; SCHEWIAKOFF, Biblioth. ZooL, v, 1889, p. 5 ; Journ. Roy. Mic. Soc., 1889, pp. 832, 833 ; ZOJA, Boll. Sci. Pavia, 1892 ; Zeit.f. wiss Mik., ix, 4, 1893, p. 485 ; LONGHI, Bull. Mus. Zool. Univ. Genova, 4, 1892 ; Zeit. f. wiss. Nik., ix, 4, 1893, p. 483 ; BRANDT, Fauna u. Flora d. Golfes v. Neapel, 1885 ; Journ. Roy. Mic. Soc., 1888, p. 665 (for Sphserozoa). 879. Demonstration of Cilia (WADDINGTON, Journ. Roy. Mic. Soc., 1883, p. 185). Solution of tannin, or a trace of alcoholic solution of sulphurous acid. 880. Stains for Flagella. The celebrated method of LOFPLER has run through several forms (Gentmlb. f. BacterioL, vi, 1889, p. 209 ; vii, 1890, p. 625 ; Zeit. f. wiss. Hik., vi, 3, 1889, p. 359; vii, 3, 1890, p. 368; Journ. Roy. Mic. Soc., 1889, p. 711 ; 1890, p. 678), of which that given here is the latest. To 10 c.c. of a 20 per cent, solution of tannin are added 5 c.c. of cold saturated solution of ferrous sulphate and 1 c.c. of (either aqueous or alcoholic) solution of fuchsin, methyl violet, or " Wollschwarz." The mixture will require for some forms the addition of a few drops of 1 per cent, solution of caustic soda ; e.g. for typhoid bacilli, 1 c.c. ; for Bacillus subtilis, 28 to 30 drops ; for bacilli of malignant oedema, 36 to 37 drops. Some other forms will require besides the addition of a trace of sulphuric acid to the soda solution : so for cholera bacteria, half a drop to 1 drop ; for Spirillum rubrum, 9 drops. 500 CHAPTER XXXV. Cover-glass preparations are made and fixed in a flame in the usual way, special care being taken not to over-heat. Whilst still warm the preparation is treated with mordant (i.e. the above- described mixture), and is heated for half a minute until the liquid begins to vaporise, after which it is washed in distilled water and then in alcohol. It is then treated in a similar manner with the stain, which consists of a saturated solution of fuchsin in anilin water, the solution being preferably neutralised to the point of precipitation by cautious addition of 0*1 per cent, soda solution. The modifications of this method by BUNGE are as follows : Firstly (see Journ. Roy. Hie. Soc., 1894, p. 640, or Zeit.f. wiss. Mik., xiii, 1, 1896, p. 96), he modifies the mordant by taking Liquor Ferri Sesquichlorati instead of the sulphate. He dilutes the Liquor Ferri Sesquichlor. with twenty vols. of dis- tilled water, takes three parts of the tannin solution and one part of the dilute iron solution, and adds to 10 c.c. of the mixture 1 c.c. of saturated aqueous solution of fuchsin. The mordant should be allowed to ripen exposed to the air for some days or weeks. In a later paper (see Journ. Eoy. Mic. Soc., 1895, pp. 129 and 248) he recommends adding to the ripened mordant a few drops of peroxide of hydrogen, until it becomes of a red-brown hue. It is then shaken up and filtered on to the prepared cover-glass, on which it is allowed to act for about a minute. The cover-glass is then mopped up, dried, and stained, preferably with carbol-gentian. TBENZMANN (Centralb., vi, 1889, p. 433 ; Zeit.f. wiss. Mik., vii, 1, 1890, p. 79) mordants for several hours at the normal temperature in a 1 per cent, solution of tannin in 0'5 per cent, hydrochloric acid, and stains for several hours in carbolic fuchsin ; and gives also two other similar methods. BEOWN (The Observer, iii, 1892, p. 298 ; Journ. Roy. Mic. Soc., 1893, p. 268) mordants for several hours in a mixture of 30 gr. tannin, 12 drops anilin oil, and 1 fl. oz. of alcohol, which may, if required, be alkalised by addition of a trace of caustic soda (so for Spirillum undula and Bacillus ulna}, or be acidified for some forms with a little hydrochloric acid. The cover is stained by the process of heating over a flame for a few minutes with any anilin-water solution of fuchsin, methyl violet, dahlia, methyl green, &c., neutralised with caustic soda as in Loeffler's process, or with a solution of rosanilin in anilin water. See also JULIEN, quoted ibid., 1894, p. 403 ; VAN ERMENGEM, ibid.. p. 405 ; SCLAVO, quoted Zeit. f. wiss. Mik., xiii, 1, 1896, p. 96 ; and HESSERT, ibid., p. 98. APPENDIX. 881. The Usual Alcohols. The following, or a similarly- spaced series of alcohols, should be kept on the table. Absolute Alcohol See 84. The so-called "absolute alcohol" of commerce is generally of about 98 per cent, strength. This grade is convenient, but not necessary for ordinary work. 9-5 per cent. Alcohol. This is the average strength of the common strong commercial alcohol, which ranges in general from 94 per cent, to 96 per cent, according to temperature. The strength of this, or of the following, should be deter- mined by means of an areometer (Gay Lussac's being very convenient), so as to form a starfcing-point for the following mixtures, which may be made by means of the subjoined table. This is the usual grade for dehydrating before clearing. It is the highest grade that should be used for de- hydrating celloidin sections. 90 per cent. Alcohol. May be made by taking 100 vols. 95 per cent, alcohol, and 5*5 vols. water. This is the usual strength of the strongest commercial Methylated Spirit, which (if free from mineral naphtha) may be taken instead of pure alcohol for common work. If naphtha be present the alcohol becomes turbid on the addition of water. Oil of bergamot will clear from this grade. 80 per cent. Alcohol. Rectified Spirit. B.P., is a little weaker than this, viz. 84' 5 per cent. 70 per cent. Alcohol. Only exceptionally powerful clearers, such as anilin oil, will clear from this grade; see 125. This if the proper grade in general for preserving organisms and tissues in (but see the remarks on pp. 4 and 5) ; higher grades should not generally be used unless it is desired to harden. This ift the proper grade for u-ashing out borax-carmine stains, nn-rusiue sublimate after fixing, &c. 50 per cent. Alcohol. This is the strength of Proof Spirit. " One-third Alcohol." Made by taking 1 vol. of 90 per cent, alcohol, and 2 vols. water. See 83. 502 APPENDIX. 882. Table for diluting Alcohol (after GAY-LUSSAC). To use this table, find in the upper horizontal row of figures the per- centage of the alcohol that it is desired to dilute, and in the vertical row to the left the percentage of the alcohol it is desired to arrive at. Then follow out the vertical and horizontal rows headed respectively by these figures, and the figure printed at the point of intersection of the two rows will show how many volumes of water must be taken to re- duce one hundred volumes of the original alcohol to the re- quired grade. Thus, if it be required to manufacture some 70 per cent, alcohol, starting with 90 per cent., we find the figure 90 in the upper column, the figure 70 in the vertical column, and at the point of intersection we read 31*05, show- ing that a fraction more than 3,1 volumes of water must be added to 1Q.O volumes of 90 per cent, alcohol. Or similarly, if we wish as before to make 70 per cent, alcohol, but start with an alcohol of 85 per cent., we find that 23' 14 volumes of water must be employed. Weaker grade required. ORIGINAL GRADE, 90 p. 100. 85 80 p. 100. i p. 100. 75 p. 100. 70 p. 100. 65 p. 100. 60 55 50 p. 100. p. 100. p. 100. p. 100. 85 6-56 80 1379 6-83 75 21-89 14-48 7'20 ! 70 31-05 23-14 15-35 7-64 65 41-53 33-03 24-66 16-37 8-15 60 53-65 44-48 35-44 26-47 17-58 8-76 55 67-87 57-90 48-07 38-32 j 28-63 19-02 9-47 50 84-.71 73-90: 63-04 52-43 41-73 31-25 20-47 10-35 45 105-34 93-30 81-38 60-54 57-78 46-09 34-46 22-90 11-41 40 130-80 117-34 104-01 9076 j 77-58 64-48, 51-43 38-46 25-55 35 163-28 148-01 132-88 117-82 102-84 87-93 ! 73-08 58-31 43-59 30 206-22 1 188-57 171-05 153-61 |l36-04 118-94 101-71 84-54 67-45 APPENDIX. 603 883. Histological Reagents and Apparatus. See 216. As regards the products of GRUBLER AND Co., so often quoted in the foregoing pages, I would add that they should either be ordered from them direct, or, if ordered through any agent, should be ordered to be sent in the original packages, signed ami dated by them. This is in order to ensure the due fre*h- /<>.v.N-of the products; many of them will not keep well for very long. Glass and other apparatus can be obtained as well as chemicals from the above-quoted houses. Want of space compels me to suppress the lists of suggested reagents given in a former edition under the headings " The Laboratory Table" and " The Zoologist's Travelling Case." Either collection may still be obtained from Griibler and Co., the latter in appropriate bottles, fitted into a case measuring 1 foot 4 inches X 5i inches X 4^ inches, at the price of about '2 os., or a case somewhat larger, yet not too heavy to be carried in the hand, at about 3. 884. Cleaning Slides and Covers. The readiest way known to me of freeing slides from balsam, damar, and cement is to wet with water and s -rape with an old knife, using afterwards, if necessary, one of the solvents mentioned below. HAX AMAN, Journ. Roy. Mic. Soc., i, 1878, p. 295 ; American Naturalist, xii, p. 573. To a cold saturated solution of bichromate of potash add % of its bulk of strong sulphuric acid (care must be taken on account of the heat uiul vapours evolved). HEXEAGE GIBBES, ibid., iii, 1880, p. 392. Place the cover-glasses in strong sulphuric acid for an hour or two, wash well until the drainings give no acid reaction ; wash first with methylated spirit, and then with absolute alcohol, and wipe carefully with an old silk handkerchief. SEILEE, ibid., p. 508. New slides and covers are placed for a few hours in the following solution : Bichromate of potash 3 ounces. Sulphuric acid ...... 3 fluid ounces. Water 25 Wash with water. The slides may be simply drained dry ; the covers may be wiped dry with a linen rag. Slides and covers that have been used for mounting either with balsam or a water medium are treated as follows : The covers are pushed into a mixture of equal parts of alcohol and hydrochloric acid, and after a few days are put into the bichromate solution and treated like new ones. The slides are scraped free of the mounting medium with a knife and put directly into the bichromate solution. FOL (Lehrb., p. 132) recommends either a solution containing 3 parts of bichromate, 3 of sulphuric acid, and 40 of water; or simply dilute nitric acid. i i ABBIXI (^lanuale, p. 31) puts slides for a day into 10 per cent, sulphuric acid, then washes, first with water and then with alcohol. 504 APPENDIX. BEJIEENS (Zeit.f. wiss. Mik., 1885, p. 55) treats slides first with con- centrated nitric acid, then with water, alcohol, and ether. JAMES (Journ. Roy. Mic. Soc., 1886, p. 548) treats used slides with a mixture of equal parts of benzin, spirit of turpentine, and alcohol. KNAUEE (Centralbl. f. Bdkt., x, 1891, p. 8 ; Zeit. f. iviss. Mik., ix, 2, 1892, p. 187 ; Journ. Roy. Mic. Soc., 1891, p. 833) recommends boiling for twenty or thirty minutes in 10 per cent, lysol solution, then rinsing with cold tap water till clear. NIAS (Journ., pag. cit.) finds it is sufficient to boil with washing soda, and rinse. In the employment of the water or alcohol section-fixing method, 186, it is extremely important to work with slides absolutely free from grease. After the slides have been cleaned by one of the processes given above, they should be rinsed with distilled water and preserved in 90 per cent, alcohol, from which they should be removed with forceps when required for use, not with the fingers, then simply drained, or wiped with a very clean cloth. 885. Gum Mucilage for Labels, &c. The Journ. of the Chemical Soc. says that the adhesive qualities of gum may be very much exalted by the addition of aluminium sulphate (the so-called "patent" alum) to the mucilage. " 2 grms. of crystallised aluminium sulphate, dissolved in 20 grms. of water, is added to 250 grms. strong gum arabic solution (2 grms, in 5 grms. water). Ordinary solutions of gum arabic, however concen- trated, fail in their adhesive power in many cases, such as the joining together of wood, glass, or porcelain ; prepared, however, according to the above receipt, the solution meets all requirements " (from Public Opinion? Feb. 19th, 1886). FOL (Lehrb., p. 148) advises that slides be prepared for labelling by spreading over one end a layer of aluminium-chloride gelatin dissolved in acetic acid, and allowing it to dry before putting on the label. Why do not the glass makers furnish slides with roughened (ground) end- surfaces for the reception of labels ? For four other receipts for gums and pastes for labels, see ELIEL, in Engl. Mechan., 1887, p. 535 ; Amer. Mon. Mic. Journ., 1887, p. 93 ; Zeit. f. wiss. Mik., v, 1, 1888, p. 69. VOSSELEE (Zeit. f. wiss. Mik., vii, 4, 1891, p. 459) recommends, for attaching protective cardboard ridges to slides, a syrup-thick solution of bleached shellac in alcohol. 886. Green Light. The suggestion of the employment of green light in microscopy is, I believe, due to ENGLEMANN (Pfliiger's Arch., 1880, p. 550). He strongly recommends the use of green light for delicate observations, as giving sharper definition, allowing finer details to be seen r and tiring the eyes less than white light. Green glass of sufficiently good quality is found in commerce. The glass is best put between the mirror APPENDIX. 505 and the object, e. g. on the diaphragm. Blue glass (cobalt or ammonio- sulphate of copper) is also useful, but less so than green. Red light is most hurtful. " The explanation of these points, so important in practice, may be found in the results obtained by Lamansky in his researches on the ' Limits of Sensibility of the Eye to the Different Colours of the Spectrum ' Arch.f. Ophthalm., xvii, p. 123, 1871)." I would add that for lamp-light work, especially fine work with high powers, either green or blue glass is, according to my experience, a sine qua non if the best attainable results be desired. I always use blue cover-glasses, putting from one to four of them on the diaphragm of the condenser. For some unexplained reason I find I get better results by means of several superposed thin glasses than by one thick one. 887. Re-staining Old Mounts (HENNEGUY, from the last edition of the Traite des Methodes techniques de I'anat. microscopique, LEE et HENNEGUT) . It is probably not generally known that balsam mounts the stain of which has faded, or which it may be desired to submit to some other staining process or mount in some other medium, may often with great advantage be re-stained and re-mounted. All that is necessary is to put the slide into a tube of xylol or benzol till the cover falls off (about two days), wash well for some hours in clean xylol, and pass through alcohol into the new stain. Since this was pointed out to me by Dr. Henneguy, I have unmounted and re-stained a considerable number of old preparations, some of them over fifteen years old, and have been most agreeably surprised at the results obtained. I have succeeded in every case with series of sections mounted on Mayer's albumen, but I doubt whether the process would be safe with sections mounted on such a fixative as Schallibaum's collodion or the like. INDEX. The numbers refer to the Pages* A. ABBE, mounting medium, 277. Absolute alcohol, uses, 4, 5 ; prepara- tion, 52. Acanthocephali, 480. Acephala, see Mollusca. Acetate of alumina, for mounting, 265. Acetate of copper, 46, 267. Acetate of lead, 61, 392. Acetate of potash, 265. Acetate of uranium, 46. Acetic alcohol, 45. Acetic acid for fixing, 44, 45, 467. Aceto-carraine, 152, 155. Acetone, for dehydration, 4. Acid anilin dyes, 179. Acid, free, test for, in tissue, see Congo red. Acid fuchsin, 219; for nerve tissue, 403, 414. Acid Magenta, see Acid fuchsin. Acidophilous cell-elements, 446. Acids, see Acetic, Chromic, Formic, Hydrochloric, &c. Acidulated alcohol, 52. Actiniaria, 13 17, 488. ADAMKIEWICS, stain for nerve-centres, 414. Adipose tissue, 35, 445. Adjective staining, 140, 141, 224. Agar-agar, section fixative, 127. AGA3Sizand\VHiTMA>-,pelagicova,346. Albumen, imbedding mass, 118; in- jection mass, 307 ; section-fixing process, 123, 126; removal of, from ova, 342 et seq. Albuminoids, reactions, 357. Alcohol, its action on tissues, 5 ; fixing, 51; absolute, 52; "one third," 51; acetic, 45; acidulated, 52; picric, 50; for hardening, 62; macerating, 313; preserving tis- sues, 4, 5, 266; the usual grades, 500; table for diluting, 501. Alcohol balsam, 280. Alcoholic carmines, 157, 158. Alcoholic cochineal, 158, 159. Alcoholic hfematein stains, 172 174. Alcyonaria, 489. Alcyouella, 14. Alcyonium, 12, 489. Aldehyde green, 198. ALFEEOW, silver impregnation, 247. Alizarin, artificial, 232. ALLEGEB, serial sections, 127. ALLEX, rnethylen blue, 207. ALT, Congo red nerve stain, 418. ALTMANN, fixing methods, 35, 364; paraffin stove, 82 ; bioblasts, 138, 364, 368 ; impregnation and cor- rosion methods, 323. Alum for fixing, 37; ammonia-alum, solubility in water, 170. Alum-carmine, 150152; ditto with acetic acid, 151; with picric acid, 152. Alum-cochineal, 150. Alumina acetate, 265. Amaraecium, 346. Amber varnish, 289. Ammonia bichromate, 60, 392 ; neutral chromate, 61. * In accordance with a wish expressed by readers in whose judgment I have confidence, the references are now given to the pages, and not as heretofore to the numbered sections. 508 INDEX. The numbers i Ammonia-alum, 170. Ammonia-carmine, 155, 415. Ammonium molybdate, for impregna- tion, 261 ; for fixing nuclei, 364 ; sulphocyanide, 315 ; vanadate, 310. Amphibia, embryology, 342 et seq. Amphioxus, 378. Amphipoda, embryology, 352. Amphiura, 488. Amphophilous cell-elements, 446. Amyl nitrite, 293. Amyloid matter, 199, 201. Anchinia, 467. Ancylus, 469. ANDEER, phloroglucin, 326. ANDRE, fixing Ancylus, 469. ANDRES, Actinia, 13, 14, 488 ; section - stretcher, 87. ANDREWS, imbedding apparatus, 77. Anilin, for clearing, 70, 109. Anilin dyes, generalities, 179 et seq. ; classification into acid and basic, 179 ; regressive staining process, 182 et seq. Anilin black, 231, 475. Anilin blue, 230. Anilin blue-black, 231, 416. Anilin green, 228. Anilin oil for clearing, 70, 109; for staining, 190. Anilin red, 196. Anilin violet, 200. Anilin water, 190. Aniseed imbedding mass, 118. Annelida, 483 485 ; embryology, 352. Anodonta, 15. Antedon, 488. Anthea, 488. Anthozoa, 488. Antipathes, 489. Antipyrin for narcotisation, 495. APATHY, celloidin imbedding, 100; preserving the blocks, 106 ; orien- tation of objects, 103; serial sections, 129, 130; cement, 289; hsematoxylin stain, 174; raethy leu- blue impregnation, 202, 205, 207 210; mounting medium, 269. APEL, Sipunculus and Halicrypius, 481. efer to the Pages. Aphides, preparation and embryology, 351. Aplysia, 468. Aqua Javelli, 322, 330. Arabin, 126. Araneiua, embryology, 351. ARCANGELI, carmines, 155. ArchH7234. Archiplasm, archoplasm, see Cyto- logical methods, Plasma stains. Arctiscoida, 475. Areolar tissue, 444. ARNOLD, critique of Flemming's mix- ture, 32, 34. ARNSTEIN, methylen-blue method, 206, 209, 210 ; gold method, 374. ARONSON, gallein stain, 436. Arsenic acid for decalcification, 326. Arthropoda, general methods, 472; clearing and softeningchitin,472 ; depigmentatiou, 473; eyes, 473 475 ; nerve and muscle, 475 ; em- bryology, 348352. Artificial alizarin, 232. Artificial fecundation, 331. Artificial oedemata, 444. Artificial pigment spots, 59, 391, 392. Artificial saliva, 315. Artificial serums, 264, 265. Ascaris, ova, 45, 368. A^cidians, 16, 346, 467. Asphalt varnish, 286. Asphyxiation as a killing method, 16. ASPINALL'S enamel, for cement, 290. Astacns, embryology, 352. Aster acanthi on, 14. Asteroidea, 486. Astroides, 489. Attraction spheres, see Cytological methods, Plasma stains. AUBERT, cements, 285. AUGSTEIN, Strongylus, 480. Aurantia, 229. Aureoline, 329. Avricularia, 487. Aves, embryology, 338 et seq. Axis cylinder, 403 et seq. ; stains, 415 et seq. Axolotl, ova, 343. AZOULAY, nerve-impregnation, 412. INDEX. 509 The numbers refer to the Pages. B. BABEH, picro-carmine, 154. BABES, safranin stain, 190 j super- saturated do., 365. BALBIANI, " noyau vitellin," 368 ; liv- ing ova, 332; ova of Phalangida, 351 ; Protozoa, 495. BALFOUB, embryology of Araneina, 351. BALLOWITZ, muscle of Cephalopoda, 383. Balsam, 277 et seq. BALZEK, elastic tissue, 450. BAKFF, boroglyceride, 272. BARNES, observation of Trichinae, 4SO. BAREOIS, embryology of Echinoder- mata, 487. Basic anilin dyes, 179. Basophilous cell-elements, 446. BASTIAN, gold method, 255. BATAILLON and KOEHLEE, borax methylen blue, 365. BAUMGAETEN, fuchsin and methylen- blue stain, 232 ; bleu de Lyon, 237. BATEEL, stain for cartilage, 236, 455 ; decalcification, 325. BEALE, digestion fluid, 320; glycerin jelly, 273 ; injections, 308 ; varnish, 289. BECHTEREFF, hardening brain, 392. BECK, cements, 285. BECKEE, microtome, 72. BEDOT, fixing process, 492. BEHX, hardening skin, 370. BEHBEXS, cements, 285; indulin and nigrosin, 228 ; amber varnish, 289 ; cleaning slides, 503. BELA HALLER, macerating mixture, 318. BELLABMINOW, injection, 310. BELLOXCI, preparation of brain, 413. BELL'S cement, 286. BEXDA, iron haetnatoxylin, 176; cop- per hsematoxylin, 177 ; double- >t;iin, 227; freezing brain, 400. BEXECKE, connective-tissue fibrils, 444. Bengal rose, 226. Benzin, for imbedding, 79. Benzol, for clearing, 70. Benzo-azurin, 197. Benzo-purpurin, 225. Benzoyl green, 228. Bergamot oil, 68. BEEQONZINI, staining mixture for plasma cells, 449. BERKLEY, cerebellum, 398; rapid Weigert stain, 411. Berlin blue, 302, 309. BEBLINEBBLATJ, F., regeneration of Weigert's hajmatoxyliri, 407. BEBNHEIM, gold process, 257; bladder of frog, 384. BEBNHEIMEB, haBmatoxylin for retina, 441. BETHE, methylen blue, 212; stain for chitin, 475. BETZ, central nervous system, 392 394. BEVAN LEWIS, see LEWIS, BEVAN. BIANCO, S. Lo, chromo-acetic acid, 45; Molluscoida (Bryozoa), 468; Mollusca, 468; Nemertina, 478; Gephyrea, 481; Chsetopoda, 484; Holothurioidea, 485 ; Medusae, 491 ; Siphonophora, 493 ; Cteno- phora, 49-i; Tunicata, 16, 467; narcotising mixture, 14. Bichloride of mercury, see Corrosive sublimate. Bichromate of ammonia, 60. Bichromate of potash, for fixing, 36, 60; for hardening, 58 et seq., 391 et seq. ; for maceration, 317. Bichromate of silver impregnation, see GOLGI. BICKFALVI, digestion fluid, 320. Eiebricher Scharlach, 226. BIEDEBMANN, methylen-blue method, 207; nerve-endings in muscle, 379. BIXET, bleaching osmic objects, 28. Biniodide of mercury medium, 275. Bioblasts, Altmann's, 138, 364, 368. BiONDi, staining mixture, 219, 223 ; methods for blood, 457, 461. Bipinnaria, 487. Bismarck brown, for regressive stain- ing, 196 ; for progressive, 200. Bitume de Judee, 286. 510 INDEX. The numbers refer to the Pages. BIZZOZEEO, gentian violet, 192, 193 ; picro-carmine, 154; blood, 458, 460, 461. BJELOUSSOW, injection, 307. Blackley blue, 228. Bladder of frog, 384. BLANC, Infusoria, 497. Blattida, embryology, 350. BLATJE, glandular epithelium, 370. Bleaching, 328 et seq. ; bichromate ob- jects, 58; Erlicki objects, 59; osmic objects, 27. Bleu alcool, 230. Bleu carniin aqueux, 231. Bleu de Lyon, 230. Bleu de nuit, 230. Bleu lumiere, 230. BLOCHMANN, ova of Amphibia, 342; nervous system of Cestodes, 433. Blood, 456461. Blue-black, 231, 416. BLUM, formaldehyde, 53, 62. BOBBETZKY, embryology of Lepido- ptera, 350. BOCCABDI, gold method, 256; motor plates, 381. BOHM, gold method, 256; Golgi's im- pregnation, 427. BOHMEB, hsernatoxylin, 170. BOHMIG, Khabdoccela, 477. Bone, decalcification, 323 et seq.; pre- paration, 451 455. Borax- carmine, GBENACHEB'S, 156; aqueous, 155; with indigo-car- mine, 235, 236. Borax-methylen blue, 365. Bordeaux-iron-hsematoxylin, 366. Boric-acid carmine, 155. BOEN, paraffin imbedding, 77 ; section- fixing, 127; reconstruction of sec- tions, 333 ; section-stretcher, 87 ; embryological method, 344. Boroglyceride, 272. BOTTCHEB-HEBMANN staining process, 182. BOUMA, stain for cartilage, 455. BOVEEI, ovum of Ascaris, 368; medul- lated nerve, 404. BOYEE, embryology of Teleostea, 345. Brachionus, 482. BEADY, chloral hydrate medium, 266. JBradynema, 480. Brain, see Central nervous system and Neurological methods. BBANDT, glycerin jelly, 273 ; Protozoa, 496, 499. BEASS, alcoholic carmine, 158; bleach- ing osmic objects, 28; paraffin imbedding, 79, 94 ; Protozoa, 497. BEAUN, Alcyonaria, &c., 489 ; Rhabdo- coela, 477 ; Neinatodes, 480. BBECKENFELD, Hydra, 490. BEEGLIA, Weigert's hroinatoxylin, 407, 411. BEEMEE, motor plates, 381; methylen blue and eosin, 227. BEIDE, MAC-, see MACBEIDE. BEISTOL, regeneration of osmic acid solutions, 25. BBOCK, macerating medium, 317. BEOSICKE, staining method, 260. BBOWN, stains for flagella, 500. BEUCKE, digestion fluid, 320 ; injec- tions, 302. BBUN, mounting medium, 270. BBUNOTTI, gelatin imbedding mass, 98. Brunswick black, 286. Bryozoa, 14, 15, 468. BUDGE, injection, 310. Bugula, 468. BUMPUS, celloidin imbedding, 110. BUNGE, stains for flagella, 500. BUBCI, elastic tissue, 451. BUECKHAEDT, nervous system of Pro- topterus, 398. BUBGER, nervous system of Nemertina, 206. BUSCH, eosiu and hjematoxylin, 237; decalcificatiou, 323, 324. BUSSE, photoxylin imbedding, 1()0 ; celloidin do., 101, 103, IOC. BUTSCHLI, paraffin imbedding, 81 ; haematoxylin stains, 171, 177. CAJAL, RAMON Y, silver chromate im- pregnation, 425; terminations of nerves and trachea;, 380; retina, 441 ; sympathetic ganglia, 425. INDEX. 511 The numbers refer to the Pages. CALBEBLA, methyl green, 197, 199; ditto and eosin, 226; indulin, 228; glycerin mixture, 272; ma- cerating mixture, 315. CALDWELL, serial sections, 123. Cambridge rocking iricrotome, 72. Canada balsam, for mounting, 279 et seq.; imbedding method, 115. CANFIELD, iris, 383. Cannel oil (cinnamon oil), 68. Caoutchouc, for section-fixing, 128 ; cement, 286. CAPPABELLI, dichro'ism of methyl vio- let, 201. CARAZZI, bleaching method, 329. Carbolic acid, for clearing, 69, 109. Carbolic f uchsin, 196. Carbolised syrup, 265. Carbonic acid narcotisation method, 17. Carchesium, 495. Carcinoma corpuscles, 192. Carmalum, 149. Carmine, analysis of, 145 ; generalities on, 145 149 ; theory of staining with, 145 ; practice of staining, 148; for what purposes useful, 148. Carmine blue, 231. Carmine stains, aqueous, 149 155 ; alcoholic, 156 160; choice of, 148 ; carmine combination stains, 235 et seq. ; and see the names of the respective authors, as also the separate entries, "Alum-carmine," " Borax-carmine," &c. Carminic acid, 146. Carminroth, 155. CABNOY, acetic alcohol, 45; chromo- aceto-osmic acid, 34 ; cement, 290 ; cytological methods, 357, 358, 364; salt solution, 263; tannin solution, 267. CABPENTEB, cements and varnishes, 284. CABBIEBE, corpuscles of Herbst and Grandry (gold method), 375 ; eyes of Gastropods, 469. CABTEB, injection mass, 300. Cartilage, 455, 456. Caryophyllia, 489. Cassiopeia borbonica, 491. CASTELLABXAU,DE,Lamellibrnnchiata, 468; Medusae, 491; Nemertina, 479 ; Ophiuridea, 486 ; Zoau. tharia, 489. Castor oil for mounting, 282. CATTANEO, corpuscles of Golgi, 381 ; Infusoria, 43, 497. Caustic potash and soda, for macera- tion, 314; for corrosion, 322. CAVAZZANI, triple stain, 239. Cedar oil, for minute dissections, 9; for paraffin imbedding, 80 ; clear- ing, 66 ; for mounting, 278. Cell granules, 368 et seq., 445450, 459. Cell researches, see Cytological me- thods. Cellepora, 468. Celloidin, 99 et seq. ; the new method, 110; the dry method, 111; clear- ing sections, 108 ; injection masses, 310. Cells, paper, for mounting, 285. Cements, 284 et seq.; generalities, 284 ; comparative tenacity of, V 285. Central nervous system, introduction, 385, 386 ; fixing, 387, 388 ; hard- ening, 388398; imbedding and cutting, 398 401 ; nerve-fibre stains, 405 414 ; axis-cylinder and protoplasm stains, 415 419 ; impregnations, 419 ; dry processes for preserving brains, 437; me- thylen blue for, 443; neuroglia, 435, 436 ; myelon of reptiles, &c., 398; and see Nerves, Retina, &c., and the names of Authors. Centrosomes, see Cytological methods, Plasma stains. Cephalopoda, eyes, 470 ; ova, 346. Cercariae, 477. Cerebellum, cerebrum, see Neurolo- gical methods. CEBFONTAINE, curarising Lumbricus, 483. CEBTES, Protozoa, 496, 497, 498. Cestodes, 47G. 512 INDEX. The numbers refer to the Pages. Chaetopoda, 483. Chaetopteridffi, 484. Chain section-cutting, 88. CHEATLE, dehydration apparatus, 4. Cherry gum, 495. Chemicals, 143. CHENZINSKY, methylen blue and eosin, 227. CHICHZOFF, Turbellaria, 478. China blue, 231, 403. Chinolin blue, 228. Ckironomus, embryology, 351. Chitin, clearing and softening, 472 et seq. ; staining, 475. Chitonida?, embryology, 348 ; eyes, 470. Chloral hydrate for narcotising, 14; preservative solutions, 265 ; for maceration, 314 ; Gilson's mount- ing medium, 275 ; Geoffroy's ditto, ibid. Chlorate of potash for maceration, 318. Chloride-of-aluminium carmine, 150. Chloride of copper fluid, 46. Chloride of gold, see Gold chloride. Chloride of magnesium for narcotisa- tion, 16. Chloride of manganese examination medium, 263. Chloride of palladium, see Palladium chloride. Chloride of platinum, 36, 42, 43, 59, 61. Chloride of vanadium, 418, 441. Chloride of zinc, for hardening brain, 396, 397 ; for impregnation, 434. Chlorine bleaching methods, 328. Chloroform, for paraffin imbedding, 80, 81; for celloidin ditto, 104; for bleaching, 330 ; for narcotisa- tion, 13 ; for clearing, 70. CHOLODKOWSKY, embryology of Blat- tida, 350. Chromate of ammonia, 61. Chromate of lead stain, 261. Chromate of silver impregnation, see GOLGI. Chromates as fixing agents, 37. Chromatin, reactions of, 357 360 ; solvents of, 358 ; stains for, 365. Chromic acid, generalities, 29 ; fixing, 29; hardening, 56; killing, 22; maceration, 317; decalcification, 324, 326; chromic acid with acetic acid, 30; with alcohol, 30, 57 ; with nitric acid, 35 ; with platinum chloride, 36 ; with osmic acid, 31 ; with acetic and osmic acid, 31 et seq. Chromic objects, action of light on, 30; bleaching, 30. Chromo-acetic acid, 30 ; chromo-aceto- osmic, 31 35, 361; chromo-for- mic, 31; chromo-nitric, 35 ; chromo- osmic, 31, 57[; chromo-picric, 36, 57 ; chromo-platinic, 36. CHBSCHTSCHONOWIC, gold method, 255. CHUN, imbedding Siphonophora, 95. CIACCIO, gold process, 256 ; motor plates, 381; corpuscles of Golgi, 382. CIAGLINSKI, spinal cord, 414. Cilia of Infusoria, 499. Ciliated epithelium, 471. Cinnamon oil, see Cannel oil. dona, 467. Cladocora, 489. CLAEKE'S spirit-proof cement, 286. Clasmatocytes, 450. Clavellina, 467. CLAYPOLE, serial sections, 124. Cleaning slides and covers, 503. Clearing, generalities, 6; practice of, 64. Clearing agents, 64 et seq. : STIEDA'S experiments on, 65 ; NEELSEN and SCHIEFFEBDECKEE'S, 65 68 ; cel- loidin sections, 108 ; paraffin ditto, 70. Clove oil, 9, 67 ; for imbedding, 79. Coal-tar chromatin stains, 180 et seq. regressive ditto, 182 197; pro- gressive ditto, 197209; coal-tar plasma stains, 215232. Coal-tar colours, see Anilin dyes. COBB'S differentiator, 4. Cocaiu for narcotising, 15. Coccids, 472. INDEX. 513 The numbers refer to the Pages. Cochineal, theory of staining with, 147; use of, 159; HEBBICK'S 151 ; CZOZOR'S, 150 ; KLEIN'S, 151 ; MATES' s, 158, 159; PAETSCH'S, 150. Cochlea, 442, 443. Ccelenterata, 488 493. COHXHEIM, gold staining, 252 ; silver staining, 668. COLE, freezing method, 116. Collagen, 444 et seq. COLLIX, Criodrilus, 483. COLLINGE, pelagic fish ova, 346. Collodion imbedding, 99 et seq.; sec- tion-fixing process, 120122. Collodionising paraffin sections, 89. Colophonium, cement, 288 ; imbedding mass, 115; mounting medium, 281. COLUCCI, retina, 441. Comatula larva?, 488. Combination stains, 233 et seq. ; and see Stains. Congelation imbedding methods, 115 118. Congo red, 225. 402, 414. Conjunctiva, 375. Connective tissue, 444 et seq. COXTEJEAN, Ranvier*s hmatoxylin, 173. Conroh'fa, 477. Copal, imbedding process, 114; varnish for mounting, 282. Copepoda, 472. Copilia, 472. Copper chloride and acetate preserva- tive mixture, 267. Copper sulphate fluids, fixing, 344, 492, 493 ; impregnation process, 434 ; haematoxylin stain, 177. Coral, 489. Corallin, 196. COBI, narcotisation, 14, 15; Flemming's mixture, 33 ; preserving osmic acid solutions, 25. Cornea, 375377. Corpuscles, tactile, 373 et seq.; corpus- cles of Golgi, 381, 382 ; of Herbst and Grandry, 374 ; of Krause, 375 ; of Meissner, 375. Corrosion, 322 et seq.; ALTMAXX'S methods, 323. Corrosive sublimate, 38 ; fixing liquids, 38 42 ; preservative liquids, 266. Cotton blue, 231. COTJPIEE'S blue, 228. Covers and slides, cleaning, 503. Cox, Golgi's mercuric impregnation, 434. Creosote, see Kreasote. Creseis, 469. Criodrilus, 483. Cristatella, 14, 468. Crocein, 219. Crystal violet, 372. Crystalline, 315, 377. Ctenophora, 493. CTTCCATT, picro-carmine, 154; hsema- toxylin, 173 ; retina, 441 ; soda- carmine, 155. Cucitmaria, 486. Cupric fixing mixtures, 492, 493 ; pre- servative ditto, 267; impregna- tion process, 434. Curare for narcotisation, 16, 354. CUBSCHMAXX, amyloid matter, 199. Cyanide of mercury, for fixing, 481. Cyanin, 228. CYBULSKY, gold method, 258. Cytological methods, 354 et seq. ; sub- jects for study, 354356 ; obser- vation of living cells, 354 ; stain- ing ditto, 356; fresh cells, 356; micro-chemical reactions, 357 360; fixing, 360 365; cytologi- cal stains, 365368 ; Nebenkern, Archoplasraakugel, sphere attrac- tive, ibid. ; nucleus of Balbiani, 368 ; cell granules, 368. CZEENY, macerating mixture, 316. CZOKOB, cochineal, 150; turpentine cement, 287. D. Dahlia, 195. Dammar, 280. DASZKIEWICZ, KOEYBUTT-, plasma- cells, 446. 33 514 INDEX. DAVIDOFF, ova of Distaplia, 346. DAVIES, injection, 300. DEANE, mounting medium, 268; gly- cerin jelly, 273. Decalcification, 323 et seq. Decapoda, 472. DECKEB, section stretcher, 87. DEECKE, hardening brain, 396; im- bedding and cutting, 400. Definition, affected by transparency of stains, 188 j green light for, 504. Degenerate nerves, 412. Dehydration, 3, 4. DEJEBINE, neurological methods, 386, 400. DEKHUYSEN, fat, 445; blood, 459; silver staining, 246. DELAFIELD, hamatoxylin, 170. DELAGE, osmium-carmine, 155 ; Rhab- docoela, 477. Deltapurpurin, 225. Dendrocoela, 478. Dendrophyllia, 489. DENDT, Geonemertes, 479. Depigmentation, 328 et seq., 472 et seq. DE QUEBVAIN, fixation of nerve- centres, 387. Desilicification, 327. DE SOUZA, pyridiu, 62. Dextrin freezing mass, 117. DEZSO, Tebhya, 494. Differentiators, 4. Diffusion apparatus, 4. Diffusion currents, to avoid, 4. Digestion, 320 et seq., 358. DIMMOCK, carminic acid, 145, 155. Dinitrosoresorcin, 403. DioiiiDorr, hardening brain, 392. DipTiyes, 493. Diptera, embryology, 351. Dissections, minute, 6, 9. Dissociation, 312 et seq. Distaplia, 346. DOGIEL, olfactive organs, 375; iris, 383 ; methylen-blue impregnation ' method, 207, 209, 212 ; corpuscles of Herbst and Grandry, 374; of Meissner, 375 ; retina, 441. The numbers refer to the Pages. DOMEBGUE FABBE, see FABEE DO- ME EGUE. DONALDSON, hardening brain, 391, DOSTOIEVSKY, iris, 383. Double imbedding in celloidin and pa- raffin, 112. Double stains, see Stains, Combination. DOYERE, nerve and muscle of Arcti- scoida, 475. DEASCH, gold staining, 252; tactile hairs, 373. DRASH, serial section method, 122. DEOOST, Mollusca, 471. DEUEBIN, blood-plates, 461. DUNHAM, clearing celloidin sections, 109. DTI PLESSIS, Nemertians, 479; Pro- tozoa, 499. DUEHAM, alcohol section-fixing pro- cess, 120. DUEIG, formaldehyde in Golgi's im- pregnation, 427. DTJVAL, silver staining, 246, 248 ; car- mine and anilin blue, 236; col- lodion imbedding, 100 et seq. ; embryology of Aves, 341 ; harden- ing encephalon, 396; purpurin, 235; paraffin sections, flattening, 91. E. Ear, inner, 442, 443. Eau de Javelle, 322, 330. Eau de Labarraque, 322, 330. EBNEE, VON, decalcification, 325. Echinodermata, 485 488. Echinorhyncus, 480. Echtgelb, 219. Echtgriin, 403. EDINGEE, nerve-centres, 59. EHLEES, fixing fluid for Annelids, &c., 31. EHEENBAUM, imbedding method, 115. EHELICH, dahlia, 195 ; ha3raatoxylin, 171 ; methyleu blue for nerves, 203 ; Mastzellen, 445, 447 ; gen- tian violet, 192; blood, 456, 459; classification of anilin dyes, 179 ; of cell granules, 446 ; neutral red, INDEX. 515 The numbers refer to the Pages. 226 ; acidophilous mixture, 229 ; mixture for eosinophilous cells, 229 ; triacid mixture, 223. EHRLICH-BIONDI-HEIDENHAIN stain, 219223. EHRMANN, structure of epithelium, 372. EICHLER, labyrinth, 443. Kr IK MAN, degenerate nerves, 412. EISIO, fixing mixture, 61 ; narcotising, 14. EISEN, serial sections, 128. EISMOND, quieting Infusoria, 495. Elastic tissue, 450, 451. Eledone, eyes, 470. KIT EL, gum for labels, 504. ELSCHNIG, celloidin imbeddfng, 101. Embryology, general methods, 331 et teq.; Amphibia, 342 344; Am- phipoda, 352; Aphides, 351; Ara- ueina, 351 ; Arthropoda, 348 352 ; Attacus, 352 ; Aves, 338 et seq. ; Blattida, 350 ; Cephalopoda, 346 ; Diptera, 351;Echiuodermata,487; Gastropoda, 347 ; Lepidoptera, 350; Mammalia, 335; Mollusca, 346 et seq.; Phalangida, 351; Pisces, 344 et seq. ; Planaria, 352 ; Reptilia, 342; Teleostea, 344 346 ; Tunicata, 346 ; Verraes, 352 et seq. Embryos, reconstruction from sections, 333. EMERY, injection, 309. Encephalon, 385 et seq.; find see Cen- tral nervous system, neurological methods, &c. Emlosinosis, to avoid, 4. EXGELMANN, ciliated epithelium, 471 . green light in microscopy, 504. ENTZ, G., Infusoria, 498. Eosin, 226; eosiu stains for blood, 458, 459. Epidermis, 370. Epithelium, 370 et seq. ; ciliated, 471 ; glandular, 370. ERLICKI, hardening fluid, 59. ERRERA, nigrosin, 197. - Krythrosin, I'L'-:. Essence, see Oil, and Clearing agents. ETERNOD, histological rings, 244. Ether, for preserving, 5. Etherisation, 14, 354. EULBNSTEIN, cement, 296. Eunice, 484. ETERARD, DEMOOR and MASSART, haematoxylic eosin, 239. Examination media, 262 et seq. EXNER, hardening brain tissue, 391; process for medullated nerve tracts, 412. Exosmosis, to avoid, 4. ETCLESHKIMER, orientation of celloidin objects, 103 ; cutting same, 110 ; clearing, 109; serial sections, 129. Eyes of Arthropoda, 473 et seq.; of Cephalopoda and Heteropoda, 470 ; of Chitonidae, 470; of Gastro- poda, 469; of Pecten, 470; of Asteroidea, 486. F. FABRE-DOMERGTIE, glucose presejjva- tive medium, 270 ; Infusoria, 497, 499. FAIRCHILD, dehydration cylinders, 4. FAJERSTAJN, nerve-endings in tongue of frog, 375. FARIS, glycero-gum, 269. FARRANT'S medium, 268. Fast green, 228 ; fast blue, 228. Fat, 35, 445. FATJSSEK, critique of Flemming's mix- ture, 32. Fecundation, artificial, 331. FEIST, methylen-blue method, 210 212 ; orientating spinal cord, 401. FEREERI, phloroglucin decalcification, 326, 327. FERRIA, elastic tissue, 451. FEHRIER, examination liquid for blood, 459. FICK, Golgi's silver chromate method, 429. FIEDLER, Spongilla, 494. FIELD and MARTIN, petroleum-ether for imbedding, 80 ; orientation in paraffin, 85 ; double imbedding, 113 ; Schallibaum's collodion, 121. 516 INDEX. The numbers refer to the Pages. FISCHEL, medullated nerve, 404. FISCHEE, soap imbedding mass, 477 ; Trematodes, 476 ; tactile corpus- cles, 373 ; motor plates, 379. FISCHEE, A., cell granules, 369. FISH, oil of thyme, 69, 109 ; celloidin section-cutting, 110; decalcifica- tion, 323, 324, 326; hardening brain, 391, 396, 397, 398 ; formalde- hyde for Golgi's impregnation, 427. Fixing, generalities, 2 et seq., 18 et seq. Fixing agents, 24 et seq.; action of, 18; choice of, 19; methods of using, 20 ; washing out, 2, 20 ; cytological, 360 365. FLATAF, Golgi's sublimate impregna- tion, 433. Flattening sections, 87, 91. Flagella, stains for, 499. FLECHSIG, gold method, 256 ; redwood nerve stain, 411; other nerve stains, 434. FLEMMING, chromo-acetic acid, 30; chromo-aceto-osmic acid, weak solution, 31 ; strong ditto, 33 ; cy- tological methods, 354, 355, 357, 360, 363; daniar solution, 281; double stains, 216, 217; eyes of Gastropods, 469 ; fixing liquids, 30, 31, 33, 50, 361 ; preservation in glycerin mixtures, 5 ; injection of molluscs, 471 ; staining method, 182 et seq., 189 et seq., 195 ; saf- ranin solution, 190; gentian violet, 192 ; dahlia, 195 ; orange method, 217 ; fatty tissue, 445 ; bone, 454; goblet cells, 463; con- nective-tissue fibrils, 444. FLESCH, MAX, chromo-osmic mixture, 31 ; dichroism of hamatoxylin stains, 165; inner ear, 443; mo- dification of Weigert's hsema- toxylin stain, 407 ; dry preserva- tive process, 439 ; dental tissue, 456 ; blood, 457. FLOGEL, serial sections, 126. FLOEMAN, celloidin imbedding, 106. Flustra, 468. FOA, fixing liquid, 42 ; hsematoxyliu and saf ranin stain, 240 ; blood,460. FOETTINGEB, chloral hydrate narco- tisation method, 14; prepared paraffin, 94. FOL, absolute alcohol, 52 ; albumen fixative, '124; bleaching osmic objects, 27, 28; chromo-aceto- osmic acid, 32 ; cleaning slides,. 503; decalcification, 326; gelatin fixative, 128 ; glycerin jelly, 295 ; gold-impregnation of marine ani- mals, 258; gumming labels, 504; imbedding in vacuo, 83 ; injection masses, 299, 303, 305, 306; In- fusoria, 499; narcotisation me- thod, 17; paraffin oven, 82; per- chloride of iron fixing and staining process, 44, 260; picro-carmine r 154 ; picro-chromic acid, 36 ; re- constructing sections, 333. Formaldehyde, properties, 53 ; for fixing, 53; hardening, 62; pre- serving, 466; macerating, 314;. hardening brain, 397, 398 ; for the Golgi impregnation, 427 ; as a> mordant, 187. Formalin, see Formaldehyde. Formalose, see Formaldehyde. Formic acid for fixing, 46. Formol, see Formaldehyde. FOSTEE and BALFOUE, embryology of Aves, 338, 340. Fowl, embryology of, 338, 341. FEANCOTTE, section-stretcher, 87 ; vacuum imbedding, 83. FEEEBOEN, staining nervous centres, 416 ; connective tissue, 444. Freezing microtome, masses for, 115 118. French cement, 287. FEKNKEL, fixing mixture, 43. FEENZEL, serial section methods, 126, 128 ; sublimate solutions, 38. FEEY, injection mass, 305 ; iodised serum, 264; indifferent liquids, 263, FEIEDLAENDEE, fixing mixture, 493; critique of Golgi's impregnation, 420. Frog, bladder, 384; tongue, 375. Fuchsin, basic, 196 ; ZIEHL'S carbolic, 196. INDEX. 517 The numbers refer to the Pages. Fuelisin S, see Saurefuchsin, and Acid fuchsin. G. GAGE, picro-caruiine, 154; chloral hEematoxylin, 173; decalcification, 325; section stretcher, 87; pre- -i-rvative fluid, 266 ; injection, 310; smooth muscle, 382; section-fixing, 122, 129; clear- ing mixture, 69; picric al- cohol, 50; preservation of potash or soda preparations, 314 ; macera- tions, 314, 317. GALEOTTI, intra-vitam staining, 139; neutral red, 226. GALLEMAEBTS, section-fixing process, 121. GALLI, stain for neuroceratin funnels, 403. GANNAL, mounting medium, 265. GABBINI, safranin staining, 191; Al- cyonaria, 489; cleaning slides, 503. GASKELL, paraffin sections, water method, 91. Gastropoda, embryology, 347; eyes, 469 ; preparation, 468 et seq. ; killing, 16, 468. GAULE, section-fixing process, 119; hardening brain, 392; fixing liquid, 40 ; choice of paraffin, 94. GAY-LrssAC, table for diluting al- cohol, 502. GKBEBG, gold method, 257 ; corpuscles of Grandry, 374. GEDOELST, digestion, 321 ; medullated nerve, 404. GEHTJCHTEN, VAN, acetic alcohol, 45; cytological methods, 364; nerve centres, 399, 403. Gelatin, imbedding masses, 97, 98, 117 ; injection masses, 293 et seq. ; cement, 285 ; section fixative, 127. GELPKE, Weijrert's haematoxylin, 406. Gentian blue, 230. Gentian violet, 192194. GEOFFBOY, mounting medium, 275. Geonemertes, 479. Gephyrea, 481. GEELACH, gold method, 255, 256; gelatin imbedding, 98 ; injection, 300; embryology of Aves, 339; nerve-endings in muscle, 379. GEZA ENTZ, Infusoria, 498. GIACOMINI, gelatin process for serial sections, 134 ; dry process for pre- serving brains, 437. GIBBES, HENEAGE, borax-carmine, 155 ; cleaning slides, 503. GIEBKE, maceration of nerve tissue, 316; staining nervous centres, 416; action of chloral on ditto, 58; uranium carmine, 155; im- pregnation, 242. GIESBBECHT, clearing method, 64; serial section method, 122; section- stretcher, 87 ; paraffin imbedding, 81, 82. GIESOX, VAN, clearing celloidin sec- tions, 109; formaldehyde, 397. GiLSON, acetate of uranium fixing liquid, 46 ; fixing with osmic acid, 26 ; bleaching bichromate objects, 58, 330 ; celloidin imbedding, 111 ; chloral hydrate jelly, 275 ; double imbedding, 113 ; mercuro-nitric fixing mixture, 40; preservative fluid, 266. Glandular structures, 461 et eq. Glucose preservative media, 270. Glue, marine, 287. Glycerin, 270 et seq.; glycerin mounts, to close, 285 ; injections, 308 et seq. ; glycerin jelly, 97, 98, 273 et seq. ; preservative mixture, 272. Glycero-gnm, 269. Glychaemalum, 169. GOADBY, preservative fluids, 267. Goblet cells, 463. GOETTE, hardening liquid, 344. Gold chloride, for impregnation, gene- ralities, 250 et seq. ; composition of the commercial salt, 251; and see the names of Authors. Gold orange, 219. Gold size, 287. 518 INDEX. The numbers refer to the Pages. GOLGI, chromate of silver impregnation, introduction, 419 ; slow method, 421423; rapid method, 423; mixed method, 424; critique of the same, 424 et seq. ; variations, 425 431 ; fixation of nerve-cen- tres by injection, 387 ; mercuric impregnation of nervous centres, 431; corpuscles of Golgi, 381, 382; nerve-endings in muscle, 381. GOODALL, spinal cord, 399. Gorgonia, 489. GORONOWITSCH, embryology of Tele- ostea, 345. GRAF, fixing Hirudinea, 485. GRAFF, VON, Turbellaria, 477. GKAM, stain for bacteria, 193. GBANDRY, corpuscles of, 374. Granule cells, 445 et seq. Granules, protoplasmic, 368, 445 et seq., 459, 460. Grape-sugar imbedding mass, 114. GKASEE, staining process, 201. GRATIS, section, fixative, 127. GRAY, serial sections, 127. Green light in microscopy, 504. GRENACHER, alum-carmine, 150 ; alco- holic carmine, 157; bleaching mixture, 330 ; borax-carmine, aqueous, 155 ; alcoholic, 156 ; castor oil for mounting, 278 ; hsematoxylin, 170; purpurin, 235 ; eyes of Mollusca, &c., 470. GREPPIN, Golgi's silver chromate method, 430. GRIEB, alum-carmine, 150. GRIESBACH, benzo-purpuriu,225; Ben- gal rose, 226; Biebricher Sclmr- lach, 226 ; blood-stains, 456, 457 ; Congo red, 225 ; elastic tissue, 450; iodine green, 228; metanil yellow, 219; methyl green, 200; gold orange, 219. GROUVEN, Ehrlich-Biondi mixture, 222. GBUBLER, histological reagents, 144; cherry gum, 495. Griinpulver, 197. Grunstichblau, 230. Guernsey blue, 228. GUIGNET, soluble Prussian blue, 301. GULLAND, flattening and mounting paraffin sections, 91, 120; blood, 460. Gum, arabic, pure, 126 ; preservative media, 268 et seq.; imbedding masses, 113, 114, 116, 117; injec- tion mass, 307; mucilage for labels,. 504 ; fixative for sections, 126. Gum damar, 280. Gum sandarac, 283. Gutta-percha, cement, 287; section- fixing process, 128. H. HACKE, hsematoxylin, 173. Hseniacalcium, 172. Hffiinalum, 167, 168. Haemateate of ammonia, 164. Haainatein, generalities, 161 167 ; properties, 163; where obtainable, 163 ; formulae for hamatein stains > 167 et seq. ; alumina-hsematein stains, 167 173 ; other haematem stains, 173 178; and see Haema- toxylin. Haematem-ammoniak, 163. Hsemateinum crystallisatum, 163. Haematoxylic eosin, 238. Hffiinatoxylin, generalities, 161 167; theory of staining with, 161 ; ripening of solutions, 161 Ki3; dichroism of the stains, 1G4, 165; affiuitiesfor plasma granules and lor mucin, 165 ; the practice of staining, 164 166; how to "blue" stains, 164,165; formulae for stains : alum solutions, 167 173 ; stains depending on the formation of other lakes, 173 178,367; combination stains, 237 et seq. ; and see the separate en- tries, such as " Ironhaematoxylin," "Haemacalcium," &c. ; and see Hiematein. Haemoglobin, 458. HAENSEL, liquid of, 36. Hairs, tactile and others, 372. I.XDEX. 519 The numbers refer to the Pages. Halicryptat t 4Sl. HALLE and BORN, orieuting celloidiu objects, 103. HALLEK, BELA, macerating fluid, 318. HALLIBURTON, chemistry of the cell, 359. HAMAXX, carmine, 155 ; Echinoderms, 486 ; Acjiuthocephali, 480. HAMILTON, freezing process, 116; hardening brain, 395; freezing ditto, 399. HAXAMAN, cleaning slides, 503. HAXSEX, haematein stain, 109; elastic tissue, 451. HANTSCH, glycerin medium, 273. Hardening, generalities, 21 ; practice of, 22 ; nerve-centres, 388, 398. Hardening agents, 56 et seq. ; choice of, 23. HARDY, Rotifers, 482. HAUJIEK, silver-staining marine ani- mals, 249. HARTIG, white injection mass, 305. HARTING, preservative fluid, 266; oemeut, 287. HARWELL'S dehydration apparatus, 4. HAUG, borax-carmine, 155; haema- toxyliu, 327; decalcification, 323, 325, 326; modification of Wei- gert's nerve stain, 412. HAYEM, blood, 456, 457. Heat, killing by, 11; fixing by, 365. HEIDEXIIAIX, M., cytological methods, 355, 364, 366; lanthaniu, 364; haematoxylin (iron), 175, 366; discussion as to the Ehrlich- Biondi stain, 220, 222; paraffin imbedding, 80; cause of " shrink- age " in paraffin sections, 80, 87; sublimate solution, 40; water sec- tion-fixing process, 120; thionin, 194; Bordeaux-iron-haematoxyliii, 366; vanadium lueinatoxylin, 3l>8. HEIDENHAIN, K., haematoxylin, 173 ; glands of small intestine, 465. H EIDER, collodionising sections, 9C. Helix, eyes, 409; ova, 347 ; narcotisa- tion, 15, 16. HELLEE and GUMPERTZ, osmic acid impregnation, 200. HENCHMAN, embryology of Limax, 347. HENKING, collodionising sections, 90 ; embryology of Diptera, 351; of Phalangida, 351; of Insecta in general, 348; examination liquid for ova, &c., 349. HENLE, staining nervous tissue, 415. HENNEGUY, alum-carmine, 151 ; em- bryology of Aves, 340; of Axolotl, 343 ; of Gastropoda, 347 ; of Te- leostea, 344 ; Protozoa, 495, 496 ; washing out carmine stains, 148 ; permanganate staining method, 186; mounting methyl green stains, 199 ; mouutipg paraffin sections, 91, 125, 127; L. John- son's platinic mixture, 59, 00; staining with thionin, 195; re- staining old mounts, 505. HEXOCQUE, gold process, 255. HEEBST, corpuscles of, 374. HEELA, stain for attraction spheres, 368. HERMANN, silver staining, 246; papilla? foliatae of rabbit, 375; platino- aceto-osmic mixture, 43, 362; osmium and pyroligueous acid impregnation, 258; cytological stains, 367; formaldehyde, 53, 54, 62, 63. HERMANN-BOTTCHBR staining process, 182, note. HERRICK, cochineal alum-carmine, 151. HERTWIG, silver staining, 246, 249; macerating methods, 318; Actinia?, 488; Medusae, 491; embryology of liana, 343; of Triton, 343; tobacco-smoke method, 12. HERXHEIMER, elastic tissue, 450; epi- thelium, plasma fibrils of, 372. HESCHL, amyloid matter, 199. HESSERT, stains for flagella, 500. Heteropoda, eyes, 470. HEUKCK, VAX, mounting medium, '277. HEYDENREICH, amber varnish, 289. HEYMONS, embryology of Biattida, 351. HEYS, balsam, 279. 520 INDEX. The numbers refer to the Pages. HICKSON, haematoxylin and eosiu, 237 ; maceration, 314 ; eye of Musca, 473. HIBOTA, blastoderm of fowl, 341. Hirudinea, 15, 208, 484. His, fixing, 35 ; cornea, 376. HOCHSTETTEE, injection, 310. HOFEE, narcotisation method, 15. HOFFMANN, vacuum imbedding, 83. HOFMANN, ovum of fowl, 340. " Hofmann's Griin," 228. HOGGAN, silver staining, 246; histo- logical rings, 244 ; perchloride of iron stain, 260. HOLL, toluol for imbedding, 79; note concerning liquid of Kleinenberg, 361. Holothurioidea, 485. Homarus, eyes, 474. HOPEWELL SMITH, decalcification, 325 ;' teeth, 453. HOPKINS, maceration, 318. Horn, 372. HOYEE, picro-carmine, 154; silver staining, 246; gold ditto, 255; gum-arabic mounting medium, 268 ; gelatin injection masses carmine, 298; Berlin blue, 303; lead chromate, 304 ; silver nitrate, 305; green, 305; shellac mass, 310; oil-colour masses, 310; chlo- ral for preserving masses, 294; water section-fixing process, 120; reactions of muciu, 461. HOTEE, jun., formaldehyde, 54. HITBER, Golgi's silver chromate method, 429 ; medullated nerve, 404. HUDSON, Rotifers, 482. HYATT, imbedding method, 114. Hydatina, 482. Hydra, 15, 490. Hydrate of chloral for narcotising, 14 ; and see Chloral. Hydrochlorate of cocain, 15. Hydrochloric acid for decalcification, 323, 325. Hydrofluoric acid for desilicification, 327. Hydrogen peroxide for bleaching, 25, 28, 30, 329. Hydroidea, 490. Hydroxylamin, 15. Hypochlorite of potassium, see Eau de Javelle. Hypochlorite of sodium, see Eau de Labarraque. I. IDE, double imbedding, 113 ; epi- thelium, 371. IGACUSCHI, gold process for liver, 464. IIJIMA, embryology of Planaria, 352. Imbedding defined, 74; generalities on, 74 et seq.; double imbedding in celloidin and paraffin, 112 ; and see also Celloidin, Paraffin, &c. Impregnation, distinguished from staining, 241 ; negative and posi- tive ditto, 241 ; primary aud secondary ditto, 242. Impregnation methods, 241 et seq. ; and see Gold chloride, Nitrate of silver, Methylen blue, &c. Inchiostro di Leouardi, 200. India-rubber cement, 287. Indian ink injection, 310. Indifferent liquids, 262 et seq. ludigen, 228. Indigo substitute, 228. Indigo-carmine, 235 ; THIEESCH'S staining method, 235 ; SEILEE'S, 235; MEEKEL'S, 236; with saf- ranin,229. Indirect staining method, 137 et seq. Indulin, 228. Indulin-aurantia-eosin mixture, 229. Infusoria, 495 et seq., and see Protozoa. Injection of Mollusca, 471. Injections, 293 et seq.; injection masses, albuminous, 307; aqueous, 309 et seq.; collodion, 310 ; fatty, 310 ; gelatinous, 293 et seq. ; glycerin, 308 et seq. ; gum, 307; resinous, 310; starch, 310; and see the names of Authors. Injections, natural, 311. Inner ear, 442, 443. Insecta, see Arthropoda. Insecta, embryology, 348 et seq. INDEX. 52] The numbers refer to the Pages. Intra-epiderniic nerve-fibres, 373. Intra-vitam staining, 138, 203. Inversion of stains, 142, 224. * lodic acid for fixation, 458. Iodide of palladium nerve stain, 413. Iodine for fixing, 46, 47 ; for harden- in-, 62; LUGOL'S solution, 46; iodine as a mordant, 141, 143. Iodine green, 228. Iodine haematoxylin, 173. Iodised serum, 263, 264; ditto for macerations, 313. Iris, 383. Iron, perchloride, for fixing, 44; for staining, 260 ; pyrogallate of, 260; iron-hfeinatoxylin stains, 175 177; with Bordeaux, 366; iron- carmine, 152 ; detection of iron in cells, 359. ISRAEL, orcein, 234. J. JACKSON, cloudiness in clearing media, 65. JACOBS, freezing mass, 117. JACQTTKT, injection of leeches, 485. JADASSOHN, structure of epithelium, 272 ; thiouin for plasma-cells, 450. JAGEB, glycerin medium, 273. JAKIMOYITCH, silver staining, 248 '> medullated nerves, 404. JAMES, cleaning slides, 504 ; damar solutions, 281. JANSSENS, carmine blue, 231. JAVELLE. eau de, see Eau de Javelle. JELGEHSMA, staining nervous centres, 416. JELINEE, extraction of picric acid, 47 ; Stabilit imbedding blocks, 108. Jellies, glycerin, 273275. .JENSEN, immobilisation of Infusoria, 496. JOHNSON, LINDSAY, keeping osmic itions, 24; sunning solutions of metallic salts, 24, 243 ; platiuic hardening mixture, 59; gold im- pregnation, 257 ; retina, 439, 440; cementing celloidin blocks, 107- JOLIET, imbedding method, 113. JOSEPH, injection mass, 307. JULIEN, cement, 289; stains for flagella, 500. JUNG, microtomes, 71. K. KADTI, imbedding mass, 95. KAES, nerve stain, 411. KAISER, glycerin jelly, 97, 274 ; uaph- thylamin brown for spinal cord, 417; Bismarck-brown stain, 196; nerve stain, 410. KALLirs, Golgi's silver chromate method, 426, 431. KANTHACK, histological reagents and apparatus, 144. KASTSCHENKO, reconstruction from sec- tions, 333. KEISEB, sublimate solution, 40; fixa- tion of Acanthocephali, 481. KEMP, blood-plates, 460. KENT, S., Infusoria, 46, 497. KENT, A. F. S., cement, 290. Kernschwarz, 233. Killing tissues and organisms, 11 17. KIXGSLET, imbedding method, 77 ; section-fixing, 123. KIONKA, blastoderm of fowl, 341. KISHINOUTE, embryology of Araneiua, 351. K i TTON, asphalt, 286; cement, 290. KLEBS, glycerin jelly, 97. KLEIN, fixing mixture, 30; cornea, 375; cochineal, 151. KLEINENBERG, colophonium for mount- ing, 281; embryology of Lum- bricu*, 352; picro-sulphuric acid, 48; haematoxylin, 173. KLEMENSIKWICS, picro-carmiue, 154. KNAUBB, cleaning slides, 504. Knotting, for cement, 287. KOCH, VON, copal imbedding method, 114. KOFOID, embryology of Agriolimax, 348. KOGANEI, iris, 383. 522 INDEX. The numbers refer to the Pages. KOLLIKEB, ovum of rabbit, 335, 337 ; bone, 454. KOLLMANX, fixing mixture, 345. KOLOSSOW, gold method, 256; osmic acid mixture, 27 ; osmic acid stain, 260 ; regeneration of osmic acid solutions, 25. KOPPEN, elastic tissue, 451. KOPSCH, formaldehyde for Golgi's im- pregnation, 427. KOBOTNEFF, killing Siphonophora, 13. KOBSCHELT, Infusoria, 497. KOBYBUTT-DASZKIEWICZ, plasma-cells, 446. KOSSEL, chemistry of the cell, 359. KOSSINSKI, double stains, 229, 230. KOTLABEWSKY, central nervous system, 32, 61, 392. KOWALEWSKY, embryology of Teleos- tea, 345. KBATISE, corpuscles of, 375; molybdate of ammonium, 261 ; motor plates, 381; silver staining, 248; retina, 441, 442; thiophen green, 228; Ehrlich-Biondi mixture, 219. Kreasote, for blenching, 329; for clearing, 70, 109. KKOMAYEB, plasma tibrils of epithe- lium, 371. KBONECKEB. artificial serum, 265. KBONIG, cement, 288. KBYSINSKY, photoxylin imbedding, 100. KUHNE, digestion fluids, 321 ; macerat- ing mixture, 318 ; motor plates, 381. KUHNE, H., aniseed freezing mass, 118. KUHNT, retina, 441. KUKENTHAL, Annelids, 14, 483, 484. KULTSCHITZKY, fixing mixture, 36 ; double imbedding, 112; preserva- tive fluids, 5; tactile corpuscles, 374 ; stain for nervous centres with hannatoxylin, 410; ueuro- glia, 436; elastic tissue, 451; " patentsaures Rubin," 219. KUPFFEB, embryological methods, 342 ; axis-cylinder, 403 ; liver, 464. KUSKOW, digestion fluid, 320. L. LABAEBAQUE, eau de, see Eau de Labarraque. Labelling slides, 504. Labyrinth of ear, 442, 443. Lacerta, ova of, 342. LACHI, formaldehyde for brain, 397. Lactic acid for decalcificatiou, 324, 326. LAMANSKY, sensibility of the eye, 505. Lamellibranchiata, 468, 471. LANDOIS, impregnation methods, 261; macerating mixture, 316. LANDOLT, retina, 442. LANDSBEBG, Infusoria, 497. LAKG, fixing liquids, 40 ; methods for Platyhelmiti, 40. LANGEBHANS, Amphioxus, 378; gum and glycerin medium, 269; tactile corpuscles, 374; fixation of ma- rine animals, 21. LANGHANS, dichroism of hseumtoxyliu stains, 165. LANKESTEK, eyes of Limulus, 473. Lanthanin, 364. LATJTH'S violet, 194. LAVDOWSKY, blood, 458 ; sandarac me- dium, 283 ; chloral hydrate me- dium, 265; macerating fluid, 314; inner ear, 442 ; formaldehyde, 55. LAWBENCE, glycerin jelly, 273. Lead, acetate, 61, 392; chromate, 261. LEBEB, impregnation methods, 261 > retiua, 441. Lecithin, reactions, 357. LECLEBCQ, stains for blood, 459. LEE, A. B., preservation in paraffin or cedar oil, 5; cedar oil for paraffin imbedding, 80; mounting celloidin sections, 128; intra-vitam stain- ing, 139, 140 ; sublimate solutions, 38, 364; formaldehyde, 53, 54; celloidin imbedding, 101, 105, 106; dry celloidin process, 111 ; Mayer's albumen, 125, 128 ; toluidin blue, 197; malachite green, 228; ni- grosin, 228; bleu de L\on, 230; Wasserblau, 231; Kernschwarz. 233; orcei'n, 235; carmine :md indigo-carmine, 235 ; impregnation INDEX. 523 The numbers refer to the Pages. with gold, 250 ; osmic acid and pyrogallol stain, 253; glycerin mixture, 272 ; resinous mounting media, 277 280; colophonium mounting medium, 282; cedar oil for mounting, 27S; paper cells for mounting, 285 ; Neiiierteans, pre- paration, 478; fixing Hirudinea, 485. Leech, 485. LEGAL, alum-carmine, 152. LKOROS, silver staining, 248. Lemon juice for fixing nuclei, 364 ; for killing, 12. LEXDEXFELD, vox, staining collar- cells, 494. LEXHOSSEK, YOX, tongue of rabbit, 375 ; nerves of annelids, 484. LKXXOX, retina, 441. Lens, ci-ystalline, 315, 377. LKOXARDI, inchiostro di, 200. Lepidoptera, embryology, 350. LEPKOWSKY, gold method for teeth and hone, 454. LETELLIER, injection, 310. LEUCKHABT, imbedding boxes, 76. LEVULOSE for mounting. 270. LEWIS, BEYAX, freezing process for brain, 388; hardening ditto, 394; staining, 416. Liehtgrun, 2i'7. Li EBERMAXX, analysis of carmine, 145 ; rarminic acid, 146. Li^'ht, action of, on alcohol containing chromic objects, 30; on metallic salts, 242 ; green, in microscopy, 504. Jjigitla, 476. LILIEXFELD, blood-plates, 461 ; che- mistry of the cell, 359. LILIENFELD and MONTI, test for phos- phorus in tissues, 359. Limajc, 469 ; embryology, 347. L'tnt'/ns, 473. LINDSAY JOHXSOX, xte JOHNSON, LlXDSAY. Linseed-oil injection, 310. Liquid of Miiller, of Krlicki, of Knlt- iichizky, of Merkel, &c., see the names of the respective Authors. Liquidambar, 283. LIST, hsematoxylin and eosiu, 237 ; methyl green and eosin, 226 ; other combination stains, 464 ; macerations, 313 ; Authozoa, 488 ; Coccidse, 472 ; goblet cells, 464. Lithium-carmine, 155. Litmus paper, to make, 297. Liver, 464. Lo BIANCO, S., see BIANCO, 8. Lo. LOCY, embryology of Agelena, 351. LOEFFLER, stains for flagella, 499. Loligo, eyes, 470. LOXGHI, eserin for Infusoria, 499. LONG WORTH, corpuscles of Krause,375. LONNBERG, cytology of the snail, 356; Trice nophorus, 476 Looss, softening cliitin, 322 ; Nema- todes, 479. LOYETT, cement, 290. LO\VE, crystalline, 377. LO\VEXTHAL, uerve-ceutres, 59 ; picro- earrnine, 154. LOWIT, gold method, 252 ; blood, 456, 457, 459. LO'WY. macerating skin, 371. Loxosoma, 468. LUGOL, solution of, 46. Lumbricus, 483 ; embryology, 352. LUSTGARTEX, elastic tissue, 195, 450. LUYS, noir Colin, 416. Lymphatics, see Impregnation methods and Injections. Lysol, properties, 319 ; for maceration, 319. 1C. MAAS, carmine and malachite green, 237. MACALLUM, Sphyran ura, 477 ; double stain, 236; detection of iron in cells. 359. MACBRIDE, development of Amphiura, 488. Maceration, 312 et seq. ; epithelium, 370 ; Mollusca, 471. Macrotoma, 475. Mugdala red, 195. Magenta, 196 ; Magenta S, 219. MAGIXI, stain for nervous centrrs, 434. 524 INDEX. The numbers refer to the Pages. Magnesium chloride, for narcotisation, 16 ; sulphate, ibid. MAHBENTHAL, VON, osmium impreg- nation, 260. Malachite green, 228. MALASSEZ, ammonia-carmine, 155. MALFATTI, chemistry of the cell, 359. MALLOEY, phospho-molybdic acid hse- matoxylin, 178, 418. Mammalia, embryology, 335 et seq. Manchester brown, 200. MANFBEDI, gold method, 256. Manganese chloride, 263. MANN, osmic acid mixture, 27 ; picro- sublimate mixtures, 41 ; toluidin blue, 197; Wasserblau and eosin, 231; fixative for sections, 126; fixing nerve-centres, 387. MAECHI,' VON, Limax, 469 ; corpuscles of Golgi, 381 ; degenerate nerves, 412. MABCUS, formaldehyde for nerve- centres, 397. Marine animals, killing, 17; fixing, 21; silver impregnation, 248; gold ditto, 258. Marine glue, 287. MASK, collodionising sections, 89 ; embryology of Limax, 347; sec- tion-fixing, 123. MABSCHALKO, VON, plasma-cells, 450. MABSH, bleaching, 328; gelatin ce- ment, 285 ; decalcification mix- ture, 326. MAESHALL, Mays' gold process, 381. MAETIN, benzoazurin, 197, 232. MAETINOTTI, safranin staining, 191 ; damar solutions, 281 ; aniliu black, 416; picro-nigrosin, 417; elastic tissue, 450 ; intra-mtam staining, 138. MASON, nervous system of reptiles, &c., 398. Mastzellen, 445450. MATSCHINSKY, bone, 453. MATTBICE and SCHULGIN, double stain, 237 ; embryology of Amaroecium, 346. Mauvein, 196. MAX FLESCH, see FLESCH. MAYEE, PAUL, acidulated alcohol, 52 ; alcoholic carmine, 157, 158; albu- men fixative for sections, 123; bleaching, 328; borax-caruime, 156; carmine staining, theory of, 145147; Carrnalurn, 149; chlo- ride of aluminium carmine, 150 ; Paracarmine, 156 ; carmine and indigo-carmine, 236 ; cochi- neal staining, theory of, 147; cochineal, old formula, 158 ; new formula, 159 ; desilicification, 327 ; general principles of preparation, y ; hffiinatoxylin (ha3inatein) stain- ing, theory of, 161167 ; Haeina- luui, 167, 168; HsBinacalciurn, 172; Glychaemaluin, 169 ; pre- paration of the ammonia-salt of hsernatein, 164 ; bluing hsema- tein stains, 164, 165; mounting haematein stains, 278 ; critique of Kleinenberg's haeinatoxylin, 173 ; injection, 309; picro-carmine, 154; picro-sulphuric acid, 48; picro-hydrochloric acid, 50 ; picro- nitric acid, 50; section-stretcher, 87; section-fixing methods, 123 125 ; shellac fixative, 123 ; theory of staining, 137 ; staining mucus, 462 ; Mucicarmine, 463 ; Muchse- matein, 463; water- bath for paraflin, 82. MAYBE, S., ' violet B." for connective tissue, 231, 444; rnethyleii-blue method, 207, 209, 212. MAYS, nerve-endings (gold process), 381. MAYSEL, Bismarck brown, 200. Medullated nerve, 403, 404; and see Neurological methods. Medusae, 490492. MEISSNEE, corpuscles of, 375. MEECIEE, haernatoxylin nerve stain, 412 ; Upson's nerve-impregna- tions, 436, 437 ; Golgi's impreg- nation methods, 419, note. Mercury bichloride, see Corrosive sub- limate; biniodide, mounting me- dium, 275 ; cyanide, for fixing, 481 ; nitrate and picrate, 364. INOKX. 525 "mbers refer to the Pages. MERE, making up chroino-aceto-osmic acid, 34. MEBKEL, chromo-platinic liquid, 36; impregnation method, 261; car- mine and indigo stain, 236; cel- loidin imbedding, 100; staining nervous tissue, 415. Metagelatin, 306. Metallic salts, action of light on solu- tions, 242. Metallic stains, 241 et seq. Metanil yellow, 219. METCALF, embryology of Chiton, 348. Methanilin green, 197. Methanilin violet, 200. Methyl alcohol for narcotising, 14; tor safranin staining, 191. Methyl blue, 230. Methyl green, 197 200 ; combination stains with, 226. Methyl mixture for maceration, 319. Methyl violet, 200. Methylal, for dehydration, 5, 211. Methylated spirit, 501. Methylen blue, for impregnation, 202 214, 443; generalities, 202; staining in toto during life, 203; staining nerve-tissue during life, 204 ; influence of oxygen and of ammonia, 205 ; staining by in- jection or by immersion, 206; the solutions employed, 206 ; pre- servation of the stain, 209; im- pregnation of epithelia, lymph- spaces, &c., 212, 213 ; of central nervous system, 443 ; polychro- matic, 202 ; with eosin, 227 ; borax-methylen blue, 365 ; stains for hardened nervous tissue, see RBHM, SAHLI, and ADAMKIEWICS ; for plasma-cells, 448. MEYEB, salicylic solutions, 267 ; silver staining, 249. METEB, SEMI, methylen blue for cen- tral nervous system, 443. MIBELLI, elastic tissue, 451. Michrochemistry of the cell, 357 360. Microtomes, 71 74. MIGTTLA, glycerised serum, 265. MILLER, injection, 305; caoutchouc cement, 286. Milne-num, 475. MlNOTjhaematoxylin stains, 178; clear- ing celloidin sections, 109 ; mace- rating skin, 371 ; carmine, 155 ; microtome, 73. MITBOPHANOW, gold process, 371 ; maceration, 371 ; sense organs of Amphibia, 375 ; "Wasserblau, 231 ;. prickle-cells, 371. MITSTTKFEI, embryology of Reptilia,. 342. MOBIUS, macerating media, 317. MOEBXEB, tracheal cartilage, 456. MOLESCHOTT, potash or soda solution, 314; MOLESCHOTT and Piso BOEME'S macerating fluid, 314. Molgula, 467. Mollusca, 468471 ; embryology, 346 et seq. Molluscoida, 11, 18, 19, 468; and see Bryozoa. Molybdate of ammonium, 364. Monobromide of naphthalin, 277. MONTI, sulphate of copper impregna- tion method, 434; test for phos- phorus in tissues, 359. MOOEE, stain for blood, 458. MOOEE, V. A., aniseed imbedding, 118* Mordants for staining, 140 143. MOBGAN, ova of Arthropods, 348, 472 ; removing albumen from ova, 343, 348 ; test-cells of Ascidians, 346. Morphia for narcotising, 16. MOSELEY, shell, 470; Chitonidse, 470. Mosso, blood, 457. Motor plates, 378 et seq. Mounds of Doyere, 475. Mounting media, see Examination media. Muchaematein, 463. Mucicarniine, 463. Mucilage for labels, 504. Mucin, 461 et seq. Mucus, removing, from Mollusca, 469. Mucus glands, 461 et seq. MULLEB, blood, 456; hardening liquid,. 58; silver staining, 247; injection mass, 309. 526 INDEX. The numbers refer to the Pages. MUNDEB, G., Instological reagents, 144. MTJNSON, chloral hydrate fluid, 266. Muse a eyes, 473. Muscle, dissociation of, 378; nerve- endings in, 379 et seq. ; smooth, 382 et seq. ; sections, 378. Myelin, 403; myelin stains, 405 414. Myelon, see Central nervous system. N. Nails, 372. NANSEN, maceration of nerve'tissue, 316. Naphtha, for clearing, 70; for im- bedding, 79. Naphthalin, monobromide, 277. Naphthylamin brown, 417. Narcotisation, 1217. NATHUSIUS, VON, hairs and nails, 373. NEALEY, bone, 453. Nebenkern, see Cytological methods, Plasma stains. NEELSEN and SCHIEFFEEDECKEB, clearing agents, 65. NEGEO, motor plates, 381. Nematodes, 479. NemeT'tina, 478. Nereis, embryology, 353. Nerves, degenerate, 412. Nerves, see Neurological methods. Nerve-endings in muscle, 378 et seq. ; in skin and others, 373 et seq. ; methylen-blue method, 204 et seq.; and see Neurological methods. Nervous system, central, see Central nervous system and Neurological methods. NESSLEB, test for ammonia, 298, note. NESTEBOFFSKY, gold process, 256. Neuroceratin, 403, 404. Neuroglia, 435, 436. Neurological methods, 385 443 ; in- troduction, 385, 386; section methods, 387 401 ; cytological methods of neurology, 401 404 ; nerve-fibre stains, WEIGEBT and others, 405 414 ; axis-cylinder and protoplasm stains, 415 419; axis-cylinder and protoplasm im- pregnations (GOLGI and others), 419 434 ; and see Impregnation methods, Retina, Inner Ear, Nerves, Central nervous system, &c., and the names of Authors. Neutralisation of carmine masses, 296. Neutral red, 226. Neutral tar coloui's, 179. Neutrophilous cell-elements, 446. New green, 228. NEYT, see VAN BENEDEN and NEYT. NIAS, cleaning slides, 504. NIETZKI, carminic acid, 146 ; ha3ma- tein, 161. NICOLLE and CANTACUZENE, impreg- nation method, 261. Nicotin for narcotisation, 13. NIESSING, fixing mixtures, 363. Nigranilin, 231. Nigrosin, 197, 228; with safranin, 230. NIKIFOBOW, boi'ax-carmine, 155; stain for nervous centres, 414 ; clearing celloidin sections, 108 ; acidophi- lous mixture, 229. NISSEN, gentian violet staining pro- cess, 193. NISSL, stains for nerve- cells, 401, 402 ; for nerves, 414. Nitrate of silver, for impregnation, 242 et seq. ; for marine animals, 248 ; and -see the names of Authors. Nitrate of uranium, fixing mixture with osmium, 27. Nitric acid for fixing, 35 ; for harden- ing, 57, 392 ; for maceration, 318 ; for decalcification, 324, 325; for bleaching, 329. Nitric and chromic acid mixtures, 35. Nitrite of amyl as a vaso-dilatator for injections, 293. Noir Colin, 231. NOLL, corrosion, 322 ; sponge spicules, 494; salicylic acid medium, 268. NOEDMANN, plasma-cells, 447. Normal salt solution, 263. NOEEIS and SHAKESPEAEE, carmine and indigo stain, 236. Nuclein, reactions of, 357 360, and see Chromatin. Nucleus, see Cytological methods. Nucleo-albuniins, 358. INDEX. 527 The numbers refer to the Pages. 0. OBERSTEIXEB, central nervous system, 386 ; hardening, 391; staining, 415. OBREGIA, Golgi's silver chrouiate me- thod, 430; serial section method, 133. Oceania, 491. Octopus, eyes, 470. ODEXIUS, tactile hairs, 319. OGATA, hardening brain, 392. OHLMACHEB, reactions of safranin with iodine and picric acid (psec.do- carciuoma corpuscles), 192; sec- tion-fixing process, 125 ; formalde- hyde staining process, 187. Oil, of aniseed, imbedding mass, 118; of bergamot, 68 ; cedar wood, 80; cloves, 9, 67; origanum, 68, 109; sandal- wood, 69; turpentine, 69; and see Clearing agents. Oinmatostrephet, eyes, 470. One-third alcohol, 51, 313. O nu phis, 484. Opal blue, 230. Ophiuridea, 486. Opisthobrauchiata, 468. Opisthotrema, 470. OP PEL, liver, 427, 464. OPPITZ, silver staining, 248. Orange G, 216, 217. Orcein, 234, 445, 451. Orchella, 234. Orchestia, embryology, 352. Orcin, 234. Orientation in paraffin; incelloidin, 103. Origanum oil, 68, 109. Orseille, 234. OETH, methyl violet, 200; lithium- carmine, 155. OSBOEN, embryology of Triton y 343; ijrain of Urodela, 400. acid, for fixing, 2429; for hardening, 57, 391 ; for macerat- ing, 317 ; regeneration of solution, 25 ; for impregnation, 258 ; blackening of preparations with, '27; how to keep solutions, 24; GOBI'S solution, 24 ; KOLOSSOW'S solution, 25, 27 ; osmic acid and alcohol, 27; and chromic acid mixture, 31; and chromic and acetic acid mixture, 31 35 ; pla- tinic mixture, 43; osmic and oxalic stain, 260; osmic and pyro- gallic ditto, 258. Osmicated cells, 28. Osmium-blackened fat, 35, 445. Osmium-carmine, 155. Osmosis, to avoid, 3, 4. Ostracoda, 472. Otocyst of Mysis, 475. Ova, see Embryology. Ovens for paraffin, 82. OVEETON, bleaching osmic objects, 28 ; fixing with iodine vapours, 47. OVIATT, injection method, 294. Ovum, see Cytological methods, and Embryological methods. ONVEX, preservative liquid, 267. Oxalic acid for maceration, 319. Oxygenated water, 25, 28, 30, 329. P. PACINI, preservative liquids, 26G. PAL, haematoxylin stain, 409; staining nervous centres, 433. PALADINO, iodide of palladium nerve stain, 413. Palladium chloride, for fixing, 43 ; de- calcifying, 323; hardening, 61; staining, 261. Palladium iodide nerve stain, 413. Palythoa, 489. Pancreatin digestion fluids, 320. PANBTH, making Weigert's liama- toxylin, 407 ; goblet-cells, 463. PAXSCH, injection mass, 310. Paper, imbedding trays, to make, 75, 76 ; paper cells, 285. Papilla foliatse, 375. Paracarmine, 156. Paraffin, imbedding processes, 79 et seq. ; review of, 92 ; paraffin masses, 93, 94; solvents of, 79, 91 ; ovens, 82 ; paraffin for luting mounts, 289. Paralinin, 359. Paris green, 198. 528 INDEX. The numbers refer to the Pages. Paris violet, 200. PAEKEE, turpentine cement, 287. PABKEE, G. H., dehydration methods, 5; methylen-blue method, 211, 474 ; eyes of scorpions, bleaching-, 329; eyes of Homarus, 474; of Astacus, 474. PABKEB and FLOYD, formaldehyde for brain, 397. Parma blue, 230. PAETSCH, cochineal stain, 150. " Patentsaures llubin," 219. PATTEN, embryology of Blattida, 350 ; orientation in paraffin, 84; eyes of Molluscs and Arthropods, 470 ; maceration of Mollnsca, 471. PAULSEN, goblet cells, 463. Pecten, eyes of, 470. Pedicellina, 468. Pelagic ova, 345. Pennatulidae, 489. Pentacrinus, 488. Pepsin digestion fluids, 320. Perchloride of iron, for fixing, 44 ; for impregnation, 260. PEEEMESCHKO, cytological methods, 354. PEBENYI, fixing fluids, 35, 342. PEEGENS, picro- carmine, 154. Periplaneta, ova of, 350, 472. PEEL, soluble carmine, 155. Permanganate of potash for washing out carmine stains, 148; for macerating, 317 ; for mordanting, 186; for reducing silver impreg- nations, 248. Perophora, 467. Peroxide of hydrogen, 25, 28, 30, 329. Peroxide of sodium, 329. PEEEIEE, narcotising Lumbricus, 483. Perruthenic acid, 464. Petroleum-ether, 80. PFITZNEE, safranin solution, 190; damar solution, 281 ; Infusoria, 497. Phalangida, 475 ; ova of, 351. Phallusia, 467. Phascolosoma, 481. Phenicienne, la, 200. Phenylen brown, 200. Phloroglucin, 326. Phloxin, 226. Phoronis, 481. Phospho-molybdic-acid hffiinatoxylin, 178, 418. Phosphoric acid for decalcification, 326. Phosphorus in tissues, test for, 359. Photoxylin imbedding method, 100. Phyllosoma, 472. Physalia, 493. Physiological salt solution, 263. Physophora, 493. PIANESE, double-stain for connective tissue, 444; rnethylen blue and eosin, 227. Picric acid, fixing fluids, 47 et seq. ,- hardening ditto, 61 ; staining with, 216; decalcification with, 324, 326. Picric alcohol, 50, 317. Picro-alum-carmine, 152. Picro-carminate of ammonia, 153. Picro-carminate of soda, 154. Picro-carmine, 153, 154. Picro-chromic acid, 36. Picro-hydrochloric acid, 50. Picro-nigrosin, 417. Picro-nitric acid, 50. Picro-osmic acid, 50. Picro-platinic mixture, 50. Picro-sublimate mixtures, 41. Picro-sulphuric acid, 48. PICTET, examination liquid, 263. PIEESOL, embryology of Mammalia, 338. Pigment spot?, artificial, 59. PINTNEB, Treniffl, 476. PISENTI, alum-carmine, 150. PlaTclna, larvae, 494. Planaria, 40; embryology, 352. Plasma-cells, 445 450. Plasma fibrils, 371. Plasma stains, 215232, 365. Plastic reconstruction of sections, 333. PLASTIN, 359. Platinic mixture, MEBKEL'S, 36, 61 ; LINDSAY JOHNSON'S, 59. Platino-aceto-osmic mixture, 43. Platino-osino-bichromate mixture, 59. INDEX. Tin.- in'inln r? rrfer io the Pages. Platiuo-subliroate mixture, 332. Platinum chloride, for fixing, 42, 43. PLAINER, Kernschwarz, 233; medul- lated nerve, 403. Platyhelmia, 476479. PLESSIS, DU, see DU PLBSSIS. Plutei'.s, 487. PODWYSSOZKI, fixing mixture, 34 ; saf raniu staining, 191 ; carcinoma corpuscles, 192. Poisoning methods, 16. POLAILLON, impregnation method, 260. POLI, serial sections, 128. POLITZEB, inner ear, 443. Polychaeta, 484. Polychromatic methylen blue, 282. POLZAM, imbedding mass, 95. Porifera, 493. Porpita, 493. Potash acetate, mounting medium. 265. Potash solution for maceration, 314. Potassium bichromate 58 et seq. Potassium hypochlorite, 322, 330. Potassium permanganate, see Perman- ganate of potash. POUCHET, bleaching methods, 329. PRECAST, safranin staining, 191; cochlea, 442. Preservative media, 4, 5, 262 et seq. Prickle-cells, 371. Primrose, 226. PRINGLE, the water section-fixing pro- cess, 120; vacuum imbedding, 83. PRITCHAED, hardening fluid, 57; re- ducing solution, 256 ; inner ear, 442. Progressive staining, 136, 197 et seq. Prosobranchiata, 468. Protula, 484. Protozoa, introductory, 495 ; quieting, 495 ; staining intra vitam, 496 ; cilia, 499; flagella, 499; other methods, 497 499. PRUDDBN, haematoxylin, 170. Prussian blue, soluble, 301 ; impreg- nation with, 261 ; injection masses, see Injections. Prussic acid, for killing, 16. Pteropoda, 469. PUECELL, eyes of Phalangida, 475. Purpuriu, 235. Pyrenin, 359. Pyridin for hardening and clearing 62. Pyrogallate of iron stain, 260. Py rosin, 226. Q. QUEEVAIN, DE, fixation of nerve- centres, 387. Quieting Infusoria, &c., 495. Quince-mucilage fixative, 127. Quinolein blue, 228. R. Rabbit, embryology, 335 et seq. RABL, fixing methods, 31, 41, 42, 332 ; embryos of Salamandra, 343; serial sections, 121; alum-co- chineal, 151 ; picro-sublimate mixture, 41 ; platino-sublimate mixture, 332; medullated nerve, 404; staining method, 239; cyto- logical methods, 363; collodion- ising sections, 90. RABL-RUCKHAED, embryology of Telostea, 345. RAMOX Y CAJAL, see CAJAX. Sana, embryology, 343. RANVIEE, alcohol, absolute, prepara- tion of, 52; one-third ditto, 51, 313; areolar tissue, 444; bladder of frog, 384 ; bone, 451 ; carmine, neutral, 155; clasmatocytes, 450; cornea, 376; corpuscles of Golgi, 381; decalcification, 325; epithe- lium, 370; gland-cells, 464 ; gold chloride impregnation, 253, 254 (and see Motor plates, Nerve- fibres, Sac.) ; haematoxylin, 173 ; injection masses, carmine, 296 ; Prussian blue, 300, 301 ; silver nitra. te,306; glycerin, 309; aqueous, 309; iodised serum, 263 ; macera- tion, 313, 319 ; medullated nerve, 404; motor plates, ?80; mucus cells, 464; nerve-fibres, intra-epi- 34 530 INDEX. The numbers refer to the Pages. dermic,373; neutral carmine, 155 ; nitrate of silver impregnation, 243, 245; perruthenic acid, 464; picro-carmine, 153, 154 ; purpurin, 235; quinolei'n blue, 228; retina, 439, 442 ; tactile hairs, 373. RANVIER and VIGNAL, osmium mix- ture, 27. RAWITZ, ha3inatein staining, 166 ; " in- vert" stain, 224; artificial alizarin, 232; GOLGI'S impregnation me- methods, 419, note, lleagents, how to procure, 143 ; selected collections of, 503. RECKLINGHAUSEN, VON, impregnation methods, 242, 246, 247. Reconstruction of objects from sections, 333. REDDING, gold process, 258. REDENBAUGH, narcotisation, 16. REGAUD, silver impregnation, 247. Regressive staining, 137, 182 et seq. REHM, nerve-tissue stains, 402, 417. REICH, silver nitrate, 246. REICHENBACH, embryology of Astacus, 352. REiNBACH,triacid mixture, 223; cover- glass preparations, 460. REINHOLD-GILTAT microtome, 73. REINKE, Flemming's orange stain, 218 ; lysolfor maceration, 319; staining horny tissues, 373. REMAK'S copper hardening fluid, 344. RENAUT, hsematoxylin, 173 ; haema- toxylic eosin, 238 ; silver impreg- nation, 247 ; cornea, 376. Reptilia, embryology, 3^2 ; myelon,398. | RESEGOTTI, coal-tar stains, 185, 189 ; staining by substitution, 185 ; on the kinds of sat ran in, and on saf- ranin staining, 189. Resins, mounting media, 277 et seq. ; imbedding masses, 114, 115. Retina, 439442. RETTERER, natural injections, 311 ; ova of rabbit, 337 ; smooth muscle, 382. RETZIUS, methylen-blue method, 209. REZZONICO, medullated nerve, 404. Rhabdocoela, 477. JlhizopJiysa, 493. R hop a lea, 467. Ribbon section-cutting, 88. RICHARD, Bryozoa, 15. RINDFLEISCH, maceration, 317. Ringing wet mounts, 285. RIPART and PETIT, preservative fluid, 46, 267. RITTER, embryology of Chironomus, 351. ROBERT, fixing Aplysia, 469. ROBERTSON, imbedding mass, 114. ROBIN, injection masses, 294, 295, 305; natural injections, 311. ROBINSKI, silver nitrate, 246. Rocking microtome, 72. ROLLETT, Carminroth, 155 ; freezing process, 118 ; cornea, 376 ; sec- tions of muscle, 378. ROOSEVELT, pyrogallate of iron, 260. Rose B. a 1'eau, 226. Rose de naphthaline, 195. ROSE, preparation of bone, 452. Rosein, 196. ROSIN, stain for nerve-cells, 402 ; for axis-cylinders and protoplasm, 417. Rossi, stain for nervous centres, 412 ; blood, 457. ROSSLER, Phalaugida, 475. Rotatoria, 482. Rouge, fluorescent, 196. ROUGET, silver nitrate, 246, 248; methylen blue, 207. ROTJSSELET, permanent preservation of Rotifers, 482. Roux, embryological methods, 344. Rubin, 196. RUFFINI, corpuscles of Golgi, 382. RUPRECHT, bone, 453. Ruthenium, impregnation method, 261. RUTHERFORD, picro-carmine, 154. RYDER, double imbedding, 112. SACERDOTTI, Golgi 's impregnation, 426. SACHS, motor plates, 381. SAEFFTIGEN, EchinorJtyncus, 480. Saflrosin, 226. 1XDKX. 531 numbers refer to the Pages. Safranin, 189 et seq.; with indigo-car- mine or nigrosin, 229, 230; with Wasserhlau, 231; with Lichtgriin or Saureviolett, 227; for elastic tissue, 450 ; for cartilage, 455. Saga rt ia, 488. SAHLI, balsam, 280; nerve-centres, hardening, 390 ; stain for, 414. SALA, Golgi's silver chromate impreg- nation, 430. Snlamatidra, embryology, 343; cyto- logy of, 354, 355 ; breeding larvae, 355. Salicylic vinegar, 267, 268. Saliva, artificial, 315. Salivary glands, 464. Salt solution, 263; for maceration, 313, 314. Salts, metallic, action of light on, 242. SAMASSA, Golgi's silver chromate me- thod, 429; sections of Ctenophora, 493. Sandal-wood oil, 69. Sandarac, gum, 283. SANFELICE, haematoxyliu, 173. SANKEY, staining nervous centres, 416. SAMORIUS, paraffin stove, 82. Sapphirina, 472. SARASIN, embryology of Reptilia, 342. Saroolemma, 378. SARGENT, bleaching, 328. SATTLER, silver staining, 248. Saurefuchsin, 219. Sauregelb, 219. Saurerubin, 219. Saureviolett, 227. SAVILLE KENT, tee KENT. SAWTSCHENKO, carcinoma corpuscles, 192. SAZEPIN, antennae, 473. SCHAFER, muscle-cells, 378. SCHAFFER, cartilage, 455 ; retina, 441 ; reconstructing sections, 333 ; bone, 451. SCHALLIBAUM, serial sections, 120. SCHENK, fixing fluid, 46; carbolic fuchsin, 196. SCHEWIAKOFF, Protozoa, 499. SCHIEFFERDECKER, cartilage, 456; cel- loidin imbedding, 100, 101, 104; injection masses, 310 ; levulose for mounting, 270; methyl mixture, 319 ; pancreatin digestion fluid, 320; serial sections, 120, 129; maceration of retina, 442 ; me- dullated nerve, 404; Mastzellen, 447; the water section-fixing pro- cess, 120. SCHMAUS, uranium - carmine, 155; staining nervous tissue, 416. SCHMIDT, embryology of Limax, 348. SCHNEIDER, aceto-carmine, 152. SCHOLTZ, intra-vitam staining, 138. SCHTJLGIN, doable stain, 237 ; embry- ology of A.mar