/Is THE METHODS OF BACTERIOLOGICAL INVESTIGATION, BY DR. FERDINAND HUEPPE, DOCENT IN HYGIENE AND BACTERIOLOGY IN THE CHEMICAL LABOBATOBT OF E. FBE8ENIUS AT WIESBADEN. TEANSLATED BY HERMANN" M. BIGGS, M.D., INSTBUCTOB IN THE CABNEGIE LABOBATORT, AND ASSISTANT TO THE CHAIB OF PATHO- LOGICAL ANATOMY IN BELLEVl'E HOSPITAL MEDICAL COLLEGE. ILLUSTRATED BY THIRTY-ONE WOOD-CUTS. NEW YORK: D. APPLETON AND COMPANY, 1, 3, AND 5 BOND STEEET. 1886. COPYRIGHT, 18S6, BY D. APPLETON AND COMPANY. DEDICATED IN THANKFUL EESPECT TO THE GEHEIMEtf KEGIERUNGSRATH, DB. ROBERT KOCH. AUTHORS PKEFACE. URGED by the wish of my highly esteemed teacher, the Gfeheimrath Koch, I have attempted in the fol- lowing work to meet the lack of a comprehensive representation of the methods of bacteria-investiga- tion. It was my endeavor, as an historical and ex- perimental critic, to sift the whole of the literature, which was extraordinarily scattered and in part very difficult of access, and to select the good from the hardly conceivable confusion of useful and useless communications, in order to give to the independent investigator a useful hand-book, and to the beginner a trustworthy introduction into this territory. THE AUTHOR. WIESBADEN, February, 1885. TEANSLATOE'S PEEFAOE. THIS translation of Hueppe's excellent work on the " Methods of Bacteriological Investigation " was suggested by the want in English of a satisfactory text-book on this subject for the use of students working under my direction in the Carnegie Labo- ratory. The preparation of the original was under- taken by Dr. Hueppe, at the request of Prof. Eobert Koch, and the work has been thoroughly and care- fully done. It shows a complete familiarity with the subject in hand, is comprehensive in character, and treats carefully all #f the approved methods of investigation. Some difficulties have been met in the transla- tion which those acquainted with this kind of work will readily understand. It is literal so far as is consistent with clearness, and no attempt has been made to attain elegance in style or diction ; but it is well known that many German terms have no exact English equivalent, and can only be ren- dered accurately by a roundabout expression. This is well illustrated by " Massenkultur " (as translat- ed by quantity-culture on page 101 et seq.\ which means a culture, whether pure or not, where a great 6 TRANSLATOR'S PREFACE. quantity or bulk of bacteria are growing ; but, since this expression can not be inserted each time in the text, quantity-culture will be used wherever it occurs. The labor of preparing the translation was per- formed in the short intervals of leisure found in the midst of numerous other duties, and was un- avoidably interrupted just before its completion, so that some errors may be found in the book, but I think that none of serious importance have escaped correction. The author has requested me to emphasize the fact "that the short, concise form chosen in the work is based on an extensive historical and ex- perimental review of the whole subject, and that if any methods, still much used, have been omitted or only briefly considered, it is because they have not now the significance or importance that has been ascribed to them by other writers." I here desire to acknowledge my indebtedness to Dr. L. W. Hubbard and Dr. S. N. Nelson, both of New York, for valuable assistance kindly ren- dered me in the translation. The work has been very favorably received in Germany, and if this translation meets only a small part of the same consideration in America, I shall feel well repaid for the labor expended. HERMANN M. BIGGS. CABNEGIE LABORATORY, NEW YORK, December 1, 1885. CONTENTS. PAGE INTRODUCTION 9 True Saprophytic Forms 11 I. SPONTANEOUS GENERATION AND THE PKINCIPLES OP STERIL- IZATION 15 II. FORMS OF BACTERIA AND MICROSCOPICAL TECHNIQUE . . 28 A. The True Endospore Bacteria 29 B. Arthrospore Bacteria 29 1. Arthro-Cocci 29 2. Arthro-Bacteria 29 3. Leptothrix 29 4. Cladothrix 30 Determination of the Presence of Bacteria Unstained . . 34 Staining Bacteria 40 General Principles of Staining 42 Preparation of Staining Fluids 48 Other Keagents and Apparatus 51 Cover-glass Preparations 55 Examination for Tubercle Bacilli in Sputum . .61 Examination of Blood for Bacteria . . . .67 Methods of Staining the Flagella 73 Methods of Staining Spores 74 Preparation of Sections 78 III. CULTURE-METHODS; PURE CULTURES 92 1. Transparent Fluid Culture-Media 92 2. Fractional Cultures 100 3. Opaque Solid Culture-Media 101 4. The Gelatin-Culture of Klebs and Brefeld . . .105 5. Method of Dilution 113 Method of Isolation by Heat 119 8 CONTENTS. PACK 6. Cultures in Capillary-Tubes, after Salomonsen . . 121 7. The Infection-Methods 125 8. The Cultures upon Transparent Solid Nutrient Media, according to Koch 128 A. Transparent Solid Media made by the Addition of Gelatinizing Substances " Nutrient Gelatin " . 132 a. Slide-Cultures 134 fc. Plate-Cultures 138 c. Test-Tube Cultures 142 Improvised Means 146 B. Transparent Solid Media, without the Addition of Gelatinizing Substances Blood-Serum . . . 148 IV. INOCULATIONS FOB THE DETERMINATION OF THE CAUSAL RELATION OF BACTERIA-GROWTH TO DECOMPOSITION AND DISEASE 160 A. Septic Bacteria 160 Anaerobiosis in Fluids 164 B. Parasitic Bacteria 172 Inhalation Experiments 174 Feeding Experiments 175 Cutaneous Inoculations 177 Subcutaneous Applications 178 Subcutaneous Injections 179 Direct Injection into the Circulation . . . .180 V. GENERAL BIOLOGICAL PEOBLEMS 183 Enzyme 185 Ptomaines 187 Behavior to Temperatures 188 Disinfection with Fluids 194 Disinfection with Gases 195 Drying 197 Action of Low Temperature and High Pressure . . .197 Electricity ' . 198 Phosphorescence 198 Light 199 VI. SPECIAL HYGIENIC INVESTIGATION 200 A. Earth 200 B. Water 202 (7. Air 205 VII. BACTERIOLOGY AS AN OBJECT OF INSTRUCTION . . 210 BACTERIOLOGICAL INVESTIGATION. INTRODUCTION. THE perfection of peculiar methods for the study of bacteria was conditioned, on the one hand, by the minuteness and rapid multiplication of these lower organisms, by reason of which the formerly approved methods scarcely at all sufficed to determine their morphology ; and, on the other, by the biological processes in which these micro-organisms take part, and which the methods for their study must also consider. In regard to their biology, the bacteria may be separated into two great groups : the septic (sapro- phytic) bacteria, which feed on dead organic bodies, and the parasitic, which are found in living organ- isms. Among the septic bacteria, the true bacteria of putrefaction should be distinguished from those which cause a more typical decomposition of life- less organic matter. In the latter it is possible to even separate chemically the products formed. These last septic forms are designated as ferment bacteria. Aside from these, the pigment bacteria also deserve special notice. 10 BACTERIOLOGICAL INVESTIGATION. Many of these septic micro-organisms need, un- der all conditions, the oxygen of the air for life and activity the aerobic forms ; some species can for the time being be deprived of this, and are able even then to bring about their specific decomposition, but they can also live and multiply with a free access of air. These may be designated as the optional anaerobic (facultativ-anaerobiotische) forms. Again, of other forms it has been asserted that the absence of the oxygen of the air is necessary for their life and ac- tivity, and that they are immediately destroyed by oxygen the obligatory anaerobic (obligat-anaerobio- tische) forms. We know of still other forms in which, directly contrary to this, a transference of the oxygen to the medium in which they grow takes place with their life activity. These cause an oxidation fermen- tation. Among the bacteria living as parasites, there are forms which do not complete their development upon the animal organism, but only occasionally appear as parasites, or pass through a portion of their exist- ence in living organisms the facultative parasites of van Tieghem; other forms find, as a rule, only in living organisms all the conditions necessary for their existence, but can occasionally, or in certain stages of development, also live as septic bacteria the facul- tative septic forms of de Bary. Still other forms, finally, seem to be fitted only for the parasitic mode of life, and appear quite incapable of passing any period of their existence as septic bacteria. These are designated as the strongly obligatory parasites of de Bary. Although it is very difficult to determine in individual cases whether a parasitic micro-organism belongs to this or that group, yet in such cases as the recognition of the minute differences also depends INTRODUCTION. more or less upon the subjective opinion of the ob- server, it thus permits this grouping to be regarded as much freer from constraint as to their real condition than the division of the pathogenic infectious micro- organisms into endogen and ectogen, which has ref- erence only to the extremes. These last designations permit only insufficiently, either a valuation of the investigations concerning the accessory causes of the infectious diseases, or a strong emphasis being placed upon one or another factor, and do not allow the at- tainment of an unprejudiced judgment upon matters all-important in the aetiology. There is also the great- est difference as to the need of oxygen among the parasitic bacteria. It is to be noted, likewise, that the parasites may be endophytic i. e., can live in the interior of organs or cells ; perhaps also epiphy- tic i. e., can live upon the surface. These different phenomena of adaptability can be theoretically deduced from the simple septic mode of life ; but it is to be remembered that direct inter- mediate links can seldom be determined, and that individual forms can produce different actions. TEUE SAPEOPHYTIC FOEMS. / I I. Ferment bacteria. II. Pigment bacteria. III. Parasitic bacteria. Aerobic. Facultative parasites. Forms produc- Facultative anaerobic. Facultative septic forms. ing oxidation | fermentation. Obligatory anaerobic. Strongly obligatory parasites. The most general problem which is presented in bacteria investigation viz., the determination of the group to which a form of bacteria belongs can now be quickly and accurately solved. I. It is to be determined whether, in decomposi- 12 BACTERIOLOGICAL INVESTIGATION. tion or disease, bacteria are present or not. This question associates itself essentially with the question of spontaneous generation and abiogenesis, teaches us the value and general principles of sterilization, and makes us acquainted with the indispensable re- quirements for reliable work in bacteriology. II. If bacteria are present, it is to be determined what forms they possess. This general morphologi- cal question demands special technical skill, as the ordinary histological technique does not suffice. III. Each form found to be present is to be culti- vated by itself, free from all chemical and morpho- logical admixtures " pure cultures." The problem to be solved by the aid of pure cultures is a double one i. e., with the help of these the general mor- phological investigation is completed and extended, and, IY. By transfers of really pure cultures to decom- posable materials or susceptible animals, it is to be determined whether the bacteria found are the cause of the decomposition or disease. By this investigation it is certainly shown to which of the described groups a form of bacteria belongs ; then, extending likewise from the pure cultures, there are yet, V. A further series of more exact biological prob- lems to be solved later, which, in union with the first questions, afford the broad basis for theoretical con- sideration and practical treatment. The solution of all these questions is to be aimed at ; but experience has shown that this is not possible in every case, and that cases may come up in which one or the other of these cardinal questions remains unanswered. For instance, as regards the parasitic bacteria which, in the highest degree, represent the INTRODUCTION. 13 parasitic adaptability, viz., the strongly obligatory parasitic forms, perhaps only the presence of the micro-organism may be determined, while pure cult- ures can not be obtained. In other forms of this group, a further step has already been taken, by which, to a limited extent, transfers to animals have been made. With individual pathogenic bacteria of the other groups, on the other hand, transfers are not successful, because as yet no species of animals has been shown to be susceptible to the disease which they produce, although pure cultures of these bac- teria have been obtained. (Compare Methods of In- fection.) In these cases the greater attention should be given to those problems that can be solved ; and it is always to be borne in mind that, with a more complete mas- tery of the methods in the seemingly most unprom- ising cases, a classical solution of all questions has sometimes been obtained. The facts ascertained by means of a perfected technique must form, in every branch of natural sci- ence, the solid foundation upon which the theories are built up. No investigator, although in possession of many facts, can dispense with the guiding ideas that these afford ; but the aversion of many investi- gators to every speculation is immediately aroused, for the reason that by many the deductions of natu- ral philosophy are stated as scientific facts. Even the subject of bacteriology has in this respect passed through an experience too sad to allow us to forget that the observations in natural philosophy can be nothing else than provisional explanations of phe- nomena not yet really understood, the value of which, however, in suggesting lines of investigation, is very great. Resolutely must we guard ourselves against 14 BACTERIOLOGICAL INVESTIGATION. placing under restraint such deductions, often con- fused, but yet in harmony with facts ; against bring- ing into discredit the solid foundation of facts by cheap badinage of the endeavors of " learned ones" in search of ''little facts"; or against citing ever anew agreeable theories or refuted statements as proved facts, as has often happened in the domain of bacteriology. Kesolutely also must we see to it that speculations do not form the foundation of the practical treatment of hygiene. Against such sad errors of speculation there is no better remedy than the familiarity to be obtained with the methods (constantly becoming more accu- rate) which are so closely united to the real advances in the knowledge and possibilities regarding the caus- al relation of micro-organisms to decomposition and disease. I. SPONTANEOUS GENERATION AND THE PRINCIPLES OF STERILIZATION.* SPALLANZANI f opened the series of scientific ex- periments concerning spontaneous generation and the principles of sterilization. He placed infusions of organic substances in flasks, which were corked and sealed, and then boiled for an hour in a water-bath. These experiments, which form the groundwork of the Appert method of preservation of organic substances, can be better made in flasks with long-drawn-out necks, which are closed during the process of heating. Now and then one of these experiments (where the dura- tion of the heating is only one hour) fails. But the chief objection to the conclusions drawn from these experiments lies in the fact that no oxygen can gain admission to the flask. In 1810 Gay-Lussac expressed the opinion that, for the production of fermentation, oxygen must have admission. The next methodical advance in this direction con- sisted in the admission of air into the vessel after heating, this air having been previously so treated * "ZusaramenfassendeDarstellungen derFrage uber Abiogenesis; Generatio spontanea finden sich bei Gscheidlen." ''Physiologische Methodik," Heft 2, 1876, S. 274; Heft 4, 1879, S. 499; und bei Tyn- dall, " Essays on the Floating Matter of the Air, v second edition, 1883. t " Physikalische und mathematische Abhandlungen," 1769. 16 BACTERIOLOGICAL INVESTIGATION. that it could contain no germs. Franz Schnlze * de- signed a flask which had a double perforated cork, in which were placed two glass tubes bent at a right angle, and which terminated close underneath the cork. The decomposing infusions or substances sus- pended in water were heated on a sand-bath until all portions had reached the temperature of boiling wa- ter. Then, while the steam was still escaping, a Lie- big's globe apparatus was fastened to each of the glass tubes. One of the globes was filled with concentrated sulphuric acid, and the other with a solution of caus- tic potash. Now, by the application of suction-force on the side to which the apparatus containing caustic potash is attached, air is drawn into the flask which before its entrance must pass through the sulphuric acid. When this is done, no decomposition takes place, notwithstanding the presence of the air ; but it soon occurs if, after the heating, ordinary air is allowed admittance. Schwannf showed that "it is not the oxygen, or at least not alone the oxygen, of the atmospheric air" which causes fermentation and putrefaction, by pass- ing the air through mercury which was heated to the boiling-point. No change occurred in the fluids after this was done. To meet the objection, that by this procedure the air might be altered chemically, Schroder and von Dusch^: passed the air through cotton, which was * " Vorlaufige Mittheilung der Resnltate einer experimentellen Beobachtung uber Generatio aequivoca." Poggendorf ? s " Annalen der Physik," 1836, Bd. 39, S. 487. t " Vorlanfige Mittheilung, betrefiend Versnche fiber die Wein- gahrung nnd Faulniss." Poggendorf s "Annalen," 1837, Bd. 41, S. 184. \ " TJeber Filtration der Luft in Beziehung anf Faulniss imd Gah- rnng." " Annalen der Chemie und Pharmacie," 1854, Bd. 89, S. 337. SPONTANEOUS GENERATION. 17 either placed in tubes connected with the right-angled glass tubes, or they stopped the neck of the flask with cotton during the process of boiling. Pasteur * boiled the infusions in flasks, the necks of which were long-drawn-out and curved in different ways, with only this precaution, that the open end always looked downward. In this way the air could enter, after the heating, unaltered and unfiltered, and no putrefaction took place. In milk, Pasteur succeeded in preventing decom- position certainly only by elevating the temperature to 110 or 112 C. in a pressure of one and one half atmospheres. Schroder also showed the inefficacy of boiling to prevent decomposition in respect to single substances, which he found could be sterilized with certainty only after a long-continued boiling, or by elevating the temperature in a digester with a press- ure of about two atmospheres. The digester, or steam-kettle, for expanded steam, is used for sterilization with a high temperature. The temperature required for sterilization ranges be- tween 110 and 112 C., corresponding to a pressure of from one to two atmospheres. Since an equaliza- tion of temperature is produced by the strong cur- rents in the fluid brought about by the differences in temperature, the best execution, reasoning a pri- ori, ought to be expected from this apparatus, since, in accordance with the dynamic theory of heat with the elevation of the temperature, the time required for the equalization of the temperature, and hence the time for sterilization, is diminished. Many * "MSmoire stir les corpuscles organises qui existant dansl'atmos- phere." " Annales de chimie et de physiques," III. Ser., T. 64, 1862, S. 66. And shorter article, " Compt. rend.," Bd. 48, 1 859, S. 337 ; and ibid., Bd. 50, S. 849. 18 BACTERIOLOGICAL INVESTIGATION. forms of apparatus* correspond to this theoretical postulate, but all f do not satisfy it in all respects. The further consideration is added to this uncer- tainty that many substances are chemically altered by the elevation of the temperature above 100 C., so that the apparent advantages of the apparatus, as compared with others, are in many cases quite illusory. Any one who possesses a reliable, tested steam-kettle (Dampfkessel) may safely use this, especially if the substances to be sterilized will bear an elevation of the temperature above 100 C. If it is desired to use a temperature above 100 C., without employing a steam-kettle, a salt-, oil-, or paraffine-bath can be employed. If boiling water is to be used, then the larger flasks, smaller flasks, and test-tubes are boiled di- rectly in the water-bath, but the water in it should have a somewhat higher temperature than the con- tents of the glasses. The equalization of tempera- ture in the water -bath is not only attained cor- respondingly slower than at a lower temperature, but it is so uncertain that it should be practically tested. If a sufliciently long boiling affords a real steril- ization, then the temperature of the boiling water can be better used in the form of streaming steam. The steam sterilization-cylinder of Koch, Gaffky, and Loffler answers this purpose (Fig. 1). This consists of a cylinder made of strong tin plate, about half a metre high and from twenty to twenty-five centime- * Fitz, " Ueber Spaltpilzgahrungen." " Berichte der deutschen chemischen Gesellschaft," Bd. XVII, 1884, S. 1188. t Koch, Gaffky, Loffler, " Versuche iiber die Tenverthbarkeit heisser Wasserdampfe zu Disinfectionszwecken." " Mittheilungen aua dem kaiser-lichen Gesundheitsamte," I, 1881, S. 322. SPONTANEOUS GENERATION. 19 IT tres in diameter, which is surrounded by an asbestos covering to prevent the loss of the heat, and is pro- vided with a copper bottom. In the interior at .#, in the lower third, is placed a grate ; the space under this is filled three fourths full of water, which is brought to the boiling-point by a number of gas -flames (three or five burners) placed under the vessel. It is closed above by a cover (H) made of block-tin covered with as- bestos, and is not hermetically sealed, so that the steam can escape around the edges. In a hole in the cover a thermometer (t) is placed. The apparatus may also have somewhat larger dimensions. But, if these dimensions are very much, increased, it is necessary to use salt solution in order to keep the temperature of the outer currents of steam at 100 C. If the escape of the steam is not quite free, and the radiation of the heat is prevented, the temperature of the interior of the cylinder in this way may be kept equal throughout ; and since the cover is not hermetically sealed, the temperature of the steam does not exceed the boiling-point, but gives the temperature of boiling water, correspond- ing to the conditions of pressure that is, with the barometer at nearly its normal height, about 100 C. The advantages of this apparatus, in comparison with the steam-kettle, are its cheapness and the im- possibility of exceeding the temperature of 100 C. when water is used, so that all substances can be sterilized with this, which will bear a temperature of 100 C. The equalization of the temperature is very soon reached, and does not undergo such oscillations 20 BACTERIOLOGICAL INVESTIGATION. as with, the steam-kettle, because the technical use of the apparatus can scarcely be simpler, and on this account this element is reduced to the minimum. The currents of steam are far superior to the water- bath, through the certainty of their action and the relatively short time required. These practical advantages render this apparatus the most desirable one for all cases in which high temperatures are used for sterilization, in spite of the fact that, theoretically, to compensate for the some- what lower temperature, it must be used longer than would be necessary for expanded steam at a tempera- ture above 100 C. The beginning of the heating not being included, the time required for the sterili- zation varies from one half to two hours, depending on the size of the object. A vessel which fits in the cylinder is included with the apparatus. In this are placed the small flasks or tubes to be sterilized, and to the handle of the vessel or to the neck of the larger flasks a string is fastened for conveniently raising and lowering them. This string is made fast to the hook 7i, to be found on the rim of the cylinder. But many substances are altered by exposure to a temperature of 100 C., and especially coagulation of the albumen is always brought about. In order to avoid this, the discontinuous sterilization of Tyndall is used. The principle of " Sterilization by Discon- tinuous Heating " (I. c., pp. 210 and 337) was founded on the observation that living bacteria are killed by exposure to a relatively low temperature, below the point required for the coagulation of albumen, while the spores are not destroyed by these low tempera- tures,* but are easily killed after germination. If * Colin, " Untersnchnngen fiber Bacterien,'' IV ; "Die Bacterien SPONTANEOUS GENERATION. 21 FIG. 2. the fluid to be sterilized is exposed for one or two hours to a temperature of from 52 C. to 65 C., only the living bacteria are in this way destroyed, and per- haps not even all of these the first time. The resistant spores possibly present in the solution thus treated germinate some on the first and second, others on the third and following days. If now the fluid is exposed to the same temperature as before on the second and third days, the living bacteria, or those that have de- veloped from the spores, are killed each time, so that, if this operation is continued long enough, it is pos- sible to sterilize with certainty all fluids below the temperature producing chem- ical alterations. In general, exposure to a temperature of about 58 C. for one or two hours, on from five to eight successive days, is recommend- ed. This may be done by the use of the water-bath, but more conveniently with the appara- tus shown in Fig. 2. This con- sists of a double-walled cyl- inder made of copper. The chamber between the two walls is about half filled with water, and the cylinder itself well closed with a double- walled cover, which is also filled with water. The cover has upon its side a hol- low tube (d), whose lumen communicates with the chamber in the cover. This is warmed by the flame (d l \ while the cylinder itself is warmed from below by a flame placed under it. There are three tubes in the cover, one of which (c) is used for filling the cover und die Urzengung." " Beitrage zur Biologie der Pflanzen," Bd. II, Heft 2, S. 249, 1876. 22 BACTERIOLOGICAL INVESTIGATION. and for receiving a thermometer which indicates the temperature of the water in it ; a second (5 1 ) receives a thermometer which passes into the air-chamber (b) of the cylinder ; and the middle tube (a 1 ) receives a thermometer which passes into a small central cylin- der (a), the cavity of which communicates with the water-chamber on the exterior. The outer water- chamber is filled by means of a tube placed on the side. The principle of discontinuous sterilization may also be used in many cases as discontinuous boiling, since there are many substances which are altered by long boiling that are not materially changed by brief but often repeated boiling. If such substances as, for example, gelatin are boiled for a short time on four or five successive days, it is possible to sterilize them with certainty. For refuting other objections which are made as to the existence of spontaneous generation, it may be desirable to use substances which have not even once been subjected to the lower temperatures i. e., tem- peratures below the coagulation-point of albumen. This object is attained in two ways : first, by free- ing the solution by filtration from possible admixt- .ures ; or, secondly, by endeavoring to obtain the same uncontaminated from the beginning. In respect to the first, Helmholz* observed that the fermentation produced by yeast did not pass through a membrane ; on the contrary, this did occur with putrefaction. Consequently such membranes are not available for this purpose. Positive results were first obtained by Tiegel,f who succeeded in separating * " Ueber das TVesen der Faulniss tmd Gahrung." Muller's " Archiv fQr Anatomic und Physiologic," 1843, S. 453. f " Correspondenzblatt fur schweizer Aerzte," 1871, S. 275 ; und SPONTANEOUS GENERATION. 23 mechanically by filtration of septic fluids through clay cells, using positive or negative pressure on one side the septic material from the quite inactive fluid. Miquel and Benoist * endeavored to remove the germs by a gypsum filter. They took glass balloons with drawn-out necks, in the narrow portion of which an asbestos-stopper was placed, and beyond this a layer of gypsum. Before use the apparatus is slowly heated to 170 C., then the fluid to be filtered is slow- ly poured upon the gypsum-stopper. The stopper consists of 1*6 asbestos, 52*1 gypsum, 46 water. The diluted juice of flesh and plants and urine filtered rapidly, serum and albuminous fluids somewhat more slowly ; all were germ free and free from life. A cap- illary tube, cemented underneath the constriction, by being united to an aspirator, permits the reduc- tion of the pressure in the balloon. Gautier f used a very long-necked flask of faience or unglazed porcelain, which tapered below into a cone. Through this porous cone, the real filter, the fluid to be filtered passed from without into the inte- rior of the porcelain flask. For rarefying the air in the neck of the flask, a glass tube, bent at a right an- gle, is fastened by vermilion-paint cement, so that the shank reaches down to the bottom of the cone while the other outer end tapers into a small cone and exactly fits into a corresponding conical expan- sion of a second tube. This second glass tube is like- wise bent at a right angle, and the end, which is " TJeber die fiebererregende Eigenschaft des Mikrosporen septicum." Dissert. Bern, 1871. Citirt nach Klebs. * " Bulletin de la societS chimique de Paris," 1881, Bd. 35, S. 652. t " Sterilization a froid des liquides fermentescibles." " Bulletin de la sociSte chimique," 1884, Bd. 42, S. 146. 24 BACTERIOLOGICAL INVESTIGATION. united with the portion of the first tube, possesses a conical expansion, while the other end reaches to the bottom of a glass flask with a narrow neck. To the side of this glass flask a tube with a conical expan- sion is cemented. The two conical expansions are closed with cotton, and then this glass balloon with its projecting portion and the glass tubes are steril- ized. In the same way the porcelain flask with its glass tube is heated, and, after removal of the cotton stopper from the cone of the first glass tube, this is inserted into the conical expansion of the second. In the conical expansion of the projection from the glass flask, after the removal of the stopper, a glass tube, extending out into a corresponding cone, is intro- duced, which is filled with heated asbestos. The joints uniting the conical expansions with the corre- sponding conical constrictions are covered with shel- lac. By aspiration at the free end of the asbestos- tube, the air in the entire apparatus is rarefied, and, when the cone of the porcelain flask is immersed in a fluid, by the existing negative pressure, fluid which is quite free from germs is aspirated into the porce- lain flask. The vermilion-paint cement, prepared with oil of turpentine, consists of Boracic acid (crystallized) 8 parts. Silicic acid 2 " Vermilion 12 " Most of the filtrates obtained free from germs in this way have , experienced no material alterations ; but some of them do not remain unchanged after the filtration, as the filter does not allow all materials to pass through equally well, and albuminous solu- tions are often quite seriously altered quantitatively and qualitatively. In order, also, to eliminate this possible error, the SPONTANEOUS GENERATION. . 25 substances should be obtained, quite uncontaminated, by the use of the greatest cleanliness. Before each manipulation the hands are cleansed with a one per cent solution of corrosive sublimate, and are then rinsed in sterilized water, or the sublimate is removed by alcohol, the alcohol by ether, and the latter al- lowed to evaporate. All vessels are well sterilized ; the manipulations and all operations are performed very rapidly and with the antiseptic precautions of modern surgery. In this manner it is possible to ob- tain blood, milk, urine, etc., without anything ever being mingled with the same, or without decompo- sition taking place. Some details follow later in the methods of infection and in the inoculation experi- ments. No organism is ever formed, not even a mi- crococcus, either from the unorganized material, or from " molecules of nitrogen," or from microscopic forms of life, or from the anamorphosis of proto- plasm, which fact certainly has not prevented many authors from mistaking, for real cocci, bodies of dif- ferent origin showing molecular motion, from which also later, bacilli, etc., should develop. Whoever has obtained contrary results, upon the ground of a few experiments, must first show that he has mastered the technique, as was the case in the many positive results of van den Broek, Pasteur, Roberts, Lister, Cheyne, and Meissner. The technical dexterity nec- essary for this is only to be obtained by many indi- vidual experiments. By these experiments, arranged for spontaneous generation (which, especially through Pasteur, have taken a form easy of mastery), and by the disinfection experiments of Koch, the still remaining principles of sterilization have been reduced to a reliable and convenient form. 26 BA CTERIOL GIGAL INVESTIGA TION. Metal objects scissors, knives, pincettes, plati- num-needles are first mechanically cleaned, then are heated in the flame, and for cooling are laid upon a sterilized glass plate and protected from dust by a bell- jar. Glass objects glass plates, slides, flasks, test-tubes are first mechanically cleaned, then, if they are very dirty, greasy, or have been used for other purposes, are dipped in concentrated sulphuric acid or hydro- chloric acid, and finally repeatedly washed in dis- tilled water until every particle of the acid has been removed. The water is first allowed to run off, and the articles thoroughly dried in the dry-oven, or it is removed by means of alcohol, in the following manner : they are dipped into alcohol, and the last particles of this are removed by ether, which is al- lowed to evaporate. According to the degree of un- cleanliness of the vessels in the beginning, either this whole procedure must be gone through with, with the greatest care, or it is sufficient to wash them in distilled water. The chemically clean vessels must then be freed from germs. For this purpose the vessels, having their necks stopped with a closely fitting mass of cotton, are immediately placed in the double- walled dry- oven (Fig. 3). This is provided with a thermo-regulator (r) and a thermometer (t). The test- tubes are more conveniently placed in a basket (d) made of wire, which will hold a large number. Catheters, syringes, pipettes, capillary- tubes, and other glass tubes are SPONTANEOUS GENERATION. 27 placed in a clean glass and then put in the dry-oven. All such glass objects should remain at least two hours * at a temperature of 150 or 160 C., the time required to raise the temperature to this point not being included. The objects are allowed to cool in the oven, so that they can be removed directly before use, or at least care should be taken to protect them from dust. Cork stoppers are to be avoided. Eubber corks, caps, and bands, etc., are sterilized in the steam- cylinder for from three quarters of an hour to one hour. Most vessels with their contents should be steril- ized once more ; after which it is advantageous to bind over the tops two layers of filter-paper, since the cotton stoppers are proof against bacteria, but not al- ways against fungi. Previous experience has shown that germs from the air are more seldom the cause of failure than the unintentional infection through unclean or insuf- ficiently sterilized vessels, and the manipulation with hand and instruments not certainly sterilized. * Practically I have found that exposure to a temperature of 150 or 160 0. for from fifteen to twenty minutes answers exactly the same purpose, and it has been repeatedly shown that neither germs nor their spores will withstand exposure to so high a temperature for this length of time. TE. II. FORMS OF BACTERIA AND MICROSCOPICAL TECH- NIQUE. IN the examination, under the microscope, of a substance containing bacteria, different forms present themselves, whose general morphological peculiari- ties, on the one hand, and whose differences or like- nesses, on the other, must be determined. The forms are determined by the use of the gen- eral microscopical technique, in its peculiar applica- tion to bacteria, while the second question can only be solved after obtaining pure cultures. The forms of bacteria (Fig. 4) are round (1, 3, 4, FIG. 4. In part from Koch and Prazmowski. BACTERIA AND MICROSCOPICAL TECHNIQUE. 29 and 5), oval (2), shorter (6) or longer (7) rod^f orm cells ; and, in addition, curved rods (11, 12) and spiral-formed organisms (13-16) are observed. These forms appear sometimes isolated, sometimes united in a definite manner (3, 4, 5). The next object to be attained is the separation of these forms and the grouping of them as naturally as possible, so that a general examination of the mor- phology of the bacteria may be made. A. THE TEUE ENDOSPOEE BACTERIA (Fig. 4). These multiply sometimes by division, sometimes also by the endogenous formation of spores. 1. Cocci. -| a ' Kound - i Cells. ) Tendency to the for- { * Ova1 ' C ination of zooglcea. rt oj. -LJ. i C a Short rods. 2. Straight rods, C ^ ^ , Tendency to the for . e. Clostridium-forms. mation of fila- ments, desmo-bac- 3. Curved rods, vibrioncs. f teria ; the filaments show no variation from end to end. 4. True spirilla-forms, spirilla, and spirocha3ta3. B. AETHEOSPOEE BACTEEIA. These forms also multiply by division, bat they do not produce endoge- nous spores. On the other hand, single individuals may separate themselves from the colonies and form new generations. These individuals appear almost constantly as round cells, gonidia, or arthrospores. 1. Arthro-Cocci. These consist only of cocci, and, by the union of the cocci, form torula. 2. Arthro-Bacteria. These form round cells simi- lar to cocci, but also short rods, long rods, and fila- ments, which show no variation from end to end. 3. LeptotJirix. These form cells similar to cocci, rods, spirals, and filaments, which show a variation from end to end. 30 BA GTEEIOL GICAL INVESTIGA TION. 4. CladotTirix (Fig. 5). FIG. 5. These form cocci, rods, filaments, and spi- rilla. The -fila- ments form false ramifications (A). / The arthro-coc- ci and arthro-bac- teria have as yet been very little studied, and are perhaps to be classed with the true bacteria. These two groups, so far as they have been studied, seem to stand nearer to the fission algae on account of their form. Zopf * class- es them directly with the bacteria, because of the ap- pearance of forms similar to these in their development. Cladothrixdichotoma; after Zopf. A, branching The next qU6S- plunt with slightly and decidedly spiral tion in the micrO- branches. J5, spiril, one end of which is more winding than the other. D, branch SCOpical with narrow and broad windings. E, spir- . ils ; , undivided, 6, divided into rods, and gatlOU IS TO at c into Zopfs cocci. F, spirochseta form; -p^^p which of at <7, undivided, at 6, schematic division into m long rods, ate, into short rods, and at din cocci, described forms are, in general, present. After obtaining a pure cult- * Compare my criticism of the work of Zopf in the "Fortschrit- ten der Medizin," 1883, No. 6. BACTERIA AND MICROSCOPICAL TECHNIQUE. 31 ure, it must then be determined what is the typi- cal form ; whether it remains the same under all conditions, so that it not only has the value of growth-form, but also constitues a form-species or form-genus ; or whether it corresponds to external conditions, being sometimes larger and broader, some- times smaller and thinner ; or whether, finally, other forms may appear in their development. Further, it ought to be ascertained whether, in the develop- ment of a species, a single form or a form- cycle ap- pears or can appear. In regard to the individual peculiarities, there re- main yet the following points to be noted, although those already referred to seem to be numerous. In the spiral forms, Fig. 4 (13, 14, 15, 16), the number and the arrangements of the typical spirils should be observed ; then it is to be noted whether, in divis- ion, the spirils separate into curved rods similar to the vibriones, and whether these fissions-products again develop into typical spirils, or whether the division extends on to the production of forms which are similar to the rod-bacteria and cocci, which finally originate new generations. If re- agents are used, it is first necessary to note the stage of development, since, either during the pro- cess of division or immediately before it, the re- agents will make visible the lines of division in place of the apparently homogeneous character of the fila- ments. On the other hand, the value of such chem- ical action should not be overestimated, since, in the height of their development, that most susceptible reagent, photography, shows not the slightest trace of union. In the case of the vibriones, Fig. 4 (11, 12) the smallest forms of which (11) Koch, on account of 3 32 BACTERIOLOGICAL INVESTIGATION. their characteristic appearance with the usual mag- nifying power, called comma-bacilli it should be noticed whether they divide into smaller vibriones, or whether they can form spherical and rod-shaped forms. Through the union of a number of vibriones, S-forms and long, slender filaments similar to spirilla (11) may be produced which the inexperienced ob- server can easily confound with the true spirilla (13). These spiral filaments, according to the degree of curvature of the individual vibriones, form sometimes slightly wavy lines, sometimes spirils with narrow windings. The spiral forms of the vibriones are to be considered as a kind of thread-formation, which, as contrasted with the true spirilla, appear only un- der certain conditions, as, for example, the partial or total exhaustion of the culture-medium, and never show the regularity of the latter. In the rod-formed bacteria it should be observed whether the division of long rods into short ones, or into oval and round cells, predominates. Some rod- iuriH2, by their union, produce long filaments, while others show a tendency to the formation of zoogloea. On account of the greater tendency of the short-rod bacteria, like the cocci, to form zoogloea, Cohn sepa- rated them from the vibriones and long-rod bacteria, which he classifies as filament-bacteria. It is to be noted whether the character of the culture medium (solid or fluid) has any influence upon this. In the cocci it should be determined whether the oval cocci produce, by their division, smaller oval cells or round cells, and whether the round cocci, be- fore their division, become oval, and, finally, whether the oval cocci become short rods previous to their division. The appearance of straight rods may be produced BACTERIA AND MICROSCOPICAL TECHNIQUE. 33 in the curved rods, when the concavity or convexity of the rod is turned upward. A straight rod stand- ing upright may present the appearance of a round cell, and a long rod placed at an angle to the plane of vision may appear like a short rod. It is only possible to decide such questions after many single observations and with the use of pure cultures. Many hundred observations of mixtures of bacteria do not serve at all for the decision of morphological questions. The consideration of spore-formation, Fig. 4 (5 5, 8, 9, 10, 12), and germination shows two different types. The rods may develop into filaments before the forma- tion of spores (8), or the spores may appear in the mobile rods (9) ; sometimes they are formed in the interior (8, 9), sometimes near the end (12). The rods often become whetstone- or club-shaped before the formation of the spores (clostridium, 10). Under other conditions, quite abnormal, so-called involution forms are observed (17). The work of Cohn * is fundamental as regards the value of form. In this he first clearly described the relations of the growth-form, the form-genus, form- species, true varieties, form- varieties, and physiologi- cal varieties. This work is not only often entirely misunderstood by his opponents, but also construed in an opposite sense by his own zealous supporters, who have repeatedly interpreted his arguments for the existence of true species among the bacteria in the sense of a belief in constancy in species and form,t which was accepted before the Darwinian theory. * " Untersnchungen fiber Bakterien." " Beitrage znr Biologie der Pflanzen," Bd. I, 2. Heft, S. 127, 1872. 2. Abdruck, 1881. t For the further investigation of these questions, consult Naegeli, 34: BACTERIOLOGICAL INVESTIGATION. DETEKMINATION OF THE PKESENCE OF BACTERIA UNSTAINED. The oldest method of examining unstained bac- teria consisted in mingling a small drop of fluid or a particle of matter containing bacteria, with a drop of indifferent fluid, placing it upon a slide, putting over it a cover-glass, and then examining it accord- ing to the method of histological procedure. The picture presented in these cases is of the same nature as in unstained tissue-preparations, especially in this : that the objects, because of their different power of refracting light from the inclosing media, or, according to Koch, u on account of the refraction of the rays of light passing through, present a picture of lines and shadows viz., the structure-picture." In these cases a diaphragm is used, as in other histological work in which it is desired to make out the structure-picture.* The diffused daylight will "Die niederen Pilze in ihren Beziehungen zu den Infectionskrankheiten und der Gesundheitspflege," 1877. Naegeli und Buchner, in Naegeli's " Untersuchungen iiber niedere Pilze," 1882. Koch, " Zur Aetiologie desMilzbrandes," " Mittheilungen aus dem kaiserlichen Gesundheits- amte," Bd. I, 1881, S. 49. Gaffky, " Experiraentell erzeugte Septi- caemie mit Rticksicht auf progressive Yirulenz und accommodative Zuchtung," ibid., S. 80. Flugge, u Fermente und Mikroparasiten," 1883, und " Deutsche med. Wochenschrift," 1884, No. 46. Zopf, " Die Spaltpilze," 2. Aufl., 1884. De Bary, " Vergleichende Morphologic und Biologie der Pilze," 1884. Htippe, " Fortschritte der Medi- zin," 1883, No. 6, und 1884, No. 6 (Kritik der Ansichten von Zopf), und " Ueber die Zersetzungen der Milch und die biologischen Grund- lagen der Gahrungsphysiologie," "Deutsche med. "Wochenschrift," 1884, Nos. 48-50. * Zur weiteren Orientirung uber das Mikroskop und die mikro- skopische Technik: Dippel, "Das Mikroskop," 2. Aufl., I, 1882-83; Frey, " The Microscope and the Microscopical Technique " ; Strass- BACTERIA AND MICROSCOPICAL TECHNIQUE. 35 not suffice to illuminate the object when the higher powers are used, so that it is necessary to employ a condenser, which does not define the structure-pict- ure, but serves to increase the illumination. For this kind of microscopical work the achromatic condens- ers of the larger English instruments answer the pur- pose better than any others. The dry system of objectives does not suffice for most bacteria ; in order to make out only approxi- mately the correct forms, one must use in this inves- tigation the immersion system, in which, according to Amici, the stratum of light, by passing through a stronger refracting medium, compensates as much as possible for the error caused by the dispersion of the rays by the cover-glass. Since the cover-glass and the front lens of the ob- jective consist of crown-glass, and water, on account of its low refractive power, does not quite correct the error, in the water-immersion systems correction-ad- justers and cover-glasses of a certain thickness are necessary. But the correction is obtained if the immersion- fluid has the same exponent of refraction as crown- glass. Such a fluid, according to Abbe,* presents an optical homogeneous union between the preparation and the objective, which prevents all refraction of the rays in front of the convex surface of the optical sys- tem. By this means the loss of light through reflec- tion at the natural joints of the different optical media is avoided, and at the same time a very con- burger, " Das botanische Practicum," 1884 ; Friedlander, " Micro- scopical Technique." * " TJeber Stephenson's System der homogenen Immersion." " Sitz- ungsberichte der Jenaiscnen Gesellschaft f. Med. und Naturw.," 1879, 10. Januar. 36 BACTERIOLOGICAL INVESTIGATION. siderable amount of spherical aberration is prevented. On account of this, the correction-adjusters necessary in the water-immersion systems can be dispensed with, and the thickness of the cover-glass is of no great moment, since, as soon as the intervening medi- um has the same index of refraction and dispersion as the cover-glass, the same result for optical pur- poses is obtained whether a thick layer of glass and a proportionally thin layer of fluid or the reverse is interposed between the object and the lens sys- tem. Anise-oil was used by Amici for the purpose of increasing the exponent of refraction ; by Spencer glycerin was employed. Stephenson * desired an en- largement of the aperture of the lens, not only to avoid the necessity of the correction of the cover- glass, but also to increase the power of differentia- tion. The union of these two postulates by Stephenson, their settlement by Abbe, the construction of lenses by Zeiss, and the introduction of this system for homogeneous immersion by Koch,f marked a new era for the microscopic side of bacteria investiga- tion. The best fluid is the ethereal oil of cedar, the index of whose refraction is the same as crown-glass, and its index of dispersion differs only in a slight degree from crown-glass. When somewhat inspissated, it is more convenient for use for most optical purposes. By mixing other stronger-refracting ethereal oils such as oil of cloves, phenol, and anise with olive- or * On a large-angled immersion objective. *' Journal of the Eoyal Microscopical Society," 1878, p. 51. t " Untersuchungen tiber die Aetiologie der Wundinfectionskrank- heiten," 1878. BACTERIA AND MICROSCOPICAL TECHNIQUE. 37 castor- oil, an immersion-fluid can be made, which is similar in its refracting power to oil of cedar, or which differs from it to a certain degree. In consequence of the removal of the troublesome cover-glass correction (attained by different lengths of the draw- tubes) which allows, in a delicate man- ner, a compensation for the influence upon the aber- ration of the varying distance of the picture, the ob- jectives for homogeneous immersion are always ad- justed for a definite length of the draw-tube. On this account it is to be noted that lengthening the draw- tube beyond this normal length acts in the way of a spherical over-correction, shortening in the way of an under-correction. In using an immersion-lens, one places a drop of oil on the cover-glass, screws down the draw- tube with the coarse adjustment, or, if this is lacking, brings it down with a rotary motion by the hand, so far that the front lens of the objective touches the oil and the picture begins to be visible. Then the fine adjustment with micrometer- screw is used. Some place a drop of oil on the front lens of the objective. Others put a drop not only upon the objective, but also upon the cover-glass. After use, the oil is carefully removed from the lens with a fine linen cloth, less satisfactorily with blotting-paper, and the system is returned to its case. If the cover-glass preparation is to be preserved, then the oil is soaked up with filter- paper, and what remains is finally removed by chloroform or ben- zine. According to histological tradition, which influ- enced the earlier use of the microscope, the magnify- ing power should be increased rather through the use of a more powerful lens than a more powerful 38 BACTERIOLOGICAL INVESTIGATION. ocular. But, according to a purely physical princi- ple, the strength of an ocular, which an objective will allow with advantage, depends upon the angle of aperture of the latter. The larger the angle of aper- ture, so much the stronger, other things being equal, can the ocular be. Our best homogeneous systems correspond to this requirement, namely, that one can use the same objective and at the same time employ the most powerful oculars. In this way, bacteria without any special prepara- tion may be observed between the slide and cover- glass. One cause of uncertainty is here noticed, i. e., almost all bacteria are in motion. This in part seems to be spontaneous motion ; in part a simple Brownian molecular movement, such as occurs in all fine particles suspended in a fluid. In proportion to the minuteness of the objects, these movements ren- der exact observation difficult. For this reason one should early eliminate this, and fix the forms of micro-organisms by narcotizing them.* For this pur- pose, a particle of spirituous or dilute alcoholic tinct- ure of opium can be added with the point of a needle to a drop of water. Moreover, von Recklinghausen f has shown that small, round, granular tissue-detritus, which is so easily confounded with bacteria, can be sharply dif- ferentiated by the fact that bacteria are pre-eminent- ly homogeneous granules, and are totally unaffected by the action of acetic acid, glycerine, or even caustic soda. BaumgartenJ succeeded in making the tubercle * Pertv, "Zur Kenntniss kleinster Lebensformen," 1852, S. 13. t " Verhandlungen der Physikal-Medizin. Gesellschaft in Wurz- burg," N. F., II. Bd., Heft 4, 1872. " Sitzunjrsberichte," S. XII. I " Centralblatt f. d. raed. Wissenschaft," 1882, No. 15. BACTERIA AND MICROSCOPICAL TECHNIQUE. 39 bacilli visible by their resistance to a diluted solution of caustic soda, though he could not recognize them by staining according to the methods in use at that time. We possess now more convenient means, both for fixation and differentiation. But it would be a seri- ous error not to examine bacteria unfixed and un- stained. It is, on the contrary, necessary throughout that the bacteria should be observed under the most natural conditions possible in order to study their motion, to follow the formation of spores and their germination, and to control the forms according to other treatment. For this purpose we do not use the previously described form of investigation, but employ the moist chamber. For this the hollow slides A and B (Fig. 6) serve. A small drop (c) of the bacteria-containing FIG. 6. L 7? fluid is placed upon a cover-glass (5). The cover-glass is quickly reversed, and, with the drop now hanging underneath (c, B\ is laid over the hollow (a) in the slide, and its edges are surrounded by vaseline, wax, paraffine, or balsam, in order to prevent evaporation of the fluid. Another and a better form is represent- ed in Fig. 7. Upon a slide, A or B, a glass plate (b) 40 BACTERIOLOGICAL INVESTIGATION. is cemented, which has a central circular opening. A chamber is formed by laying a cover-glass (a) over FIG. 7. c e the opening. This room, in place of the almost half- circular cavity in Fig. 6, is bounded by parallel walls. The drop is prepared in a similar manner, and in the same way hangs within the chamber. These chambers can be improvised if, instead of cementing a piece of glass, a thin piece of paper of corresponding size, with a circular aperture, is fastened upon an ordi- nary slide. The warm stage may be employed for di- rect observation at higher temperatures. STAINING BACTERIA. In examining unstained bacteria the diaphragm is used in order to make the structure-picture clear ; but small objects and particles, the size of bacteria, imbedded in the tissue made visible in the struct- ure-picture, are hidden by the shadows of the struct- ure-picture. If these particles are stained, and if they are of a certain size, they will be visible in spite of the shadow; but under this size, notwith- standing their color, they are concealed by the BACTERIA AND MICROSCOPICAL TECHNIQUE. 41 shadows. Therefore it is desirable to stain the bacteria and to so arrange the light that the struct- ure-picture does not further interfere, and that the color-picture, as pure as possible, be presented to the observer. Koch (loc. cit.) succeeded in pro- ducing this isolation of the color-picture by re- moval of the diaphragm. In this way so weak a structure-picture was produced that the minutest particles of the color-picture became distinct. In the stronger structure-pictures, after the remov- al of the diaphragm, he used a condenser which threw so intense a cone of rays upon the ob- ject that the diffraction appearances were entirely avoided. With such a method of illumination, in which the preparation is permeated in all directions by the penetrating rays, only those elements remain visible which produce an absorption of the rays on account of their staining. Further, Abbe in this way has shown that, although the illumination in name re- mains central, yet the important advantages of the oblique illumination are obtained through the co- operation of the rays passing at a greater inclination to the axis of the microscope. On account of this co-operation of the oblique rays for the isolation of the color-picture, and for the complete development of the capacity for differentiation of the oil-immer- sion objective necessary for this, the condenser must furnish a cone of light of a size at least equal to the aperture of the objective, which is made, according to Stephenson, with a large angle of aperture. This is as yet attained, in a manner that fulfills all the re- quirements, only by an Abbe condenser. In order that the structure-picture can be brought out in spite of this condenser, an arrangement for interposing a 42 BACTERIOLOGICAL INVESTIGATION. diaphragm is added, which is furnished with open- ings of different sizes. An Abbe condenser belongs to the system for homogeneous immersion in bacteria investigation. Now and then it is useful to place a drop of water or immersion-fluid between the condenser and the under- side of the slide, so that below and above a continuous union is formed. These form immersion-condensers which were formerly used before the Abbe condenser was constructed. GENERAL PRINCIPLES OF STAINING. Since Hartig, in 1854, and Gerlach, in 1858, showed, by the systematic use of carmine in histo- logical work, that in employing coloring-matters cer- tain elements of the tissues become more distinct and can be differentiated from other elements, staining has been recognized as equivalent to a chemical re- action. Weigert * first succeeded in staining the zoogloea masses of the micrococci with the nuclei, by the use of the nuclei-staining ammoniac- carmine solution, and the subsequent treatment with hydrochloric - acid glycerine. This staining, it is true, was first used by Weigert as a staining of cement substance ; but later it was corrected by him, and it was in this way shown that the bacteria can be brought into view by other characteristics than their greater resistance to acids and alkalies. In the following year Eberth and Wagner suc- ceeded in staining micrococci, but not bacilli, with hgematoxylin. * u Ueber Bakterien in der Pockenhaut." " Centralblatt f. d. med. Wissenschaft," 1871, No. 49. BACTERIA AND MICROSCOPICAL TECHNIQUE. 43 Then Weigert * showed that the micrococci, espe- cially in zooglcea masses, can be stained by different nuclei-staining materials. For this purpose he used at that time methyl-violet, an aniline-dye which stains nuclei. By the subsequent treatment of prep- arations stained with haematoxylin (in which the micrococci and nuclei are stained blue), with diluted caustic potash and strong acetic acid, he first suc- ceeded in procuring an isolated staining of the mi- crococci. Weigert f further observed that the larger bacilli, which were not stained by hsematoxylin, could be made visible by certain aniline-dyes. Soon after this, KochJ found that the bacteria take up aniline-dyes with such certainty, and so quickly and completely, that " these dyes can be used as reagents for differentiation of bacteria from crystalline and amorphous precipitates, or from the smallest fat- drop or other minute bodies." Koch* then succeeded in obtaining the isolated staining of the bacteria by washing out the section with a solution of carbonate of potassium, by which all the elements except the bacteria were decolorized. Finally Weigert || obtained a double staining, when he subsequently treated with picro-carmine prepara- tions that had been stained with a blue aniline-dye, whereby the bacteria appear blue and the nuclei red ; * " Sitzung der Schlesischen Gesellschaft fur vaterlandische Cul- tur," vora 10. December, 1875. t "Berl. klin. Wochenschrift," 1877, Nos. 18-19; und "Berichte tiber die Munchener Naturforscherversammlung," 1877, S. 288. | "Verfahren zur Untersuchung, zum Conserviren und Photo- graphiren der Bakterien." "Beitrage zur Biologie der Pflanzen," Bd. II, 3. Heft, 1877, S. 399. * " Wundinfectionskrankheiten," 1878, S. 39. || "Zur Technik der mikroskopischen Bakterien untersuchungen." Virchow's " Archiv," 1881, Bd. LXXXIV, S. 275. 44 BACTERIOLOGICAL INVESTIGATION. and Koch* observed that certain forms of bacteria stained differently from the nuclei and from other bac- teria which were present in the same preparations. What coloring-matters ought to be used for stain- ing bacteria ? Weigert's observation that the micrococci, but not the bacilli, are stained by carmine, and a similar ob- servation by Eberth and Wagner concerning hsema- toxylin, seemed to show, according to Weigert, that there are essential chemical differences between the single-group forms of Cohn. Safranine, one of the best reagents for the staining of nuclei, is also of more value for micrococci than for other forms of bacteria. Further, Obermeyerf observed that the spirilla are less resistant to the action of acids and alkalies than other bacteria. But these differences are not essential, since some micrococci and bacilli are less resistant to the action of alkalies and acids than others, and since some bacilli stain as well with hsematoxylin as micrococci, while others take this dye badly. However, the basic aniline-dyes have shown them- selves to be available staining materials, under all conditions, both for the dried cover-glass prepara- tions and for sections, so that we assign to them a first rank among materials for staining bacteria, and give to other dyes a second place. Ehrlich,$ partly in conjunction with his pupils Schwarze and Westphal,* undertook to classify the * " Berl. klin. Wochenschrift," 1882, No. 15. t "Berl. klin. Wochenschrift," 1873, S. 391. I "Zeitschrift fur klin. Med.," Bd. I, 1880, S. 553; and kleinere gelegentliche Mittheilungen. * Schwarze, " Ueber eosinophile Zellen," Dissert., Berl., 1880. Westphal, " Ueber Mastzellen," Dissert., Berl., 1880. BACTERIA AND MICROSCOPICAL TECHNIQUE. 45 dyes used in microscopic work. The principle under- lying this theoretical study of dyes rests on the ob- servation that the different elements of the tissues and cells possess the capacity of taking certain dyes only, or of holding them with a greater tenacity than other elements. This " election," this affinity of the dyes for certain elements, lends to staining the value of a chemical reaction ; or, more correctly (in the want of a precise chemical reaction), shows to the eye the presence of differences otherwise impercep- tible or distinguishable only with difficulty. Many staining materials color at first many ele- ments of a tissue quite diffusely, so that the individual elements are not recognizable. If, then, certain de- colorizing agents are used, some of these individual elements give up their color, while, on the other hand, other elements retain it persistently. In this way, by an indirect method, it is possible to attain a maximum staining of certain elements, while others remain as much as possible uncolored. Ehrlich called this method, which was first used in another way by Friedlander, * " the principle of the maximal decolorization." Histologically, one must differentiate in every col- oring-material two peculiarities : first, the affinity to certain elements ; and, secondly, the staining power. In respect to the election or affinity for certain ele- ments, Ehrlich divides the aniline colors into two groups (a) the acid, (b) the basic aniline-dyes ac- cording as the coloring principle is the acid or base- color. In this histological sense it is of equal import whether the acid in its use acts as a free acid or a salt ; also whether the base acts as such or as a salt. * " Studien uber automische Herzbewegnng," in " Untersuchungen aus dem physiologiscben Institut zu Wiirzburg," I, 1867. 46 BACTERIOLOGICAL INVESTIGATION. The acid aniline colors are divided into four classes : 1. Fluorescin e. g., fluorescin and eosin. 2. Nitrogenous bodies e. g., martius yellow, pic- ric acid, and aurantia. 3. Sulphuric acid e. g., tropseolin. 4. Primary dye-acids e. g., rosol acid, alizarin, and purpurin. Of the basic aniline-dyes, the following are found to have the most value : Fuchsin (muriate of rose- anilin), methyl- violet (muriate of trimethyl rose-ani- lin), gentian-violet, methyl-blue, and vesuvin. Of less value are methyl-green, cyanin, safranin, magdala, and dahlia. Of these, especially the violets (methyl- violet, gentian-violet, iodine-violet, and dahlia) have sometimes the valuable capacity of double staining ; that is, they stain certain elements in a color varying from the fundamental color e. g. , methyl- violet does not stain the amyloid substance violet, as it does the bacteria and nuclei, but red ; methyl-green stains the nuclei green, the amyloid substance violet. The members of the first group show altogether the same elective peculiarities i. e., they act as acid dyes and stain all the accessible elements, but in a differing degree. The basic aniline-dyes also show the same elective peculiarities, since they stain the accessible elements in a basic color ; but also in these there is a difference in the intensity of the staining. This intensity of the staining is dependent upon the coloring power, and the coloring power is conditional on the fact that the different coloring-matters are re- tained in different degrees of intensity in the tissues or cell-elements, in the presence of the individual groups of decolorizers, such as alcohol, acetic acid, and glycerine. For example, methyl-green in a short BACTERIA AND MICROSCOPICAL TECHNIQUE, tf time is completely extracted from a preparation by alcohol, while vesuvin is scarcely at all affected. In respect to this coloring power, the basic aniline-dyes arrange themselves in the following order : Vesuvin, bismarck-brown, and aniline-brown theoretically take the first place, because these coloring-matters are not extracted by glycerine, and they are at the same time suitable colors for photography. After these follow in a descending scale fuchsin, methyl-violet, gentian- violet, and methyl-blue ; but these should be placed generally before the brown, as they are to most per- sons more agreeable and satisfactory colors. The re- maining dyes have as yet found no general use. This scale, as I will remark, to avoid misunder- standing, has a conditional value, since certain colors are produced only when certain fluids are used for solution and when the preparations are subsequently treated in a special manner, as will be described later more in detail. At the same time it is not impos- sible that some one of the remaining dyes may prove^ to be more valuable than these. The basic aniline colors are soluble in water, and for the most part in one or all of the decolorizing agents. In use, a weak watery solution colors at first the intercellular substance and the cell-body, while the nuclei remain unstained. Through the subse- quent treatment with alcohol, glycerine, or acetic acid an inversion of the staining takes place, by which the elements previously colored become colorless, while the previously colorless nuclei are stained. In the use of the stronger solutions the staining follows (without any discernible inversion) directly and quick- ly ; and, in general, its intensity is in proportion to the concentration of the solution. In a quite concen- trated watery solution overstaining may occur, which 48 BACTERIOLOGICAL INVESTIGATION. in sections can be reduced to the proper degree by subsequent decolorization. Methyl-blue alone does not overstain, according to Ehrlich,* even after a long action, and it is conse- quently to be used if for a special reason no decolor- izing agents should be employed. If the dyes are dissolved in the decolorizing agents such as absolute alcohol, acetic acid, or thick gly- cerine they stain slightly or not at all. Instead of using some decolorizing agent subsequently, to re- duce the intensity of the staining to the proper degree, in preparations which have been overstained in watery solutions, in many cases a solution of the dye-stuff in a mixture of water with alcohol (Herrmann), gly- cerine (Schaefer), or acetic acid (Ehrlich) may be used. PKEPAKATION OF STAINING FLUIDS. The basic aniline-dyes are used in the following solutions : 1. Concentrated watery solutions. These are either used directly or after dilution to the desired degree with distilled water. The solu- tions are prepared with distilled water (which has been previously boiled), so that an excess of the col- oring-matter remains undissolved. They must be often filtered. Only a small quantity of these watery solutions should be made at a time. 2. Concentrated alcoholic solutions. The solution of an excess of the coloring-material is brought about in the best way by absolute alcohol, or, in want of this, by the officinal 90 per cent spirit of the Pharmacopeia. In general, one can calculate about 20 to 25 grammes of the dye-stuff to 100 grammes of the spirit + " Zeitschrift f. klin Med.," Bd. II, 1881, S. 710. BACTERIA AND MICROSCOPICAL TECHNIQUE. 49 or alcohol. These solutions are kept prepared, and are not used directly for staining, but are mixed with a certain amount of distilled water. In place of con- centrated watery solutions, these can be used if five or six drops are added to a small watch-glass of dis- tilled water. This mixture I shall briefly designate in the future as the diluted alcoholic solution. 3. Vesuvin, bismarck-brown, and aniline-brown can not be used in alcoholic solution, nor in a watery solution, even if filtered each time ; so that a concen- trated solution in equal parts of glycerine and water is prepared.* 4. Alkaline solutions. a. Weak. Koch.f Concentrated alcoholic solution methyl-blue. . 1 c. cm. Aq. destil 200 c. cm. 10 per cent solution caustic potash 0*2 c. cm. b. Strong, i Concentrated alcoholic solution methyl-blue . 30 c. cm. Solution caustic potash, 1 to 10,000 100 c. cm. 5. Aniline-water solution (according to Ehrlich).* Pure aniline-oil in excess is shaken with distilled water for one half to one minute (about 5 c. cm. of oil with 100 c. cm. of water). Then, after allowing it to stand five minutes, the mixture is filtered through a filter which has been previously moistened with dis- tilled water. The filtrate must be perfectly clear, and serves in place of water as a menstruum. Since this saturated aniline solution very quickly becomes un- * Koch, " Verfahren zur Untersuclmng." " Beitrage zur Biologie der Pflanzen," 1877, Bd. II, 3. Heft, S. 5. t " Berl. klin. Wochenschrift," 1882, No. 15 ; " Mittheilungen aus d. k. Gesuadheitsamt," 1884, Bd. II, S. 5. I " Mittheilungen," 1884, Bd. II, S. 439. * " Deutsche med. Wochenschrift," 1882, No. 19. 50 BACTERIOLOGICAL INVESTIGATION. stable, it is better to prepare it fresh each time that it is used. If it is desired to make this permanent, according to B. Fraenkel, 5 to 10 per cent of alcohol is added, or 3 c. cm. of aniline-oil is dissolved in 7 c. cm. of alcohol and 90 c. cm. of distilled water is added. Fuchsin, methyl-violet, and gentian-violet are the best dyes for use in this menstruum. In most cases, according to Ehrlich, it is more con- venient to add a saturated alcoholic solution of fuch- sin or methyl-violet to the clear aniline-water until a distinct cloudiness of the fluid is present, which indi- cates that the fluid is saturated with the coloring- matter. For certain purposes the following modifi- cation of the Ehrlich solution, according to Weigert and Koch,* recommends itself for common use, but this must be renewed after ten or twelve days, be- cause its coloring power is generally diminished : Saturated aniline- water 100 c. cm. Concentrated alcoholic solution, methyl- violet, or f uchsin 11 c. cm. Absolute alcohol 10 c. cm. 6. In place of aniline, toludin, prepared in the same manner, can be used as a menstruum (B. Fraen- kel). f Also turpentine (Prior) ; and a five per cent watery solution of carbolic acid (Ziehl),* or one half per cent ammonia (Weigert) [ [Liq. ammon. caust., 0'5 c. cm. ; aq. destil., 90 c. cm. ; alcohol abs., 10 c. cm. ; gentian-violet, 2 grm.]. For double staining, the nuclei-staining carmine * " Mittheilungen aus dera kaiserlichen Gesundheitsamt," Bd. II, 1884, S. 6. t " Berl. k. Wochenschrift," 1884, No. 13. I " Berl. k. Wochenschrift," 1883, No. 33. *" Deutsche med. Wochenschrift," 1882, S. 451; 1883, S. 12 und 247. | " Deutsche med. Wochenschrift," S. 351. BACTERIA AND MICROSCOPICAL TECHNIQUE. 51 and hsematoxylin* can also be used; the first for blue or violet, the latter for red-stained bacteria. In place of the ordinary nuclei-staining carmine, picro- carmine can be used for preparations of bacteria stained blue, which stains the nuclei an intense red, the fibrillar substance of the connective tissue pink, and the protoplasmic substance a more or less yellow, so that a threefold staining results. Hsematoxylin is best used in the following solution : Hsematoxylin 2 parts Alcohol 100 " Aq. destil 100 " Glycerine 100 " Alum, sulph 2 " This hsematoxylin stains micrococci, many bacilli, and at the same time the zooglcea masses, with the in- tercellular substance. The staining of the bacteria is paler than that produced by the blue or violet basic aniline-dyes, which, on the other hand, do not stain the zoogloea masses. In order to obtain the threefold staining after the bacteria are stained red by fuchsin, it is better to stain the nuclei with hsematoxylin, and then afterward stain the protoplasm with a satu- rated solution of picric acid or eosin. The rose-red eosin can be added to the above solution of hsema- toxylin in the proportion of one half per cent. (To retain the yellow color of the picric acid, picric -alco- hol and damar resin must be used.) OTHER REAGENTS AND APPARATUS. Other solutions are often required : (a) Iodine 1 part Potas. iodid 2 parts Aq. destil 100 " * Cf. die citirten Handbftcher, besoD tiers Friedlander. 52 BACTERIOLOGICAL INVESTIGATION. (b) In the use of the basic aniline-dyes it is sel- dom necessary to extract the fat. If it is desirable to do this, the sections are first washed thorough- ly in absolute alcohol (three to ten minutes), then are transferred to a watch-glass containing ether and chloroform for a few minutes ; after this they are again placed in alcohol, cleared up in acetic acid (for the solution of the coagulated albumen), and are then examined immediately or after previous staining. (c) Nitric acid, diluted in the proportion of one part of the officinal acid to three or four parts of wa- ter, is sometimes used. (d) For the removal of the lime salts, according to von Ebner, the following solution may be used, but must be often renewed : Ac. muriat 5 parts Alcohol 100 " Aq. destil 20 " Sodii chlorid 5 " (e) Acetic acid is employed in from one half to one per cent solution for obtaining the maximal decol- orization and for the examination of unstained bac- teria. (/) Chromic acid is used in a one half per cent solution, or as Muller's fluid : Potas. bichrom 2 parts Sodii sulphat 1 part Aq. destil 100 parts (g) The hydrates of potassium or sodium in one to three per cent solutions, or a solution made by adding one to two drops of the thirty-three per cent solution (so much used in histology) to a watch-glass of water, may be employed for rendering the un- stained bacteria visible. BACTERIA AND MICROSCOPICAL TECHNIQUE. 53 (7i) Glycerine and alcohol are always to be used only in pure form completely free from acid. (i) The distilled water which is used in bacterio- logical work should always be sterilized by boiling for an hour over a flame or in a sterilization appa- ratus, since the ordinary distilled water always con- tains bacteria and their germs. All the solutions used in bacteriology should be made with sterilized distilled water, and all solutions and reagents must be tested for the possible presence of bacteria. For the preservation of the solutions, flasks with ground stoppers are necessary, and for daily use small glass bottles with hollow-ground stoppers, having above a rubber cap and below a capillary tube, are very con- venient. These capillary pipettes serve to draw up many or few drops as is desired. For the preservation of the bacteria preparations, glycerine can be used only with the brown dyes, since it more or less rapidly extracts the other aniline col- ors. The glycerine gelatin of Klebs can be used for the brown colors. The saturated solution of potas. acetat. (1-2) can be often advantageously used for the preservation of the stained and unstained bacteria ; but the most univer- sally valuable material for mounting is Canada bal- sam, which is used most conveniently from a tube (the artisfs ordinary paint-tub^) after having been dissolved. For diluting Canada" balsam, turpentine or xylol must be used, because chloroform extracts the basic aniline-dyes. For the same reason, the precaution should be taken not to warm the balsam. The much- loved oil of cloves should not be employed for clearing up on account of the same objection ; but in place of it oil of turpentine, cedar, or bergamot must be used. 5i BACTERIOLOGICAL INVESTIGATION. In the way of apparatus the following articles are necessary : good slides and cover - glasses ; watch- glasses ; ordinary porcelain dishes, and those with plain ground bottoms ; crystallization-glasses of differ- ent sizes, preferably two sizes, of which the greater can be used at the same time as a cover for the smaller ; beakers of different sizes ; small, square wire baskets for holding test -tubes; test-tubes; wash-bottles; graduates and pipettes; a plate of black glass or porcelain to place under white or unstained objects ; a plate of white glass or porcelain to place under stained objects; glass tubes, some drawn out to a capillary-point ; glass rods, some having from 3 to 5 cm. of platinum wire of different sizes melted into one end (to be used straight or bent into a loop) ; spider-forceps ; and many slide-pincettes. On one of these latter instruments the ends should be bent out somewhat, and small pieces of flat cork fastened to them for taking hold of cover-glasses. The slide pincettes can be improvised by placing over ordinary pincettes the narrowest possible ring of cork. Of metal instruments, it is necessary to have some teasing-needles, scissors, knives, and a wide spatula of nickel, steel, or platinum, on which the sections can be spread out in transferring them from one fluid to another. New slides and cover-glasses are first cleaned with warm water and dried with a linen cloth. But this is not sufficient, and they must then be carefully rubbed with a cloth which has been dipped in spirit. For the preparation of the flat hanging-drop it is often necessary to allow the apparently clean cover-glass to lie some hours in absolute alcohol ; to remove the remains of the alcohol by ether, and then, finally, to dry by evaporation. Slides and cover-glasses which BACTERIA AND MICROSCOPICAL TECHNIQUE. 55 have been used are laid in concentrated muriatic, ni- tric, or sulphuric acid, and, after cleaning and removal of the acid, are treated with water until all acid reac- tion has disappeared, and are then further treated as the new. COYER-GLASS PREPARATIONS. After it was observed that the morphological ele- ments in the blood, dried in a thin layer, were not materially altered by drying, Koch * first employed these casual observations methodically in bacteria investigation. He spread out upon a cover-glass, in a very thin layer, a drop of fluid containing bacteria, so that the individual elements were brought very nearly on the same plane. This thin layer was then fixed by simply drying in the air. In order to elimi- nate the slight alteration produced by this, it is nec- essary afterward to cause again a swelling up of the bacteria. If the layer which has been dried in the air remains too long in the water or glycerine used for this purpose, it is entirely dissolved, instead of only partially swelling. If the cover-glass with the dried layer is la^d in absolute alcohol, or a one half per cent solution of chromic acid, the layer is rendered insoluble in water and glycerine, and no longer swells up. But if the layer which has been made insoluble is put into po- tassium acetate, it swells sufficiently without being entirely dissolved, and all the forms seem to be in a natural condition. The solutions of the aniline-dye have the same action and cause the same swelling without removing the layer, and at the same time stain the bacteria. * " Verfahren zur Untersuchung:," etc. " Beitrage zur Biologie der Pflanzen," 1877, II, 3. Heft, S. 399. 56 BACTERIOLOGICAL INVESTIGATION. In the use of this method in the investigation of the blood, Ehrlich * found that the rapid drying pre- vented coagulation of the cell-albumen, and retained the natural staining capacity of the elements. Only the haemoglobin was extracted by aqueous and gly- cerine solutions of the dyes. But if the prepara- tions were kept for a few hours at a temperature of 115-125 C., the elements of the blood, without any important alteration and without the appearance of artificial products, retained their elective affinities for dyes. In following up these observations, Koch f discovered that, in place of the fixation by alcohol, the application of heat for only a few minutes an- swered the same purpose. A drop of the fluid containing bacteria, either un- diluted or after the addition of a drop of distilled water (according to the amount of its morphological elements), is spread out in a thin layer upon the cover-glass, by means of a pointed scalpel or platinum wire, and the excess of fluid soaked up with filter- paper ; or a drop is placed upon one cover- glass and a second is applied to this, which, through its press- ure, spreads out the drop in an even layer. If, then, the two cover-glasses are drawn apart with pincettes, we have two similar preparations. The cover-glasses, protected from dust, are allowed to remain until com- pletely dry, or they can be dried in a dry-oven some- what more rapidly. The drying can also be hastened by holding the cover-glass with the prepared side upward high above the gas-flame, and moving it to and fro to prevent the direct action of the flame. Upon the dried preparation a drop of the staining * " Zeitschrift f. klin. Mod.," Bd. I, S. 553. t " Znr Untersuchung von pathogenen Organismen." " Mitthei- Inngen aus dem kaiserlichen Gesundheitsamte," 1881, Bd. I, S. 1. BACTERIA AND MICROSCOPICAL TECHNIQUE. 5T solution can then be placed to stain the elements, but only in case the fluid is free from albumen and the staining follows quickly, since, by the prolonged ac- tion of the staining solution, the layer is completely loosened. If the dried layer consists of an albumi- nous substance, such as blood, tissue-fluids, or sputa, on the addition of the staining solution, precipitation occurs. On this account it is especially necessary that the preparation, after drying in the air, should be more securely fixed by heating. For this purpose the cover-glass may be placed in a drying-box or upon a copper plate. The copper plate is laid upon a tri- pod, and one end is heated by a gas-flame, so that the different portions, at different distances from the flame, have varying degrees of temperature. A few minutes' exposure to a temperature of 125 C., or ten to twenty minutes at 110 C., is sufficient to thor- oughly dry bacteria preparations. This may be done more conveniently, and in some cases also more cer- tainly, according to Koch-Loefller, if the cover-glasses with the dried layer are drawn rather rapidly three times through a gas- or spirit-flame. The reason for heating in exactly this manner, ac- cording to Koch,* is this: because in preparations which have not been heated the above-described pre- cipitation occurs, while in preparations which have been passed through the flame only once or twice, the fixation of the elements, especially in those containing much albumen, is not sufficient for all cases. In those passed through the flame three times, while the forms themselves are not materially altered, the capacity for staining is retained, and the albuminoid material has become so insoluble that precipitation no longer * " Mittheilungen," 1884, Bd. II, S. 7. 58 BACTERIOLOGICAL INVESTIGATION. takes place. Passing the preparations through the flame a yet greater number of times destroys the susceptibility of the bacteria to the staining fluid. The want of success in making preparations, which many beginners experience, seems to be especially due to the fact that the preparations are generally heated before they have been completely dried in the air. If the preparation still contains any water, coagulation of the albuminoid material occurs when heated, while in those completely free from water this does not hap- pen, and the albumen is rendered homogeneous by heating. The preparations, dried in the air and then drawn three times through the flame, are now stained. The cover-glasses, with the prepared side upward, are laid on a piece of filter-paper, and, by means of a glass rod, a cap pipette, or the glass stopper with the capil- lary-tube, a few drops of a staining solution are placed upon the preparation. The staining fluid should remain about twenty minutes, or until it is seen, by an inclination of the cover-glass, that the preparation has already taken up the color. If the action of the staining solution ought to be prolonged, then it should not be placed drop by drop on the cover-glass, because, in drying, the staining solution forms a ring of color at the edge which it is difficult to remove. In this case a sufficient quantity of the staining solution is placed in a watch-glass or crys- tallization-glass, and the cover-glass is then taken, with the prepared side downward, between the thumb and index or middle finger, and allowed to fall flat upon the surface of the staining solution, so that it swims with the prepared side upon the surface of the fluid. To prevent evaporation, the dish is covered with a glass plate. BACTERIA AND MICROSCOPICAL TECHNIQUE. 59 For the removal of the excess of coloring-matter, a stream from a wash-bottle is thrown obliquely from above upon the cover-glass, taking care not to strike the surface of the preparation directly ; or the cover- glass, held in pincettes, is moved to and fro in a beaker filled with distilled water ; or the excess of the fluid may be soaked up with filter-paper, a few drops of water added to be soaked up anew, and so on until none of the coloring-matter is given up to the filter- paper. Then the cover-glass preparation is examined in a drop of distilled water. The upper side of the cover-glass is freed from every particle of water by soaking it up with filter- paper, because on it must be placed a drop of oil for the homogeneous immersion-lens. If the cover-glass preparations are to be preserved, the oil is removed with filter-paper and chloroform, the water by careful warming or evaporation (pro- tected from dust), and the dried preparation is di- rectly imbedded in Canada balsam. Different dyes are used for each variety of bacteria, since some stain only the bacteria ; others at the same time the fine gelatinous sheath ; others the cap- sule. On this account the corresponding pictures in all the methods of staining are not absolutely similar; so that it ought to be self-evident that al- ways, in comparison, only preparations should be used which have been treated in exactly the same manner. These considerations must guide one in the choice of the staining solution. We must therefore distinguish between staining for a special purpose i. e., for the establishment or employment of coloring methods which have been described or proved to be best in particular cases and the investigation staining used especially to prove the presence of bacteria. 60 BACTERIOLOGICAL INVESTIGATION. Since in cover-glass preparations almost all bac- teria can be stained by watery solutions of the basic aniline-dyes, saturated watery solutions or the equal- ly valuable alcoholic solutions are first employed. The saturated watery solutions have for this testing an advantage, because all basic aniline-dyes are known to be applicable ; so, with a few preparations, the dif- ferent colors can be tried. If no bacteria come to view in this way, notwith- standing their supposed presence, then aniline-water, with methyl- violet or f uchsin, is used, or the stronger alkaline solution of methyl-blue. The trial examina- tion as to the presence of bacteria resolves itself, in the larger number of cases, into the following pro- cedure : 1. Drying in a thin layer. 2. Fixation by passing the cover-glass three times through the flame. 3. Staining by placing a few drops of a watery or dilute alcoholic solution of a basic aniline-dye upon the preparation. 4. Removal of the excess of the coloring-matter by washing or soaking up with filter-paper. 5. Examination in a drop of distilled water. For the isolated staining of bacteria in cover-glass preparations they can be laid for about one minute in a half -saturated solution of potassium carbon- ate ; or, if they are stained in aniline-water-gentian- violet, the remaining elements can be decolorized ac- cording to the method of Gram.* The stained cover- glass preparations are for this purpose laid for about one minute in the solution of potassium iodide (vide page 51), and then placed in absolute alcohol until * " Ueber die isolirte Farbung der Schizomyceten." " Fortscliritte der Medizin," II, 1884, No. 6. BACTERIA AND MICROSCOPICAL TECHNIQUE. 61 they appear decolorized. The alcohol is soaked out and the preparation examined in water. For double staining the cover-glass preparations, after being decolorized according to the Gram meth- od, they can be taken from the alcohol and placed in a weak watery solution of vesuvin. Then the bac- teria remain blue, often almost blue-black, while the nuclei are stained brown. The preparations stained red or blue can also afterward be stained with car- mine or hsematoxylin ; yet this double staining has much less value in the cover-glass preparations than in sections. EXAMINATION FOR TUBERCLE BACILLI IN SPUTUM. These preparations can be stained according to the Gram method; but by this both the tubercle bacilli and other bacteria are stained blue in contrast with the brown nuclei. For the differential diagnosis this is not sufficient, and for this purpose the principle established by Koch must be exclusively observed i. e., that the tubercle bacilli should be stained in a different color from other bacteria and the nuclei. Koch succeeded in doing this, in preparations stained for twenty-four hours in a weak alkaline solution of methyl-blue, and then placed for a short time in a watery solution of vesuvin. In this way the tubercle bacilli (and the bacilli of leprosy) are stained blue ; all other bacteria and nuclei, brown. After this im- portant principle was discovered, Ehrlich showed that aniline-water was still a better agent for increas- ing the intensity of the color, and that in the prepara- tions stained with aniline-water colors the tubercle bacilli withstood decolorization by nitric acid, while all other bacteria were decolorized by this mineral acid. But the preparations can not be left so long in 62 BACTERIOLOGICAL INVESTIGATION. the acid that complete decolonization occurs, because then also many or all of the tubercle bacilli are de- colorized. They should remain in the acid until the red (fuchsin) or blue (methyl- violet) hue has changed into a yellow-red or greenish blue. At this stage the preparations are placed in water, and again a red or blue color appears. By the action of the acid the simple acid union (red or blue) is changed into a triple acid (yellow-red or blue-green), and, by the addition of the water, the triple acid union is de- stroyed and the red or blue hue reappears. The preparations decolorized by the acid are not washed in water, but in 50 or 60 per cent alcohol ; then they are stained in a dilute solution of methyl-blue (or vesuvin). After washing away the methyl-blue (or vesuvin) the preparations are examined in water, or, after removal of the water, preserved in Canada balsam. After this whole procedure, the tubercle bacilli retain their red or blue color, and are easily recog- nized among the other elements. Aside from this differential diagnostic action of the double staining, the subsequent staining in another color has an ad- vantage by affording an easier examination of the specimens. Concerning the choice of the material containing bacteria, it is to be noted that the cheesy masses are to be spread out thin with a sterilized scalpel. Nod- ules of tubercle must be crushed with a scalpel or between two scalpels, and then be pressed flat upon the cover-glass. The tough, yellowish masses from the sputum are used. One of these particles is taken and spread out in a thin layer on the cover-glass, or flattened by pressing one cover-glass upon another, so that, after separating the two cover-glasses with BACTERIA AND MICROSCOPICAL TECHNIQUE. 63 pincettes, two preparations are obtained. The entire method is, according to Koch (after the adoption of the previously described aniline-water staining of Ehrlich), briefly as follows : 1. Pass the dried cover-glass preparations three times through the flame. 2. Stain with the Weigert-Koch solution of methyl- violet or fuchsin for twelve hours. 3. Treat with dilute nitric acid (1 to 3 or 4) for a few seconds. 4. Wash in a 60 per cent solution of alcohol by a to-and-fro motion. 5. Stain in a dilute solution of vesuvin or methyl- blue. 6. Wash and examine in water or mount in bal- sam. This method is the best thus far discovered, and serves as a control in all doubtful cases. For the differential diagnostic decolorization and subsequent staining of preparations of tubercle ba- cilli, the following reagents have been used : 1. Other aniline-dyes (vesuvin by Koch). 2. Acids (nitric by Ehrlich, hydrochloric by Orth, acetic by Petri). 3. Acid alcohol (weak nitric by Rindfleisch, and hydrochloric by Orth). B. Fraenkel combined these three variations by preparing an acid-alcoholic solution of methyl-blue and of vesuvin. a. For blue : Alcohol 50 parts. Water 30 u Mtric acid 20 u Methyl-blue to saturation. To be filtered. 5 64: BACTERIOLOGICAL INVESTIGATION. &. For brown : Alcohol 70 parts. Nitric acid 30 " Vesuvin to saturation. To be filtered. For the use of these solutions the following method of Fraenkel is recommended : About 5 c. cm. of aniline-water are heated in a test-tube to boiling and poured out in a watch-glass ; to this hot aniline- water a concentrated alcoholic solution of fuchsin or methyl-violet is added, drop by drop, until the solu- tion of the dye assumes a cloudy appearance. Upon this warm solution the cover-glass preparations are allowed to swim, and even in two or three minutes most of the tubercle bacilli are stained ; but, as a precaution, they should be left five or ten minutes. From this staining solution, the preparations stained red or blue are passed into the blue or brown acid- alcohol solution. After remaining one or two min- utes in the latter, the preparations seem colored and are washed in water or 50 per cent alcohol, to which one half per cent acetic acid has been added, and are then examined in water. Those who prefer the hydrochloric acid of Orth can employ the following method of Kaatzer: stain as before, and then decolorize with a mixture of Alcohol (90 per cent) 100 c. cm. Water 20 c. cm. Concentrated hydrochlor. acid 20 gtt. wash with 90 per cent alcohol to remove the acid, and then stain with a concentrated watery solution of methyl-blue or vesuvin. In sputum, according to Celli and Guarnieri, some- times very fine fat-crystals are found, which react to the staining almost as the tubercle bacilli, "pseudo- BACTERIA AND MICROSCOPICAL TECHNIQUE. 65 bacilli," but which, on careful examination, are not to be confounded with them, on account of their vary- ing size and because they are dissolved by ether and chloroform. The published modifications of the Ehrlich meth- od, founded on the Koch principle, are so numerous, but without anything having been added, that I must on this account simply refer to some of the compre- hensive descriptions,* and content myself with giving the underlying method of Koch (which is now best employed according to Ehrlich- Weigert- Koch), and two practical modifications of it. In these methods the bacilli of leprosy react as the tubercle bacilli, from which, moreover, they are morphologically not easy to differentiate. The differ- ential diagnosis by staining is founded on this fact : that the bacilli of leprosy do not stain so deeply as the tubercle bacilli, and that, in respect to the ease with which they give up the dye, they stand between these and most other forms of bacteria. According to Baumgarten,f the dried cover-glass preparations are allowed to float for six or seven minutes in a dilute alcoholic solution of f uchsin (5 or 6 drops of a con- centrated alcoholic solution in a watch-glass of dis- tilled water), decolorized fifteen seconds in acid alco- hol (1 part nitric acid to 10 parts alcohol), washed in distilled water, afterward stained in a watery solu- tion of methyl-blue, washed again, and examined in water. * Kaatzer, " Die Tecbnik der Sputumuntersuchung auf Tuberkel- Bacillen," 1884. B. Fraenkel, "Ueber die Farbung des Kocb'scben Bacillus," " Berl. klin. Wocbenscbrift," 1884, No. 13. Baumgarten, "Beitrage zur Darstellungsmethode der Tuberkel-Bacillen," "Zeit- sehrift ftir wissenscbaftliche Mikroskopie," I, 1884, S. 51. t " Ueber TJntersuchungsmethoden zur Unterscheidung von Lepra- ond Taberkel-Baeillen," ibid, S. 367. 66 BACTERIOLOGICAL INVESTIGATION. The bacilli of leprosy seem then as red rods upon a blue ground, while the tubercle bacilli during this time, by this treatment, have as yet taken up no color. The reaction of the tubercle bacilli, both in the method of staining discovered by Koch with its differ- ent modifications, and in the method of Baumgarten (the recognition of these bacteria in unstained condi- tions), seems at first to separate these bacilli qualita- tively from all other bacteria. Further studies, how- ever, have shown that these differences are not quali- tative, but are especially quantitative. The tubercle bacilli are stained with the greatest difficulty; but they retain the color persistently. It has been shown since by Lichtheim,* and especially by the work of Baumgarten, previously cited, that the tubercle bacilli in dried cover-glass preparations are stained in about one hour, both in dilute alcoholic and strong aqueous solutions of methyl-violet, gentian- violet, and fuchsin, or by simultaneous warming, in about five minutes. (For sections the time required is about twelve hours, or about ten minutes with elevation of temperature.) These stainings withstand the decolorization by acids for some time, although after a longer action of the same the tubercle bacilli are also decolorized. The tubercle bacilli remain unchanged after treatment with carbonate of potassium, as do other bacteria. If the stained cover-glass preparations are laid for de- colorization for about one minute (sections, five min- utes) in alcohol, and afterward for five minutes (sec- tions, fifteen to twenty minutes) in a concentrated aqueous solution of vesuvin or methyl-blue, a double staining is obtained. Therefore, neither the addition of the alkali nor the aniline-water is absolutely essen- * "Zur diagnostischen Verwerthung der Tuberkel-Bacillen." " Fortscbritte der Medizin," 1883, S. 1. BACTERIA AND MICROSCOPICAL TECHNIQUE. 67 tial to the staining; the reaction to acids is not a qualitative differentiation from other bacteria, and the use of the acid is not entirely necessary for ob- taining the double staining. A satisfactory explanation of the theory of stain- ing, which seems to lie so near, has not yet been dis- covered, on account of the varying quantitative re- action of the tubercle bacilli. The possibility of a better understanding is offered by the following fact : The addition of an alkali, as well as the feebly alka- line aniline-oil, renders the staining easier. Other aromatic bodies and ammonia act in a similar man- ner. The addition of an acid to the aniline-water does not arrest its action, so that probably the favor- able action of carbolic acid is reduced to the aromatic element, and is effectual in a similar manner in spite of the acid reaction. In addition, Gibbs * showed that in the simulta- neous action of two aniline-dyes the bacilli are stained differently from the other elements, while by another method these are stained in both of the dyes. A so- lution of 3 c. cm. of aniline-oil in 15 c. cm. of spir- its is added slowly to 2 grammes of fuchsin and 1 gramme of methyl-blue, and, after the solution of the dyes, 15 c. cm. of water are added. This solution is warmed, the cover-glass preparation is laid upon it for five minutes, and then washed in spirits till color is no longer given up. The bacilli then appear red upon a blue ground. This method is, unfortu- nately, not sufficiently reliable. EXAMINATION OF BLOOD FOR BACTERIA. The examination of blood for bacteria offers very great difficulty, because in the normal blood within * "Lancet," 1883, p. 771. 68 BACTERIOLOGICAL INVESTIGATION. the vessels, and in the normal disintegration of the healthy blood, granular elements are present, or are formed, which, under certain pathological conditions, in anaemic states and in fever, are increased in num- ber, and can be easily mistaken for micrococci. They have already been often confounded with micrococci, and are almost daily mistaken for them e. g., the re- nowned syphilitic corpuscle, and the so-called organ- isms of the venom of serpents. Here belongs also much of what has been spoken of as the development of bac- teria from nitrogen molecules, from microzymen, or from the anamorphosis of protoplasm. An exact study of these granules of the blood is on this account an indispensable desideratum in bacteria investigation. These granules form, further, a constituent part of the cellular elements of the blood, and on this ac- count again are of interest in setiology, because there are parasites which are similar to the amoeboid cells e. g., those monads found by Lewis in the blood of rats, by Koch in the blood of marmots. The ele- ments of the blood, which directly or through their granules may be confounded with micro-organisms (with the exception of the red blood-corpuscles, and the products of their disintegration), are divided, ac- cording to Ehrlich,* into 1. Lymphoid elements. (a) Small lymph-cells. () Large lymph cells. 2. Myeloid cells (eosinophile). 3. Undetermined (spleen and [or] marrow). (a) Large mononuclear cells. () Transitional forms, (c) Polynuclear. * Cfr. the work of Ehrlich, "Westphal, Schwarze, aqd Spilling, u Ueber Blutuntersuchnngen bei Leukamie," Dissert., Berlin, 1880; and Einhorn, u Ueber das Verlialten der Lympliocyten zu den weis- sen Blutkorperchen," Dissert., Berlin, 1884. BACTERIA AND MICROSCOPICAL TECHNIQUE. 69 The small lymphoid elements are somewhat small- er than the red blood-corpuscles, possess a very large nucleus, so that there is very little or no protoplasm to be seen. The large lymph-elements are a further development of the first, and are only to be differen- tiated from them in this, that they possess around the large nuclei a distinct border of protoplasm. The myeloid elements are large, round cells, with large, oblong nuclei. The mononuclear cells are about three times the size of the red blood-corpuscles, and possess round or oval nuclei of large size, and a con- siderable mass of protoplasm. The mononuclear transitional forms are to be differentiated from these cells only in this, that the nuclei are no longer round or oval, but have become indented. The polynuclear elements are somewhat smaller, but still are always larger than the red blood-corpuscles, and their nuclei show, as a further differentiation, a polymorphous form. These are the true white blood- corpuscles. The granular elements, or granules, which are pres- ent in the cells, and which become free in the de- struction of the same, are divided with respect to their reaction to aniline-dyes. The a, or eosinophile granule, is coarsely spher- ical, strongly refracting, and can be stained in all the acid aniline-dyes. It is present in the myeloid ele- ments, seldom in the normal blood, and its number is greatly increased in leucsemic processes. The /3, or amphophile granule, is found especially in the marrow, very often in the leucocytes in the blood of rabbits and guinea-pigs, and can be stained by acid and basic aniline-dyes. The 7, or basophile plasma cell granule, can be stained, like the bacteria, by the basic aniline-dyes. These granules are coarse, slightly refracting, almost 70 BACTERIOLOGICAL INVESTIGATION. completely wanting in normal human blood, increased in leucsemic processes, and are present normally in the blood of the lower animals, especially the white rat. The S, or basophile granule, is fine, and can be stained in basic aniline-dyes, and forms a constituent part of the large mononuclear elements. The e, or neutrophile granule, is very fine, and fills the polynuclear elements of the human blood quite thickly, is present sparsely in the transitional forms, and very seldom in the mononuclear ele- ments. It can be stained by the neutral dyes. Without recourse to staining, these granules, as a whole, and also the products of the disintegration of the red blood-corpuscles, may be confounded with micrococci. By systematic staining with the aniline- dyes, the a, /3, and e granules can be excluded. An error is then possible only with the y and & granules, because these are stained in the basic aniline- dyes, the same as the bacteria. These last, on account of their fine grain, can, with comparative ease, be differ- entiated from micrococci, and have not, as yet, been confounded with them. The plasma cell granules, on account of their medium size, come so near to the known forms of cocci, that not only the individual free granules in the blood have been considered as cocci, but even the same so-called plasma cells in the tissues have been described as colonies of cocci. They can, on purely morphological grounds, be dif- ferentiated from them in this way, viz., that they do not all have the symmetrical appearance of the cocci, but present the greatest differences in the size of the granules. If it is desired to examine the blood for bacteria, a small drop is rapidly spread out in a thin layer, dried, fixed, and then passed three times through the BACTERIA AND MICROSCOPICAL TECHNIQUE. 71 flame, and then stained in the ordinary manner. In such preparations the bacteria are sufficiently stained, but not the granules. The small drop of blood, at most the size of a pin- head, must be taken with the greatest care. In case of a haemorrhage, a small drop is taken with a steril- ized platinum wire, or, for obtaining the blood, the skin may be pricked with a previously sterilized needle, the end of the finger being the best spot. The skin is first cleansed with a brush and soap, and then washed with a solution of corrosive sublimate, 1 to 1,000. The sublimate is removed with alcohol, the alcohol with ether, and the last allowed to evapo- rate. The first drop of blood which wells up is re- moved with a sterilized platinum wire, and the fol- lowing drop used, a cover-glass being lightly pressed upon it with the pincettes, without coming in contact with the surrounding skin. Upon this first cover- glass a second is laid, which, by pressure, spreads out the blood in a thin layer, in which the elements are not materially altered. The two cover-glasses are then drawn apart by pincettes, so that two cover-glass preparations are obtained. Some of the cover-glasses are used as above in the examination for bacteria, after the layer has been dried in the air, and heated only for a short time ; but others are heated for one hour at a temperature of 120 C., and then treated with a basic aniline-dye, in order to study more ex- actly the basophile granules. Other preparations for the determination of the eosinophile elements are treated with the acid aniline- dyes after heating a short time, and also after an hour's heating. A mixture is made of the yellow, red, and black dye-stuffs of the strongest staining power, each of which alone stains all of the acid- 72 BACTERIOLOGICAL INVESTIGATION. forming elements, and both, the simultaneous action and the elective staining are valuable, since the three eosinophile elements are stained at the same time in different colors. One part of a saturated glycerine solution of aurantia is diluted with two parts of glycerine; then aniline-black (sulphate of indolin) and eosin are added in excess, and, by long shak- ing, dissolved to saturation. This saturated glyc- erine solution stains all the parts containing hae- moglobin intense orange, the nuclei gray-black to black, and the eosinophile granules red to red- black. For staining the neutrophile granules the neutral dyes are used, which are formed by the union of the basic and acid dyes e. g., if acid-fuchsin and orange (Gr) are mixed with the basic methyl-green. Accord- ing to Ehrlich, 125 c. cm. of a saturated aqueous solu- tion of orange are mixed with 125 c. cm. of a saturated solution of acid-fuchsin in 20 per cent alcohol ; 75 c. cm. of absolute alcohol are then added, and after- ward 125 c. cm. of a saturated aqueous solution of methyl-green with shaking. The solution remains standing for some time, and both a precipitation occurs and a film forms on the surface. In order to obtain the solution quite clear, a pipette is introduced into the middle of the solution and a quantity of the clear fluid is withdrawn. The pipette as well as the vessel must be absolutely dry, or otherwfse a further cloudiness appears. By this mixt- ure the heemoglobin is stained yellow to orange, the nuclei green, neutrophile granules violet, and the eosinophile dark gray with a tinge of blue. For the demonstration of the cells in the blood the following mixture is used : * * Ehrlich, "Deutsche med. Wochenschrift," 1883, No. 46. BACTERIA AND MICROSCOPICAL TECHNIQUE. 73 Water 100 c. cm. Glycerine 100 " Absolute alcohol 100 " Hsematoxylin 1-2 grm. Eosin 1 " Glacial acetic acid 10 c. cm. Alum to saturation. The red blood-corpuscles show an intense red color, the nuclei of the lymph oid and polynuclear cells are stained intensely blue, *and the nuclei of the mono- nuclear cells bluish green. The protoplasm of the large lymphoid and polynuclear cells is reddish, that of the mononuclear cells dark green. The importance of this method of examination for micro-organisms in the blood has been lately pointed out by Koch ; * but it has not as yet received neces- sary attention. The descriptions given here are all the more needful because our good text-books on histological technique do not sufficiently treat this subject, for the reason that the original works are so difficult of access. METHODS OF STAINING THE FLAGELLA. The flagella (Fig. 4 ; 7, 9, 14, 16), which become visible in the hanging- drop at one or both extremi- ties of the bacteria by forming an eddy, can be best stained in dried cover-glass preparations, according to Koch,f by the addition of a concentrated aqueous solution of campechianum. The flagella are stained brown, but the staining in this manner is not durable. On this account the stained preparations are laid for some time in a 5 per cent solution of chromic acid or * " Mittheilungen," Bd. I, 1881, S. 7. t " Verfahren zur Untersuclumg, etc." "Beitrage zur Biologic der Pflanzen," 1877, Bd. II, 3. Heft, S. 419. 74 BACTERIOLOGICAL INVESTIGATION. in Muller's fluid. Then there is formed an insoluble brownish black union of the extract of hsematoxylin with the chromic acid. After washing, these prepa- rations can be directly preserved in glycerine, or, after drying, in Canada balsam. METHODS OF STAINING SPORES. The spores of bacteria were first observed and de- scribed, but not rightly understood, by Perty.* Then Pasteur f made a sharp distinction between the biol- ogy of an organism and its spore without quite solv- ing the question morphologically. Cohnij: was the first to describe biologically and morphologically the formation and germination of spores. Further pecul- iarities were observed by Koch, Brefeld, Buchner, and especially Prazmowski,* who clearly described the different forms of the germination of spores (Fig. 4 ; 18 and 19). By his observations this process of fructification derived a heightened significance. The observation of the spores in the unstained con- dition finally followed (Fig. 4 ; 5 b, 8, 9, 10, 12). They appear, especially in the hanging - drop, as strongly refracting round or oval bodies either within the less refractive bacteria, or free near these. Sometimes they are situated near the middle, sometimes at the end. The cells in which they appear are sometimes unaltered, sometimes peculiarly swollen. Since con- * "Zur Kenntniss kleinster Lebensformen," 1852. Taf. XV, Fig. 26, and following. f "Etudes sur la maladie des vers a soie," 1870, T, S. 228. | "Beitrage zur Biologie der Pflanzen," 1876, Bd. II, 2. Heft, S. 263. * " Ontersuchungen ftber die Entwicklnngsgeschichte und Fer- mentwirkung einiger Bakterien-Arten," 1880, und " Ueber den gene- tischen Zusammenhang der Milzbrand und Heubakterien." " Biolog. Centralblatt," 1884, No. 13. BACTERIA AND MICROSCOPICAL TECHNIQUE. 75 densed bacteria-protoplasm, according to Prazmow- ski, strongly refracts light, it is to be concluded that a body still more strongly light-refracting is to be re- garded as a spore. Here belong, together with the foregoing morpho- logical changes which regularly appear under certain biological conditions in spores, their great resistance to chemical agents, and especially to high temperature, and the fact that in the use of watery or dilute alco- holic solutions they are not stained, but appear as un- stained refracting spaces within the stained bacteria. An accidental observation showed how the spores could also be brought to view stained. Koch * saw, in staining the tubercle bacilli with aniline- water- methyl-blue, that the spores of a species of large bac- teria were stained blue at the same time, while the bacteria themselves were stained brown by the subse- quent treatment. Gaifky was not able to stain the spores of other bacteria in the same manner. On the contrary, Neisser succeeded in staining the spores red and the bacilli blue when he used warm aniline-water- fuchsin and subsequently stained with methyl-blue. Bienstock f also used this staining. Further, Buch- nerj discovered a means for the isolated staining of spores. Because the staining of the living bac- teria was not successful, but those killed by dry- ing and heating were readily colored, Buchner thought that the reason the spores were not stained was because of the greater resistance of the spore- membrane. He endeavored, therefore, to destroy the membrane of the spores of the bacillus subtilis, and * "Mittheilungen," 1884, Bd. II, Tafel V, Fig. 23. t "Zeitschrift fur klin. Med.," 1884, S. 1. I "Ueber das Yerhalten der Spaltpilzsporen zu den Anilinfar- ben/' " Aerztliches Intelligenzblatt," 1884, No. 33, S. 370. 76 BACTERIOLOGICAL INVESTIGATION. thus make them accessible to the staining fluid. In this manner he succeeded in staining spores in dried cover-glass preparations which had been heated from one half to one hour at a temperature of 210 C. in a dry-oven, or one hour in a steam-kettle at 120 C. A successful result was also obtained when the prepara- tions were dipped in concentrated English sulphuric acid for fifteen seconds and afterward carefully washed, or when they were subjected for a longer time to a concentrated solution of caustic soda. In preparations thus treated, especially in the use of methyl-blue, the spores alone are stained, while the bacteria themselves no longer take up color. Even before the publication of the brief article of Buchner, I had endeavored to bring the spores into view by both isolated and double staining. Although these investigations are not yet in all directions con- cluded, still I shall now describe the general results, because this part of the technique is as yet little known and used. If bacteria are examined in the hanging -drop shortly before the formation of spores, in many of them refracting bodies are found which have not yet a size equal to that of spores. When the prepa- rations, dried and fixed by passing them through the flame three times, are stained with an aqueous or di- lute alcoholic staining solution, the bacteria in this stage are found to be stained, not so equally as before, but some parts are more deeply colored. This con- densed protoplasm takes the dye more readily than the protoplasm not thus concentrated. (I had pre- viously noticed intimations of this in the involution forms of the bacillus cyanogenus.) Then follows the stage in which the refracting corpuscles become much more equal in size, but they still stain well ; and finally BACTERIA AND MICROSCOPICAL TECHNIQUE. 77 a stage in which similar refracting corpuscles are pres- ent in the unstained preparations, but these no longer take up the color. Now the spores have first ob- tained an insusceptibility to the dye through the formation of a membrane difficult of penetration, which prevents the absorption of the coloring-matter. In the cover-glass preparations, which have been passed through the flame three times, the bacteria and nuclei are stained well. If they are passed through the flame more times, say six, the bacteria are stained successively worse, but the nuclei still absorb the dye well, as does also the condensed protoplasm of the bacteria which has not yet formed spores. At this stage, besides the nuclei, granular elements can often be seen which may easily impress one as belonging to the badly stained bacteria. If the preparations are passed through the flame still more times, say ten, then both the nuclei and the condensed proto- plasm lose their susceptibility to the dye, and the spores gain it. In the case of some of the bacteria of putrefac- tion, it is sufficient to pass the preparation through the flame only seven times, but in others ten times are required. (In the dry-oven a similar stage is reached in from fifteen to thirty minutes at 180 to 200 C.) The spores then take up aqueous solutions of red, violet, blue, brown, and green basic aniline- dyes. This isolated staining for the proof of the resist- ance of the spores, as Buchner intended, is perhaps sometimes to be used; but, since in this way noth- ing is learned concerning the relation of the spores to the bacteria, it is better to use a double staining. The procedure is almost the same, quantitatively in- creased, as that for staining the tubercle bacilli. T8 BACTERIOLOGICAL INVESTIGATION. Either the preparations, passed through the flame three times, are stained with a strong alkaline solu- tion from twelve to twenty-four hours (less advan- tageously by warming for one hour), and afterward stained with vesuvin, or the aniline-water-dye solu- tions are used, of which that of Neisser, previously described, has proved to be the most convenient and satisfactory. Stain in hot aniline-water f uchsin, de- colorize with nitric acid, then stain with methyl-blue. Some spores are stained, moreover, by saturated aqueous or dilute alcoholic solutions, if they are at the same time heated. The difference of spores, in respect to their susceptibility to dyes, seems to be scarcely less than that of the bacteria themselves. PEEPAKATION OF SECTIONS. Pieces of fresh organ can be cut with the freez- ing microtome. The sections are placed in a one half per cent solution of sodium chloridum, and in part examined fresh, in part stained. For the latter pur- pose, the section, according to Weigert,* is spread out upon a section-lifter in the salt solution with the help of a needle, is then taken out, and the excess of the fluid removed with filter-paper. Then the section is placed in absolute alcohol, introducing it slowly, where it remains at least until the disappearance of all the air-bubbles arising from the thawing of the frozen specimen. Then the section is placed in a staining solution, for which purpose vesuvin is most recommended, because for staining in the other dyes the sections must remain longer in alcohol. Accord- ing to Friedlander,f the section can also be taken out * Virchow's " Archiv fttr pathologisclie Anatomie," Bd. LXXXIV, 1881, 8. 290. t " Mikroskopische Technik," 2. Aufl., S. 119. BACTERIA AND MICROSCOPICAL TECHNIQUE. 79 of the salt solution and placed directly in the brown staining fluid, then for a short time in alcohol, and finally in glycerine, or oil of cloves and Canada balsam. For the study of bacteria, it seems to me that the preparation of fresh sections is entirely superfluous work. In the same time in which available sections for this purpose can be made, a dozen dried cover- glass preparations of the tissue-juice can be obtained, which furnish better information as to the presence and import of bacteria. For the exact study of the presence and distribu- tion of bacteria in the tissues, it is necessary to harden the tissues thoroughly, and from the hardened speci- mens to prepare with the microtome a series of deli- cate sections. These sections are partly examined un- stained and partly after staining. Since both the staining itself, as well as the maximal decolorization, is dependent upon the condition of the preparation produced by hardening, it is of the first importance for bacteria specimens to use absolute alcohol as a hardening agent. The susceptibility to dyes, of tis- sues hardened by other agents, as chromic-acid salts, is inconstant ; but possibly these hardening agents are for other parasitic micro-organisms as valuable as alcohol. (Of. page 68.) The susceptibility of the albuminates to dyes de- pends very much upon the presence of water ; and since the albumen coagulated by alcohol retains a certain amount of water, which in the course of a few days is entirely removed by the absolute alcohol, the hardening in alcohol must continue until this condi- tion is constant. For this purpose a small piece of the organ, about the size of a hazel-nut, is allowed to remain at least three days in a large quantity of fre- quently changed absolute alcohol. 80 BACTERIOLOGICAL INVESTIGATION. In unstained sections from fresh or hardened tis- sue, the bacteria are made visible by their resistance to acids and alkalies.* The sections are fully cleared up by 50 per cent acetic acid or 1 to 3 per cent caustic soda or potash. The bacteria withstand the action of these reagents, as was shown by von Recklinghausen (page 38). Old spirit preparations are heated in these solutions to near the boiling-point. In preparations which have thus been made transparent, the bacteria can be sometimes recognized by the characteristic forms of the individual germs, as was found by Baumgarten (page 38) for the tubercle bacillus, and by Friedlander for the typhoid bacillus. In the case of bacteria not distinguishable by the characteristic form of the single individuals, especially in the cocci, their grouping as diplococci, sarcina, torula, or zo- ogloea is often characteristic. These bodies, of equal size, withstand the action of ether and chloroform, unlike fat-drops, which may be confounded with them. In the vessels the masses of cocci are characterized, according to von Recklinghausen, by the production in the course of their growth of a varicose condition of the vessels. If we find, in a section of fresh or hardened tissue, masses or chains of small bodies which are of a simi- lar size, and which withstand both the action of al- cohol and ether, and the treatment with concentrated acetic acid and the alkalies after warming, these are to be considered, according to Friedlander, f as micro- organisms. More important is the determination, by staining, of the presence of bacteria in sections hardened in al- * Friedlander's " Zusammenstellung in der mikroskopischen Tecli- nik," 2. Aufl., S. 45. t " Mikroskopische Technik," 2. Aufl., S. 46. BACTERIA AND MICROSCOPICAL TECHNIQUE. 81 cohol. The necessary duration of the hardening of tissues in alcohol, to produce constant reaction to dyes, diminishes the susceptibility of many elements to the dyes, so that the staining of the hardened sec- tions is as a rule, a quantitative increase of the meth- ods described for the dried cover-glass preparations. The sections are first overstained, and then, by a sec- ond maximal decolorization, reduced to a proper de- gree of color for the nuclei and bacteria. The sections which have been cut in alcohol and afterward placed in the same fluid are put into a saturated aqueous or diluted alcoholic solution of the dye, in which they should remain from five to thirty minutes. By warming to 40 or 50 C., the time re- quired can be shortened somewhat, and often also the intensity of the staining be increased. Gentian-vio- let, according to Weigert, is used very advantageous- ly in a 1 per cent aqueous solution. The sections are stained diffusely in this. In this method of staining the previously much- used decolorization by acetic acid is omitted, because many bacteria, such as the bacillus of typhoid fever and glanders, give up their color more or less com- pletely in a short time. The sections are placed in distilled water to remove an excess of the color. They are then carefully spread out upon a section-lifter, and with this slowly dipped in absolute alcohol for differentiation of the bacteria and nuclei and for de- hydration. They remain for a few minutes in alco- hol, which must be absolutely free from acid, then are cleared up in oil of turpentine or cedar, and can be immediately examined in this medium. For pre- serving, the oil is removed with filter-paper, and they are then mounted in Canada balsam. Since the bal- sam is soluble in the immersion-oil, it must be suf- 82 BACTERIOLOGICAL INVESTIGATION. ficiently hard to prevent solution by the shifting of the cover-glass. If it is desired to examine or send away preparations freshly mounted in balsam, it is advisable to fix the cover-glass upon the slide with shellac or gold-size, which are not soluble in the im- mersion-oil. Many different dyes are also available for use in staining sections. In the use of the method of staining just de- scribed, the typhoid bacillus stains badly ; the spiril- lum of relapsing fever only in brown, and then not well ; and the bacillus of leprosy badly in brown, but well in red and blue dyes. For these cases the greatest intensity is obtained by the use of the strong alkaline solution (page 49), which, on this account, according to Loffler, up to the present time has proved the most universally valuable solution for sections. The sections are placed in this solution for a few minutes, then for a few seconds in to 1 per cent acetic acid, and moved to and fro in order to remove the excess of color from the tissue and to differenti- ate the bacteria and nuclei ; then are dehydrated in alcohol, cleared up in oil of cedar, and preserved in Canada balsam. By this treatment many forms of bacteria, otherwise difficult to stain, are well stained ; the tubercle bacillus as well as by other methods, also the bacillus of glanders (which in the aniline-water solutions is decolorized by the acetic acid) ; the ty- phoid bacillus and the spirillum of relapsing fever, which had been previously stained only very defec- tively in any other manner. In the case of most other bacteria, no great difference has been noticed as to the value of this method. The comparative staining by the use of aqueous and alkaline solutions sometimes allows the recognition of pure microscop- BACTERIA AND MICROSCOPICAL TECHNIQUE. 83 ical differences which are available for the differen- tial diagnosis in morphologically similar forms. For the isolated staining of bacteria in sections, according to Koch, the sections are brought from the staining solution into a solution of potassium carbon- ate, which is prepared by the mixture of equal parts of distilled water and a saturated solution of the salt. The sections remain in this solution for about five minutes, and then are transferred with a section- lifter to alcohol, cleared up in oil of cedar, and pre- served in balsam. In this method different dyes can be used. If the sections are stained in the aniline- oil-gentian-violet solution, the isolation, according to the Gram method (page 60), is still more beautiful for most bacteria. The sections are taken from the al- cohol in which they are placed after cutting, and put from one to three minutes (tubercle bacilli in sections twelve to twenty-four hours) in an aniline- water solu- tion of gentian- violet. Then, either without washing or after gently washing in alcohol, they are trans- ferred to the iodine and potassium iodide solution, in which they remain one to three minutes. In the iodine solution a precipitation occurs, and the sec- tions are stained a blackish purple ; they are then placed in alcohol until completely decolorized, cleared up in oil, and preserved in balsam as usual. The bacteria appear dark blue, the nuclei and tissue pale yellow. The capsule-cocci of pneumonia (at least as a rule), and the typhoid bacilli are decolorized like the nuclei. Double staining can be obtained in sections which have been treated, according to the Gram method, by placing them in an aqueous solution of vesuvin after the decolorization in alcohol. They are then dehy- drated in alcohol, cleared up in oil, and mounted in 84 BACTERIOLOGICAL INVESTIGATION. balsam. The nuclei are stained brown, while the bacteria remain blue. As already stated, carmine and hsematoxylin do not stain all bacteria, and even those stained are not as well stained as by the basic aniline-dyes ; but they are nuclei-staining agents of the first order. Sec- tions in which the bacteria are stained blue, accord- ing to the Koch method, can afterward be placed in a solution of carmine or hsematoxylin for about ten minutes, in order to stain the nuclei. They are then likewise treated with alcohol, oil, and balsam. If the sections are diffusely stained in concentrated aqueous or diluted alcoholic solutions of dyes, they are first put into alcohol for the differentiation of the bacteria and nuclei ; then, for the removal of the alcohol, for a moment in water, and afterward into a solution of carmine or hsematoxylin, according as the first staining was with a blue or red aniline-dye. For these cases, according to Weigert, a 1 per cent solution of gentian-violet and a subsequent staining with picro- carmine is especially recom- mended. The time required for the ordinary nuclei staining (about ten minutes) must here be somewhat extended, because the carmine must displace the gen- tian-violet from the nuclei. The time required is from a half -hour to an hour. By a still longer action the carmine is also partially substituted for the aniline- dye in cocci. These different elective affinities of the bacteria and nuclei for dyes can be used, perhaps, yet more advantageously if the sections are first placed for ten or fifteen minutes in a solution of carmine or hsema- toxylin. In this time a good and durable staining of the nuclei is obtained, while the bacteria are either unstained or badly stained. After washing in water, BACTERIA AND MICROSCOPICAL TECHNIQUE. 85 the sections are then transferred to a solution of one of the basic aniline-dyes in which the bacteria are stained, while the first dye-stuff is not displaced from the nuclei. They are then treated as usual with alco- hol, oil, and balsam. If picro-carmine is used in place of the ordinary carmine, a threefold staining is ob- tained (page 51). The so-called plasma-cells in sections can be easily confounded with cocci. They are spherical or spin- dle-shaped cells with a coarsely granular protoplasm, the granules of which react to the basic aniline-dyes in the same manner as most cocci. The nuclei of these cells are not stained, so that the appearance of colonies of cocci is closely simulated, with which they have often been confounded. These granules do not show the same resistance to acids and alkalies, and are not of quite such equal size as the cocci. Further, they are especially to be recognized by their position, as they are commonly found on the walls of the vessels. A careful study of these cells is in the highest degree essential, and the ear of the white mouse is especially recommended for this purpose. An exact differentiation is often only to be obtained by the isolated staining of the bacteria, in which the plasma-cell granules remain unstained. For the simultaneous staining of most cocci and the plasma-cells, the following solution of Ehrlich and Westphal is recommended, which also permits of the differentiation of other bacteria from the nuclei : Part sch- Grenadier's carmine (carmine pur., prts. 2; aq., 200 ; alum, 5 ; boil for fifteen minutes ; fil- ter ; add carbolic acid, 1 part) 100 c. cm. Glycerine 100 c. cm. Cone, alcoholic sol. dahlia 100 c. cm. Glacial acetic acid. . 20 c. cm. 86 BACTERIOLOGICAL INVESTIGATION. In this solution the sections remain twenty-four hours, then are transferred for a few minutes to alco- hol, afterward treated with oil and balsam. In this solution the nuclei take a red, the plasma-cell gran- ules and bacteria a blue or violet color. For the demonstration of the capsule cocci of pneumonia, Friedlander now uses : Cone, alcoholic solution gentian-violet 50 c. cm. Aq. destil 100 c. cm. Ac. acetic 10 c. cm. The sections remain in this solution twenty-four hours, and then are placed for differential decoloriza- tion in 1 per cent acetic acid for a few minutes ; after- ward, as usual, in alcohol, oil, and balsam. A contingent differential diagnosis is possible mi- croscopically from the fact that by the use of the Gram method these capsule cocci are decolorized, while the remaining cocci all (?) retain the color. Babes* recommends safranin for the staining of bacteria in sections. The sections are allowed to re- main a half -hour in a mixture composed of equal parts of a concentrated aqueous and a concentrated alcoholic solution of the dye. Then they are put for a short time in water, and a few minutes in alcohol ; after- ward in oil of turpentine and balsam. The bacteria are stained red, and sometimes have almost an iso- lated staining. This method offers no advantage, since the cocci are well stained, but other bacteria partly not at all and partly much worse than by other methods. The typhoid bacilli are ordinarily more difficult to stain than most other bacteria, even if the solution is warmed. According to Gaffky,f it is best to allow * " Archiv f. mikroskopische Anatomie," Bd. XXII, S. 359. t " Mittheilungen," 1884, Bd. II, S. 378. BACTERIA AND MICROSCOPICAL TECHNIQUE. 87 the sections to remain from twenty to twenty-four hours in a deep-blue opaque solution, which is pre- pared fresh each time by the addition of a saturated alcoholic solution of methyl-blue to distilled water. They are then washed in distilled water entirely free from acid, dehydrated in absolute alcohol, cleared up in oil of turpentine, and preserved in balsam. They are also weU stained in the alkaline solution of methyl-blue, while by the use of the Gram method they are decolorized. The bacilli of glanders are decolorized, after stain- ing in an aniline aqueous solution, by water contain- ing acetic acid. However, they are well stained in an alkaline solution of methyl-blue. The bacilli of leprosy in sections are stained the same as tubercle bacilli. For differential diagnosis Baumgarten recommends the following (page 65) : The sections are placed for twelve, or at most fifteen, minutes in a dilute alcoholic solution of fuchsin, then thirty seconds in acid alcohol (nitric acid one part, alcohol ten), to decolorize them ; washed in dis- tilled water, dehydrated in alcohol, then treated with oil and balsam. In this time the bacilli of leprosy are well stained, while the tubercle bacilli are not stained at all. The tubercle bacilli can be brought out if the sec- tions are placed for twelve hours in a weak alkaline, or for one hour in a strong alkaline solution of methyl- blue (page 49), then for a few minutes in a concen- trated aqueous solution of vesuvin, and finally in al- cohol. They can also be made visible by the Gram method. For the differential diagnosis the sections are stained with the greatest certainty, according to the previously described principle, in the aniline- water-dye solutions of Ehrlich or Weigert - Koch. 88 BACTERIOLOGICAL INVESTIGATION. The sections remain twelve to twenty-four hours in an aniline-water, methyl- violet, or fuchsin solution, then a few minutes in dilute nitric acid (1 to 3 or 4) ; are washed in 60 per cent alcohol for a few minutes, stained in a dilute aqueous solution of vesuvin or methyl-blue, washed in 60 per cent alcohol, dehy- drated in absolute alcohol, cleared up in oil of cedar, and finally imbedded in Canada balsam. The vibriones of cholera Asiatica can be stained in sections like most bacilli. The spirilla of relapsing fever are stained in aque- ous and glycerine solutions of vesuvin, but not well in other dyes. On the other hand, they are stained well in strong alkaline solutions of methyl-blue. A few remarks are still necessary concerning the epiphytic bacteria and the other epiphytic micro- organisms similar to them. (Whether the parasitic bacteria are to be classed with the epiphytic has not yet been certainly determined.) The chief difficulty in this investigation is due to the presence of fat. Balzer* washed the scales of the epidermis with ether and alcohol, and examined them after staining with an alcoholic solution of eosin, or without pre- vious staining, in a 40 per cent solution of caustic soda. According to von Sehlen,f the hairs, after the removal of the fat, are placed for a short time in alcohol, and then in a dilute aniline- oil solution of fuchsin. They are afterward washed in acid alcohol (concentrated hydrochloric acid 1 part, 75 per cent alcohol 99 parts) ; the excess of the acid is removed with distilled water, and a double staining is pro- * " Contribution a l'6tnde de T&ryt&me tricophytique." "Arch, de physiol.," 3. s6r., 1883, Bd. I, S. 171. f " Mikrokokken bei Area Cilei." " Fortschritte der Medizin," 1883, No. 23. BACTERIA AND MICROSCOPICAL TECHNIQUE. 89 duced by the use of a concentrated aqueous solution of gentian- violet, and then alcohol is used for differ- entiation as before. According to Bizzozero,* the epidermis is touched with a cover-glass, which is afterward passed through a flame three times. The fat is removed by chloro- form, and the preparation is stained by fuchsin or gentian-violet. For the removal of fat from the epi- dermic scales, they are placed for a few seconds in alcohol, then for a day or two in ether, and afterward again in alcohol. After the fat has been removed there are three methods of procedure : 1. A drop, con- sisting of equal parts of water and acetic acid, or of a 10 per cent solution of caustic potash, is placed upon a slide. The epidermic scales from which the fat has been removed are placed in this drop and allowed to swell, then a cover-glass is laid upon them. For the preservation of these preparations, glycerine is intro- duced from the edge. 2. The epidermic scale is spread out with a needle in glycerine, slightly tinged with methyl-blue. In this the fungi are stained blue, while the epidermic cells remain unstained. 3. A drop of 50 per cent acetic acid is put upon a cover-glass, and the epidermic scales, with the fat removed, are placed in it. After fifteen minutes or more, if the scales are well swollen, they are spread out with a needle ; then the acetic acid is evapo- rated by gentle heat. (For this purpose, the cover- glass may be held high over a flame and moved to and fro.) After drying, the preparation is passed through the flame three times, and then stained for ten to thirty minutes by adding a drop of an aque- * " Ueber die Mikrophyten der normalen Oberhaut des Menschen." Virchow's " Archiv," 1884, Bd. XCV1II, S. 441. 90 BACTERIOLOGICAL INVESTIGATION. ous solution of methyl- violet, gentian- violet, vesuvin, methyl-blue, or of a dilute alcoholic solution of fuch- sin. It is then carefully washed, dried, and preserved in balsam. Methyl-blue is preferred by Bizzozero, because only the fungous elements are stained with it. I have used most advantageously the strong alka- line solution of methyl-blue, allowing it to act for about five minutes. With this slight modification, it seems to me that this third method of Bizzozero is at present most serviceable for the determination of bacteria. Presently I will show that in true mycosis the mycelial filaments of the mold fungus are best brought out by the method of Loffler (page 82). The presence of radiating fungi in tissue - sections can generally be determined without special prepara- tion. But in the frequent calcification of the glands in actinomycosis it is often necessary to first decal- cify, by hardening them in alcohol containing hydro- chloric acid, and then to place them in absolute alcohol. The staining of Weigert * is also useful. According to this the sections are placed for one hour in a solu- tion of orchilla (rock moss). Pure orchilla, which has previously lain for a long time in the air to allow the escape of the ammonia, is dissolved, according to Wedl, in such quantities, in a mixture of 20 c. cm. absolute alcohol, 5 c. cm. acetic acid, and 40 c. cm. of distilled water, that the fluid becomes dark red, and after filtering appears ruby-red. Then the section is washed in alcohol, placed in a 1 per cent solution of gentian-violet and treated as for the staining of bac- teria. In these sections the nuclei are stained blue- violet, the connective tissue a pale orange, the inte- rior of the refracting fungi a pale blue, and the outer * Vircbow's " Archiv," 1881, Bd. LXXXIY, S. 245. BACTERIA AND MICROSCOPICAL TECHNIQUE. 91 parts a ruby-red, often separated by a colorless zone from the central portion. For free amoeba and cells without membranes (page 68) Brass * recommends a solution of 1 part chromic acid, 1 part platinum chloride, 1 part cone, acetic acid, and 400 to 1,000 parts of water. For hard- ening the tissues, in which the cells ought to be as little altered as possible (vide page 79), Brass recom- mends a to \ per cent solution of chromic acid, to which a few drops of a concentrated solution of the same salt is added later. After some time the prepa- ration is placed in 30 per cent alcohol, and generally, for the complete removal of the water, in alcohol gradually made stronger, and finally in absolute al- cohol. The mixture of chromic acid, platinum chlo- ride, and acetic acid also produces excellent harden- ing, especially if four to six drops of 1 per cent osmic acid is added to 100 grammes of the solution. The staining is effected by borax, or ammonio-carmine, or a solution of hsematoxylin. * " Die Methoden bei der Untersuchung thierischer Zellen." " Zeit- schrift f. wissenschaftl. Mikroskopie," 1884, S. 39. III. CULTURE-METHODS; PURE CULTURES. EHRENBERG first, and later Cohn and Schroder, expressed the belief that there were true species among bacteria. On the grounds of the investigations of Cagniard-Latour and Schwann, Turpin reached the conclusion concerning fermentation, and Henle concerning the infectious diseases, that specific de- compositions and diseases are produced by specific micro-organisms ; and Pasteur, by inoculation experi- ments, proved experimentally that specific and differ- ent micro-organisms underlie specific decompositions. These views, as well as those of their opponents, who argue for inconstancy of form and action among micro-organisms, can only be conclusively proved when they are observed pure and free from all ad- mixtures. For this reason it has been the endeavor of many investigators, for a long time, to establish methods of obtaining pure culture of bacteria, which would be free from all objections. 1. TRANSPARENT FLUID CULTURE-MEDIA. This method is the oldest, and underlies all the experiments of the earlier time on fermentation. It was used for the cultivation of bacteria by Pasteur * * "Memoire sur la fermentation appellee lactique." "Compt. rend.," 1857, Bd. XLV, S. 913. CULTURE-METHODS; PURE CULTURES. 93 in his first work on the vital fermentation theory. The principle of this method, consists in transferring a particle of the original fluid containing the micro- organisms of fermentation, or a particle of yeast (which contains them in itself), to a fluid compounded to resemble as nearly as possible the original one ; from this again a particle is transferred to a third solution, etc. The school of Pasteur has adhered almost exclusively to the use of the transparent fluid media, even up to the present time, often in its origi- nal form ; also often with its modification, the meth- od of dilution, later to be described. The important morphological investigations of Cohn were also joined closely to this method. In fluids which undergo decomposition through the action of bacteria, sometimes a diffuse cloudiness appears ; sometimes precipitation occurs ; more often a mouldy scum is formed. In transfer experiments a particle is transferred to a second solution from each of the portions appearing differently. This transfer- rence to a new solution, briefly spoken of as inocu- lation, is performed as follows : a platinum-needle, previously heated and cooled, straight or bent into a loop, is brought in contact with the mycoderma (the mouldy scum), or is dipped into the cloudy fluid, and then the particle, thus taken up, is introduced into the new solution. In place of the platinum -needle, a drawn-out capillary- tube can be used, with which a drop of the fluid is transferred. Such, a particle or drop consists chiefly of a great mass of bacteria. Following this inoculation, that species develops first which finds in the new solution the best condi- tions for its existence. When the culture-material is exhausted for one species, one or another of the forms, that at first were suppressed, may develop, 94: BACTERIOLOGICAL INVESTIGATION. provided the particle or drop transferred contained several species. It is observed, in the course of these experiments, that small differences in conditions, such as placing the inoculated solution at a lower or higher temperature, favor the development of certain' germs. Finally, following out the conditions favorable to the development of a single germ, a more or less pure culture of this organism is obtained in some one of the transfers. But this is not always, in fact it is seldom, the organism which it is desired to isolate. Ordinarily, the result is a pure culture of one of the common septic species which, under the chosen con- ditions, supplanted the other more susceptible forms. On account of this it happened that, in the use of this method of culture, the bacterium termo and bacillus subtilis often appeared. These play a much greater part in the older bacteria literature than now. In this way the dependence of the growth of bac- teria on the culture-media was learned. This led to the choice of such fluids as would offer approximately equally favorable conditions for development to the largest possible number of micro-organisms. Pasteur's* fluid is the oldest of these artificial media for the cultivation of bacteria. This consists of one part tartrate of ammonia, ten parts sugar, and the ashes of one part of yeast, to one hundred parts of water. A. Mayer f used in place of the yeast-ash a solu- tion of the salt contained in it. Then Cohn,^: after he had used the culture-fluid of Mayer with mineral * " Annales de Chimie et de Physique," Bd. LVIII, S. 323. Deutsch von Griessmayer, "Die Alkohol-Gahrimg," 1878. t "Unters. fiber die Alkohol-Gabrung," 1869. "Lebrbuch der Gahrungs-Chemie," 3. Aufl., 1879. \ "Beitrage zur Biologie der Pflanzen," I, 2. Heft, S. 195. CULTURE-METHODS; PURE CULTURES. 95 nutrient salt, omitting the sugar, devised the follow- ing normal culture-fluid : Calcii phosphati -5 gramme. Magnesii sulph. (crys.). . . *5 " Calcii phosp. (tribas) *05 u Aq. destil 100 c. cm. In this 1 gramme ammon. tartrate was dissolved. Naegeli * discovered that for the lower fungi and the fission fungi, nitrogen can be best assimilated if it is present as NH a , not as well as NH, still not as well as NO, and not at all if it is in combination with other elements, such as hydrogen and oxygen, so that a de- scending scale may be constructed from the soluble albuminates, to ammonia and nitric acid. For car- bon he arranged the following scale : 1. The forms of sugar. 2. Mannite ; glycerine ; the carbon group in leu- cin. 3. Tartaric acid ; citric acid ; succinic acid ; the carbon group in asparagin. 4. Acetic acid ; ethyl-alcohol ; quinic acid. 5. Benzoic acid ; salicylic acid ; the carbon group in propylamin. 6. The carbon group in methylamin ; phenol. Naegeli established the following descending scale for the capacity of assimilation of nitrogen and carbon from their compounds : 1. Albumen (peptone) and sugar. 2. Leucin and sugar. 3. Tartrate of ammonia, or sal. ammon., and sugar. 4. Albumen (peptone). 5. Leucin. * " Ernahrung der niederen Pilze durch Kohlenstoff- und Stick- stofiFverbindungen." " Untersudmngen liber niedere Pilze," 1882, S. 1. 96 BACTERIOLOGICAL INVESTIGATION. 6. Tartrate of ammonia ; succinic ammonia ; as- paragin. 7. Acetate of ammonia. In regard to 1, it is to be noted that the bacteria must be able to transform the albumen into peptone, and to hydrate the milk and cane-sugar, so that pep- tone and grape-sugar are used to the best advan- tage. Naegeli recommends as the best for the mineral elements Potassii phos 1 gramme. Magnesii sulph '02 Calcii chlor -01 " Fluid 100 If the reaction is to be acid, Naegeli uses acid potassi phos. ; for neutral and alkaline solution, po- tassii biphos. The poorer the nutrient elements of the N and C group are, the less concentrated the salt solution should be, while, with good nutrient elements of the N and C group, this normal degree of concen- tration may be exceeded. From this Naegeli devised the following normal fluids for fission fungi : 1. Aqua 100 c. cm. Ammon. tart 1 gramme. Potassii biphos *1 Magnes. sulph '02 " Calcii chlor 01 " 2. Aqua 100 c. cm. Albumen-peptone 1 gramme, Potassii biphos '2 " Magnes. sulph *04 " Calcii chlor '02 " 3. Aqua 100 c. cm. Cane-sugar 3 grammes. Ammon. tart 1 gramme. CULTURE-METHODS; PURE CULTURES. 97 Potassii biphos *2 gramme. Magnes. sulph *04 " Calciichlor -02 " In place of the tartrate of ammonia, in the 3d for- mula, an equal amount of another of the ammonia salts may be used, or *5 gramme of ammon. nitrat. , or *7 gramme of asparagin, or '4 gramme of urea. In place of the nutrient salts, one tenth per cent beef -extract may often be more conveniently used. For the fermentation-experiments, according to Fitz,* solutions may be employed which contain three per cent sugar, glycerine, or mannite, etc., and one tenth per cent beef-extract, to which a small quantity of calcii carb. has been added. According to previous experiments with cultures in fluids, as was also the case in the experiments of Fitz, it is well to choose the culture-fluid (normal fluid) with special reference to the individual case. In the lack of other data at the beginning, that fluid should be selected (as was the case in the first experi- ments of Pasteur) in which the bacteria are observed to grow spontaneously. For micro-organisms which are observed on solid substances, a decoction or in- fusion of this substance is prepared : fresh soil, sweet dried fruit, hay, roots, etc. "A nutrient fluid which holds in solution such substances as in a solid state furnish naturally a pabulum upon w r hich a fungus develops," according to Brefeld,t "in all probability will also form a suitable fluid for the development of this fungus."" Solutions for fungi are kept, as a rule, slightly *"TTeber Spaltpilzgahrung," VII. "Berichte der deutschen chemischen Gesellschaft," 1882, XV, S. 867. t " Kulturmethoden zur Untersuchung der Pilze." " Botanische Untersuohungen liber Schimmelpilze," Heft IV, 1881, S. 5 98 BACTERIOLOGICAL INVESTIGATION. acid ; for bacteria they are rendered neutral or slightly alkaline by the addition of ammonia or car- bonate of soda, and then are boiled and filtered. " In the use of a clear nutrient fluid thoroughly sterilized, in which the examination of the fungus by direct observation is made with the same ease as if they lived in clear water, the mycological investiga- tion is turned into an algological i. e., those condi- tions are artificially prepared in the nutrient solutions for the development of the fungus, under which we naturally find the algse, as they for the most part live in water." These words from Brefeld (I. c., page 7) also forcibly point out the chief advantages of the nutrient solu- tions for the culture of bacteria. These clear, usually neutral reacting, fluids must be carefully sterilized after filtration. For this pur- pose the sterilized cotton stopper of the sterilized flask or test-tube is removed with clean, previously heated pincettes, and laid upon its side so that it can not be soiled. Then, by the aid of a sterilized funnel or pi- pette, the fluid is transferred into the flask until it is about half filled, or into the test-tube until it is about one third filled. Then the stopper is again replaced. It is often desirable, in addition, to cover the mouth of the vessel with a double layer of thick filter-paper, which is held in place by a rubber band and which prevents the dust from falling directly upon the cot- ton. The cotton plug is proof against the admission of bacteria, but not always proof against fungi, as is to be observed when the sterilized vessels are pre- served for a long time in a moist chamber. The sterilization should be accomplished accord- ing to one of the methods described in Chapter I, preferably in most cases by the use of the steam ster- CULTURE-METHODS; PURE CULTURES. 99 ilizing cylinder. Small volumes of fluid, as those in test-tubes, are sterilized by an exposure for a half to three quarters of an hour, larger flasks in one or two hours. The time required to bring the water in the cylinder to the boiling-point should not be included. The test-tubes, after being filled, are placed in a wire basket (Fig. 3, d\ and then in the steam cylinder. The manipulations with the fluids should be car- ried on in a place as free as possible from micro-or- ganisms, in order to prevent infection by air-germs. This may be obtained in a laboratory by the prepa- ration of a glass case (similar to those arranged over delicate balances), in which the inoculations and transfers are made. The walls of this case are rubbed with moist cloths, and the air is kept moist by vessels of warm water. The inoculations are made by a platinum-needle, straight or looped, which is previously heated in a flame and again cooled, or by a capillary pipette. By means of one of these a particle or drop of the material or fluid to be used for inoculation is intro- duced as quickly as possible into the sterilized fluid, after removal of the cotton plug. This is then imme- diately replaced. If the solutions have stood for a long time without the mouths of the vessels being pro- tected by filter-paper, so that germs of bacteria and fungi may have collected upon the cotton, it is better to burn the upper layer of cotton in a flame before removal of the plug. A large number of single ex- periments should be made simultaneously. The fur- ther inoculations are made in the same manner. The inoculated tubes are, according to the case, kept at the temperature of the room or exposed to a higher temperature. For the higher temperatures a brood- or culture-oven is ilsed. This is made of metal with 100 BACTERIOLOGICAL INVESTIGATION. double walls for the reception of water (Fig. 8), and is covered with felt or asbestos to prevent the loss of heat : or the oven is made TTr Q 7 with a third wall, and the outer chamber is filled with a layer of sand. The dimen- sions vary according to need ; the form, quadrangular or cylindrical, is of little mat- ter. In the quadrangular the height and breadth of the interior is about 25 cm. , and the length varies from 50 to 75 cm. A thermometer (t) and therm o-regulator (r) serve for the regulation of the temperature. These last can be dispensed with if a small gas-pressure regulator is interposed between the principal supply- pipe and the culture-oven. It is heated by petro- leum or gas flames, which vary in number, accord- ing to the size of the oven. These are surrounded by glass chimneys to protect them from draughts of air. 2. FKACTIOI*AL CTJLTUKES. The inoculations of fluids with pathogenic bacteria were first made by Klebs,* and the particle or drop containing bacteria (" Bakterientropfen " of other au- thors) was designated by him as a "fraction." Klebs proceeded in this manner (1. c., page 46) : "He intro- duced a recently drawn-out and closed pointed cap- illary-tube to the bottom of the fluid containing bacteria, and then broke off the point. The tube, after withdrawal, was again sealed, washed with strong alcohol, introduced into a sterilized culture-fluid, and * 4< Beitrage znr Kenntniss der Mikrokokken." " Archiv fur ex- perimentelle Pathologic," Bd. I, 18T3, S. 31. CULTURE-METHODS; PURE CULTURES. 101 was then again broken. The fluid-medium was con- tained in a stoppered flask and was covered with a layer of oil." This procedure was often repeated, and "in this manner it is possible to eliminate any im- purities, which may be contained in the original fluid, and to obtain those organisms pure which are present in this in preponderating number." This method has furnished no important discov- eries, notwithstanding the skillful working and the sharp emphasis laid upon the development of many germs from the one originally present in preponderat- ing numbers. In this complication of the method of Pasteur it is evident that the objection also holds good that, as a rule, after a series of fractional cul- tivations, the desired pathogenic organism is not present pure. But instead of this almost any form may be present which at the beginning may have been perhaps entirely overlooked on account of their small number ; or else some ordinary species of septic bac- teria, introduced by infection from the air or by manipulation, has been obtained pure, because, un- der the chosen conditions, it thrived better than the more susceptible pathogenic micro-organisms, and quickly supplanted these. By the methods thus far described pure cultures may be finally obtained, without successfully ac- quiring those forms pure which it was desired to obtain. These methods are on this account only now to be used when it is desired to secure an organism in pure or quantity-culture, the source and action of which are of no importance. 3. OPAQUE SOLID CULTURE-MEDIA. Aside from fluids, bacteria are observed develop- ing spontaneously on solid substances. If, for exam- 102 BACTERIOLOGICAL INVESTIGATION. pie, a slice of cooked potato is allowed to stand in the air, different slimy masses are seen to spread themselves out upon the surface of the potato. Small slimy points of different colors form upon the sur- face, which for some time are quite distinct from each other, but become confluent in their further growth. These observations were first methodically made use of by Schroeder * for the pure cultures of pig- ment bacteria. A particle from one of these slimy points, while it is yet quite isolated, is transferred with a platinum-needle to the middle of the cut sur- face of a freshly cooked potato, placed in a moist chamber in order to prevent infection from the air. The method of cultivation on potato was material- ly improved by Koch. The potatoes were first care- fully cleaned from the coarse dirt by scrubbing, then laid for one half to one hour in a one to five per cent solution of corrosive sublimate, and finally washed in water. The potatoes, thus cleansed, with the germs adhering mostly destroyed by the sublimate, are then heated to secure certain sterilization. This is done in the steam cylinder. After reaching the boiling- point, the potatoes remain at this temperature for about an hour. While the potatoes are cooling, a moist chamber is prepared. Large bell-jars, of the form seen in Fig. 9, are cleansed by rinsing in a one FIG. 9. *"Ueber einige durch Bakterien gebildete Pigmente." Cohn's "Beitrage zur Biologie der Pflanzen," Bd. I, Heft II, 1872 (2. Ab- druck, 1881), S. 109. CULTURE-METHODS; PURE CULTURES. 103 per mille solution of corrosive sublimate. Upon the bottom of the jar a number of layers of filter-paper, moistened with sterilized water or a solution of sub- limate, are placed. Then the potatoes are held in the left hand, between the thumb and index finger, cut with a sterilized knife, and laid in a jar with the cut surface upward. Before this operation the hands should be washed in a one per mille solution of sub- limate. The ordinary kitchen-knife is used for cut- ting, and, previous to use, is sterilized by heating in the flame, and while cooling is protected from the dust. For each potato a fresh knife should be used. Then, by means of a platinum-needle which has been sterilized in the flame, a particle from one of the slimy points which is still entirely isolated, a so- called colony, is taken, and is either placed near the middle of the section of potato, or several lines of inoculation are lightly drawn entirely across the sur- face. In the first case the colonies develop in the middle of the section, and gradually extend toward the edge ; in the other, more or less isolated colonies develop along the lines of inoculation, which later unite and extend throughout the lines. For new inoculation only those colonies are used which are recognized as pure with the naked eye or lens, and from which a particle has been used to make a cover- glass preparation for microscopical control. Some of the cultures can be kept at the tempera- ture of the room, and others at a higher tempera- ture in a culture-oven. The great advantage of this method, as compared with the previously described methods with trans- parent fluid media, consists in this : namely, that each germ, whether it has been intentionally inocu- lated upon the surface of the potato, or is the result 104 BACTERIOLOGICAL INVESTIGATION. of air-infection, is isolated at its point of contact with the surface, and there develops into a colony, while in fluids the different germs are mingled together. Pure cultures may also be obtained in the fluid media, but that organism is not always present of which pure cultures are desired. In the potato-cul- tures it is possible to transfer the desired micro- organism, and in this way separate it from others, and after several transfers obtain pure cultures. How- ever, the transfers must be made early, while the small colonies developed from a single germ are yet entire- ly isolated, and are solitary and pure, as the result of the isolation. The limitations of the method depend on the fact that potatoes do not furnish favorable conditions for the existence of all organisms, and, on this account, many species that develop on potato do not grow sufficiently to be visible. This is important, because in the solid opaque media the naked eye or the magnifying-glass can alone be used. The inoculations on potatoes are more useful when it is desired to determine whether pure cultures obtained elsewhere, especially of pathogenic micro- organisms, possess the capacity of developing on vegetable material. Then the sterilized potatoes are inoculated in the same way with these pure cultures. The sections of the potatoes should be kept in the same manner in moist jars, and subjected to different degrees of temperature ; or, as a greater precaution, a potato-section is placed upon a small glass plate, which is then lowered (by the help of a strip of nickel bent at a right angle) to the bottom of a cylin- drical glass vessel about 18 cm. high and 6 cm. in diameter. The glass cylinder previously closed by a cotton plug the glass plate, and nickel strip should be sterilized in a dry-oven before being used. CULTURE-METHODS; PURE CULTURES. 105 In place of the sections of potatoes which, on ac- count of their yellow or white color, are used in prefer- ence to other tubers, as carrots, etc. ground potato may be used. For this purpose, the boiled and ground potatoes are placed in flasks (preferably in the so-called Eiienmeyer flasks), and sufficient water is added to form a thick broth. This potato-broth is sterilized in the steam apparatus, and is then inocu- lated in the usual way with a platinum-needle. The potato-broth can be made into a very good culture- media for many bacteria by the addition of starch, sugar, peptone, and beef-extract, and can then be very advantageously used to obtain fractional-cul- tures of certain forms of bacteria. 4. THE GELATIN-CULTUKE OF KLEBS AND BKEFELD. Origin from One Germ. Moist Chambers. When Klebs (I. c.) sought to fix a coccus under the microscope in order to directly observe its division, and so follow back the entire pure culture to a sin- gle germ, he was unable to do this in a fluid-drop, first, because of the movement which occurred in the drop, and second, because after the admission of air the fluid was altered by the concentration produced by evaporation. This also changed the value of the fluid as a culture-medium. In order to prevent the evaporation of the fluid, or at least to limit it and to avoid the motion, instead of the ordinary culture - solutions, Klebs used isinglass as a culture-medium, which became stiff on cool- ing. Now, in order to fix a single coccus present in the isinglass, Klebs used the chamber of von Reckling- hausen and Geissler. In this (Fig. 10) a tube leads to and a second away from a middle room made of 106 BACTERIOLOGICAL INVESTIGATION. glass of the thickness of cover-glass, the upper and lower sides of which almost touch in the middle, so FIG. 10. that here is formed a small capillary- chamber. If the chamber is then filled with water, culture-fluid, or liquefied gelatin, and these solutions are again poured out, a capillary- drop remains suspended in the narrow place. If these solutions at the same time contain germs, and in such numbers that each drop holds about one germ, then, in many cases, it will happen that the drop remaining will contain a germ, which can be tolerably well fixed with a high-power dry system and thus observed. Sometimes Klebs filled the entire chamber with gelatin or isinglass, so that the entrance of air to the central portion was prevented ; sometimes he left only one drop in the chamber, but then took care that the air was filtered through cotton before its admission. In other cases Klebs used, in place of the chamber with the capillary - room, a chamber the walls of which ran parallel and whose side-tubes were placed somewhat lower than the upper wall. These cham- bers (b in Fig. 11) (the description of which is after those used by myself) were filled (L 30< tube, 70 or 80 cm. long, which leads to a glass ball by about seven gently ris- ing, parallel spirals. The apparatus is filled with 20 or 25 c. cm. of a nutrient solution, and the air is drawn through by aspiration, which, in small bubbles from the opening for entrance, passes out by the long way through the spiral, so that it comes in intimate contact with the solution, and, on this account, can easily surrender its germs to it. Since, also, in the complete absorption of the germs by the sterilized nutrient solution, serving as a wash-fluid, an isolated development of the germs, and thus a direct enumeration, is impossible, the practicability of all aeroscopes with the use of fluid ceases where really a beginning should be made. The possibility of testing a fluid laden with germs as to its contents in germs is rendered practicable by mingling these solutions with sterilized nutrient gelatin, so that then the individual germs isolated can develop into colonies in the gelatin. * " Archiv ftir Hygiene," Bd. I, 1883, S. 169. SPECIAL HYGIENIC INVESTIGATION. 207 FIG. 31. After Koch * had demonstrated in principle the value of nutrient gelatin for the examination of air also, Hesse f constructed the following apparatus, which consists of a long glass tube and an aspirator. The glass tube (B, Fig. 31) has a length of about 70 cm. and a diameter of 3J cm. The end (a) is closed by a closely fitting rubber cap, provided with a cen- tral round perforation, and over this a second similar cap is placed, w.hich has no perforation, and, on this account, com- pletely closes the end of the tube. The end (b) is provided with a tightly fitting rubber stopper, in the center of which is a hole about 1 cm. wide. Through this a glass tube, about 10 cm. long, is in- troduced, the free end of which is connected with the aspirator (A and A'}. In this glass tube two cotton stoppers are placed, one of which lies near the center, while the other projects inward somewhat into the lumen of the tube. After removal of the rubber stopper the tube is filled with about 50 cm. of a sterilized nutrient gela- tin (containing 5 to 10 per cent of gelatin), the end is closed again, and the tube with the gelatin placed * " Mittheilungen arts dem kaiserlichen Gesundheitsamte," Bd. I, 1881, S. 32. f " Ueber quantitative Bestimmung der in der Luft enthaltenen Mikroorganismen." " Mittheiltmgen," Bd. II, 1884, S. 182. 14 208 BACTERIOLOGICAL INVESTIGATION. for one to two hours in hot steam. After the tube has cooled enough, so that it can be handled readily, the entire inner surface of the tube to B is coated with a thin layer of gelatin, while the bottom, upon which the germs will almost exclusively settle, is clothed with a greater mass of gelatin. For this purpose the tube is cooled under the water-faucet by successively bringing it in its entire length hori- zontally under the stream, and then rotating it rap- idly upon its axis. When the gelatin becomes quite consistent the rotation is immediately ceased, and it is then moved only horizontally. Thus on one side, in its entire length, a somewhat thicker layer of gelatin is formed, which, in putting the tube on its support, is placed downward as the floor layer. At the place for the observation the outer cap is removed from the end (a), the aspirator is set in motion, and the air is slowly drawn through. In the experiments in the open air two separated litre-flasks can be used as an aspirator. In order, on the one hand, to meet the objection that not all the germs cling to the surface of the gela- tin, but especially for the improvisation of an appa- ratus which unites the advantages of the gelatinizing substances with a wash-fluid, recourse may be had to the following procedure : Sterilized test-tubes are pro- vided with rubber stoppers that have been sterilized in steam, and in which are two perforations. In both of these, sterilized glass tubes, bent at right angles, are introduced ; one of the tubes ends directly under- neath the stopper, while the other passes almost to the bottom. The test-tubes, thus armed, are filled with nutrient gelatin, or, according to von Sehlen,* with agar-agar which has been carefully sterilized. The * " Studien tiber Malaria." " Fortschritte der Medizin," 1884, ISTo. 18. SPECIAL HYGIENIC INVESTIGATION. 209 free ends of the glass tubes are closed by sterilized cotton stoppers, which are removed at the spot from which the air is to be taken. The gelatin is liquefied by placing it in water at 30 C., the agar-agar at 40 C., and during the aspiration it is held in solution at this temperature, so that, as a* nutrient solution, it re- ceives the germs. Care must be taken to join two or three such test- tubes with one another, since if a large amount of air is drawn in, the first tube will not contain all the germs of the air. After completion of the experi- ment the cotton stoppers are replaced and the solu- tions solidified by removing them from the warm bath. In order to prevent, in these cases, any fur- ther infection, the germs are allowed to develop iso- lated in the test-tubes. VII. BACTERIOLOGY AS AN OBJECT OF INSTRUCTION. IF the methods thus far developed for the inves- tigation of bacteria should obtain the significance which the mass of physicians ascribe to these things, then our high-schools must furnish the opportunity for instruction in this important subject. Up to this time this has been done in a quite in- sufficient manner, from the lack of suitable instructors and institutes. Great praise is due the University of Copenhagen for going in advance, breaking the way, and for having established in 1883, in connection with the Botanical Institute, an institute for medical bac- teriology, the direction of which was discreetly placed in the experienced hands of Salomonsen. Of the German universities, as yet only Munich in connec- tion with the pathological institute, Gottingen in the hygienic, and Breslau in the botanical institute, fur- nish the opportunity for learning the methods. In addition to these there is still the hygienic bacterio- logical department of the chemical institute of Frese- nius at Wiesbaden, directed by myself, which takes teaching into special consideration. The laboratory of the office of the Imperial Board of Health is only accessible in a civil and diplomatic way, so that for the general purpose of teaching it does not come at all into consideration. BACTERIOLOGY AS AN OBJECT OF INSTRUCTION. 211 The zeal with which the work on this subject is now done at all high-schools would be exceedingly fruitful, and would render many a fiasco impossible, if an opportunity for work in the methods were af- forded in a manner sufficient for the existing needs. The demand, as such institutes are constituted, is at the moment scarcely to be answered in a sufficient manner, from the want of suitable instruction and from pecuniary considerations. Botany is interested in the solution of many mor- phological and biological questions in bacteriology. (But a distinguished botanist has sorrowfully com- plained of the disgrace that most botanists do not concern themselves with bacteriology.) These ques- tions can be referred to the botanical institutes, of which there is no lack. It is impossible for physiology to be satisfied with the present condition of the subject of digestion, but it must now give quite a different consideration than formerly to the biological processes in the intestines. The solution of these problems, which are associated in part with the physiology of fermentation, can be referred exclusively to the richly endowed physiologi- cal and physiologico-chemical institutes. Also pathology, sufficiently provided with insti- tutes among the many attractions with which it has to do, can not fail to give greater consideration to bacteria as a further field for investigation. Whoever has really interested himself in bacteria investigation is rejoiced when the subject is under- taken from the most different standpoints. But such divergent standpoints do not suffice for the investi- gation, if they do not succeed in giving to bacteri- ology its due place as an object of study. For a definite basis for bacteriology it seems to me 212 BACTERIOLOGICAL INVESTIGATION. that only two ways offer a prospect for lasting re- sults : first, special bacteriological institutes can be founded, as at Copenhagen ; or, secondly, these insti- tutes ought to be united with the hygienic institutes to be erected. The latter seems to me to be the most promising plan. Botany, physiology, and pathology have always an actual interest only on a certain side of the inves- tigation, while to the other questions they are placed in nearly equal relation. The botanical questions possess an important in- terest for hygiene, because the formation in the sys- tem, the constancy and the variability of forms, etc., can have a direct influence upon the hygienic treat- ment. Hygiene shares this interest in botanical questions in part with physiology and pathology. Hygiene must know the general biology of bacte- ria, because the general processes of decomposition brought about by septic micro-organisms are of sig- nificance in the consideration of the aetiology of dis- ease. Botany and physiology in part share in this interest. Hygiene must be very familiar with the patho- genic bacteria, since these have not only the value perhaps of an external attraction for the aetiology, but because, as the conditio sine qua non, they form the hinge of the entire aetiology, which pathology can consider only in an insufficient manner, because the processes in the body lay claim sufficiently to their activity. The problems of combining the experimental inves- tigations concerning the parasites of disease with the results of the investigations as to the accessory causes of epidemic diseases (basing the hypothesis of the soil and drinking-water theories upon the biology of BACTERIOLOGY AS AN OBJECT OF INSTRUCTION. 213 micro-organisms and facts, and no longer exclusively upon reflections and probabilities) belong only to hygiene, and are to be solved in a satisfactory man- ner only by looking well to all sides of the investiga- tion, few parts of which are, after all, only sufficiently recognized by botany, physiology, and pathology. It seems to me to follow, from these considerations, that that branch of science which from its very na- ture must be accountable to the whole field is best adapted in the fullest sense to do justice to the sub- ject in hand as an object of teaching. In the same manner as the teacher of hygiene, in another direction, is required to familiarize himself with bacteriology to enable him to impart the same to his pupils, so must we also require of the bacteri- ologist intrusted with hygiene that he should master the other chapters of hygiene to become a teacher. Hygiene can not and must not be lost sight of in the importance of bacteriology, and it would be a sad error to consider only the bacteriological side in fill- ing the chair of hygiene. Bacteriology as an object of study, as seen in some institutions, would be more practical if combined with hygiene. It is self-evident that, in the methods of teaching, two things must be kept well separated i. e., the in- troduction into the methods by practical courses, and independent working after preparatory instruction ; the latter will only be employed by a few especially interested, but it must be sufficiently observed in the equipment of the institution. The actual every-day necessities lie at present more in the preparatory courses. Every practicing physician, and especially every medical officer, is desirous of so far informing himself concerning these methods as to be able to fol- low intelligently the recent investigations, since the 214: BACTERIOLOGICAL INVESTIGATION. morphological and biological foundations of the gen- eral processes of decomposition and the aetiology of infectious diseases have assumed tangible form through the development of the methods. Courses may be arranged in the form of practical exercises lasting from four to six weeks, in which the most important methods are practiced with par- ticular regard to those most important, considered from a hygienic standpoint. This plan alone might at the time be adequate to meet the necessities of medical officers and any surgeons who can absent themselves from duty for only a few weeks, but is far less agreeable to the teacher than if he could dis- tribute the practical introduction into the methods over a semester. The latter plan, which must be- come the rule for the young medical man and the naturalist, permits the teacher to take cognizance of all methods, and thus to render the perception more acute for the development and for the future prob- lems of investigations. As a result, this mode of introduction into the methods becomes an important aid, which well pre- pares for independent work, and may bring about the entire disappearance of over-hasty publications in bacteriology. INDEX. ABBE, condenser of, 41. immersion-fluid of, 35. Acetate of potash for preservation, 53. Acetic acid for maximal decoloriza- tion, 52. Acid aniline-dyes, 46. Acromatic condenser, 35. Actinimycosis, 90. Aerobic bacteria, 10. Agar-agar for cultures, 144. Air, bacteria in the, 205. Anaerobiosis in fluids, 164. Aniline-dyes for staining bacteria, 43. Aniline-water solution, 49. Anise-oil for immersion system, 36. Anthrax bacteria, attenuation of, 190. effect of drying, 197. accidentally parasitic, 158. ARSONVAL, D', thermostat of, 1 88. Arthro-bacteria, 29. Arthro-cocci, 29. Arthrospore bacteria, 10. Bacilli, 29. Bacteria, forms of, 28. Basic aniline-dyes, 46. BAUMGARTEN, leprosy bacillus, 65. Biological problems, general, 183. Blood, examination for bacteria, 67. inoculation from, intra vitam, 155. Blood-serum for cultures, 148. Brood-oven for high temperatures, 99. BREFELD, gelatin to prevent evapora- tion of culture-fluids, 129. studies from a single germ, 107. transparent fluid media of, 128. Canada balsam for mounting, 63. Capillary -tubes for cultures, 121. Capsule cocci of pneumonia, staining of, 86. Caraghen for cultures, 144. Carbonate of soda to neutralize nu- trient gelatin, 132. Carmine for staining micrococci, 42. CARTER, infection of monkeys with relapsing fever spirillum, 126. Causal relation of bacteria to disease, 160. Cedar oil for immersion system, 36. Cement, vermilion paint, 24. Chamber, moist, for pure cultures, 135. Chicken cholera bacillus, attenuation of, 190. Cholera Asiatica bacilli, effect of dry- ing, 197. staining in sections, 88. Chromic-acid solution, 52. Cladothrix, 30. Clostridium, 29. Cloves, oil of, 53. Cocci, 29. COHN, artificial culture-fluid of, 95. transparent fluid media of, 128. Colony from a single germ, 131. Colonies, isolated, 130. Comma-bacilli, 32. Condenser of ABBE, 43. Copenhagen, University of, 210. Counting-slide of THOMA, 116. Cover-glass preparations, 65. Culture-methods, 92. 216 INDEX. Culture-oven for high temperatures, 99. Cutaneous inoculation, 177. Decalcifying solutions, 52. Desmo-bacteria, 29. Dilution, method of, 113. for plate cultures, 141. Discontinuous sterilization, 20. Disinfection with gases, 197. with fluids, 195. Distilled water, use of, 53. Drying, influence of, on bacteria, 197. Earth, bacteria in, 200. EHRLICH-WEIGERT-KOCH method of staining tubercle bacilli, 65. Electricity, effect on bacteria, 198. Endospore bacteria, 29. Enzyme, 185. Eosin for staining, 46. Epiphytic bacteria, 88. Erysipelatous skin, inoculation from, 156. Eye, inoculation of anterior chamber of, 179. Fat crystals are pseudo-bacteria, 64. Feeding experiments, 75. Ferro-cyanide, double, 169. Filter warmed with hot water, 133. FITZ, development from one germ, 128. Flagella, staining of, 73. Fluid culture-media, 92. Fluorescin for staining bacteria, 46. Fractional cultures, 100. FRIEDLANDER'S pneumococci, staining of, 86. Fuchsin for staining bacteria, 46. GAY-LUSSAC, experiments on spon- taneous generation, 15. GEISSLER'S culture-chamber, 105. Gelatin for solid transparent culture- media, preparation of, 130. General biological problems, 183. Gentian- violet for staining bacteria, 46. Glanders, bacilli of, staining, 86. Gonidia, 29. GRAM'S method of decolorization, 61. Granular detritus vs. bacteria, 88. Htematoxylin for staining, 42. Hanging-drop, 39. Heat, method of isolation by, 119. HESSE, apparatus of, for examination of air, 207. Homogeneous immersion-lens, 37. Hydrobiosis, 163. Hygienic investigation, special, 200. KLEBS, use of gelatin to prevent evap- oration of culture-fluids, 129. method of inoculation, 100. KOCH, attenuation of anthrax bacilli, 191. blood-serum for cultures, 148. the color- vs. the structure-picture, 41. pure culture of tubercle bacilli, 128. examination for tubercle bacilli, 61. homogeneous immersion system, 36. infection of monkeys with spirilla of relapsing fever, 126. modification of PRAVAZ hypodermic syringe, 179. steam sterilization-cylinder of, 18. Lactic-acid bacteria, 190. Leprosy bacilli like tubercle bacilli, 65. staining of, in sections, 87. Leptothrix, 29. Light, effect on bacteria, 199. Liquefaction of gelatin by bacteria, 137. LISTER, development from one germ, 128. LOFFLER, meat-water peptone gelatin, 133. Malignant oedema, bacilli of, 201. Meat-water peptone gelatin, 132. Methyl-blue for staining bacteria, 46. Methyl-violet for staining bacteria, 46. Mica, use for cultures of anaerobic bacteria, 163. Mice, field- and house-, septicaemia of, 126. Microscopical technique, 28. MIQUEL, examination of the air for bacteria. 205. Moist chamber for pure cultures, 135. Mould-fungi, cultivation of, 158. Movements of bacteria, 38. INDEX. 21T MTTLLER'S fluid, 52. Mycosis, 90. NAEGELI, artificial culture-fluid, 96. development from one germ, 128. Nail-cultures of FRIEDLANDER, 143. Nutrient gelatin, 132. Obligatory anaerobic bacteria, 10. Optional anaerobic bacteria, 10. Oil of cloves, cedar, etc., 53. Opaque solid culture-media, 101. Origin from one germ, 105. Parasitic bacteria, 9, 170. PASTEUR, attenuation of bacteria of anthrax and chicken cholera, 190. artificial culture-fluid of, 94. examination of the air for bacteria, 205. experiments on anaerobiosis in fluids, 166. spontaneous generation experi- ments of, 17. Pathology of plants, bacteria in, 181. Peptone added to nutrient gelatin, 133. Phosphorescence, effect of, on bacte- ria, 198. Picric acid for staining, 46. Picro-carmine for staining, 43. Plants, pathology of, 181. Plate-cultures, 138. Pneumococci, staining of, 86. Potassium, acetate of, for preserva- tion, 53. Potato-cultures, 102. PRAYAZ subcutaneous syringe, 179. Pressure, high, effect of, on bacteria, 197. Pseudo-bacilli. 64. Ptomaines, 187. Pyrogallic solution for experiments in anaerobiosis, 166. Quantity-culture, 101, 105, 118, 121, 157, 158, 165. Rabbit septicaemia, 126. RECKLINGHAUSEN, VON, culture-cham- ber of, 105. Relapsing fever, spirillum of, stain- ing, 88. spirillum of, in monkeys, 126. Safranin for staining, 46, 86. SALOMONSEN, at Copenhagen, 210. capillary-tubes of, for cultures, 121. isolation of pure colonies in blood 123. Saprophytic bacteria, 9, 11. SCHWANN, spontaneous generation ex- periments of, 16. Sections, preparation of, 78. Septic bacteria, 9, 160. Septicaemia of mice, 126. of rabbits, 126. Serum, blood-, for cultures, 148. Slide-cultures, 134. Solid culture-media, opaque, 101. transparent, 128. SPALLANZANI, spontaneous generation experiments of, 15. Spirilla, 29. Spirochaetae, 29. Spontaneous generation, 15, 25. Spores, relation of, to temperature, 191. method of staining, 74. Sputum examination for tubercle bacilli, 61. Staining bacteria, methods of, 40. ! Sterilization, by discontinuous heat- ing, '20. steam-cylinder for, 18. steam-kettle for, 17. principles of, 15. Sterilizing oven, 26. " Structure-picture," 34. Subcutaneous applications, 178. injections, 179. syringe of PRAVAZ, 179. Technique, microscopical, 28. Temperature, behavior of bacteria to, 188. effect of low, 197. relation of spores to, 191. Test-tube cultures, 142. Thermostat of D'ARSONVAL, 188. THOMA, counting-slide of, 116. TIEGHEM, VAN, infection of plants, 125. Torula, 29. Transparent fluid culture-media, 92. solid culture-media, 129. Triangle to level plate-cultures, 135. Trichinosis, 125. 218 INDEX. Tubercle bacilli demonstrated by caustic potash, 38. examination of sputum for, 61. in sections, 87. Tuberculous material, inoculation from, 154. TYNDALL, method of discontinuous sterilization of, 20. Typhoid bacilli, staining of, 86. Unstained bacteria, examination of, 34. Vermilion-paint cement, 24. Vesuvin for staining, 46. Vibriones, 29. Water, bacteria in, 202. WEIGERT, methods of staining of, 42. Yeast, cultivation of, 158. ZAHN, pipette of, for blood-cultures, 124. ZEISS, immersion-lenses, 36. Zooglea, 29. THE END. THE NEW YORK MEDICAL JOURNAL, A WEEKLY REVIEW OF MEDICINE, PUBLISHED BY A. 4yjj|j ijrf 'f ~. p. EDITED BY 0, Appleton & Co,, fflO^^a |M Frank P. Foster, M, D,, Has for its principal features LECTURES, ORIGINAL COMMUNICATIONS, CLINICAL REPORTS, CORRESPONDENCE, BOOK NOTICES, LEADING ARTICLES, MINOR PARAGRAPHS, NEWS ITEMS, LETTERS TO THE EDITOR, PROCEEDINGS OF SOCIETIES, REPORTS ON THE PROGRESS OF MEDICINE, and MISCELLANY. So rapidly has the JOURNAL gained in public favor that it is now acknowl- edged to be the Leading Journal of America. It numbers among its contributors many of the most eminent men in the profession, whose papers are valuable additions to medical literature. The Society Reports, of which each number of the JOURNAL contains several, are full of interesting and instructive matter. The Reports on the Progress of Medicine, written by men espe- cially qualified in their respective branches, cover the most recent advances in medical science. Current events of the day are treated under the head of News Items, from a medical stand-point. Everything new and useful in materia medica is chronicled under the bead of Miscellany in " Therapeutical Notes," which appear weekly, and are alone well worth the subscription price of the JOURNAL. The arrangement of the matter in the JOURNAL enables us to furnish a greater amount of reading-matter in its twenty-eight double-columned pages than is given by any other journal of its class in America. Illustrations are more freely given than in most journals, and are, as a rule, much better executed. To the physician who desires to keep abreast of the times in medicine and sur- gery, 66 The New York Medical Journal" is an indispensable requisite, and to the ADVERTISER who wishes to reach the medical profession it is a medium second to none, as its rapidly increasing circulation, distributed throughout the entire Union, will abundantly prove. TERMS, PAYABLE IN ADVANCE. One Year .......... .$5 00 Six Months - - - . . . . - .-850 (No subscriptions received for less than Six Months.) Binding Cases (for permanent binding), Cloth, per volume - 50 The Popular Science Monthly and The New York Medical Journal t~t the same address, 89. OO per Annum (full price, $10.00). New York : D. APPLETON & CO., 1, 3, & 5 Bond Street. THE POPULAR SCIENCE MONTHLY. CONDUCTED BY E, L AND W, J, YOUMANS, THE POPULAR SCIENCE MONTHLY will continue, as heretofore, to sup- ply its readers with the results of the latest investigation and the most valuable thought in the various departments of scientific inquiry. Leaving the dry and technical details of science, which are of chief concern to specialists, to the journals devoted to them, the MONTHLY deals with those more general and practical subjects which are of the greatest interest and importance to the public at large. In this work it has achieved a foremost position, and is now the acknowledged organ of progressive scientific ideas in this country. The wide range of its discussions includes, among other topics: The bearing of science upon education ; Questions relating to the prevention of disease and the improvement of sanitary conditions ; Subjects of domestic and social economy, including the introduction of better ways of living, and improved applications in the arts of every kind; The phenomena and laws of the larger social organizations, with the new standard of ethics, based on scientific principles ; The subjects of personal and household hygiene, medicine, and archi- tecture, as exemplified in the adaptation of public buildings and private houses to the wants of those who use them ; Agriculture and the improvement of food products ; The study of man, with what appears from time to time in the depart- ments of anthropology and archeology that may throw light upon the development of the race from its primitive conditions. Whatever of real advance is made in chemistry, geography, astrono- my, physiology, psychology, botany, zoology, paleontology, geology, or such other department as may have been the field of research, is recorded monthly. Special attention is also called to the biographies, with portraits, of representative scientific men, in which are recorded their most marked achievements in science, and the general bearing of their work indicated and its value estimated. Terms : $5.OO per Annum, in advance. The New York Medical Journal and The Popular Science Monthly to the same address, $9.00 per Annum (full price, $10.00). New York : D. APPLETOX & CO., 1, 3, & 5 Bond Street, PEBKUAKT, 188& D. APPLETON & CO.'S RECENT MEDICAL PUBLICATIONS, A Text-Book of Ophthalmoscopy. By EDWARD G. LORING, M. D. Part I. THE NORMAL EYE, DETERMINATION OF REFRACTION, AND DISEASES OP THE MEDIA. 8vo, 267 pages, with 131 Illustrations, and Four Chromo-Lithograph Plates, containing Fourteen Figures. Cloth, $5.00. The Diagnosis and Treatment of Diseases of the Ear. By OREN D. POMEROY, M. D., Surgeon to the Manhattan Eye and Ear Hospital, etc. With 100 Illustrations. New edition, revised and enlarged. 8vo. Cloth, $3.00. A Treatise on Nervous Diseases : Their Symptoms and Treatment. A Text-Book for Students and Practitioners. By SAMUEL G. WEBBER, M. D, 8vo. Cloth, $3,00. The Use of the Microscope in Clinical and Pathological Exami- nations. By Dr. CARL FRIEDLAENDER. Translated from the enlarged and improved second edition, by HENRY C. COE, M. D., etc. With a Chromo- Lithograph. 12mo. Cloth, $1.50. Elements of Modern Medicine, including Principles of Pathology and Therapeutics, with many Useful Memoranda and Valuable Tables for Ref- erence. Designed for the Use of Students and Practitioners of Medicine. By R. FRENCH STONE, M. D. 12mo. Leather, with pocket and tuck, $2.50. A Practical Treatise on the Diseases of Children. By ALFRED VOGEL, M. D. Translated and edited by H. RAPHAEL, M. D. Third Ameri- can from the eighth German edition, revised and enlarged. Illustrated by Six Lithographic Plates. One volume, 8vo. Cloth, $4.50. The Science and Art of Midwifery. By WILLIAM THOMSON LUSK, M. A., M. D. Second edition, revised and enlarged. Complete in one volume, 8vo, with 246 Illustrations. Cloth, $5.00; sheep, $6.00. A Manual of Dermatology. By A. R. ROBINSON, M. B., L. R. C. P. and S. (Edinburgh). Revised and corrected. 8vo. Cloth, $5.00. The Curability and Treatment of Pulmonary Phthisis. By S. JAC- COUD, M. D. Translated and edited by MONTAGU LUBBOCK, M. D. 8vo. Cloth, $4.00. A Hand-Book of the Diseases of the Eye, and their Treatment. By HENRY R. SWANZY, A.M., M. B. Crown 8vo, with 122 Illustrations, and Holmgren's Tests for Color-Blindness. Cloth, $3.00. Osteotomy and Osteoclasis, for the Correction of Deformities of the Lower Limbs. By CHARLES T. POORE, M. D. 8vo, with 50 Illustrations. Cloth, $2.50. Practical Manual of Diseases of Women and Uterine Therapeu- tics. For Students and Practitioners. By H. MACNAUGHTON JONES, M. D. 12mo, with 188 Illustrations. Cloth, $3.00. Elements of Practical Medicine. By ALFRED H. CARTER, M. D. Third edition, revised and enlarged. 12mo. Cloth, $3.00. An Atlas of Clinical Microscopy. By ALEXANDER PEYER, M. D. Translated and edited by ALFRED 0. GIEARD, M. D M Assistant Surgeon United States Army. First American, from the Manuscript of the second German edition, with Additions. Ninety Plates, with One Hundred and Five Illustrations, Chromo-Lithographs. Square 8vo volume. Cloth, $6.00. "All who are interested in clinical microscopy will be pleased with the design and execution of this work, and will feel under obligation to the author, trans- lator, and publishers for placing so valuable a work in their hands. The plates in which are figured the various urinary inorganic deposits are especially fine, and the various forms of tube-casts, hyaline, waxy, epithelial, and mucous, are depicted with great fidelity and accuracy." Philadelphia Medical Times. " To those students and practitioners of medicine who are interested in micro- scopical work and who are familiar with the use of this valuable aid to human vision in the study of nature, the present work will prove of incalculable value, since it represents the original work of an accomplished microscopist and artist. Accompanying the plates is a text of explanatory notes showing the various methods of working with the microscope and the significance of what is observed. The plates have been most handsomely printed. We have seen nothing in this special line of study that will compare in point of accuracy of detail and artistic effect to the work under consideration." Maryland Medical Journal. The Use of the Microscope in Clinical and Pathological Examinations. By DE. CARL FRIEDLAENDER, Privat-Docent in Pathological Anatomy in Berlin. Translated from the enlarged and improved second edition, by HENEY 0. COE, M. D., etc. With a Chromo-Lithograph. 12mo, 195 pages, with copious Index. Cloth, $1.00. " We are very much pleased to see Dr. Friedlaender's little book make its appearance in English dress. As we have a practical acquaintance of the Ger- man edition since its appearance, we can speak of it in terms of unqualified praise. . . . Every one doing pathological work should have this little book in his possession. The translator has done his work well, and has certainly conferred a great favor on all microscopists by placing within the reach of every one the work of so accomplished a teacher as Dr. Carl Friedlacnder." Canada Medical and Surgical Journal. " Much good has been done in placing this little work in the hands of the profession. The technique of preparing, cutting, and staining specimens is given at some length ; also rules for the examination of the various bodily fluids in both health and disease. The use of the microscope with high powers, immer- sion lenses, and other accessories, is explained very clearly. It is a very readable volume, even for those not engaged in actual laboratory work. A chromo-litho- graph shows the various forms of disease-germs which have been definitely iso- lated." Medical Record. New York: D. APPLETON & CO., 1, 3, & 6 Bond Street. January, 1886. MEDICAL AND HYGIENIC WORKS PUBLISHED BY D. APPLETON & CO., 1, 3, & 5 Bond Street, New York BARKER (FORDYCE). On Sea-Sickness. A Popular Treatise for Travelers and the General Reader. Small 12mo. Cloth, 75 cents. BARKER (FORDYCE). On Puerperal Disease. Clinical Lectures delivered at Bellevue Hospital. A Course of Lectures valuable alike to the Student and the Practitioner. Third edition. 8vo. Cloth, $5.00 ; sheep, $6.00. BARTHOLOW (ROBERTS). A Treatise on Materia Medica and Therapeutic?. Fifth edition. Revised, enlarged, and adapted to " The New Pharmacopoeia." 8vo. Cloth, $5.00; sheep, $6.00. BARTHOLOW (ROBERTS). A Treatise on the Practice of Medicine, for the Use of Students and Practitioners. Fifth edition, revised and enlarged. 8vo. Cloth, $5.00; sheep, $6.00. BARTHOLOW (ROBERTS). On the Antagonism between Medicines and be- tween Remedies and Diseases. Being the Cartwright Lectures for the Year 1880. 8vo. Cloth, $1.25. BASTIAN (H. CHARLTON). On Paralysis from Brain-Disease in its Common Forms. 12mo. Cloth, $1.75. BASTIAN (H. CHARLTON). The Brain as an Organ of the Mind. 12mo. Cloth, $2.50. BELLEVUE AND CHARITY HOSPITAL REPORTS. Edited by W. A. Ham- mond, M. D. 8vo. Cloth, $4.00. BENNET (J. H.). Winter and Spring on the Shores of the Mediterranean ; or, The Riviera, Mentone, Italy, Corsica, Sicily, Algeria, Spain, and Biarritz, as Winter Climates. With numerous Illustrations. New revised edition. 12mo. Cloth, $3.50. BENNET (J. H.). On the Treatment of Pulmonary Consumption, by Hygiene, Climate, and Medicine. Thin 8vo. Cloth, $1.50. BILLINGS (F. S.). The Relation of Animal Diseases to the Public Health, and their Prevention. 8vo. Cloth, $4.00. BILLROTH (THEODOR). General Surgical Pathology and Therapeutics. A Text-Book for Students and Physicians. Translated from the tenth German edition, by special permission of the author, by Charles E. Hackley, D. M. Fifth American edition, revised and enlarged. 8vo. Cloth, $5.00 ; sheep, $6.00. BRAMWELL (BYROM). Diseases of the Heart and Thoracic Aorta. Illus- trated with Lithographs, Ohromp-LithographB, and numerous Wood-Engrav- ings. 8vo. Cloth, $8.00 ; sheep, $9.00. BUCK (GURDON). Contributions to Repnrative Surgery, showing its Applica- tion to the Treatment of Deformities produced by Destructive Disease or Injury; Congenital Defects from Arrest or Excess of Development; and Cicatricial Contractions following Burns. Illustrated by Thirty Cases and fine Engravings. 8vo. Cloth, $3.00. CARPENTER (W. B.). Principles of Mental Physiology, with their Applica- tion to the Training and Discipline of the Mind, and the Study of its Morbid Conditions. 12mo. Cloth, $8.00. CARTER (ALFRED II.). Elements of Practical Medicine. Tbird edition, re- vised and enlarged. 12mo. Cloth, $3.00. CHAUVEAU (A.). The Comparative Anatomy of the Domesticated Animals. Translated and edited by George Fleming. Illustrated. 8vo. Cloth, $6.00. COMBE (ANDREW). The Management of Infancy, Physiological and Moral. Revised and edited by Sir James Clark. 12mo. Cloth, $1.50. COOLEY. Cyclopaedia of Practical Receipts, and Collateral Information in the Arts, Manufactures, Professions, and Trades, including Medicine, Pharmacy, and Domestic Economy. Designed as a Comprehensive Supplement to the Pharmacopoeia, and General Book of Reference for the Manufacturer, Trades- man, Amateur, and Heads of Families. Sixth edition, revised and partly re- written by Richard V. Tuson. With Illustrations. 2vols.,8vo. Cloth, $9.00. CORNING (J. L.). Brain Exhaustion, with some Preliminary Considerations on Cerebral Dynamics. Crown 8vo. Cloth, $2.00. CORNING (J. L.). Local Anaesthesia in General Medicine and Surgery. Being the Practical Application of the Author's Recent Discoveries in Local Anaes- thesia. Small 8vo. volume of about 50 pages, with several Illustrations. (Nearly ready.) DAVIS (HENRY G.). Conservative Surgery. With Illustrations. 8vo. Cloth, $3.00. ELLIOT (GEORGE T.). Obstetric Clinic: A Practical Contribution to the Study of Obstetrics and the Diseases of Women and Children. 8vo. Cloth, $4.50. EYETZKY (ETIENNE). The Physiological and Therapeutical Action of Ergot. Being the Joseph Mather Smith Prize Essay for 1881. 8vo. Limp cloth, $1.00. FLINT (AUSTIX). Medical Ethics and Etiquette. Commentaries on the National Code of Ethics. 1 21110. Cloth, GO cents. FLINT (AUSTIN, JR.). Text-Book of Human Physiology; designed for the Use of Practitioners and Students of Medicine. Illustrated by three Litho- graphic Plates, and three hundred and thirteen Woodcuts. Third edition, mi*'d. Imperial 8vo. Cloth, $6.00; sheep, $7.00. FLINT (AUSTIN' JR.). The Physiological Effects of Severe and Protracted Muscular Exercise; with Special Reference to its Influence upon the Excre- tion of Nitrogen. 12mo. Cloth, $1.00. 3 FLINT (AUSTIN, JR.). Physiology of Man. Designed to represent the Exist- ing State of Physiological Science as applied to the Functions of the Human Body. Complete in 5 vols. Vol. I. Introduction; The Blood ; Circulation; Respiration. 8vo. Vol. II. Alimentation ; Digestion ; Absorption ; Lymph ; and Chyle. 8vo. Vol. III. Secretion; Excretion; Ductless Glands; Nutrition; Animal Heat; Movements; Voice and Speech. 8vo. Vol. IV. The Nervous System. 8vo. Vol. V. (With a General Index to the live volumes.) Special Senses; Generation. Per vol., cloth, $4.50 ; sheep, $5.50. *** Vols. I and II can be had in cloth and sheep binding ; Vol. Ill in sheep only. Vol. IV is at present out of print. FLINT (AUSTIN, JR.). The Source of Muscular Power. Arguments and Con- clusions drawn from Observation upon the Human Subject under Conditions of Rest and of Muscular Exercise. 12mo. Cloth, $1.00. FLINT (AUSTIN, JR.). Manual of Chemical Examinations of the Urine in Disease; with Brief Directions for the Examination of the most Common Varieties of Urinary Calculi. Revised edition. 12mo. Cloth, $1.00. FOTHERGILL (J. MILNER). Diseases of Sedentary Life and Old Age. 8vo. Cloth, $2.00. FOURNIER (ALFRED). Syphilis and Marriage. Translated by P. Albert Morrow, M. D. 8vo. Cloth, $2.00; sheep, $3.00. FREY (HEINRICH). The Histology and Histochemistry of Man. A Treatise on the Elements of Composition and Structure of the Human Body. Trans- lated from the fourth German edition by Arthur E. J. Barker, M. D., and revised by the author. With 608 Engravings on Wood. 8vo. Cloth, $5.00 ; sheep, $6.00. FRIEDLANDER (CARL). The Use of the Microscope in Clinical and Patho- logical Examinations. Second edition, enlarged and improved, with a Chrorno-lithograph Plate. Translated, with the permission of the author, by Henry C. Coe, M. D. 8vo. Cloth, $1.00. GAMGEE (JOHN). Yellow Fever a Nautical Disease. Its Origin and Preven- tion. 8vo. Cloth, $1.50. GROSS (SAMUEL W.). A Practical Treatise on Tumors of the Mammary Gland. Illustrated. 8vo. Cloth, $2.50. GUTMANN (EDWARD). The Watering-Places and Mineral Springs of Ger* many, Austria, and Switzerland. Illustrated. 12rao. Cloth, $2.50. GYNAECOLOGICAL TRANSACTIONS, VOL. VIII. Being the Proceedings of the Eighth Annual Meeting of the American Gym-ecological Society, held in Philadelphia, September 18, l!, and 20, 1883. 8vo. Cloth, $5.00. GYNAECOLOGICAL TRANSACTIONS, VOL. IX. Being the Proceedings of the Ninth Annual Meeting of the American Gynecological Society, held in Chicago, September 30, and October 1 and 2, 1884. 8vo. Cloth, $5.00. HAMILTON (ALLAN McL.). Clinical Electro-Therapeutics, Medical and Surgical. A Hand-Book for Physicians in the Treatment of Nervous and Other Diseases. 8vo. Cloth, $2.00. HAMMOND (W. A.). A Treatise on Diseases of the Nervous System. Seventh edition, rewritten, enlarged, and improved. 8vo. Cloth, $5.00; sheep, $6.00. HAMMOND (W. A.). A Treatise on Insanity, in its Medical Relations. 8vo. Cloth, $5.00; sheep, $6.00. HAMMOND (W. A.). Clinical Lectures on Diseases of the Nervous System. Delivered at Bellevue Hospital Medical College. Edited by T. M. B. Cross, M. D. 8vo. Cloth, $3.50. HART (D. BERRY). Atlas of Female Pelvic Anatomy. With Preface by Alexander J. C. Skene, M. D. 37 Plates with 150 Figures, and 89 pages descriptive text. Large 4to. Clotb, $15.00. (Sold only % subscription.) HARVEY (A.). First Lines of Therapeutics. 12mo. Cloth, $1.50. HEALTH PRIMERS. Edited by J. Langdon Down, Henry Power, J. Mortimer- Granville, and John Tweedy. In square 16mo volumes. Cloth, 40 cents each. I. Exercise and Training. V. Personal Appearance in Health II. Alcohol : Its Use and Abuse. and Disease. III. Premature Death : Its Pro- VL Baths and Bathing. motion or Prevention. VII. The Skin and its Troubles. IV. The House and its Surround- VIII. The Heart and its Functions. ings. IX. The Nervous System. HOFFMANN-ULTZMANN. Introduction to an Investigation of Urine, with Special Reference to Diseases of the Urinary Apparatus. By M. B. Hoff- mann, Professor in the University of Gratz; and R. Ultzmann, Tutor in the University of Vienna. Second enlarged and improved edition. 8vo. Cloth, $2.00. HOWE (JOSEPH W.). Emergencies, and how to treat them. Fourth edition, revised. 8vo. Cloth, $2.50. HOWE (JOSEPH W.). The Breath, and the Diseases which give it a Fetid Odor. With Directions for Treatment. Second edition, revised and corrected. 12mo. Cloth, $1.00. HUEPPE (FERDINAND). The Methods of Bacteriological Investigation. Written at the request of Dr. Robert Koch. Translated by Hermann M. Biggs, M. D. (Nearly ready.} HUXLEY (T. H.). The Anatomy of Vertebrated Animals. Illustrated. 12mo. Cloth, $2.50. HUXLEY (THOMAS HENRY). The Anatomy of Invertebrated Animals Illustrated. 12mo. Cloth, $2.50. JACCOUD (S.). The Curability and Treatment of Pulmonary Phthisis. Trans- lated and edited by Montagu Lubbock, M. D. 8vo. Cloth, $1.00. JOHNSON (JAMES F. W.). The Chemistry of Common Life. A new edition, revised and brought down to the Present Time. By Arthur Herbert Church. Illustrated with Maps and numerous Engravings on Wood. 12mo. Cloth, $2.00. JONES (H. MACNAUGHTON). Practical Manual of Diseases of Women and Uterine Therapeutics. For Students and Practitioners. 188 Illustrations. 12mo. Cloth, $3.00. KEYES (E. L.). The Tonic Treatment of Syphilis, including Local Treatment of Lesions. 8vo. Cloth, $1.00. KINGSLEY (N. W.). A Treatise on Oral Deformities as a Branch of Mechani- cal Surgery. With over 350 Illustrations. 8vo. Cloth, $5.00 ; sheep, $6.00. LEGG (J. WICKHAM). On the Bile, Jaundice, and Bilious Diseases. With Illustrations in Chromo-Lithography. 8vo. Cloth, $6.00 ; sheep, $7.00. LETTERMANN (JONATHAN). Medical Recollections of the Army of the Potomac. 8vo. Cloth. $1.00. LITTLE (W. J.). Medical and Surgical Aspects of In-Knee (Genu-Valgum) : its Relation to Rickets, its Prevention, and its Treatment, with and without Surgical Operation. Illustrated by upward of Fifty Figures and Diagrams. 8vo. Cloth, $2.00. LORING (EDWARD G.). A Text-Book of Ophthalmoscopy. Part I. The Normal Eye, Determination of Refraction, and Diseases of the Media. With 131 Illustrations, and 4 Chromo-Lithographs. 8vo. Cloth, $5.00. LUSK (WILLIAM T.). The Science and Art of Midwifery. With 246 Illustra- tions. Second edition, revised and enlarged. 8vo. Cloth, $5.00 ; sheep, $6.00. LUYS (J.). The Brain and its Functions. With Illustrations. 12mo. Cloth, $1.50. McSlIERRY (RICHARD). Health, and how to promote it. 12mo. Cloth, $1.25. MARKOE (T. M.). A Treatise on Diseases of the Bones. With Illustrations. 8vo. Cloth, $4.50. MAUDSLEY (HENRY). Body and Mind : an Inquiry into their Connection and Mutual Influence, specially in reference to Mental Disorders. An enlarged and revised edition, to which are added Psychological Essays 12mo. Cloth, $1.50. MAUDSLEY (HENRY). Physiology of the Mind. Being the first part of a third edition, revised, enlarged, and in great part rewritten, of u The Phys- iology and Pathology of the Mind." 12mo. Cloth, $2.00. MAUDSLEY (HENRY). Pathology of the Mind. Third edition. 12mo. Cloth $2.00. MAUDSLEY (HENRY). Responsibility in Mental Disease. 12mo. Cloth $1.50. NEFTEL (WM. B.). Galvano-Therapeutics. The Physiological and Therapeuti- cal Action of the Galvanic Current upon the Acoustic, Optic, Sympathetic and Pneumogastric Nerves. 12mo. Cloth, $1.50. NEUMANN (ISIDOR). Hand-Book of Skin Diseases. Translated by Lucius D. Bnlkley, M. D. Illustrated by 66 Wood-Engravings. 8vo. Cloth $4.00 ; sheep, $5.00. THE NEW YORK MEDICAL JOURNAL (weekly). Edited by Frank P. Foster, M. D. Terms per annum, $5.00. GENERAL INDEX, from April, 1865, to June, 1876 (23 vols.) 8vo. Cloth, 75 cents. NIEMEYER (FELIX VON). A Text-Book of Practical Medicine, with particu- lar reference to Physiology and Pathological Anatomy. Containing all the author's Additions and Revisions in the eighth and last German edition. Translated from the German edition, by George H. Humphreys, M. D., and Charles E. Hackley, M. D. 2 vols., 8vo. Cloth, $9.00; sheep, $11.00. NIGHTINGALE'S (FLORENCE). Notes on Nursing. 12mo. Cloth, 75 cents. OSWALD (F. L.). Physical Education ; or, The Health Laws of Nature. 12mo. Cloth, $1.00. PEASLEE (E. R.). A Treatise on Ovarian Tumors: their Pathology, Diagnosis, and Treatment, with reference especially to Ovariotomy. With Illustra- tions. 8vo. Cloth, $5.00 ; sheep, $6.00. PEREIRA'S (Dr.) Elements of Materia Medica and Therapeutics. Abridged and adapted for the Use of Medical and Pharmaceutical Practitioners and Students, and comprising all the Medicines of the British Pharmacopoeia, with such others as are frequently ordered in Prescriptions, or required by the Physician. Edited by Robert "Bentley and Theophilus Redwood. Royal 8vo. Cloth, $7.00; sheep, $8.00. PEYER (ALEXANDER). An Atlas of Clinical Microscopy. Translated and edited by Alfred C. Girard, M. D. First American, from the manuscript of the second German edition, with Additions. Ninety Plates, with 105 Illustrations, Chromo-Lithographs. Square 8vo. Cloth, $6.00. POMEROY (OREN D.). The Diagnosis and Treatment of Diseases of the Ear. With One Hundred Illustrations. New edition, revised and enlarged. 8vo. Cloth, $3.00. POORE (C. T.). Osteotomy and Osteoclasis, for the Correction of Deformities of the Lower Limbs. 50 Illustrations. 8vo. Cloth, $2.50. QUAIN (RICHARD). A Dictionary of Medicine, including General Pathology, General Therapeutics, Hygiene, and the Diseases peculiar to Women and Children. By Various Writers. Edited by Richard Quain, M. D., In one large 8vo volume, with complete Index, and 138 Illustrations. (Sold only . 00. RANNEY (AMBROSE L.). Lectures on Electricity in Medicine, delivered at the Medical Department of the University of Vermont, Burlington. Nu- merous Illustrations. 12mo. Cloth, $1.00. RIBOT (TIL). Diseases of Memory: an Essay in the Positive Psychology Translated from the French by William Huntington. 12mo. Cloth, $1.50. RICHARDSON (B. W.). Diseases of Modern Life. 12mo. Cloth, $2.00. RICHARDSON (B. W.). A Ministry of Health and other Addresses. 12nio. Cloth, $1.50. ROBINSON (A. R.). A Manual of Dermatology. Revise;! and corrected. 8vo. Cloth, $5.00 ROSENTHAL (I.). General Physiology of Muscles and Nerves. With 75 "Wood- cuts. 12mo. Cloth, $1.50. ROSCOE-SCHORLEMMER. Treatise on Chemistry. Vol. 1. Non-Metallic Elements. 8vo. Cloth, $5.00. Vol. 2. Part I. Metals. 8vo. Cloth, $3.00. Vol. 2. Part II. Metals. 8vo. Cloth, $8.00. Vol. 3. Part I. The Chemistry of the Hydrocarbons and their Derivatives. 8vo. Cloth, $5.00. Vol. 3. Part II. The Chemistry of the Hydrocarbons and their Derivatives. 8vo. Cloth, $5.00. SAYRE (LEWIS A.). Practical Manual of the Treatment of Club-Foot. Fourth edition, enlarged and corrected. 12mo. Cloth, $1.25. SAYRE (LEWIS A.). Lectures on Orthopedic Surgery and Diseases of the Joints, delivered at Bellevue Hospital Medical College. New edition, illus- trated with 324 Engravings on Wood. 8vo. Cloth, $5.00 ; sheep, $6.00. SCHROEDER (KARL). A Manual of Midwifery, including the Pathology of Pregnancy and the Puerperal State. Translated into English from the third German edition, by Charles H. Carter, M. D. With 26 Engravings on Wood. 8vo. Cloth, $3.50 ; Sheep, $4.50. SIMPSON (JAMES Y.). Selected Works: Anaesthesia, Diseases of Women. 3 vols., 8vo. Per volume. Cloth, $3.00; sheep,, $4.00. SMITH (EDWARD). Foods. 12mo. Cloth, $1.75. SMITH (EDWARD). Health: A Hand-Book tor Households and Schools. Illustrated. 12mo. Cloth, $1.00. STEINER (JOHANNES). Compendium of Children's Diseases: a Hand-Book for Practitioners and Students. Translated from the second German edition, by Law son Tait. 8vo. Cloth, $3.50 ; sheep, $4.50. STONE (R. FRENCH). Elements of Modern Medicine, including Principles of Pathology and of Therapeutics, with many Useful Memoranda and Valuable Tables of Reference. Accompanied by Pocket Fever Charts. Designed for the Use of Students and Practitioners of Medicine. In wallet-book form, with pockets on each cover for Memoranda, Temperature Charts, etc. Roan, tuck, $2.50. STRECKER (ADOLPH). Short Text-Book of Organic Chemistry. By Dr. Johannes Wislicenns. Translated and edited, with Extensive Additions, by W. H. Hodgkinson and A. J. Greenaway. 8vo. Cloth, $5.00. SWANZY (HENRY R.). A Hand-Book of the Diseases of the Eye, and their Treatment. With 122 Illustrations, and Holmgren's Tests for Color- Blind- ness. Crown 8vo. Cloth, $3.00. TRACY (ROGER S.). The Essentials of Anatomy, Physiology, and Hygiene. 12mo. Cloth, $1.25. TRACY (ROGER S.). Hand-Book of Sanitary Information for Householders. Containing Facts and Suggestions about Ventilation, Drainage, Care of Con- tagious Diseases, Disinfection, Food, and Water. With Appendices on Dis- infectants and Plumbers' Materials. 16ino. Cloth, 50 cents. 8 TRANSACTIONS OF THE NEW YORK STATE MEDICAL ASSOCIA- TION, VOL. I. Being the Proceedings of the First Annual Meeting of the New York State Medical Association, held in New York, November 18, 19, and 20, 1884. Small 8vo. Cloth, $5.00. TYNDALL (JOHN). Essays on the Floating Matter of the Air, in Relation to Putrefacation and Infection. 12mo. Cloth, $1.50. ULTZMANN (ROBERT). Pyuria, or Pus in the Urine, and its Treatment. Translated by permission, by Dr. Walter B. Platt. 12mo. Cloth, $1.00. VAN BUREN (W. H.). Lectures upon Diseases of the Rectum, and the Sur- gery of the Lower Bowel, delivered at Bellevue Hospital Medical College. Second edition, revised and enlarged. 8vo. Cloth, $3.00 ; sheep, $4.00. VAN BUREN (W. H.). Lectures on the Principles and Practice of Surgery. Delivered at Bellevue Hospital Medical College. Edited by Lewis A. Stim- son, M. D. 8vo. Cloth, $4.00 ; sheep, $5.00. VAN BUREN AND KEYES. A Practical Treatise on the Surgical Diseases of the Genito-Urinary Organs, including Syphilis. Designed as a Manual for Students and Practitioners. With Engravings and Cases. By W. H. Van Buren, M. D., and Edward L. Keyes, M. D. 8vo. Cloth, $5.00; sheep, $0.00. VOGEL (A.). A Practical Treatise on the Diseases of Children. Translated and edited by II. Raphael, M. D. Third American from the eighth German edi- tion, revised and enlarged. Illustrated by six Lithographic Plates. 8vo. Cloth, $4.50 ; sheep, $5.50. WAGNER (RUDOLF). Hand-Book of Chemical Technology. Translated and edited from the eighth German edition, with extensive Additions, by William Crookes. With 336 Illustrations. 8vo. Cloth, $5.00. WALTON (GEORGE E.). Mineral Springs of the United States and Canadas. Containing the latest Analyses, with full Description of Localities, Routes, etc. Second edition, revised and enlarged. 12mo. Cloth, $2.00. WEBBER (S. G.). A Treatise on Nervous Diseases: Their Symptoms and Treatment. A Text-Book for Students and Practitioners. 8vo. Cloth, $8.00. WEEKS (CLARA S.). A Text-Book of Nursing. For the Use of Training- Schools, Families, and Private Students. 12mo. With 13 Illustrations, Questions for Review and Examination, and Vocabulary of Medical Terms. 12mo. Cloth, $1.75. WELLS (T. SPENCER). Diseases of the Ovaries. 8vo. Cloth, $4.50. WYLIE (WILLIAM G.). Hospitals: Their; History, Organization, and Con- struction. 8vo. Cloth, $2.50. ^^ YC oc RETURN TO * LOAN PERIOD 1 2 3 4 5 6 ALL BOOKS MAY BE RECALLED AFTER 7 DAYS DUE AS STAMPED BELOW jNTPR! IRRAKY LOAM APR :? IPS 7 UrtiV. OF CALIF., 8ER / \, UNIVERSITY OF CALIFORNIA, BERKELEY FORM NO. DDO, 15m, 2/84 BERKELEY, CA 94720 $ YC 88546 Micr. Soo G THE UNIVERSITY OF CALIFORNIA LIBRARY