IC-NRLF 
 
 BACTERIOLOGICAL 
 
 TECHNOLOGY 
 
 - 
 
LIBRARY 
 
 OF THK 
 
 UNIVERSITY OF CALIFORNIA 
 
 OIPT OR 
 
 Received 
 Accession No. 
 
 Class No. BIOLOGY 
 
^ v. 
 
BACTERIOLOGICAL TECHNOLOGY 
 
 FOR PHYSICIANS 
 
 WITH SEVENTY-TWO FIGURES IN THE TEXT 
 
 BY 
 
 DR. C. J. SALOMONSEN 
 
 Authorized Translation from the Second Revised Danish Edition 
 
 BY 
 
 WILLIAM TRELEASE 
 
 - 
 s 
 
 ~-\ - 
 
 
 NEW YORK 
 
 WILLIAM WOOD & COMPANY 
 1890 
 

 BIOLOGY 
 R* 
 
 COPYRIGHT, 1890, 
 
 BY 
 
 WILLIAM WOOD & COMPANY 
 
AUTHOR'S PREFACE. 
 
 THE first edition of this text-book was issued in 1885, as a 
 reprint from the " Nordiskt Medicinskt Arkiv." The increased 
 size of this new edition is partly due to the addition of an in- 
 troduction to the microscopic investigation of bacteria; but 
 the active development of bacteriology in the last few years 
 has also necessitated a re-elaboration and extension of all of 
 the chapters. 
 
 In the work, I have had two objects in view: the prepara- 
 tion of an outline adapted to bacteriological courses for physi- 
 cians and veterinary surgeons, and a guide for those who are 
 obliged to take up the subject at home without the assistance 
 of an instructor, yet wish to carry out for themselves the 
 fundamental experiments which are most important for path- 
 ology and hygiene. I have, therefore, not attempted to give 
 an exhaustive presentation of the entire technology of the 
 subject, but rather to describe the simplest and most easily 
 managed apparatus and methods, so that the equipment of a 
 home laboratory need not appear too expensive; while I have 
 tried to describe these in a sufficiently elementary and de- 
 tailed manner. 
 
 Those who wish a more comprehensive treatise, are re- 
 ferred to the last edition of Hueppe's " Methoden der Bakte- 
 rienf orschung " (Wiesbaden, 1889). 
 
 C. J. S. 
 
TRANSLATOR'S PREFACE. 
 
 So many works on bacteriology are now accessible, that a- 
 new one must possess decided merit to justify its appearance. 
 The scope of the present little volume is such as to satisfy me 
 that it should fill a place as yet vacant ; and the careful treat- 
 ment of the subject gives rise to the hope that it may find a 
 larger field of usefulness in the English language than could 
 be possible while it was confined to the less-used Danish. 
 
 The physician who wishes to read a well-written account 
 of the more important pathogenic bacteria, is further referred 
 to Fraenkel's " Bakterienkunde." The fullest and most re- 
 liable attempt at a bacteriological flora, is to be found in 
 Fluegge's " Ferment e und Mikroparasiten," which contains 
 much additional information. 
 
 W. T. 
 
BACTERIOLOGICAL TECHNOLOGY. 
 
 CHAPTER I. 
 
 STERILIZATION. 
 
 BACTERIOLOGICAL technology must in the first place put 
 means in our hands for separating- the different kinds of bac- 
 teria from one another, and cultivating- each species in a state 
 of complete purity. Pure cultivation is a necessary condition 
 for a trustworthy study of the morphology and physiology 
 of these microscopic organisms. Unless one has this con- 
 stantly in mind during his work, he is certain to end in sad 
 confusion; and the short history of bacteriology is only too 
 full of worthless investigations and wrong- results, which are 
 to be attributed to the untrustworthiness of the methods em- 
 ployed. 
 
 It is well known that the pure cultivation of a single kind 
 of bacteria is surrounded by quite peculiar difficulties, due to 
 the extreme smallness, the large numbers, and the extraordi- 
 nary distribution, one might almost say omnipresence, of bac- 
 teria. Everything which is allowed to stand uncovered in the 
 air for a short time is likely to receive spores capable of g-er- 
 mination, which fall on its surface with the dust. Every con- 
 tact with hands or clothing- is attended with the same dang-er. 
 We must learn first of all to meet and control this accidental 
 and invisible source of contamination, for it is self-evident that 
 all vessels, instruments, fluids, etc., which are to be used in 
 cultures must be clean, not only in the sense in which chemists 
 use the word, but also in the bacteriological sense, or sterile, 
 that is, free from living germs. 
 
 Heat is the most generally used means of sterilization, but 
 1 
 
2 Bacteriological Technology. 
 
 it must be used in different ways, and of different degrees, 
 according* to the nature of the material to be sterilized, a 
 platinum needle, for example, is best sterilized by heating it 
 to incandescence directly in the flame; but a knife blade, or a 
 pair of scissors, can clearly not be heated to this point. These 
 can only be " flamed/' that is, drawn slowly back and forth 
 through the flame, until one is sure that all of their surface 
 has been heated to at least 200 C., so that any germs have 
 been burned. 
 
 But it is only exceptionally that glowing or flaming can be 
 employed. If one has to deal with glass-ware, metallic arti- 
 cles, cotton, paper, etc., these are most frequently sterilized in 
 air heated to about 150 C. In the preparation of most fluid 
 and solid culture-media, one can most rapidly and surely 
 reach the desired end by the help of a Papin digester, an 
 " autoclave," in which the sterilization is effected by steam 
 under pressure, at 110 to 120 C.; or one contents himself with 
 using the boiling temperature, either by directly cooking the 
 substance, or by heating it to 100 C., or about that point, by 
 the aid of streaming steam. Occasionally, however, one may 
 have to use a temperature lower than this (60 to 70 C.), to 
 kill germs, for instance where serum is used. In some cases 
 filtration and disinfecting solutions are used as means of ster- 
 ilizing. 
 
 The entire collection of instruments needed for sterilizing, 
 the apparatus and culture-media to be used in the investiga- 
 tions that we shall describe, are, in addition to a common 
 Bunsen gas burner (or alcohol lamp), the following : 
 
 1. A sterilizing oven in which articles made of glass and 
 metal, cotton, paper, etc., are heated to about 150 C., by means 
 of hot air. 
 
 For this purpose one uses in the laboratory a double-walled 
 sheet-iron box 30 cm. high, 35 cm. broad, and 23 cm. deep, with 
 a tube in the top for inserting a thermometer, a door in the 
 side, and with a loose middle piece in the bottom against 
 which the large gas jet constantly plays, and which conse- 
 quently will burn through after long use, when it is only 
 necessary to put in a new piece of iron. 
 
 One can usually get along with less expense by making a 
 serviceable sterilizing oven (Figs. 1, 2) of one of the common 
 cracker boxes (about 24 cm. high, 22 cm. wide, and 24 cm. 
 
Bacteriological Technology. 
 
 deep) to be obtained cheaply of any grocer. In the cover a 
 round hole is to be cut in which a bored cork is fitted to carry 
 a thermometer, registering- to 200 C. For ventilation, a small 
 hole is punched in each of the four side-walls close to the top 
 and bottom. In the box is set a four-sided piece of strong 
 sheet-iron, with the sides bent down so as to hold it about 
 2 cm. from the bottom, so that the objects to be sterilized will 
 not come into immediate contact with the latter. The box is 
 inclosed in a substance which is a poor conductor of heat, felt 
 being usually chosen. One piece covers the lid, and is perfor- 
 
 FlG. 1. 
 
 FIG. 2. 
 
 FIGS. 1 and 2. Oven for sterilization at 150 C., formed of a cracker box surrounded 
 with felt and provided with thermometer and support of iron netting. 
 
 ated for the passage of the cork and thermometer. Another 
 piece is chosen long enough to be wrapped around the four 
 sides of the box and wide enough to cover the upper three 
 quarters of these, excepting- a small space where the air-holes 
 occur. The lowermost quarter is best left uncovered to avoid 
 scorching- or burning the felt. This strip of felt is fastened 
 simply by tying around it tightly a few pieces of cord or wire. 
 The box is set on one or two of the common iron tripods, such 
 as are used in chemical laboratories, and is heated by one or 
 more gas burners. Between these and the bottom of the box, 
 a loose piece of sheet-tin can be placed to prevent burning 
 
4 Bacteriological Technology. 
 
 through the bottom. If gas is not at hand, a'small oil stove 
 with one or two burners can be used. 
 
 In comparison with the larger and more expensive ovens 
 provided with ventilators and t her mo -regulator, this primitive 
 apparatus offers some inconveniences, but these do not count 
 for much in comparison with its perfect usefulness and ex- 
 treme cheapness. 
 
 Care must be taken to guard the sterilized objects from 
 fresh contamination when they are taken out of the sterilizer. 
 Vessels which are guarded from the penetration of germs, by 
 cotton plugs or otherwise, when it is not especially important 
 to keep the outside free from germs (e.g., flasks and test-tubes) 
 can simply be taken from the oven after they have been heated 
 to 150 C. for half an hour or an hour, and set aside without any 
 other precaution than covering or wrapping them up in paper 
 to prevent them from becoming very dusty. But objects 
 which must be perfectly sterilized, such as watch-glasses, 
 slides, large glass plates, etc., must be carefully wrapped in a 
 couple of thicknesses of thin, firm paper before they are put in 
 the oven. Germs from the air cannot pass through this, and 
 the objects can thus be kept satisfactorily sterile for a long 
 time. It is well to wrap each object in several layers of thin 
 paper, and then to wrap these in larger parcels. 
 
 Paper and cotton should be kept as far as possible from 
 the bottom of the oven to prevent them from becoming 
 charred by long heating. Paper and cotton (but not absorb- 
 ent cotton) assume a yellowish-brown color after being kept at 
 from 140 to 150 C. for a long time, and this discoloration con- 
 sequently shows that the heat in the sterilizing oven has been 
 sufficient even without the testimony of a thermometer. 
 
 2. The Steam Sterilizer of Koch. Sterilization at the boil- 
 ing temperature can evidently be effected by cooking in water, 
 but since this method is not only inapplicable in many cases, 
 but always more laborious and unreliable than sterilization 
 by streaming steam as introduced by Koch, the latter is now 
 the principal method used when it is desired to sterilize at 
 100 C. 
 
 It will be remembered that two advantages are gained by 
 this: the heat penetrates rapidly into the objects to be steril- 
 ized, and steam of this temperature (99 to 100 C. according 
 to altitude) sterilizes much more rapidly than atmospheric air 
 
Bacteriological Technology. 
 
 5 
 
 of the same or a much higher temperature, probably for 
 chemical reasons. 
 
 The partly diagrammatic illustration shows the Koch steam 
 apparatus of a simple and cheap form. It consists of a tin 
 
 FIG. 3. 
 
 FIG. 4. 
 
 FIGS. 3 and 4. Koch cylinder in its simplest form, with two tin shelves and pails. A, 
 Hook for suspending long objects ; #, tin pail with sieve-bottom, containing plugged test- 
 tubes ; C, tin cylinder for elongating the apparatus. 
 
 cylinder 50 cm. high and 17 cm. in diameter, and is provided 
 with a small glass tube (a) fastened by rubber tubing in which 
 the height of the water can be seen from the outside. The 
 absolute size, as well as the proportion between the height 
 
6 Bacteriological 'Technology. 
 
 and diameter, can naturally be varied within wide limits and 
 the distance between the shelves is to be adapted to the 
 height of the apparatus, etc., that are to be most frequently 
 sterilized. The bottom, which is to be exposed to the action 
 of the flame, is preferably made of somewhat thicker tin than 
 the sides. The top of the apparatus is closed by a conical lid 
 (b) the centre of which is pierced by a short tube (c) for the 
 reception of a cork and thermometer. The lid and the side 
 walls, to within 9 cm. of the bottom, are usually covered with 
 felt, though this is not absolutely necessary. Within the 
 cylinder, at a distance of 14 and 30 cm. from the bottom, are 
 placed projecting 1 ledges, on which can be placed two loosely 
 fitting shelves of heavy tin perforated with large holes (del). 
 These serve to support tin pails (ee) about 12 cm. high and 14 
 cm. in diameter, the bottom of which is best made of wide- 
 meshed galvanized iron netting (Fig. 4, B). These hold the 
 objects to be sterilized, e.g., test-tubes plugged with cotton 
 in the figure. When it is to be used, the apparatus is placed 
 on a tripod (/) of stout strap-iron. The pails are taken out 
 and the cylinder is filled with water to a depth of about 10 
 cm. The pails with their contents having been set in and the 
 lid with its thermometer put in place, the water is heated to 
 the boiling point. After some time steam will escape freely 
 around the loosely-fitting lid, and the thermometer will also 
 show about 100 C., after which the objects are left in this 
 streaming steam for a longer or shorter time (as a rule, 
 twenty minutes), the time being counted from the moment 
 when the thermometer indicates 100 C. (or the steam begins 
 to escape freely). Most objects which are to be sterilized by 
 steam can be set on the tin shelves without difficulty. Longer 
 objects can be hung in the apparatus after the pails and 
 shelves have been removed. This is most easily done by using 
 a piece of stout brass wire, bent and brought into the opening 
 of the lid (Fig. 4, A). 
 
 The apparatus can be lengthened when this is necessary 
 by using a tin cylinder, with a projecting collar near its base 
 (Fig. 4, D C) to be inserted between the lid and the top of the 
 cylinder. A stop-cock, near the bottom, for drawing off the 
 water, increases the expense of the apparatus somewhat, but 
 adds to its convenience. Moreover, one can easily form such a 
 steam sterilizer from a common kitchen pot. It is only neces- 
 
Bacteriological Technology. j 
 
 sary to fit to it a tin cylinder open at the bottom, and fur- 
 nished with a lid and felt covering 1 , but it is essential to pro- 
 vide this cylinder with a projecting- collar at a couple of 
 centimetres from the bottom, by which it rests on the edge of 
 the pot; and it is best to provide it with a couple of rings, by 
 which the cylinder may be tied to the ears of the pot. 
 
 A word of explanation should be offered for the omission 
 of the Papin digester in speaking- of necessary apparatus for 
 sterilization. There are, indeed, bacillus spores which are 
 able to survive a cooking for several hours in water or free 
 steam, while they are killed after a few moments' heating- to 
 120 C. in the digester, consequently this is the only apparatus 
 in which one can rapidly and surely effect sterilization by a 
 single attempt. Still it is a very expensive piece of apparatus, 
 which is the only reason that it has been omitted. Koch's 
 steam cylinder, on the other hand, is very simple, cheap, and 
 efficient when it is used with care. ' As a rule the spores of 
 bacilli which always get into our culture media, from water, 
 the air, etc., are not so resistant as those which have been 
 mentioned. Generally, streaming 1 steam at 100 C. will kill 
 them, and experiments from a large number of bacteriological 
 laboratories have long since" shown the adequac}^ of Koch's 
 apparatus. However, in the preparation of his culture appar- 
 atus one must always have in mind that some very resistant 
 form may have escaped destruction in the steam, and must 
 make sure of absolute freedom from germs in his culture 
 media by: 
 
 a. Cleanliness in the preparation. It is not sufficient to 
 trust to the final sterilization of the materials, but at every 
 step of the preparation one should work cleanly, to lessen the 
 chances of resistant g-erms entering. 
 
 b. Discontinuous Heating. This method was first applied 
 by Tyndall in 1877 for the sterilization of hay infusion, which 
 is known to contain some of the most resistant bacillus spores, 
 the extraordinary vitality of which renders it impossible to 
 sterilize this substance even by cooking- it for some minutes 
 (c/. Chapter IV., Section 2). Tyndall reached his method of 
 sterilizing- by the following train of thought : When a hay 
 infusion has been boiled for some minutes all of the rod -shaped 
 bacilli, unable to endure this treatment, are dead, while the 
 spores of bacilli (more exactly, of some of them) are still alive. 
 
Bacteriological Technology. 
 
 If now all of these surviving- spores are given time to germi- 
 nate and the infusion is again cooked, it will be completely 
 sterilized, because, when it is heated it contains no spores but 
 only rods. However, as one cannot be sure that all spores 
 have germinated at the same time, this process must usually 
 be repeated several times. Hence it is recommended to submit 
 the filled culture apparatus to steaming twice, with a day in- 
 tervening. This is especially important for those culture 
 media which, like gelatin, bear only a short cooking. 
 
 c. Waiting and Watching. For greater certainty, the 
 materials treated in this way are allowed to stand for some 
 time before they are used, so that one may be sure that they 
 contain nothing capable of germination. If they are placed 
 in the thermostat at about 30 C., a delay of a couple of days 
 may be looked on as sufficient. 
 
 3. Water Bath for Sterilizing at Temperatures below the 
 Boiling Point. Various fluids will not bear being sterilized at 
 100 C., because at so high a temperature they undergo chem- 
 ical changes which it is wished to avoid for one reason or 
 another. In such cases the short exposure to a high temper- 
 ature can be replaced by long maintenance at a lower tem- 
 perature, until no germ capable of growth remains in the 
 fluids. This method was discovered by Pasteur, and by his 
 advice applied on a large scale in the fabrication of wine 
 (" Pasteurization ") to secure sterilization of the wines without 
 at the same time injuring their quality. In what follows we 
 shall make use of the preparation of blood serum according to 
 Koch's directions. Rather complicated and expensive appar- 
 atus is often used for this, but one can get along very well 
 with a simple cylindrical water-bath made of tin, such as 
 Koch himself used in his first preparations. The tin receptacle 
 (Fig. 5) is 22 cm. high, and 13 cm. in diameter. At the top it 
 is provided with a collar 1.5 cm. broad. A piece of fine-meshed 
 flexible galvanized iron netting (a) serves as a lid, and is fast- 
 ened by bending its edges under the collar. The temperature 
 of the water is read off on the thermometer (6) which projects 
 through a hole in the lid. The bulb of the thermometer is 
 held up several centimetres above the bottom of the cylinder, 
 by another loose piece of wire cloth (c) resting upon its edges, 
 which are bent down; when the water has reached a suitable 
 temperature, usually 60 to 70 C., it is easy to hold it at about 
 
Bacteriological Technology. 
 
 the same point for a long time, e.g., a couple of hours, "by the 
 use of a small gas or alcohol flame. Though a range of a few 
 degrees can usually be allowed, it is best to keep the tempera- 
 ture under observation. Further directions for the prepara- 
 tion of serum are given later. 
 
 4. Porcelain Filters. Even a slight heating often causes 
 in the culture fluid chemical changes which it may be import- 
 ant to avoid. In such cases the germs have been removed by 
 filtration through burnt clay (Klebs), plaster (Pasteur and 
 Miquel), porcelain (Chamberland), or sheet asbestos (Hesse), 
 
 FIG. 6. 
 
 Fio. 5. Water-bath for Sterilization at Lower Temperatures. 
 
 FIG. G. A, Chamberland Filter (X 1/6); B, bulb-pipette for collecting the contents of 
 the filter; E, small porcelain filter (x 1 /6); F, reservoir, which can also be used as a cul- 
 ture-vessel; C and D are explained in the text. 
 
 which keep back all of the cells contained in the fluid. As 
 filtration plays a prominent part in the separation of bacteria 
 from their soluble products, we shall here dwell somewhat 
 more fully on the use of 
 
 Ckamberland's Filter. This consists of a hollow porcelain 
 cylinder (Fig. 6 A\ which is closed at the bottom and at the 
 upper end provided with a funnel-shaped glazed end-piece, 
 over which a rubber tube can be pushed. The filter is im- 
 mersed in the fluid to be sterilized and the upper (open) end is 
 connected with some kind of aspirator. If the fluid to be fil- 
 
10 
 
 Bacteriological Technology. 
 
 tered is in too great quantity to be contained in the cavity of 
 the filter, another receptacle is inserted between the aspirator 
 and filter. An ordinary wash-bottle (Fig-. ?) can be used for 
 this purpose; and when the fluid to be sterilized is not too 
 hard to filter, a common bottle aspirator (Fig-. 7, a) can be 
 employed. The details of the process are then as follows: 
 The open end of the filter is closed by a plug- of cotton batting-. 
 It is then wrapped in paper and sterilized at 150 C. The open 
 ends of both tubes of the wash-bottle are plugged with cot- 
 ton, wrapped in paper and sterilized with the filter, as is the 
 plugged wash-bottle. The rubber stopper of the latter (bored 
 
 FIG. 7. Chamberland Filter set up for use, with Aspirator and Wash-bottle Reservoir. 
 
 with two holes for the tubes), and the two pieces of firm-walled 
 rubber tubing- for connecting the flask with the filter and the 
 aspirator, must be sterilized in some other manner. They are 
 first laid for fifteen minutes in a -^ per cent sublimate solu- 
 tion, rinsed in sterilized distilled water, plug-g-ed with cotton 
 that has been previously sterilized by dry heat, and after 
 wrapping- in paper they are steamed for fifteen minutes. After 
 this has been done, the filter is put together as rapidly as pos- 
 sible, and with the ulmost cleanliness, the fingers being first 
 washed in sublimate. The glass tubes are fixed in the rubber 
 stopper, and this is fitted to the wash-bottle after removing 
 the plug from the latter with sterilized forceps. The long 
 
Bacteriological Technology. 1 1 
 
 tube is then joined to the filter by one piece of rubber tubing- 
 after removal of its cotton plug 1 . The only plug- left in place 
 is that at * of Figure 7. The entire apparatus is now steamed 
 for ten minutes, after which it can be used. For additional 
 safety, it is usually customary after the last sterilization to 
 bind the rubber tubing 1 fast to the filter and glass tubes by 
 cord, or better still by previously glowed coarse brass wire, 
 which is tightly wound by the use of pliers. 
 
 If instead of the common wash-bottle, a so-called Pasteur 
 flask, made entirely of glass, is used (cf. Chapter XL), the pre- 
 parations for filtering 1 are very much simplified. It is also 
 evident that if an autoclave is at hand the preparatory steril- 
 ization, as well as the rapid and careful setting- up of the 
 apparatus can be dispensed with. Without any especial pre- 
 cautions the glass and rubber parts can be joined and then 
 sterilized in a few moments at 120 C. 
 
 Figure 7 shows the apparatus in use. The filter is im- 
 mersed in a relatively narrow glass cylinder, which contains 
 the fluid to be filtered, as this sinks in the glass it is tipped so 
 as to use all of the filtering- surface and to prevent air from 
 being sucked through, though this is also sterilized in the pas- 
 sage through the filter. The simple aspirator (a) is sufficient, 
 when one has not to deal with fluids that are hard to filter. 
 The bubbling- of air back through the aspirator, and the con- 
 sequent hindrance to filtration, is avoided by reg-ulating- the 
 size of the escape tube by a pineh-cock. In other cases, or 
 when the filtering must be done rapidly, more powerful suction 
 is employed, e.g., the small air pump recommended by Cham- 
 berland (Fig. 8, a), or a filter-pump worked by water. If the 
 filtration is difficult and a powerful pump is used, care must 
 be taken that the walls of the flask are strong enough to bear 
 the atmospheric pressure. In such cases it is customary to 
 use a thick- walled flask instead of a common wash-bottle, and 
 under these circumstances exceptional use may be made of a 
 lead tube within the rubber tubing. After repeated sterilizing- 
 by steam, even thick-walled rubber tubing- becomes too much 
 softened for further use. 
 
 When the wash-bottle (b) is filled with the clear filtrate, it 
 is first loosened from the aspirator, then the tube (c) is fast- 
 ened by a clamp or glass plug, after which the flask can be 
 separated from the filter and its contents kept sterile. The 
 
12 Bacteriological Technology. 
 
 filtrate remaining- in the cavity of the filter can be saved, by 
 pouring 1 it into one or more sterile vessels, e.g., culture tubes, 
 as soon as the filter is opened. This is best done by using a 
 pipette (Fig. G, B), plugged at the top with cotton, while the 
 tube below the bulb is long enough to reach the bottom of the 
 filter and slender enough to pass through its opening. Before 
 use, it is hermetically sealed, sterilized, and passed through the 
 flame like an ordinary Pasteur pipette, of which, indeed, it is 
 only an enlarged edition (c/. Fig. 22). 
 
 After being used, the Chamberland filter is cleaned accord- 
 ing to circumstances, by simply brushing off the surface and 
 washing- it in water, or by the additional use of chemicals, 
 e.g., disinfectants. Occasionally it may also be desirable or 
 necessary to rinse its pores out thoroughly with water, before 
 it is dried and again sterilized. This is most quickly accom- 
 plished (Fig. 6, C), in the following way: the filter is filled 
 with water and immersed in a large vessel of water, which is 
 placed quite high, as on a shelf or cupboard. A rubber tube 
 several feet long is attached to its open end. Somewhere in 
 this a glass tube (Fig. 6, D), filled with water is inserted. 
 Before putting together, the filter and tube arc both filled 
 with water. 
 
 When one has to do with small quantities of fluid the small 
 thick- walled filter (Fig. 6, E] can be used. This is 15 cm. 
 long, with a cavity 2 mm. in diameter. These were first used 
 in Pasteur's laboratory, inserted in a filtering apparatus of 
 special construction, but they can be used in the same manner 
 as the larger ones, by fastening a rubber tube to the upper 
 end with copper wire. A strong suction apparatus is needed 
 for this. The small bulb (Fig. 6, F) is intended to be used as 
 a receiver of filtered fluid (Fig. 8) and also without special 
 cnange, as a culture vessel, with tubular plug (cf. Chapter 
 IL, p. 16). 
 
 It must be remembered that even filtration through porce- 
 lain does not always avoid the chemical modification of fluids, 
 since the filter may hold back certain compounds, as well as 
 the gerrns. If organic culture fluids must be absolutely un- 
 changed, and at the same time free from g-erms, it is neces- 
 sary to do without all sterilization, in which case they are to 
 be collected or prepared with such care that all contamina- 
 tions from their surroundings are avoided. Blood and urine. 
 
Bacteriological Technology. 1 3 
 
 for instance, are drawn directly from the heart, veins, or blad- 
 der with antiseptic precautions. The preparation of origin- 
 ally sterile infusions is further briefly discussed in Chapter XI. 
 5. Disinfecting Solutions. As a help in sterilization, it is 
 usual to have at hand a large quantity of corrosive sublimate 
 solution (1 or 2 : 1,000), which is not only used in preparing some 
 sorts of culture media, e.g.,. potatoes (see Chapter III.), but in 
 many other cases, for cleansing the hands, instruments, mor- 
 bid material, etc. If well water is used instead of distilled 
 water in making this solution, care must be taken that no 
 precipitate of insoluble mercurial salts is formed, or the fluid 
 
 FIG. 8. Small Porcelain Filter joined to Bulb-reservoir and Air-pump (a). 
 
 will possess no disinfecting value. To prevent this, a small 
 quantity of acetic acid may be added to the well water. Fiir- 
 bringer recommends 0.5 gram to each litre of 0.1 per cent solu- 
 tion. Carbolic acid solutions of various strengths are also to 
 be kept ready for use. 
 
CHAPTER II. 
 
 COMMON CULTURE-APPARATUS. 
 
 THE principal requirements of a culture-apparatus in which 
 pure cultures are to be conveniently and surely obtained are 
 the following 1 : Besides being- durable, simple, handy, and 
 cheap, it must be easily sterilized with its contents, and so 
 that it can be opened for inoculation and again closed without 
 much danger of infection from the air. It must also be so 
 closed that the entrance of foreign germs is impossible, while 
 free access of the air is permitted (except when anaerobic 
 forms are to be cultivated). This is effected by the use of cot- 
 ton batting-, which allows a sufficient circulation of air while 
 filtering the germs from this. 
 
 The apparatus to be recommended for simply keeping a 
 pure culture is the following- : 
 
 1. Test-tubes and flasks plugged with cotton. One can 
 commonly use : 
 
 a. Test-tubes of any size. The use of small tubes (e.g., 13 
 cm. long, and 1 cm. in diameter) is to be recommended. A 
 considerable quantity of the culture material is then saved. 
 Since the heating- of these glasses is generally effected in the 
 steam sterilizer and not directly over the flame, their walls 
 need not necessarily be so thin as those of chemical test-tubes, 
 and consequently it may be best to order those with especially 
 thick walls, entirely without a flange, so that the cotton plug- 
 may fit better to the edge. 
 
 The test-tubes are first carefully cleaned with common 
 distilled water, and then plugg-ed in the following manner: 
 Good cotton batting (not absorbent cotton) is pressed with a 
 pair of forceps (or twisted by the hand) into the mouth of the 
 test-tube in sufficient quantity to form a firm, tightly fitting 
 plug, which reaches a couple of centimetres into the tube, 
 while part of it projects and frays out over the edge. The 
 
Bacteriological Technology. 
 
 5 
 
 plug- must be both firm and tightly fitting, yet capable of re- 
 moval and reinsertion (by a twisting motion) without much 
 difficulty. When the test-tubes have been plugged, they are 
 put in a small four-sided basket, made of wire-cloth, of a suita- 
 ble size for the oven, and sterilized at 150 C. 
 
 If it is wished to make use of a larger surface than is avail- 
 able when a test-tube is used, even when it is obliquely placed 
 (Fig. 9, II.), recourse is had to : 
 
 b. Small Conical Flasks. (Erlenmeyer flasks) of the form 
 shown in Fig. 9, VII., and holding about 100 cc. 
 
 FIG. 9. -Various Culture-glasses with Cotton Plugs (/., IL, VII.), or Plugged Caps (III. FI.) 
 
 c. Small Medicine Bottles (30 gm.) are conveniently used 
 in the same way, and. the surface of the gelatin in them can 
 also be increased by placing them in an oblique position while 
 it is hardening. They have the advantage over test-tubes 
 that they may be stood upright without the use of a rack. 
 
 Any of these receptacles can be used for cultures in either 
 solid or fluid media. A fault common to them is that they 
 are all relatively wide mouthed, so that the removal of the 
 plug leads to a certain danger of contamination, especially 
 from dust which has settled on the plugs when they have 
 stood for some time. This can be obviated by tying a couple 
 
1 6 Bacteriological Technology. 
 
 of layers of filter paper over the cotton plug 1 (Fig. 9, VIL), 
 which is especially recommended when large flasks are used. 
 For greater safety, the part of the cotton projecting beyond 
 the tube can be singed off just before use. Both these precau- 
 tions are less necessary when solid media are used, so that the 
 tube or flask can be inverted while the plug is removed and 
 inoculation effected. They are more important when the con- 
 tents of the vessels are fluid. Much greater safety is secured 
 by the tubular cotton plug of the author which is described 
 later. 
 
 2. Vessels with Tubular Plugs. When I began in 1879-80 
 the pure culture of a large number of putrefactive bacteria, 
 which had been isolated by the capillary-tube method, I first 
 used stoppers in the form of short pieces of rubber tubing, in 
 one end of which small sterilized tampons of cotton were fixed. 
 The vessels which were closed by these tubular stoppers were 
 larger and smaller flask-shaped or test-tube-shaped glasses, 
 which had a short and broad slightly conical neck, with rela- 
 tively narrow mouth (2 to 4 mm.) over which the rubber tube 
 could be slipped (Fig. 9, III.-V.). 
 
 The tubular stoppers are prepared as follows : The cotton is 
 sterilized at 150 C. The rubber tube, 6 to 8 cm. long, is steril- 
 ized by steam, wrapped in paper. It should be a little larger 
 than the top and a little smaller than the bottom of the conical 
 neck it is to fit. After cooling, the tubes are unwrapped, and 
 by the aid of small forceps, half filled with tampons of the 
 sterilized cotton, which must be so large and firm that they 
 bulge the tube slightly (Fig. 9, V.). The advantages of these 
 stoppers are, obviously : a. In opening and closing the flasks 
 the cotton and the dust that has collected on it are not 
 touched. 6. The apparatus is opened and closed at a point 
 where its surface can always be easily, freed from dust. c. 
 The opening through which inoculation is effected is smaller 
 than with test-tubes. 
 
 The same principle was afterward applied in a more ade- 
 quate form in the so-called Pasteur or Chamberland flasks 
 (Fig. 10) which are much used in Pasteur's laboratory. The 
 cap of these is not made of rubber but of glass, and is closely 
 fitted to the neck by grinding. Unlike the flasks with rubber 
 tubes, they can be sterilized entire at 150 C., and hence, as a 
 rule, are preferable to the latter. The flasks and caps, which 
 
Bacteriological Technology. 17 
 
 belong together, are numbered correspondingly with Bruns- 
 wick black (Fig. 10). 
 
 Notwithstanding the decided superiority of the tubular cap 
 over the simple cotton plug, the latter as a rule is used even 
 in fluid cultures; but the rubber cap is always useful when a 
 narrow tube is to be plugged with cotton (cf. Fig. 56 and Fig. 
 58). By means of a bored rubber stopper, a tubular cap can 
 be adapted to any flask (Fig. 9, VI.). 
 
 3. Other Culture Apparatus. A pair of small glass trays 
 (3 to 5 cm. in diameter) can be used for cultures, one serving as 
 a lid for the other (Fig. 13). They have the advantage over 
 
 FIG. 10. Pasteur-Chamberland Flask. 
 
 Fio. 11. Glass Box for Soyka's 
 Museum Cultures. 
 
 vessels plugged with cotton, that their contents are more 
 easily accessible, even for microscopic examination, but they 
 are less secure against foreign germs. 
 
 A far more reliable security against contamination is 
 afforded by glass boxes with overlapping and ground covers 
 (Fig. 11), such as Soyka uses for his hermetically sealed "mu- 
 seum cultures" (cf. Chapter VI.). 
 
 A number of .other contrivances for cultures adapted to 
 particular purposes will be described later. The apparatus 
 for cultivating anaerobic bacteria, and for cultivating bacteria 
 under the microscope, are for convenience left for treatment 
 in later chapters, devoted to these subjects. 
 
CHAPTEE III. 
 
 CULTURE-MEDIA, AND THEIR INTRODUCTION INTO TEST- 
 TUBES, FLASKS, ETC. 
 
 IT would be useless to mention the enormous number of 
 solutions, infusions, etc., which have been applied advantage- 
 ously in various bacteriological investigations, and moreover 
 it may obviously be necessary for each experimenter to use 
 the most dissimilar culture-media, and to vary them, within 
 wide limits, by the addition or removal of one substance or 
 another according- to the object of the investigation. Direc- 
 tions are given here for the preparation of only such fluid and 
 solid media as have been found especially useful in the study 
 of pathogenic micro-organisms during the last few years. 
 They are commonly made in the conical Erlenmeyer flasks 
 (Fig. 10, VII.). These conical flasks will be frequently men- 
 tioned in the following pages, where they are designated as 
 large, medium, and small, holding respectively 1,500, 500, or 
 100 gin. 
 
 A. FLUID MEDIA. 
 
 1. Flesh Water. Even the simple decoction, made by cook- 
 ing meat in water, with an acid reaction, affords a good nutri- 
 ent substance for a large number of bacterian forms, but 
 when it is neutralized by the addition of a little sodium carbon- 
 ate, a fluid is obtained in which a large number of the most 
 important pathogenic germs yet known, also thrive. For the 
 preparation of broth, different kinds of meat are used accord- 
 ing to circumstances, and the procedure is somewhat different 
 in the various laboratories. This is worth noting, because 
 we must be prepared to recognize in our present imperfect 
 knowledge concerning the nutrition of bacteria, that slight 
 variations in the preparation may give rise to differences in 
 the results of cultures. 
 
Bacteriological Technology. 19 
 
 The two most common methods of preparation are the 
 following- : 
 
 a. Bouillon (B). A pound of lean, scraped, or finely 
 chopped beef, is placed in an enamelled kettle, or a large flask, 
 with a litre of distilled water. The mixture is cooked for half 
 an hour and filtered. The filtrate is neutralized or rendered 
 slightly alkaline by adding- to it, drop by drop, a solution of 
 carbonate (or phosphate) of sodium. It is then again boiled 
 for about an hour, by which time the insoluble albuminoids 
 are all coagulated (C. Fraenkel), after which it is allowed to 
 cool, when the fat solidifies. Having been once more filtered, 
 the clear broth is filled into small vessels (usually medicine 
 bottles [or test-tubes], plugged with cotton, c/. p. 21), in 
 which it is finally sterilized in the steam-cylinder for a quarter 
 of an hour. For greater security, it is again sterilized on the 
 following day (disconnected sterilization, c/. p. 7). With 
 each new cooking, the clear filtered broth may become clouded, 
 but the turbidity disappears on cooling, otherwise it is neces- 
 sary to repeat the cooking and filtration several times, until 
 the fluid is obtained perfectly clear. This decoction of meat 
 is known in the laboratory as bouillon (B) in contrast with the 
 following. 
 
 b. Flesh water (K), in the more restricted sense. A pound 
 of chopped lean beef is covered with a litre of water and set 
 in an ice-chest for twenty-four hours, after which it is thor- 
 oughly shaken and filtered through muslin, the juices being 
 well wrung out from the meat. In this way a litre of flesh 
 extract is obtained, which is then cooked, filtered, rendered 
 slightly alkaline, etc., as before. 
 
 The addition of 0.5 per cent of table salt increases the value 
 of broth and flesh extract as a culture fluid for a number of 
 bacteria (Miquel). 
 
 The addition of five per cent of glycerin (before the last 
 neutralization) gives an excellent medium for the tubercle 
 bacillus (Roux and Nocard). 
 
 Obviously, there may also be occasion for varying the com- 
 position of the bouillon by the addition of various other sub- 
 stances, e.g., peptone, cane or grape-sugar, acetic acid, etc. 
 
 A useful flesh-water can also be obtained by the solution in 
 water of a suitable quantity of some meat-extracts, followed 
 by very careful " disconnected " sterilization (because of the 
 
2O Bacteriological Technology. 
 
 numerous resistant germs often present in these extracts), 
 neutralization, filtration, etc. 
 
 c. Liebig's extract (E), 5 gm. to a litre of water, is to be 
 recommended, and especially 
 
 d. Cibil's extract (C), 20 gm. to a litre of water 
 
 Of other nutrient fluids, only the following-, of proved and 
 recognized value, will be named : 
 
 2. Aqueous decoction of liver, lungs, and other viscera. 
 
 3. Neutralized or slightly alkaline wine (used at one time 
 by Pasteur) in the pure cultivation of the bacillus of splenic 
 fever, on a large scale. 
 
 4. Infusion or decoction of wheat, hay, cabbage. 
 
 5. Yeast- water, a filtered and sterilized decoction of 100 
 parts water to 10 parts compressed yeast. 
 
 For the cultivation of yeast and moulds, the following are 
 especially adapted : 
 
 6. Beer-wort, a decoction of dried and pulverized malt, ob- 
 tainable at every brewery. This must be cooked for an hour 
 and then filtered, before use, but it is hard to obtain it clear. 
 
 7. Decoction of horse dung (and of the excrement of other 
 herbivora). One part of fresh horse dung is mixed, by the use 
 of a glass rod, with three parts of water, set in a cool place for 
 twenty-four hours, after which the mixture is cooked for an 
 hour, and filtered through a double filter, which is a very slow 
 process (unless a filter pump is used). The filtrate is again 
 cooked for some time and if necessary refiltered, after which 
 it is filled into small receptacles, sterilized in the steam- 
 cylinder, and boiled twice for fifteen minutes, with a day be- 
 tween. 
 
 8. Decoction of prunes, which is best prepared as follows : 
 The prunes are allowed to stand for a day in a little water, in 
 w r hich they are then cooked carefully, so that they remain 
 unbroken. The fluid is afterward filtered and boiled down 
 somewhat. In some cases it may be desirable to reduce the 
 acidity of the decoction by the addition of sodium phosphate. 
 
 9. Decoction of other dried fruits, e. g., raisins, dried pears, 
 etc. 
 
 The horse dung, prunes, raisins, etc., used in making these 
 decoctions, may be preserved in a sterile condition, for use as 
 solid media in the culture of moulds, yeasts, etc. 
 
 A neutral or very slightly acid beer-wort is especially 
 
Bacteriological Technology. 2 1 
 
 adapted to the culture of Mucorini; while for the various 
 species of Aspergillus a simple acid mixture of wort and 
 prune-juice is an especial!}^ good medium, as O. Joh. Olson 
 has told me. A, mixture of the three liquids numbered 6, 7, 
 and 8, may at times be useful. The reader is referred to 
 Chapter XL for directions for collecting- and preparing prima- 
 rily sterile nutrient fluids. 
 
 When a large quantity of culture fluid has been prepared, 
 it is distributed in several medicine bottles (or Erlenmeyer 
 flasks) holding one or two hundred grams, previously plugged 
 with cotton and sterilized at 150 C. In these smaller vessels 
 the fluid is finally sterilized in the steam cylinder for five to 
 fifteen minutes on two sucessive days. It can then be pre- 
 served as long as is wished, in a dry place, provided the flasks 
 are well plugged. The safest plan is to use a large cotton 
 plug, and to tie over it several layers of filter paper (or to 
 cover it with a sterilized thin rubber cap, such as the Germans 
 now use extensively). 
 
 Formerly "Pasteur's fluid" (pure rock-candy, 10 gm; am- 
 monium acetate, 0.1 gm.; and the ashes of 1 gm. yeast, all dis- 
 solved in distilled water), " Mayer's fluid," or " Cohn's fluid " 
 (potassium phosphate, 0.5 gm.; magnesium sulphate, 0.5 gm.; 
 tribasic phosphate of potassium, 0.5 gm. ; acetate of ammonium, 
 1 gm. ; water, 100 gm.) were used. These fluids are little suited 
 to the cultivation of bacteria. It must also be remembered 
 that Pasteur by no means introduced such mineral solutions as 
 suitable nutrient fluids for microbes, but to show (1858) that 
 yeast cells can produce albuminoids from a carbo-hydrate and 
 an inorganic nitrogenous compound, when the necessary ash- 
 constituents are also present. 
 
 B. SOLID MEDIA. 
 
 The systematic use of solid nutrient media, especially of 
 nutrient gelatin (Robert Koch, 1881), marks a turning point in 
 the history of bacteriological technology. Brefeld had previ- 
 ously employed nutrient gelatin, but essentially only to check 
 the drying of slide-cultures. The starting point for. Koch's im- 
 portant discovery was the long-known fact that the cut surface 
 of a cooked potato, laid away for some time exposed to the 
 air, becomes the seat of large and small colonies of mould, 
 
22 Bacteriological Technology. 
 
 yeast, and bacteria, the last two of which often occur as 
 small distinct, slimy colonies of various colors. Each colon}^ 
 as a rule, contains only one form of yeast or bacteria, which 
 has developed from a germ that fell from the air and found 
 in the potato a favorable soil for its growth (first noticed by 
 Hoffmann, in 1869, and utilized by Schroeter in 1872, in his 
 cultivation of pigment bacteria). If we imagine these germs 
 to have fallen, not on the solid surface of the potato, but on 
 an equally large surface of some fluid in which they could 
 thrive as well, it is easily seen that the several forms would 
 have run together after a short time, motile and quiescent 
 being mixed together. Some of the germs which succeeded 
 in developing upon the potato, where they found space un- 
 disturbed by other colonies, would, perhaps, have failed to 
 develop at all in the fluid, yielding to others in the struggle 
 for existence. The same germs which in the fluid produced 
 a motley tangle of intermingled forms, gave a series of well- 
 separated colonies, on the solid medium. 
 
 When Koch had become aware of the extraordinary ad- 
 vantage offered by cultures on solid media over those in fluids, 
 he sought to give various useful culture fluids a solid form, 
 and he succeeded in doing this by gelatinizing them, in the 
 manner to be described. The "nutrient gelatins" so pre- 
 pared, have the advantage over potato, that their chemical 
 composition can be varied within wide limits, so that solid 
 culture media may be produced for such bacteria as cannot be 
 found on potato. They are, further, transparent, which ren- 
 ders possible the observation of the growth of bacteria within 
 the gelatin, as well as the microscopic examination of the 
 culture. Finally, they are liquefiable at a low temperature, 
 which is of decided value for their application to the isolation 
 of the different bacterian germs. 
 
 A low melting point, however, limits in some ways the use- 
 fulness of gelatins, since it makes it impossible to employ 
 them for cultures at much above 20 C. Koch therefore in- 
 troduced for such cultures a second gelatinizing substance, 
 agar-agar, which remains solid at the highest temperatures 
 used for culture investigations. The same remark applies to 
 sterilized blood-serum, which Koch found a method of pre- 
 paring. 
 
 It was said above that the introduction of solid culture- 
 
Bacteriological Technology. 23 
 
 media marks a turning- point in the history of bacteriological 
 technology. This is doubly true. In the first place, it has be- 
 come possible by them to surely and easily isolate, and culti- 
 vate in a state of purity, the various bacterian forms, as is 
 evident from what has been said, and as will be shown more 
 in detail in the next chapter. But the introduction of solid 
 media has also in many ways simplified bacteriological work, 
 making it possible to work with far simpler apparatus, and at 
 the same time with far better control and far greater cer- 
 tainty, aside from other reasons, because any accidentally in- 
 troduced foreign germ manifests itself more readily when it 
 forms a limited colony on or in the gelatin, than when its 
 progeny in a fluid become scattered and mixed among- all the 
 other bacteria. 
 
 It must not be forgotten, however, that there are also limits 
 to cultivation upon solid media, and that it by no means neces- 
 sarily renders fluid cultures superfluous; so that the same 
 care is due to the technique of the latter as formerly. To men- 
 tion a single instance of many : it is obvious that experiments 
 concerning the fermentation products of bacteria, and their 
 nutrition, may demand the use of culture fluids which have a 
 very simple chemical composition, but which would be changed 
 into very troublesome and complex mixtures by the addition 
 of gelatin or agar-agar. In addition to this, the usefulness 
 of a culture fluid for certain bacteria is at times lessened or 
 destroyed by the addition of gelatin, etc. 
 
 1. Boiled Potato. According- to circumstances, the pre- 
 paration and application of potatoes varies a little, so that one 
 either : 
 
 1. Simply cuts them in two, and lays them in a moist 
 chamber. The problem in preparing- them is first and chiefly, 
 to remove or sterilize the dirt adhering to them, which always 
 contains larg-e numbers of particularly resistant bacillus spores. 
 To this end as clean and smooth-skinned potatoes as possible 
 are selected. Then they are repeatedly washed in water, and 
 any remaining- dirt is carefully scrubbed off under water, with 
 a brush. By the use of a pointed knife any diseased or injured 
 pa^ts of the skin, as well as the changed parts of the under- 
 lying- parenchyma, are removed. The potatoes are then laid 
 for some time in a 0.1 per cent, sublimate solution, wrapped 
 singly, without being- dried, in thin wrapping paper, and ex- 
 
24 Bacteriological Tcclinology. 
 
 posed for half an hour to streaming- steam at 100 C. After 
 twent3^-four hours the steaming- is repeated for fifteen minutes 
 and the potatoes are ready for use. They are taken from the 
 paper one at a time, held between thumb and one finger of 
 the left hand, which has previously been washed in sublimate, 
 and are halved by a table knife that has been carefully steril- 
 ized by being drawn several times through the flame, or by 
 prolong-ed heating- at 150 C. (wrapped in paper in the steril- 
 izing- oven). The halves are then quickly laid with the cut 
 faces up, in a moist chamber, e.g., under a bell glass set on an 
 earthen plate (or in the flat pairs of trays similar to Fig-. 13, 
 but about 20 cm. in diameter which, though they cannot be 
 so easily obtained, are safer and have the advantage that 
 they can be set away one on top of the other). Dish and bell 
 glass have first been carefully cleaned in water and rinsed in 
 1.0 per cent sublimate; and one or two thicknesses of filter 
 
 FIG. 12. FIG. 13. 
 
 FIGS. 12 and 13. Shallow and Deep Glass Trays in Pairs, the Larger serving as a Lid for 
 
 the Smaller. 
 
 paper moistened in the same solution have been laid in the 
 bottom of the plate. A potato cooked and divided in this 
 manner, placed under a bell glass is one of the simplest ar- 
 rangements for cultivating bacteria, but it is always exposed 
 to contamination by germs from the air. This is avoided by: 
 
 2. Dividing the potatoes into prismatic pieces (or cylinders, 
 by the use of a small-sized tin cutter made like common apple 
 corers) and putting these in plugged test-tubes. In this case 
 it is only necessary to cleanse the surface of the potato with 
 brush and sublimate solution. After the potatoes have been 
 steamed once for five minutes, they are pared and with sterile 
 instruments cut into pieces which are quickly placed in the 
 test-tubes already sterilized in the usual way, after which they 
 are steamed for a quarter of an hour. 
 
 3. Nicer cultures are obtained by cutting the cooked and 
 pared potato into round discs, which are laid in the bottom 
 of a small pair of glass trays (Fig. 13, cf. Fig. 11, as well as 
 Soyka's museum cultures), which after being wrapped in paper 
 
Bacteriological TecJmology. 2 5 
 
 are then steamed for fifteen minutes. Such discs are best cut 
 out by aid of a small tin ring 1 . 
 
 4. Potato broth prepared by mashing- pared boiled pota- 
 toes and adding- a proper amount of water can be occasionally 
 used with advantage, if an especially large culture surface is 
 desired. 
 
 2. Gelatinized Media. If we desire, according to Koch's 
 directions, to change our different culture fluids into solid and 
 transpsarent but liquefiable substances we make use of the- 
 f olio wing: 
 
 a. Q-elatin. The finest French gelatin, which comes in 
 thin oblong sheets of about 2.5 gm. weight, is used. Five, or 
 commonly ten, per cent of this is added to the nutrient fluid, 
 dissolved, cooked, rendered slightly alkaline, cleared, filtered, 
 poured into smaller vessels and steamed twice with a day's 
 interval. 
 
 b. Agar-agar (The Asiatic name of several peculiar gela- 
 tinous alga 2 ;, which grow in the Indian Archipelago and come 
 into the market, dried in yellowish cartilaginous strips [or 
 spongy prisms] ). When cooked in water this forms a stiff 
 jelly and can be added to the various culture fluids in a quan- 
 tity of 1 to 2 per cent, precisely like gelatin. As a rule we use 
 1.5 per cent, which is dissolved lay long cooking and rendered 
 slightly alkaline, after which it is cleared, filtered, etc. 
 
 In passing to a fuller description of the preparation of the gel- 
 atinizing substances, we must dwell briefly on the advantages 
 and disadvantages attending the use of each. Agar-agar was 
 introduced b}' Koch, as above indicated : (a) because it melts 
 at a much higher temperature than gelatin, and can there- 
 fore be used for cultures on a solid medium at a higher tem- 
 perature (30-40 C. or higher) ; (b) it has also the advantage 
 as compared with gelatin that it endures cooking for a longer 
 time without diminution of its gelatinizing power; (c) there 
 are many bacteria which liquefy gelatin in their growth, but 
 do not affect agar in this manner, which in many cases is a 
 great advantage, especially in isolation -cultures. On the 
 other hand, it must be said: (a) that because of its low melt- 
 ing point, gelatin is' better adapted to the isolation of germs ; 
 (6) it gives a filtrate as clear as water, while it is very diffi- 
 cult, not to say impossible, to get perfectly clear nutrient agar; 
 (c) the difference between the mode of growth of different 
 
26 Bacteriological Technology* 
 
 bacteria shows far more clearly in gelatin than in agar, so 
 that two species which give in gelatin colonies of very differ- 
 ent appearance, sometimes appear identical when they have 
 grown in agar. 
 
 c. Agar-Grelatin. To Jensen is due the credit of combin- 
 ing the good qualities of both media, and avoiding their disad- 
 vantages, by adding to culture fluids 5 per cent of gelatin, 
 and 0.75 per cent of agar. The introduction of this mixture 
 marks a real advance, and it is worthy of use as almost the 
 chief culture-medium, since it is easily filtered clear, and lique- 
 fiable at so low a temperature that it can be used without 
 difficulty for plate-cultures, though it remains solid at 30- 
 40 C. 
 
 d. Irish-Moss (Chondrus crispus), rarely used. Neisser 
 recommends a strength of 2.5 per cent. 
 
 The preparation of gelatins, agars, and agar-gelatins, is 
 effected essentially in the same manner, so that a single de- 
 scription will suffice for all. But the length of time during 
 which the solutions can be kept at the boiling point for steril- 
 ization, cleaning, etc., must be relatively short for those con- 
 taining gelatin, which otherwise loses its powder of solidifying. 
 Agar, on the other hand, endures long cooking, while for ag'ar- 
 gelatin, a golden mean is kept. Gelatin, as a rule, is cooked 
 ten minutes before filtration, and ten minutes after. Agar is 
 best cooked forty-five minutes before, and a like time after; 
 and agar-gelatin twenty minutes before and thirty minutes 
 after filtering. Further than this, only clarifying and filter- 
 ing demand special mention. 
 
 Clarifying. When the" nutrient jelly has cooked long 
 enough, it is allowed to cool to somewhere about 50 C. An 
 egg is then broken into 100 gm. water, and gradually added 
 to the cooled but still fluid mixture, with which it is thor- 
 oughly incorporated. For a litre of jelly, the entire egg is 
 used, a correspondingly smaller quantity being used for less 
 than a litre. When the mixture is again heated to the boil- 
 ing point, the white of egg is precipitated in a large yellowish 
 curd, floating in a perfectly clear. fluid. After cooking for 
 some time, the next step is proceeded to 
 
 Filtering. This must necessarily be done while the jelly 
 is still warm. If little is to be filtered, it is merely necessary 
 to heat it up well before pouring it over the filter, so that,. 
 
OF THB 
 
 DIVERSITY 
 
 Bacteriological Technology. 
 
 27 
 
 notwithstanding- the cooling, it will remain fluid long- enough 
 for all to run through. But in this case, both the funnel and 
 flask need to be first warmed. It is very easy to warm and 
 sterilize flask, funnel, and filter, as well as to moisten the 
 latter, in the manner shown in Fig. 14. A layer of water one 
 or two centimetres deep is poured in the flask, and the funnel 
 and filter are set in its mouth, the top of the funnel being 
 covered with several thicknesses of filter paper, over which a, 
 plate of zinc or asbestos is laid (glass is apt to break). By 
 heating the water to the boiling point for a few minutes, 
 everything is sterilized, warmed, and moistened, in a single 
 operation. While the water is still hot, 
 it is poured out, and filtration can begin ; 
 the funnel being kept covered with a zinc 
 or asbestos plate which prevents cooling 
 to a considerable extent. 
 
 When larger quantities (e.g., a litre) 
 are to be filtered at once, there is danger 
 that the gelatin may stiffen before the 
 completion of the process, even though it 
 was at first almost boiling. This is pre- 
 vented by using a hot- water funnel (Fig. 
 15) a double-walled water bath with a 
 projecting arm at b. The apparatus is 
 FIG. i4. simple Filtering filled with water through the hole a, and 
 Arrangement. ig kept vvarm throughout the filtration by 
 
 means of a flame set under b. Such a double-walled funnel 
 can be made by any tinner, and is far more convenient than 
 the single- walled Plantamour funnel supplied by dealers in 
 apparatus, since the latter must be plugged with a perforated 
 rubber stopper at c, as the glass funnel limits the water on 
 the inner side. To prevent too many germs from falling from 
 the air into the flask, a little sterile cotton is stuffed into the 
 mouth of the latter, about the tube of the funnel. 
 
 As examples, the preparation of the three most frequently- 
 used nutrient jellies is given in detail. Other combinations 
 (e.g., C. A. G.) are also to be recommended. 
 
 C. G. Cibil's Gelatin. 20 gm. Cibil's extract is added to 
 1 litre of distilled water, in which 100 gm. gelatin is then 
 dissolved. Heat till all is dissolved. Render slightly alka- 
 line by addition of sodium carbonate. Boil for ten minutes. 
 
28 
 
 Bactcriolog ical TccJinology. 
 
 Cool to 50 0. Clarify, as indicated above. Cook again for 
 ten minutes. Filter through two thicknesses of paper in the 
 hot-water funnel. Pour into smaller vessels, and sterilize for 
 a short time in the steam cylinder on two or three successsive 
 days. 
 
 E. P. A. Peptonized Agar. 5 gm. Liebig's extract; 30 gm. 
 peptone; 5 gm. cane-sugar; 15 gm. agar; 1 litre distilled water. 
 Cook for an hour, render slightly alkaline, and cool to below 
 60 C. Clarify, cook again for at least an hour, fill into bottles 
 or test-tubes, and steam for ten minutes on each of two or 
 
 FIG. i5. Plantamour Hot-water Funnel, for use with Gelatin and Agar. 
 
 three successive days. If 5 per cent of sterile glycerin is 
 added to this agar, and the whole neutralized, it forms the 
 glycerin B. P. A. recommended by Roux and Nocard for the 
 cultivation of tubercle bacilli. This is far easier to prepare 
 than serum, which is used for the same purpose, cf. infra, p. 
 29 et seq. and 32. 
 
 K. P. A. G. Peptonized Agar-Gelatin.To a litre of fil- 
 tered flesh-water (pp. 18, 19) add 5 gm. table salt, 10 gm. pep- 
 tone, 50 gm. gelatin, and 7.5 gm. agar. Treat like E. P. A., ex- 
 cept that it is to be cooked each time, only 20 to 30 minutes. 
 It is safest before final filtration to filter a little into a test- 
 tube and see if it remains clear after boiling 10 to l^minutes. 
 
Bacteriological TecJinology. 29 
 
 From my experiments, jellies made according- to Mahn, by 
 the use of two per cent of Cibil's extract (C. G. C. A. C. A. 
 G.) afford quite as good a medium for many sorts of bacteria 
 as the far more expensive peptonized flesh-water or meat 
 gelatins, with or without sugar. 
 
 For the cultivation of moulds or yeasts, jellies are prepared 
 by adding G. or A. to the nutrient fluids named above. Espe- 
 cially to be recommended, are : 
 
 Beer- Wort Agar (B. A.), prepared of equal parts wort and 
 water, with 1.5 per cent agar. 
 
 Raisin Gelatin (R. G.), a decoction of 250 gm. raisins in a 
 litre of water, to which is added 10 per cent of gelatin. 
 
 3. Serum. Some large glass jars (e.g., pickle-jars cf. Fig. 
 63) are tied up in three layers of paper, and sterilized at 140 C. 
 They are filled with blood from oxen, calves, or horses (lambs' 
 blood is not to be recommended), collected during the slaugh- 
 tering of the animal, with as great cleanliness as can be 
 obtained in a slaughter house. Care must be taken to avoid 
 shaking the jars, and they are at once set away to coagulate, 
 preferably in cold water. After this, they are kept in a cold 
 place (best of all an ice-box), for 24 to 36 hours. The serum 
 which has now separated out, is transferred in small quanti- 
 ties into plugged and sterilized test-tubes, by aid of a pipette 
 that has been carefully sterilized. The greatest possible care 
 is taken throughout, since any contamination will be more 
 fatal here than in preparing the other culture media, because 
 the final sterilization must be effected at a low temperature 
 and consequently in an incomplete manner. Wetting the in- 
 side of the test-tubes toward the top with serum is especially to 
 be avoided, as this part of the tube cannot be immersed in the 
 water during the subsequent heating. The water bath (Fig. 4) 
 is now filled with the test-tubes containing- serum, and heated 
 until the thermometer indicates 58 to 60 C., at which tem- 
 perature it is kept for a little over an hour, by means of a 
 small flame. This is repeated daily, for a week. The serum 
 treated in this manner has become perceptibly clearer than it 
 was originally, a small amount of whitish precipitate has 
 separated, and a thin oily layer (of cholesterin) floats on the 
 surface. All of the less resistant germs are destroyed by the 
 heating. 
 
 It remains to solidify the serum without loss of its trans- 
 
30 Bacteriological Technology. 
 
 parency. Koch effects this by prolonged heating- at 68 C. 
 The water bath is brought up to this point and carefully kept 
 there during the process. According to the peculiarities of 
 different lots of blood, the time required for solidifying serum 
 varies from one to several (6 to 8) hours, so that it is necessary 
 to examine a couple of tubes from time to time to see if the 
 coagulation has begun. The heating is stopped when it be- 
 comes easy to invert the test-tube without loosening the serum, 
 which is now an amber-yellow jelly, in all cases sufficiently 
 clear in transmitted light to permit observation of the pecu- 
 liarities of cultures growing along inoculation punctures made 
 in it. The fewer red corpuscles originally present in the 
 serum, the clearer and more attractive it becomes after coagu- 
 lation; but use can be made of a somewhat red serum, espe- 
 cially for cultures on the surface. 
 
 The last heating at about 70 C. not only gelatinizes the 
 serum, but also naturally contributes to a complete steriliza- 
 tion; yet one can onty be sure of having killed all germs by 
 convincing himself that the tubes remain free from bacteria 
 after standing for some time, a couple of weeks, at the tem- 
 perature of the room, or three or four days in the brood-oven 
 at 30 C. (Cf. Chapter VI.) 
 
 An inconvenience in the use of the water bath for steriliz- 
 ing and coagulating serum, already indicated, is that the cot- 
 ton plugs and the upper part of the tubes, which project above 
 the water, are not exposed to so high a temperature as the 
 thermometer shows. This is avoided by using, instead of the 
 water bath, a common hot chamber of the sort indicated in 
 Chapter VI., such as it is always necessa^ to have for use as 
 a brood-oven. 
 
 If it is desired to solidify the serum with a large oblique 
 surface (Fig. 5, II.), the test-tubes must be laid in the chamber 
 in a nearly horizontal position. They are best put in a flat 
 box which can be obliquely set in the brood-oven. 
 
 Koch uses for this purpose a shallow, quadrangular ther- 
 mostat covered with glass and felt. This is shown in section in 
 Fig. 16. The bottom of this is 30 cm. square (inside measure) 
 and the layer of water between the double walls is 6 cm. deep 
 in the bottom (aa) and 3 cm. wide at the sides (bb). The pro- 
 jecting edge of the outside wall (c) rises a cm. above the rest 
 and so incloses a space for a cover consisting of a square 
 
Bacteriological Technology. 3 1 
 
 plate of glass (d) (which renders possible a quick observation 
 of the temperature and the condition of the serum), and a 
 sheet of felt (e) which is a poor conductor. Water is poured 
 in through the tube (/), while air is allowed to escape through 
 a similar tube at the other end. The apparatus is set obliquely 
 by putting a couple of blocks (g) under two of its legs. This 
 size accommodates two rows of test-tubes between which a 
 thermometer is laid. 
 
 This combination of "Pasteurization" and "disconnected 
 heating," which has been outlined (cf. pp. 7, 8) first used by 
 
 FIG. 16. Shallow Thermostat for Solidifying Serum Obliquely. 
 
 Koch in preparing serum for the cultivation of the tubercle 
 bacillus, must always be used when the blood has not been 
 collected with the greatest possible cleanliness. But when the 
 occasion offers for collecting blood by skilfully bleeding the 
 animal, with sterile instruments, after carefully washing the 
 neck of the horse or cow with 0.1 per cent sublimate, it is 
 usually safe to omit the tedious and difficult preliminary steril- 
 ization and immediately proceed to solidify it jat 68 to 70 C. 
 after putting it into the test-tubes. 
 
 If it is desired to keep serum sterile for some time in a 
 large vessel, the method that has been described cannot be 
 used, but it is best to filter it through a Chamberland filter 
 
32 Bacteriological Technology. 
 
 (under low pressure) into a Pasteur flask (Fig-. 67), the tube of 
 which is then quickly sealed by melting-, and is first opened 
 when the fluid is distributed into test-tubes. 
 
 Serous accumulations, such as those of ascites, pleuritis, or 
 hydrocele, which can also be used as culture media (c/. Chap- 
 ter XL), are treated exactly like blood serum. 
 
 If 6 to 8 per cent of sterilized glycerin is added to the 
 fluid serum and this is solidified at a slightly higher tempera- 
 ture than that which has been given (75 to 78 C.) solid gly- 
 cerin serum is obtained, which, according to the observation 
 of Roux and Nocard, is a much better culture medium for the 
 tubercle bacillus than serum alone. 
 
 Serum can also be applied as a gelatinizing constituent of 
 culture media, like gelatin or agar. Toeffler has advanta- 
 geously used a mixture of three parts of sterilized fluid serum 
 and one part of neutralized bouillon, containing one per cent 
 of peptone, one per cent of grape sugar and 0.5 per cent of table 
 salt. The mixture was solidified at 66 C. 
 
 4. Softened White Bread. White bread is cut in slices 
 and the soft part is broken from the crust in small pieces, 
 which are laid in a thin layer under filter paper, to dry in the 
 air. When, after a few days, it is dry enough to crumble, it 
 is finely ground in a coffee-mill, and this dry bread powder is 
 kept in a jar covered only with paper. When it is to be used, 
 the desired quantity of the powder is weighed out, and poured 
 into a sterilized flask, plugged with cotton, so that it forms a 
 level layer in the bottom of this (about 8 gm. are used for one 
 of the smallest Erlenmeyer flasks). By the use of a pipette, 
 about two and a half times its weight of sterilized water is 
 slowly poured over it. The bread broth formed in this way is 
 sterilized twice by heating it half an hour in streaming steam, 
 with an interval of a day. 
 
 White bread can also be used in slices which, either with 
 or without previous sterilization by dry heat, are moistened 
 with sterile water and then sterilized in the steam cylinder. 
 But by pulverizing it, a more uniform and handy preparation 
 is obtained. 
 
 This moistened bread is particularly adapted to the culti- 
 vation of moulds, but many different bacteria also thrive upon 
 it. It can be improved and varied by moistening it with 
 bouillon, prune juice, decoction of manure, etc. (Brefeld). 
 
Bacteriological Technology. 33 
 
 5. Rice Milk. Very recently, Soyka has recommended the 
 following- mixture, which he has employed with success for his 
 sealed museum cultures (c/. Chapter VI.) : Rice meal, 10 gm. ; 
 milk, 15 cc.; neutral bouillon, 5 cc., mingled very thoroughly, 
 filled into glass trays by a pipette, and sterilized by discontin- 
 uous heating in streaming steam on two successive days, 
 during which it solidifies in the bottom of the tray as a white 
 opaque mass. 
 
 6. Other Culture Media. Of these, but three will be 
 named: a. According to Soyka, other things being- equal, 
 spore formation in bacillus anthracis is hastened considerably 
 by the addition of a certain quantity of sterilized sand to the 
 nutrient bouillon ; 2 to 4 cc. of bouillon to 25 gm. of sand gives 
 a suitable degree of moisture to this " artificial soil." x 
 
 b. Cultivation upon moistened blocks of plaster was first 
 used by Engel for inducing the so-called spore-formation in 
 yeasts. Hansen recommends blocks in the form of truncated 
 cones, 3 cm. high, 4 cm. in diameter at top, and 5 cm. at bot- 
 tom, which are placed in small glass trays 5 cm. deep, loosely 
 covered by similar inverted trays. Sterilized water is poured 
 into the trays so as to reach to the middle of the block. 2 It is 
 also possible to make these blocks of any desired form. I 
 have, for instance, used successfully small plaster cylinders 
 (moulded in glass tubes), that were placed in ordinary cotton- 
 plugged test-tubes. It is to be observed that a mixture of 
 eight parts of plaster and three parts of water is used in 
 making the blocks. The mould in which they are cast must 
 not be oiled. Before use they are sterilized by dry heat of 
 115 C., since a higher temperature robs' the plaster of too 
 much of its water of crystallization. 
 
 c. Eggs are shaken up so as to mix the white and yolk, 
 and the surface disinfected with sublimate solution, after which 
 a s.mall hole is made through the shell at one end, by use of a 
 needle, through which the inoculation needle is introduced, 
 and the hole closed by a little cotton and collodion. 
 
 d. Colored nutrient gelatin. Noeggerath prepares the fol- 
 lowing- mixture of concentrated aqueous solutions of aniline 
 dyes, representing approximately the spectral colors: meth- 
 ylene blue, 2 cc.; gentian violet, 4 cc.; methyl green, 1 cc.; 
 chrysoidin, 4 cc.; fuchsin, 5 cc., diluted with 200 cc. water. 
 This is allowed to stand 10 to 14 days. Of the blue-black or 
 
 3 
 
34 Bacteriological Technology. 
 
 dark gray fluid, 7 to 10 drops are added to each 10 cm. of pep- 
 tonized gelatin (K. P. G.), which is cooked a couple of times, 
 poured out on white porcelain plates, and infected by scratch- 
 cultures (as in Fig. 29). The bacteria by their growth produce 
 color changes in the gelatin, which can be used for diagnostic 
 purposes. 3 
 
 FILLING THE CULTURE VESSELS. 
 
 So far as serum, bread, and rice-milk are concerned, the 
 process has been already described. Only the gelatinizing 
 media need closer consideration. These are stored, like culture 
 fluids, in cotton-plugged medicine bottles holding 150 to 250 cc. 
 each. It is convenient to have, in addition to these, 
 smaller reservoirs, e.g., large test-tubes, so that 
 when only a little gelatin is needed it will not be 
 necessary to open a large flask, since each time 
 that one is opened, what remains in it must be re- 
 sterilized for safety. 
 
 When the contents of a storage bottle are to be 
 distributed into culture glasses they are melted in 
 the water bath, or better, the steam cylinder. Test- 
 
 d 
 * 
 
 I 
 tubes and other relatively wide-mouthed vessels ' 
 
 can be filled by pouring directly from the bottle, 
 or better, by the use of large pipettes (Fig. 17). 
 These are cleansed, dried, wrapped in paper, and 
 sterilized in the usual manner, if their size allows, in 
 the dry oven at 150 C., otherwise they are drawn 
 back and forth for some minutes through a gas 
 or alcohol flame. To avoid contamination of the 
 pipettes when they are laid down, they may be sup- 
 ported on small knife-benches (Fig. 18), such as are 
 frequently used by housekeepers, the upper side of 
 which is cleansed by flaming before they are used. 
 The only other precautions to be taken are, to close 
 the tubes as quickly as possible, and to see that 
 each tube is filled for at least 3 cm. with gelatin, 
 and that the top of the tube is not moistened where it comes 
 in contact with the cotton plug, or the latter will become 
 fastened to the glass. When flasks with a narrow neck are 
 to be filled, this is best done by aid of a common wash-bot- 
 tle, sterilized by steam. 
 
Bacteriological Technology. 3 5 
 
 After filling 1 , the glasses are finally sterilized in the steam 
 cylinder for five or ten minutes. If this apparatus cannot be 
 used, each test-tube or flask is boiled by itself. When the 
 gelatin has solidified, the test-tubes are best kept by wrap- 
 ping* a few together in paper, by which the surface, and espe- 
 cially the cotton, is kept from becoming dusty. 
 
 After standing- for some time, the g-elatin begins to dry 
 out, as is shown by the sinking of its surface at the middle of 
 the tube. When a needle is thrust into such gelatin, it does 
 not close after the needle is withdrawn, but a crack forms, 
 which may materially modify the appearance of the culture 
 when bacteria eventually develop along the puncture. Before 
 
 FIG. 18. Pipette on Knife-rest. 
 
 it is used, such gelatin should always be melted and allowed 
 to solidify again. 
 
 To prevent the drying out of cultures on gelatin or agar, 
 which is especially rapid when they are kept at an elevated 
 temperature, the cotton-plugged ends of the tubes may be 
 covered with small rubber caps. Care should be taken that 
 both the caps and the cotton plugs are absolutely sterile, or 
 there is danger of the cultures becoming contaminated. Mould 
 spores, especially, which are unable to germinate in the un- 
 capped and therefore dry plugs, germinate readily in the moist 
 cotton, and grow through it. For this reason, it is necessary 
 to singe the top of the plugs, and to carefully disinfect the 
 rubber caps in sublimate immediately before putting them in 
 place. The drying out of culture can also be prevented by 
 singeing the cotton, and then dipping the top of the test- 
 tubes in melted paraffin, which quickly hardens in an air-tight 
 layer. 
 
OHAPTEE IY. 
 
 PURE MATERIAL FOR CULTURES. 
 
 IT has been shown in the preceding- chapters that it is 
 relatively easy to sterilize apparatus and nutrient media, as 
 well as to keep them sterile for an unlimited time. The only 
 serious difficulty in the cultivation of bacteria has always been 
 connected with another point the procuring- of pure material 
 for starting- the cultures. The ubiquity and small size of bac- 
 teria have necessitated the application of quite peculiar and 
 complicated methods. Their omnipresence makes it hard to 
 avoid foreign germs, while their minuteness interferes with 
 the sowing of a single germ, as is done in the cultivation of 
 higher plants. 
 
 For certain forms of fungi this may be attained. Thus, 
 Brefeld long since (1874) postulated and fulfilled the require- 
 ments for his mould cultures, that they should originate from 
 a single spore, planted under microscopic control; and in 1883 
 Emil Chr. Hansen, in his work on yeast, which is so full of 
 importance for breweries, prepared his cultures, which demon- 
 strably start from one yeast-cell, by the use of other and 
 better methods than those used by Brefeld. 
 
 On the other hand, the evanescent, small, and little charac- 
 terized germs of bacteria cannot, as a rule, be handled in this 
 way. It has been necessary to seize upon other means of get- 
 ting pure inoculation material for cultures of these, and in 
 1881 Koch succeeded in a simple and ingenious manner in 
 overcoming the difficulties and indicating a way of isolating- 
 bacteria germs, which will surely remain in the future as the 
 chief method. 
 
 Before we proceed to a description of Koch's method, 
 which, with various modifications, will be constantly used in 
 the work that follows, we must briefly consider the earlier 
 methods of securing pure cultures, not only because of their 
 
Bacteriological Technology. 37 
 
 historical interest, but also because under certain circum- 
 stances, notwithstanding the discovery of the far more perfect 
 process of Koch, we may be obliged to use them, or may find 
 it advantageous to do so. 
 
 All of the methods which have thus far been used for se- 
 curing pure inoculation material may be arranged in two 
 principal groups: A, those in which use has been made of 
 physiological differences between the bacteria for separating 
 them; and B, those in which the actual separation of the germs 
 from one another is used as the means of isolation. 
 
 A. USE OF PHYSIOLOGICAL DIFFERENCES. 
 
 If, for instance, one virulent form exists among a number 
 of non-parasitic forms, in a putrid fluid, it may be obtained in 
 a state of purity by the inoculation of a suitable animal, in 
 the blood of which only the virulent form will come to devel- 
 opment while the others die. In this way, from putrid blood, 
 have been obtained the microbes of Pasteur's rabbit-septicae- 
 mia, Koch's mouse-septicaemia, and Davaine's rabbit-septicae- 
 mia. Or, for another example : according to Pasteur, in the 
 blood of an animal dead of splenic fever, which has been kept 
 at a high temperature, or which, from the size of the animal, 
 has cooled slowly, there is found, so soon as putrefaction has 
 set in, besides the anthrax bacillus, that which he has called 
 the vibrion septique (which is identical with Koch's bacillus 
 ceclematis maligni). Pasteur succeeded in isolating these two 
 pathogenic forms by utilizing their different behavior with 
 respect to free oxygen; for as the first is anaerobic while the 
 second is aerobic, and both grow in broth, he obtained a cul- 
 ture of one or the other by sowing the blood in receptacles 
 from which all air was removed and kept out by carbonic acid 
 (c/. Chapter VIIL), OF in an ordinary culture vessel. These 
 examples might be increased by many more, such as cultures 
 in acid and alkaline media, with or without the addition of 
 certain antiseptics, at higher or lower temperatures, etc.; but 
 only two of the methods of this class will be dwelt upon a 
 little more fully, viz. : 
 
 1. Klebs' Fractional Cultures (1873). A fluid in which a 
 large number of different bacteria live and thrive together, 
 will necessarily not contain an equal mixture of all these 
 
38 Bacteriological Technology. 
 
 forms at every point. Some of the quiescent forms will sink 
 to the bottom or adhere to the sides, while others can move 
 about everywhere in the liquid. Some forms will form super- 
 ficial pellicles, others thrive only below the surface, where 
 there is less oxygen. Moreover, according 1 to the nature of 
 the fluid, some forms will fill it with great rapidity, and far 
 surpass in numbers others which only manage to lead a re- 
 stricted existence in the liquid. If a very small quantity of 
 such a fluid containing bacteria is sown in a culture glass, in 
 all probability only a few forms will be transferred, and these, 
 less equally distributed, will develop in unequal numbers. If 
 an extremely small quantity of this new mixture, containing 
 fewer forms, is again transferred to a new culture flask, and 
 this process is repeated several times, in all probability a point 
 will finally be reached where the material used for the transfer 
 is absolute^ pure, i.e., includes only a single kind of bacteria. 
 
 This method has only slight value, and it is especially to 
 be observed that such fractional cultures are by no means to 
 be counted on as finally giving a pure culture of any specified 
 form among those that were found in the original fluid, not 
 even that form which was most abundant there. The final 
 product is often only one of the most w T idely distributed of the 
 common putrefactive bacteria, which, even though originally 
 present in small quantity, easily gains the advantage of the 
 others. Still this method of fractional cultures may serve a 
 useful purpose now and then, as supplementary to others. 
 
 2. Cohn's Heating Method (1876). It had long been known 
 that various organic fluids (milk, hay-infusion, infusion of 
 peas, etc.), notwithstanding rather long cooking and the ex- 
 clusion of germs from the air, became the seat of bacterian 
 development, and adherents of the theory of spontaneous gen- 
 eration had repeatedly taken this fact as favoring their doc- 
 trine; but it was first shown by Cohn in 1876, in a series of 
 experiments with hay infusion, that this must depend upon 
 the extraordinary resistance of certain bacillus-spores to heat. 
 The boiling heat rapidly destroyed all bacteria without spores, 
 but the latter (in Cohn's experiments those of bacillus sub- 
 tilis) where not killed a fact which Brefeld also showed later 
 by direct microscopic observation of the germination of the 
 cooked spores. This afforded a means of obtaining pure ma- 
 terial of certain spore-forming bacilli, and, as a rule, a pure 
 
Bacteriological Technology. 39 
 
 culture of bacillus subtilis can be obtained without difficulty 
 in the following 1 manner: A small quantity of water is poured 
 over some hay in a flask, which is kept for hours at 30 to 40 
 C. (either in the brood-oven, or directly over the flame, while a 
 thermometer is placed in the fluid). The dark reddish-brown 
 infusion is diluted with distilled w r ater until it reaches a clear 
 golden color (s. g. 1.006), when it is filtered through muslin, 
 neutralized with sodium carbonate, distributed in several ves- 
 sels plugged with cotton, and cooked for ten minutes, after 
 which it is set away in the brood-oven at 30 to 40 C. In the 
 course of a couple of days, a larger or smaller number of the 
 vessels will contain a growth of bacillus subtilis, which may 
 be known by the fact that it forms a firm rigid film on the 
 surface of the liquid. 
 
 It must be remembered that among- the various resistant 
 spores of bacilli there is a great difference in their ability to 
 endure heat of 100 C. Some survive only a very few moments 
 of cooking, while others bear this temperature for hours (cf. 
 Chapter XIL). The limitations of this method are indicated 
 by what has been said. It is possible to isolate by it only 
 those bacilli which form very resistant spores, and a really 
 pure culture is obtained with certainty only when the boiled 
 fluid does not contain several different forms with the same 
 power of resistance. Still there will be exceptional cases in 
 which there is use for cooking as an aid in isolation. 
 
 B. METHODS BY SEPARATION. 
 
 1. The Capillary-tube Method (Salomonsen, 1876). Greater 
 success has been reached by the use of methods in which an 
 actual separation of the original germs is secured. The oldest 
 of these is the following-, the starting- point for which was an 
 observation of the color changes in putrefying- blood. During 
 decomposition, bright red ox blood, defibrinated by whipping-, 
 assumes a dark red or red-brown color, which is partly due to 
 the removal of their color from the red corpuscles, partly to 
 deoxidation of the oxyhgemoglobin, and partly to other chemi- 
 cal processes. If the blood, immediately after being defibrin- 
 ated, is set away in a cylindrical glass where it can remain 
 entirely undisturbed at a comparatively low temperature, e.g., 
 10 C., it is seen that the change of color begins in spots here 
 and there in the mass of blood. A closer investigation shows 
 
Bacteriological Technology. 
 
 that these putrefactive blotches are due to bacteria (or moulds 
 or yeasts). Next the bottom (Fig 1 . 19), where the crowded 
 blood discs form a solid mass, in which the bacteria grow 
 equally in all directions, the spots are round, clearly denned, 
 and dark red. Above (6) they are elongated, often club- 
 shaped, less clearly outlined, and by no means of so dark a 
 color. Here the blood discs are suspended 
 in a relatively large quantity of serum, 
 and in this fluid the bacteria have been 
 able to sink uninterruptedly to the bottom, 
 marking their path by elongated spots or 
 streaks. 
 
 If this is to be utilized in obtaining pure 
 material of the various putrefactive bac- 
 teria which grow in blood, it is only neces- 
 sary to draw the defibrinated ox blood into 
 long capillary glass tubes (50 to 60 cm. 
 long and 0.5 to 1 mm. in diameter), and to 
 attach these to strips of card-board about 
 3.5 cm. wide, by means of a drop of varnish FIG. 19. Cylindrical 
 at each end, which at the same time seals Glass containing whipped 
 
 ' . Ox Blood showing Purifl- 
 
 them. The following can be recommended cation Blotches. a,serum 
 as a suitable cement: 8 parts of rosin, layer; 6, elongated disooi- 
 
 orations; c, round spots 
 
 melted with 2.5 parts of wax while the an d precipitated biood- 
 mass is constantly agitated. Of 1 part of cor puscies. 
 turpentine, enough is added, little by little, so that a drop of 
 the melted mass quickly solidifies when allowed to fall on a 
 glass plate. Another good cement is the "Cire Galoz" (to 
 be had of Alvergniat Freres, 10 Rue de la Sorbonne, Paris). 
 
 They are then laid away at the ordinary temperature of 
 the room, for daily observation. The putrefaction blotches 
 will soon appear ; some shortly after the drawing of the blood, 
 others many days later; some spreading with extraordinary 
 rapidity, others growing only slowly. The number of each 
 spot in sequence, the time of its appearance, as well as its 
 growth from day to day, are easily noted upon the card. The 
 growth is best indicated by pencil marks drawn every morn- 
 ing and night after the fashion shown in Fig. 20. This illus- 
 tration, which shows one end of a card to which is cemented 
 a capillary tube containing three putrefactive spots, renders 
 further description unnecessary. 
 
Bacteriological TccJmology. 41 
 
 These putrefaction spots come from the bacterian germs 
 which have chanced to get in the blood after its removal from 
 the animal, so that in one lot there may be many of them, in 
 another few; but each spot contains only a single sort of bac- 
 teria, developed from one germ. Consequently, when the clis- 
 colorations are relatively far apart in the tubes so that they 
 do not readily become confluent, each of them gives a small 
 pure culture, from which pure material may be obtained of a 
 specific form of bacteria. By this method, a mixed putrefac- 
 tion-flora was first separated into its elements, so that the 
 number of germs, the time of their development, rapidrty of 
 growth, etc., could be graphically represented. 
 
 2. By Dilution (Lister, 1878, Naegeli, 1879). This method 
 consists in diluting a fluid containing bacteria with sterile 
 
 FIG. 20. One end of a Pasteboard Bearing a Capillary Tube Filled with Blood. On the 
 card are noted the time (10 A. afternoon of 10th day; 9 M. morning of the 9th day), 'when 
 the spots appeared, the rapidity of their growth (indicated by the length of the lines), and 
 their number in series (/., //., V.) 
 
 water to such a degree that a given quantity of the mixture 
 holds only erne germ, and in using such an initial quantity 
 for inoculation, In the culture-flask, the offspring of this 
 single germ forms a pure culture. 
 
 Counting the germs in the original fluid is effected under 
 the microscope, by the aid of instruments similar to those 
 used for counting blood-discs. From this the number of 
 germs in, for example, 0.05 cc. of the fluid, is estimated, and by 
 aid of a sterilized graduated pipette, this quantity is added to 
 enough sterile water so that the mixture, after careful shak- 
 ing, must average half a germ for each 0.05 cc. When by 
 means of a sterilized graduated pipette, this mixture is inocu- 
 lated into a series of culture-vessels, so that 0.05 cc., is placed 
 in each, half of the glasses will not show any development of 
 bacteria, having received no germs, while in all probability 
 but one germ will have been placed in each of the others, 
 which will then contain a pure culture. Lister devised and 
 
42 Bacteriological Technology. 
 
 used this method for obtaining- pure cultures of lactic acid 
 bacilli. Naegeli, independently, cultivated micrococcus ureas 
 by aid of the method of dilution. 
 
 3. By the Use of Solid (Especially Gelatinized) Media 
 (Koch, 1881). This method consists in the isolation of the 
 germs upon or in solid culture-media, where, in their later de- 
 velopment, they give rise to separated colonies. If, in a flask 
 like that shown in Fig-. 9, VII., the bottom is covered with a 
 thin layer of nutrient g-elatin, and, after melting this at about 
 30 C., a very small drop of fluid containing bacteria is added 
 and thoroughly distributed through the gelatin by careful 
 shaking (cf. Chapter VIL), and after solidifying, the mixture 
 is set aside at room temperature, after a time small colonies 
 will appear at the surface of the gelatin. Each comes from 
 one germ, so that pure material for inoculation is obtained. 
 
 This is but one of the many forms in which the method of 
 Koch may be used. In a subsequent chapter, dealing with 
 bacteriological analysis, the method will be treated particu- 
 larly and in detail. 
 
 By aid of these methods, pure inoculation material can 
 comparatively easily be obtained of all aerobic bacteria (cf. 
 Chapter VIII.) which it is chiefly necessary to cultivate. It 
 remains only to consider the best way of transferring this to 
 the culture apparatus. 
 
CHAPTER V. 
 
 INOCULATING CULTURES. 
 
 THE transfer of pure material to one of the culture vessels 
 that have been described, is comparatively easy. The neces- 
 sary opening- of the different vessels, and the transportation 
 of the inoculation material through the air necessarily entails 
 danger of contamination. But this danger is very small, and 
 if the work is done rapidly and carefully, the transfer is un- 
 successful in a very insignificant number of cases. 
 
 The instruments needed are: 1, Platinum needles (Koch), 
 long capillary tubes of glass, Pasteur pipettes, and glass 
 needles. 
 
 1. The platinum needle consists of a glass rod about 27 
 cm. long and 4 or 5 mm. thick, in one end of which a piece of 
 platinum wire about 3 cm. long is melted as shown in Fig. 21. 
 Just before use, the surface of the glass rod is flamed, and the 
 platinum wire brought to redness throughout its entire length. 
 When, after a few seconds, it has cooled sufficiently, this is 
 brought in contact with the material to be transferred, and 
 quickly brought into the culture medium. 
 
 It is usual to have at hand some quite thin needles, and 
 some which are a little thicker. An advantage of the former 
 is that they cool quickly, while the latter are less flexible and 
 can for this reason be used when the needle is to be thrust 
 into more resistant substances, e.g., liver or lung (or when it 
 is necessary to apply considerable lateral pressure, as in dis- 
 tributing a colony of the tubercle bacillus over agar, etc.). 
 
 For certain cases, the platinum wire may be specially 
 shaped. Small loops (Fig. 21, b) insure the adhesion of larger 
 quantities of the inoculation material. A large loop (c), 
 catches a good-sized drop. It is bent at a right angle (d), 
 when one wishes to scratch the material into the surface of the 
 gelatin (cf. what is said elsewhere about cultures on potato, 
 slides, etc.). 
 
44 
 
 Bacteriological Technology. 
 
 When the inoculation must occur through a relatively 
 small opening-, through which the 
 glass rod can be passed with diffi- 
 culty or not at all, a sufficiently long- 
 piece of platinum wire can be used, 
 or small pieces (5 or 6 mm. long) are 
 employed by seizing them with ster- 
 ilized forceps, glowing- them, bringing 
 them in contact with the inoculation 
 material, and dropping them through 
 the small opening- into the culture 
 flask. Capillary tubes and glass nee- 
 dles can, of course, also be used in 
 such cases. 
 
 2. Capillary tubes usually have a 
 length of 20-25 cm. and are closed by 
 melting at both ends. Before being 
 used, one end is broken off, the surface 
 is flamed, and the open end dipped into 
 the fluid to be sown, when the other 
 end is broken, upon which the fluid 
 rises into the tube. The contents are 
 blown out into the culture-vessel to 
 be used ; care being- taken not to blow 
 all out, but to leave a little fluid in the 
 tube, thus preventing- contamination 
 from the air blown in. 
 
 3. Pasteur pipettes consist, as is 
 shown in Fig. 22, of a piece of glass 
 tubing, one end of which is closed by 
 a cotton plug, which must not project 
 be3^ond the glass, while the other end 
 is drawn out into a capillary tube 
 fused at the end. The plugged tubes 
 are sterilized at 150 C. Before being 
 used, the sealed end of the tube is 
 broken off, and this part of the glass 
 is flamed. The tube is filled by suc- 
 tion, and emptied by bio wing the fluid 
 out. These pipettes have the advan- 
 tage over capillary tubes of being more capacious, and they 
 
Bacteriological TecJinology. 45 
 
 can be used as culture-glasses by reclosing the capillary 
 extremity by heat immediately after filling-. See also Chap- 
 ter XL 
 
 4. Glass needles, i.e., glass rods drawn out into a very 
 slender thread at one end for about 15 cm., are better than 
 platinum needles in their perfect smoothness and rigidity, 
 which may occasionally be of importance, as in making- thrust- 
 cultures of anaerobic forms, where it is wished to avoid intro- 
 ducing small air-bubbles into the gelatin. 
 
 One can easily make these inoculation instruments for 
 himself by using- an ordinary Bunsen burner or a good spirit 
 lamp. A glass-blower's outfit naturally lessens the work, but 
 it is not necessary. 
 
 For platinum needles, a glass rod about 4 mm. in diameter 
 is divided into pieces 25 or 30 cm. long, by slightly marking- it 
 at the proper points with a triangular file, when by steadily 
 pulling- the glass in opposite directions at each side of the 
 scratch, with a slight side motion, it is easily and evenly 
 broken at the desired spot. The end of a piece into which the 
 needle is to be fastened is rotated in the flame with the left 
 hand, until it becomes red and soft, while a piece of wire of 
 the right length, grasped some 5 mm. from one end in a pair 
 of forceps, is brought to a white heat at this end and carefully 
 thrust lengthwise into the softened glass, which is then al- 
 lowed to cool gradually, being held for a few seconds close to 
 the flame. The other end of the glass is finally rounded off 
 by heating it to redness, rotating- it meantime. 
 
 A glass tube 6 or 8 mm. in diameter is easily drawn into 
 capillary tubes in the following manner : The tube is heated 
 to redness for a short distance (1-2 cm.), while being rapidly 
 revolved about its axis, until it becomes thoroughly softened, 
 when it is removed from the flame and drawn into a tube 
 about six feet long, which is melted off from the thicker glass 
 at each end. In the same way, by aid of the flame, the long 
 capillary tube is divided into pieces of the length indicated 
 above, which are at the same time hermetically sealed at the 
 ends. The strong heating of the glass, and the sealing of the 
 tube which immediately follows, insure freedom from germs. 
 The larger the original tube the greater the length for which 
 it is melted, and the slower it is drawn out or the shorter the 
 length of the capillary tube the coarser this will be, and con- 
 
Bacteriological Technology. 
 
 versely. This being- borne in mind, it is easy to experiment 
 until a tube of the diameter wished in a given case is obtained. 
 
 Pasteur pipettes are made by dividing a glass tube into 
 pieces 15 cm. long, by aid of a file. Each piece is then heated 
 in the middle while being revolved, and drawn out in the 
 manner already described into a capillary portion about 30 cm. 
 long, which is then melted and sealed at the middle, making 
 two pipettes. The sharp edges of the other ends are rounded 
 off by briefly glowing in the flame. After they are cooled, 
 they are plugged with cotton, and finally sterilized at 150 C. 
 
 Glass needles are drawn out in the 
 same way, solid rods being used instead 
 of tubing. 
 
 To indicate the many little manip- 
 ulations necessary in making a pure 
 transfer, a detailed account is here given 
 of (a) the inoculation of g-elatin in a 
 test-tube by use of the needle, and (b) 
 the inoculation of fluid in a narrow- 
 necked flask, by the use of small pieces 
 of wire. 
 
 a. Several tubes of gelatin that has 
 not dried out too much (cf. p. 474) are 
 chosen, and the cotton plugs are tested 
 by twisting them several times to be 
 sure that they are not glued fast to the 
 glass. The needle is sterilized as indi- 
 cated above. After allowing it to cool 
 for a couple of minutes, the tube from which the transfer is to 
 be made is opened by removing the plug by a twisting motion 
 which causes any fibres that may have adhered to the glass to 
 lie out of the way of the needle. The tube is held with the top 
 upward, or inverted, according as the bacteria growing in it 
 have liquefied the gelatin or not. The removed plug is held 
 so that the part of it which comes in contact with the inner 
 face of the glass shall not touch anything by which it might 
 be contaminated. The needle is now quickly plunged into the 
 culture, removed, and the plug replaced. (As a rule only the 
 tip of the needle is brought in contact with the culture, a suffi- 
 cient number of germs will always adhere to it. If for any rea- 
 son a large quantity is to be transferred, loops such as are 
 
 FIG. 23. Inoculating a Test- 
 tube of Gelatin. 
 
Bacteriological Technology. 47 
 
 shown in Fig-. 22, b c are used.) The tube to be inoculated, 
 held in an inverted position (Fig 1 . 23), is now quickly opened 
 with the same twisting- motion, the needle is rapidly thrust 
 into the gelatin at one or more points, the plug* replaced, 
 and the needle at once sterilized by glowing. A label contain- 
 ing- record of date, source of material used, etc., is then pasted 
 upon the tube. 
 
 b. The surface of the flask is carefully dried, after which 
 the lower part of the rubber tube and the contiguous part of 
 the neck are flamed by use of a burner or alcohol lamp, and 
 the rubber is slipped up a little, so that it can easily be re- 
 moved by one hand. Sterilized forceps are placed upon glass 
 benches (Fig-. 18) or in a shallow glass. Small pieces of plati- 
 num wire are kept ready in a glass tray. One of these is 
 grasped by one end with the forceps, at right angles to its 
 axis. The wire is broug-ht to a red heat, allowed to cool, and 
 plunged into the colony of bacteria, etc., from which the trans- 
 fer is to be made. With the left hand, the flask is now quickly 
 opened, the wire dropped into the narrow opening, the cap 
 firmly replaced, and a label prepared. 
 
OHAPTEE VI. 
 
 BROOD-OVENS AND THERMO-REGULATORS. 
 
 A MAJORITY of the bacteria known and cultivated up to the 
 present time thrive at the ordinary temperature of a room 
 (15 to 20 C.). Consequently if it is only desired to keep cul- 
 tures going 1 and to observe their growth, it usually suffices to 
 set them aside in a living room. For preserving cultures for 
 a long time, especially as museum specimens, Soyka recom- 
 mends inoculating solid media in the small glass boxes shown 
 in Figure 11, which are then carefully sealed with paraffin. By 
 this means he succeeds in checking the growth of the colony 
 at a certain point of its development, while the capability of 
 germination is preserved, and drying out is hindered. For 
 details see "Zeitschr. f. Hygiene/' 1888. After a shorter or 
 longer incubation period, they will be seen to start into 
 growth, and even by the naked eye it is possible to observe 
 the great dissimilarity which often exists between cultures of 
 different bacteria. Even in 1880, when fluid cultures were 
 almost exclusively used, I noticed in detail the microscopic 
 differences that cultures often present, and called attention 
 to their indications in judging of the purity of a culture, and 
 the diagnosis of bacteria, which was sometimes more, easily 
 made by the naked eye than microscopically. When, later, 
 Koch's gelatin cultures came into use, still more occasion 
 was offered for the observation of evident microscopic differ- 
 ences between cultures of bacteria, such, for instance, as are 
 shown in Figures 42 and 69. Sometimes it is very necessary 
 to cultivate bacteria at a higher and more uniform tempera- 
 ture than that usual in our living rooms, for some forms de- 
 velop quickly only at higher temperatures, others never thrive 
 below 30 C., and some species demand heat for the produc- 
 tion of spores. Moreover, indeed, as Pasteur first showed, 
 cultivation at a high temperature may change the physiolog- 
 
Bacteriological Technology. 
 
 49 
 
 ical character of bacteria, and is used in the fabrication of 
 " vaccines." It may also be frequently desirable to cultivate 
 a micro-organism for a long" time at a constant temperature, 
 whatever this may be. 
 
 Hence are used thermostats or brood-ovens, in which the 
 temperature is held unchanged for weeks or months at the same 
 
 FIG. 24. Brood-oven Covered with Felt, and Controlled by a Thenno-regulator. 
 
 point. The maintenance of a constant temperature is beset 
 with certain difficulties. The changes in temperature of the 
 room in which the thermostat stands is disturbing 1 ; to coun- 
 teract them the brood-oven is inclosed in as thick a layer of 
 some non-conducting- material as possible, and set up in a cellar 
 or similar place, where the temperature changes but little. 
 Variations in the strength of the source of heat used also act 
 in the same way, as is especially evident when gas is used, 
 4 
 
50 Bacteriological Technology. 
 
 since the gas pressure may vary much in the course of the day. 
 This may be obviated by the use of pressure-regulators and 
 thermo-regulators of various construction. 
 
 A serviceable small thermostat, comparatively cheap, and 
 self-regulating, is shown in Figure 24. It is a quadrangular 
 box of zinc or -copper, set upon a strap-iron support with legs 
 about 24 cm. high. The top and bottom, as well as three of 
 the side walls are double, and the space between them, which 
 is 2 to 2.5 cm. wide, can be entirely filled with water through 
 the opening (a), which is afterward closed by <a cork. The 
 fourth side is single, and serves as a door.. In the centre of 
 the top, an open tube 3 cm. in diameter is fastened into the 
 wall, piercing it. Into this by aid of a perforated cork, a ther- 
 mometer (b) is set, which projecting into the interior of the 
 box, can be examined without the necessity of opening the 
 thermostat. Usually a thermometer is also kept entirety in 
 the thermostat, standing in a vessel filled with oil, which 
 prevents the mercury from sinking too rapidly when the glass 
 is removed to observe the temperature. On the inner face of 
 the two side walls are fastened a number of small ledges (cc) 
 on which glass or tin shelves may be laid to provide support 
 for a large number of low objects. The outside of the box is 
 covered with felt, and provided with a glass tube for showing 1 
 the height of the water (d), and a faucet (e). It is heated b} T 
 a Bunsen burner, the tube of which has been removed, so that 
 it gives a pointed white flame, which permits the flame to be 
 reduced to a minimum with no danger that it will "snap 
 back." [If the brood-oven is to be kept but little above the 
 room temperature, there is some danger that the flame may 
 occasionally go out, allowing the escape of a large quantity 
 of gas into the room before it is discovered. For this reason, 
 the safety burner devised by Koch, and to be had of dealers 
 in bacteriological apparatus, though somewhat expensive, is 
 to be strongly recommended, since it promptly and automati- 
 cally cuts off the supply of gas in case such an accident 
 occurs. W. T.] The temperature is held at a constant point 
 by the use of the regulator (g), which is passed into the water- 
 filled space through an opening at b. Two generally used and 
 good types of thermo-regulator are shown in Figures 25 and 
 26, Rohrbeck's (Fig. 25) is especially to be recommended. 
 
 Reichert's latest regulator is shown in Figure 26. The gas 
 
Bacteriological Technology. 
 
 enters at a, passes through the opening- b, and out toward the 
 burner at c. The column of mercury is adjustable by the 
 screw d. In case this should rise so as to entirely close the 
 opening- &, the flame is not extinguished, for a minute opening- 
 is provided in the T-shaped tube at e, which permits the pas- 
 sage of just enough gas to keep the flame alive, without heat- 
 ing the thermostat appreciably. The right size of this pin- 
 
 FIG. 25. Rohrbeck's FIG. 26.- Reichert's 
 Thermo-regulater. Thermo-regulator. 
 
 FIG. 27. Bohr's Thermo-regulator. 
 
 hole is reached when the gas passing through it burns with a 
 perfectly blue flame. . . 
 
 Rohrbeck's modification of the Lothar-Meyer regulator, is 
 shown in Fig. 25. It is much more sensitive than the Reicherfc 
 instrument, since it is regulated by mercury and ether, the 
 vapor of which changes in tension comparatively strongly 
 with slight changes of temperature. The gas enters at a, 
 traverses the tube d, in which at the bottom an acute, sharp- 
 
5 2 Bacteriological Technology. 
 
 angled triangular opening (b) is made, and passes out toward 
 the burner at c. The mercury is indicated by line-shading, 
 vapor of ether fills the chamber cj [which is limited by a glass 
 diaphragm hi, prolonged downward into a tube open at the 
 bottom, which plunges well into the mercury]. When the ap- 
 paratus is warmed, the mercury is forced by the expanding 
 ether-vapor into the tube hh, say to the dotted line i, so as to 
 partly close the triangular opening, which is then enlarged 
 or diminished according as the mercury falls orrises. From 
 the form of the opening, and its -sharp angles, this adjustment 
 occurs very uniformly. The first adjustment of the apparatus 
 is effected by sliding the tube d up or down through the bored 
 cork by which it is adapted to the larger tube. Fig. 25 show 
 the regulator in its cheapest fprm. There is also a more im- 
 proved form, in which the tube d is of steel, and adjustable by 
 a fine screw, (Fig. 24, g). 
 
 A regulator constructed by Bohr (Fig. 27), offers consider- 
 able advantages as compared with these commonly used 
 models. The reservoir a, filled with air, is brought, with its 
 stopcock b open, into the chamber that is to be kept under 
 control. Shortly before the desired temperature is reached, 
 the cock b is closed, after which every rise of temperature, 
 however small, will cause an expansion of the air in a and a 
 displacement of the column of mercury, which closes the open- 
 ing d, allowing the gas to pass only through the reserve open- 
 ing e. It must be seen that the inside of the reservoir a is 
 not damp when the apparatus is put in use. Enough mercury 
 is poured in through / to reach as high as c7, so that this open- 
 ing is entirely closed by a slight displacement of the mercury. 
 It is advantageous to have the U-shaped tube constricted at c. 
 
 In addition to its great simplicity, the Bohr regulator is 
 superior to those previously described in that the same in- 
 strument can be used at any desired temperature below the 
 melting-point of the glass, and is adjusted for any tempera- 
 ture with extreme ease. It is also equally sensitive for all 
 temperatures. The influence that considerable barometric 
 changes exert on it (as well as on the Rohrbeck model) is 
 readily compensated by opening the cock (b) for a moment. 
 It further deserves mention, that by Bohr's regulator a con- 
 stant mean temperature can be maintained in larger spaces, 
 since the reservoir a may be given any desired form and size; 
 
Bacteriological Technology. 53 
 
 or a long- lead tube with one end hermetically sealed (pinched 
 or melted together) may be used as a reservoir, passing- into 
 various parts of the chamber. 
 
 A thermostat of this construction, with regulator, will gen- 
 erally be sufficient even for finer experiments, which demand 
 a constant temperature during months. In most cases, how- 
 ever, we need only to have a chamber in which bacteria can 
 be grown at something above 30 C., and for this a thermo- 
 stat is perfectly good and useful, even if it varies a few degrees 
 in the course of the day. If too much is not required, the reg-- 
 ulator may be entirely dispensed with, and an ordinary gas, 
 petroleum, spirit, or oil flame used. Of these four sources of 
 heat, the first and last are unconditionally to be recommended. 
 In case the apparatus is to be kept in our working room, 
 petroleum is less satisfactory because of its ill-odor, independ- 
 ently of the greater danger from fire. Alcohol is better than 
 [lard]-oil only in giving a flame free from smoke, while oil is 
 cheaper and requires less care in its use. 
 
 It is naturally most convenient to use gas when this is at 
 hand in the room. Since the gas flame as a rule needs to be 
 only small, when a small thermostat standing- in an inhabited 
 room is to be kept at 30 to 40 C., a pointed yellow flame is 
 always to be used (c/. p. 489). By a few days' experimenting, 
 the proper height of the flame is ascertained, and when the 
 temperature of the surrounding air does not vary too much 
 this affords a good guide. A regular periodical change in the 
 gas pressure, as at night, is easily compensated for by slightly 
 raising- or lowering the burner each day at the necessary 
 time. 
 
 If one is unable or unwilling to use gas, [lard]- oil is to be 
 recommended, especially if used as shown in Fig-. 28. A large 
 basin is filled to within a couple of centimetres of the top with 
 water (d). Over this is poured carefully a layer of oil 2 cm. 
 deep (c), upon which are placed one or several small floats (/) 
 with wicks, such as are used on the continent for night-lamps. 
 Such a floating burner gives an especially constant heat. As 
 the flame cannot be chang-ed in size, it is necessary to find 
 the right distance between it and the thermostat; and since 
 this must be kept as nearly constant as possible throughout 
 the day, it is usual to employ a large bowl and a thin layer of 
 oil, so that the wick shall not recede too far from the bottom 
 
54 
 
 Bacteriological Technology* 
 
 of the brood-oven as the oil is used up. The small wicks are 
 renewed morning- and evening-. 
 
 In Figure 28 is shown a still cheaper thermostat than that 
 represented in Fig. 24. It has no regulator, no tube for show- 
 ing the height of water, no faucet for removing this, and no 
 attached covering- of felt. But it can at any time be easily 
 
 FIG. 28. Simple Brood -oven Heated by an Oil-lamp 
 
 covered by rectangular pieces of felt or flat- sheets of cotton- 
 batting, of which four can.be sewed together at their edges, 
 while the fifth, over the door, is fastened only by its top to the 
 upper piece, so that it can be lifted when the door is to be 
 opened, while it is tied to the side pieces when the door is 
 closed. 
 
OHAPTEE VII. 
 
 BACTERIOLOGICAL ANALYSIS OF FLUID, SOLID, AND 
 GASEOUS SUBSTANCES; ESPECIALLY OF WATER, THE 
 SOIL, AND AIR. 
 
 MICROSCOPIC investigation shows very incompletely what 
 living- germs are present in a preparation. Different appear- 
 ing micro-organisms may represent different stages in the de- 
 velopment of a single species, and bacteria which look quite 
 identical may belong to entirely different species. Moreover, 
 all germs of bacteria are by no means recognizable as such 
 under the microscope; and the living are not certainly distin- 
 guishable from the dead. If the question is merely whether 
 some specified virulent form occurs in a sample of earth or 
 water, in case this form is pathogenic for animals, the most 
 direct answer will be obtained by inoculation. Water is sub- 
 cutaneously injected into a suitable animal. Earth is placed 
 in a little pocket made in the subcutaneous tissue. Bacteria 
 from the air are first collected in a sterilized filter of sand (cf. 
 analysis of air), which is then introduced into such a pocket. 
 But a satisfactory bacteriological analysis cannot to-day be 
 made without recourse to cultures. It is of especial interest 
 for the physician to know these methods, since they afford 
 the most important introduction to hygienic investigation of 
 water, earth, and air. Since the discovery of the cholera 
 spirillum they have also been included among the best 
 methods of clinical investigation. An exhaustive bacteriolog- 
 ical investigation includes a determination of the number of 
 bacteria present, as well as their identification; but for easily 
 understood reasons, in both hygienic and clinical investiga- 
 tions, qualitative analysis is most frequently of entirely 
 greater importance than quantitative. 
 
5 6 Bacteriological Technology. 
 
 A. LIQUIDS. 
 (With especial reference to water analysis.) 
 
 All of the methods indicated in Chapter IV. may find ap- 
 plication in the bacteriological analysis of fluids. Of these, 
 we usually resort to dilution, and isolating- the g-erms in nutri- 
 ent gelatin, as the two principal methods. 
 
 The latter, first indicated by Koch, and developed by him 
 to a hig-h degree of completeness, is alone sufficient in by far 
 the greater number of cases, and consequently will be treated 
 here in detail. Koch's method consists in mingling the fluid 
 containing- germs with liquefied gelatin, which is then spread 
 in a thin layer and allowed to solidify, being- protected from 
 atmospheric contamination. The mixing- and spreading can 
 obviously be effected in a great variety of receptacles, among 
 which are four deserving- especial attention. 
 
 a. Conical Flasks. A medium-sized flask (Fig. 9, VII.) 
 containing- a thin layer of g-elatin in the bottom is warmed just 
 enough to melt the gelatin (over-heating may destroy the 
 bacterian g-erms). By aid of a needle or pipette, a small 
 quantity of the liquid to be analyzed is then added, with the 
 greatest possible rapidity and care. The two are carefully 
 mingled, but without shaking, which might cause the forma- 
 tion of bubbles. To avoid this, the flask is held upright, and 
 several times carried around a large circle in a horizontal 
 plane. The fluid is set in so active movement by centrifugal 
 force that the commingling is perfect, without the introduc- 
 tion of bubbles into the gelatin, which is then allowed to solid- 
 ify. This is one of the simplest and surest methods by which 
 a Koch isolation-culture can be made. One disadvantage 
 attends it, viz. : the isolated colonies are not accessible, in the 
 flask, for microscopic investigation, and less accessible for ex- 
 amination by the naked eye or with a lens than in the follow- 
 ing cases. 
 
 b. Test-tubes. The mixture can also be effected in a test- 
 tube, on the inside of which the gelatin is then allowed to 
 harden in a thin layer (von Esmarch). For such "roll-cul- 
 tures," wide tubes are best, with about 10 cc. of nutrient gela- 
 tin. Naturally, one may add the fluid as indicated in Chap- 
 
Bacteriological Technology. 57 
 
 ter V., and subsequently melt the. gelatin over a water bath, 
 or this may be done first. The mingling of the two is effected 
 as in a. The distribution of the gelatin over the wall of the 
 tube is secured simultaneously with its hardening in the fol- 
 lowing manner: The projecting portion of the cotton plug is 
 clipped or singed off, and the whole covered with a tight-fitting 
 rubber cap to keep the cotton dry, and the tube laid in a dish 
 of cold water, preferably ice-water. While the tube floats on 
 the water, it is kept with one hand as nearly horizontal as 
 possible, while with the other it is rotated until the gelatin 
 has become solid in a uniform layer over the entire inside of 
 the tube, which is then dried off and the rubber cap removed. 
 Occasionally the cotton plug becomes covered with so thick a 
 layer of gelatin as to prevent the access of air to the culture 
 in an injurious manner. In this case, cotton and gelatin may 
 both be pierced by a sterile platinum needle. The advantage 
 of the test-tube over a flask is that it takes up less room, and 
 its contents are somewhat more readily accessible for investi- 
 gation. 
 
 c. Q-lass Trays. It is convenient to use a pair of shallow 
 trays (Fig. 12) about 20 cm. in diameter and 1.5 cm. deep, which 
 are, of course, first to be sterilized, well wrapped in paper, 
 from which they are removed only immediately before being 
 used. Mingling the fluid which contains bacteria and the 
 gelatin, is effected in a test-tube as in the last case. The cot- 
 ton plug is removed and the mouth of the tube flamed [it is 
 better to clip the cotton off close to the edge of the tube, push 
 the plug down a little way, and then flame until the cotton 
 browns a little. After cooling, the plug is easily removed 
 with sterile forceps, and the gelatin poured. W. T.], and as 
 soon as it has cooled the contents are poured as rapidly as 
 possible into the lower (smaller) tray, while the other, serving 
 as a cover, is pushed a little to one side. Since the tube is 
 never entirely emptied, but a small quantity of gelatin re- 
 mains in it, it is best to label and preserve it as complemen- 
 tary to the tray culture. 
 
 A uniform layer of gelatin is seldom secured in these trays. 
 The bottom is not plane, but as a rule a little elevated in the 
 middle. But the consequent difference in thickness of differ- 
 ent parts of the gelatin has its advantage, for when many 
 germs are present in the fluid to be analyzed, and they are 
 
5 8 Bacteriological Technology. 
 
 too closely crowded in the thick peripheral part of the layer, 
 they are more isolated in the central thin portion. 
 
 The colonies in such tray-cultures are much more easily 
 accessible for examination with the naked eye or lens than in 
 a or b. Examination is frequently rendered somewhat difficult 
 because the under side of the cover becomes clouded. In this 
 case it may be replaced by a dry and sterile glass plate during 
 the necessary inspection. [Non-liquefying species are readily 
 examined by inverting the trays.] These cultures are also 
 far better for microscopic examination than those in flasks or 
 test-tubes, especially if smaller trays (5 or 6 cm. in diameter) 
 are employed; but they are far from being as good in this re- 
 spect as plate-cultures. 
 
 d. Glass Plates. This method, the "plate method" of 
 Koch, of which the three preceding are only modifications, dis- 
 
 a b 
 
 FIG. 29. Slide Coated with Gelatin Crossed by Fourteen Inoculation Scratches bearing 
 many Colonies. At 6, a mould colony due to accidental contamination from the air. 
 
 tinguishes itself from these not only by a certain elegance, 
 but especially in allowing a microscopic examination of the 
 colonies anywhere on the gelatin, with high magnification. 
 Koch's first plate-cultures were undertaken in the following 
 manner: The gelatin was spread in an elongated drop on a 
 common microscope slide, by the use of a pipette. When it 
 had become solid, it was inoculated by a bent platinum needle 
 (Fig. 21, d) dipped in the fluid containing germs, and then 
 used to make a series of transverse scratches on the gelatin 
 (Fig. 29). In the course of a few days colonies of the bacteria 
 appear in the inoculation furrows, closest and often entirely 
 coalescent in those made first (a), fewer and well separated in 
 the last. Assistance in recognizing contamination of such 
 scratch-cultures from the air, is afforded by the fact that they 
 almost always appear between the scratches, as is the case 
 with the mould colony &, in Fig. 29. In two respects the 
 
Bacteriological Technology. 59 
 
 plate method is inferior to the three preceding. From the 
 nature of the inoculation, the plates are more exposed to at- 
 mospheric germs; and the distribution of the gelatin over the 
 plates takes more time and care, since it must be kept from 
 running over the edges, which is best avoided by the use of 
 suitable cooling appliances which cause more rapid solidifica- 
 tion. 
 
 The apparatus needed, and the method of starting a plate 
 culture are, in detail, the following: 
 
 Rectangular glass plates 6.5 x 12 cm. are washed, polished 
 with chamois skin and sterilized at 150 C., well wrapped in 
 paper from which they are first removed immediately before 
 use. [For convenience, the sheet-iron boxes used by the Ger- 
 mans to hold sterilized plates, are to be recommended. W. T.] 
 
 The fluid containing bacteria is mingled with the gelatin 
 as explained above (p. 56), in a cotton-plugged test-tube con- 
 
 FIG. 30. A Simple Cooling Apparatus for Plate-cultures. 
 
 taining about 5 cc. of peptonized gelatin, a quantity adapted 
 to the size of plate recommended ; especial care being taken 
 to warm the gelatin only a little above the melting point. 
 
 A serviceable cooling apparatus is easity made as show^n 
 in Fig. 30. On an ordinary pie-plate, (a) is set a preparation 
 glass (b) with ground rim, several pieces of ice are placed in 
 this and water is carefully poured in until the vessel is full, 
 with a slightly convex surface, but not running over. A well- 
 cleaned square plate of glass (d) is now let down on the sur- 
 face of the water so as to wet it everywhere and thus avoid 
 air-bubbles, a little water running over into the plate as this 
 is done. Finally, a sterilized glass dish (e) is inverted on the 
 plate to serve as a bell-glass. 
 
 One of the glass plates is unpacked and quickly laid under 
 the bell-glass (Fig. 30, /), where it is rapidly cooled by the ice 
 water. The plug is removed from the infected tube, the mar- 
 gin of which is quickly flamed [here, as above, p. 47, it is 
 
60 Bacteriolog ical Technology. 
 
 preferable to flame the tube before removing 1 the cotton plug-. 
 W. T.], after which the bell-glass is raised, and the still fluid 
 g:elatin carefully poured over the glass plate, care being taken 
 not to let it flow too near the margin. The bell-glass is re- 
 placed to protect the plate while the gelatin solidifies. The 
 more exactly horizontal the plate stands, the better. If the 
 gelatin runs in a given direction, because the plate is not level, 
 it is possible to carefully lift the apparatus with both hands 
 and check this by adjusting its position, until the gelatin is 
 sufficiently hardened. It is naturally more convenient to level 
 the plate beforehand by using a tripod and level (Fig. 31). If 
 it solidifies too quickly, before spreading over a sufficiently 
 large part of the giass, it may be spread by use of a sterile 
 glass rod, warmed, but not too hot. The test-tube, in which 
 
 FIG. 31. Leveling Tripod with Round FIG. 32. Bench of Sheet Zinc for Support- 
 
 Level (6) and Plate. ing a Plate-culture. 
 
 a few drops of gelatin always remain, is again plugged with 
 the original cotton; labelled, rotated for several minutes and 
 laid horizontally, so that this vestige solidifies in a thin layer, 
 and kept as supplementary to the plate-culture, which, as 
 soon as it has hardened, is labelled and quickly removed to the 
 moist chamber. 
 
 For a single plate, a pair of shallow glass trays (Fig. 12) 
 may be used as a moist chamber. But as a rule it is neces- 
 sary to make several plate-cultures of the same fluid. In this 
 case a pair of larger deep trays are used, or a chamber may 
 be improvised. Before use, it is to be rinsed with 0.1 per cent 
 sublimate solution, and a couple of sheets of filter paper, well 
 wet with the same solution, are laid in the bottom. The 
 plates are set in such a moist chamber, on small glass or 
 metal benches which can be set over one another. Sheet-zinc 
 benches of the form shown in Fig-. 32 are very suitable because 
 of their durability. They can easily be sterilized at 150 C. 
 before use. 
 
 The examination of the plates by the naked eye is usually 
 
Bacteriological Technology. 61 
 
 made over either a white or black background (sheets of paper 
 or tiles), as many differences in color between the colonies are 
 only seen well in this way. It is also to be remembered that 
 colonies of the same species and of equal age may present 
 great differences in size, form, and color, according- as they 
 grow at the surface or immersed in the gelatin. Counting- the 
 colonies is best effected by the use of a lens of low power, and 
 is much easier if the plate is laid on another divided into 
 square centimetres. [When it can be afforded, one of the sim- 
 ple counting- appliances used by the Germans in water-analy- 
 sis, is to be strongiy recommended. W. T.] 
 
 The microscopic examination of the plate is not difficult, 
 even with rather high powers. To be able to examine every 
 part of the plate, a smaller size than the one recommended 
 may be necessary, if the stage of the microscope is not quite % 
 larg-e. Since it is impossible to calculate beforehand the 
 number of germs added to the gelatin, in each case it is cus- 
 tomary to prepare three plate-cultures at a time of three de- 
 grees of dilution. This is best done as follows : The gelatin 
 is liquefied in three test-tubes, which are numbered 1, 2, and 3. 
 After 1 has been infected by use of needle or pipette, and the 
 added material well distributed, 2 is infected from it ; and, in 
 the same way, 3 from this. The gelatin is poured on the 
 plates as above. In this way, it is highly probable that one 
 of the three plates will contain a suitable number of colonies, 
 whether these are wanted so numerous that the culture can 
 be used for a tolerably reliable estimate of the number of 
 germs present in the fluid (c/. water analysis, p. 64), or so 
 few that a nearly absolute guarantee is given of the purity of 
 each colony, and transfers may be made from them without 
 difficulty. . 
 
 It should be remembered, as advised above, to save the 
 three test-tubes after distributing the gelatin in a thin layer 
 by rolling them several times. Where the object is to get an 
 exact determination of the number of germs in the original 
 fluid, it is necessary to include those remaining in the tube, 
 and, independently of this, it is always an advantage to have 
 two isolation-cultures of each dilution. 
 
 Soyka obtains a large number of very small plate cultures 
 by using pairs of trays which differ from those shown in Fig. 
 12 in being made of glass 1.5 mm. thick, with a bottom 50 
 
62 Bacteriological Technology. 
 
 square cm. in area, and walls only 3 to 5 mm. Seven round 
 hollows are ground in the lower tray, like those in hollow- 
 ground slides (Fig-. 57). Before being used, they are slightly 
 warmed, gelatin is placed in each of these depressions by using 
 a pipette, one of the drops is quickly inoculated with a looped 
 platinum needle, carefully stirred, and a transfer made from 
 this to the second, etc., the upper tray or lid being put in place 
 after all are inoculated. If the pipette is accurately gradu- 
 ated, and the quantity of fluid carried b} r the platinum loop 
 previously determined by weighing it dry and again with its 
 drop of water, it is claimed by Soyka that a sufficiently relia- 
 ble determination .may be made of the number of germs. This 
 modification of the customary Koch method of plating- out 
 cultures appears to me excellent in its plan, but the form of 
 ,the trays and the limited size of the hollows do not seem so 
 good. For microscopic examination, I would recommend 
 rectangular glass trays (e. g., 12x5 cm.), the lower one divided 
 by elevated ridges into eight quadrangular spaces, so that the 
 surface of the several cultures should be larger. 
 
 In the preceding descriptions we have always assumed 
 that the isolation was to be effected in either gelatin or agar. 
 
 1. Gelatin. This is most easily used, since it may be melted 
 and kept fluid at a relatively low temperature; yet the fact 
 that many bacteria and moulds liquefy (peptonize) the gelatin 
 is a disadvantage that often becomes very evident in such 
 analyses. 
 
 2. Agar- Agar. Most bacteria cause no liquefaction of this 
 substance, which is nevertheless not so good as gelatin for 
 plate-cultures, because of the difficulty of melting it and its 
 somewhat slighter transparency, and its tendency not to 
 adhere to the glass; still, it may be used well for analyses of 
 this character if the following precautions are taken. When 
 the ag-ar has been melted in the test-tubes, which does not 
 occur below 90 C., these are set aside to cool for a few mo- 
 ments and, before their contents begin to solidify, placed in a 
 water-bath kept at 40 0., at which point the agar remains 
 fluid, while the liquid containing bacteria can safely be added, 
 without danger of killing any of the germs. 
 
 While it is usually necessary when gelatin is used to hasten 
 its solidification by cold (or iced) water, this must commonly 
 l>e retarded when agar is employed, by placing the plates 
 
Bacteriological Technology. 63 
 
 over, or rolling 1 the tubes in, lukewarm water, to prevent the 
 agar from forming 1 lumps and as a result hardening with an 
 uneven surface. Since ag-ar adheres so poorly to glass that it 
 occasionally entirely separates from the plate and slips off, 
 Fraenkel advises the use of a drop of sealing- wax on each cor- 
 ner of the plate. ' [Where ag-ar must be used in plating 1 out 
 bacteria, it is much better to do this in trays (supra, c), by 
 which the curling of the ag-ar is avoided. W. T.] 
 
 3. Agar-Gelatin, prepared after Jensen's formula (supra, 
 pp. 25, 26) combines some of the g-ood qualities of "both g-ela- 
 tin and agar. It is very conveniently used for plate-cultures, 
 and, as a rule, is to be recommended in preference to either. 
 The surest way of keeping 1 it fluid during 1 the process of inoc- 
 ulation, is to set the test-tubes in a water bath at 30 to 40 C. 
 
 4. Serum cannot be used in this manner, since it is coagu- 
 lated only by prolonged heating 1 to a degree likely to prove 
 fatal to the g-erms. If it must be employed, one of two 
 methods must be adopted: Scratch-cultures may be succes- 
 sively made with an infected bent platinum needle, in a larg-e 
 number of test-tubes, the colonies being- so few in the last of 
 the series as to be separate (c/. the original scratch-cultures 
 on slides, p. 58) ; or the fluid to be used may be diluted with 
 sterile water, and a small drop of the mixture allowed to flow 
 about over the surface of the serum, in the hope that the 
 germs will settle on it at a distance from one another. But 
 it should be borne in mind that this is an exceptional method 
 which does not give nearly so complete a separation of the 
 g-erms as the careful distribution in melted gelatin. 
 
 Water Analysis. The bacteriological analysis of drinking- 
 water is undertaken according- to the rules given above. The 
 following- precautions are also to be taken : The water is col- 
 lected in sterilized glass flasks, with cotton or glass stoppers. 
 If it is taken from, a faucet or hydrant, it is allowed to run 
 for some minutes before any is g-athered, and care must be 
 taken to prevent the contamination of the stopper from any 
 source (by placing- it in a sterile glass box, Figs. 11 and 13, 
 etc.). Samples are taken from accessible bodies of water in 
 sterilized pipettes, and quickly distributed in sterile flasks. 
 In case of deeper, less accessible supplies, a weighted sterilized 
 flask is lowered and drawn up when full, its contents being- at 
 once divided between several smaller bottles. The analysis 
 
64 Bacteriological Technology. 
 
 must be made as soon as possible after the sample is taken; 
 even a few hours' delay at ordinary room temperature effects 
 a great increase in the number of germs. If it is necessary to 
 send samples of water to any considerable distance, or to let 
 them stand for some time before they can be analyzed, they 
 must be packed in ice. 
 
 The quantity of water to be added to each tube of gelatin, 
 depends upon the number of germs present in it. When few 
 are present, an entire cubic centimetre may be used. Occa- 
 sionally -gV cc - suffices. Water that contains germs in especial 
 abundance must be diluted with occasionally several thousand 
 times its bulk of sterilized water, and a drop may even then 
 give a large number of colonies on the gelatin plate, Botton 
 puts 10 colonies to the plate as a minimum, and 5,000 as a 
 maximum, if good results are to be secured. With fewer than 
 10, accidental contaminations easily vitiate the results. If 
 the plate contains too many, the task of counting them is 
 more difficult, and some of them may not develop enough to 
 become visible. It is best to keep the plates at 20 to 22 
 C., and to count the colonies on the third or fourth day. 
 
 B. SOLID SUBSTANCES. 
 
 The bacteriological analysis of solids is quickly described 
 after the detailed account of fluid analysis. It may be carried 
 out exactly like the latter, by first quickly dropping the sub- 
 stance into the melted gelatin, in which the germs are washed 
 out and distributed as far as possible in the manner indicated 
 for the analysis of water. If it is not certain that the germs 
 can be sufficiently washed out even by continued agitation in 
 the liquefied gelatin, this may be effected by violent and pro- 
 longed shaking- in sterilized water or bouillon, which is after- 
 ward investigated according to the rules given for fluids. It 
 is also possible, after such treatment, to add an equal quan- 
 tity of melted 20-per-cent nutrient gelatin to the water, and, 
 after careful mixing, to spread this out in one of the described 
 methods. 
 
 Fraenkel recommends the first method for soil analyses, 
 as the result of a large number of comparative experiment. 4 
 Because of the great difference between different samples of 
 soil, as regards the amount of water present, he further ad- 
 
Bacteriological TecJmology. 65 
 
 vises measuring- the samples in preference to weighing 1 them, 
 using- for this purpose a sharp-edged spoon holding about -^ 
 cc. A level spoonful of dirt is poured into a test-tube contain- 
 ing melted gelatin, and broken up and distributed in this as 
 thoroughly as possible by the use of a stout platinum needle, 
 and by agitating the glass, after which the procedure is the 
 same as for analysis of fluid. The same writer advises the 
 use of von Esmarch's tube-method (supra, p. 56), and counts 
 the colonies two days after preparing the tubes. But the 
 common plate -method is equally applicable and occasionally 
 even preferable, especially when the samples contain many 
 liquefying forms, as is generally the case with the surface soil. 
 A great deal of the difficulty from this cause may be avoided 
 by the use of agar-gelatin. On the other hand, the tube- 
 cultures admit of prolonged observation, and in this way of 
 the detection of those very slowly developing forms which 
 frequently occur in earth (c/. Chapter XII., on disinfection). 
 Samples from the deeper soil, which, as the studies of Koch 
 and Fraenkel show, always contain very few germs, can only 
 be obtained with the certain exclusion of dirt from above by 
 the use of a special boring apparatus (made by Muencke, of 
 Berlin). Like water, soil must be investigated very soon after 
 collection, since, especially in samples from some depth, there 
 is a very rapid multiplication of the bacteria, which may be- 
 come a thousand-fold more numerous than at first, in the 
 course of a few days (Fraenkel). The reason for this increase, 
 which cannot be prevented by the use of ice, is still unknown. 
 In his first soil analyses, Koch used the simple method of 
 sprinkling the earth over the surface of gelatin. Naturally, 
 no certain separation of the germs is possible by this means, 
 but a good notion is obtained of the kinds of bacteria and 
 moulds present in the sample. It is best for this purpose to 
 pour the sterile nutrient gelatin upon plates or in trays, 
 where it is allowed to harden. The earth is collected with 
 the usual precautions in sterile test-tubes or flasks plugged 
 with cotton. Immediately before use, the cotton is removed 
 and a layer of filter paper quickly tied over the mouth of the 
 glass. Holes are made in the paper with a flamed pin, and 
 through these the dirt is dredged upon the gelatin, so that 
 the fine particles do not lie too close together. 
 
66 
 
 Bacteriological TecJinology. 
 
 a 
 
 C. Am. 
 
 The micro-organisms which float in the atmosphere, and 
 compose an important part of its so-called dust, have been in- 
 vestigated for a long- time in many different ways, and with 
 more zeal than those of water and the soil; on the one hand with 
 reference to the theory of spontaneous generation, and on the 
 other, because too much weight was laid upon the importance 
 of the air as a carrier of nearly all contagious matters. 'But 
 the time and energy be- 
 stowed are sadly out of pro- 
 portion to the small result- 
 ant gain in knowledge of the 
 life histor3 r and mode of 
 transportation of pathogenic 
 "bacteria. 
 
 Two modes of collecting 
 air and its organisms are in 
 use: the dust is allowed to 
 settle by its own weight, or 
 it is drawn into a suitable 
 apparatus by using an aspi- 
 rator. 
 
 Aspirators. A simple 
 aspirator, easy of transpor- 
 tation, is that shown in Fig. ' 
 
 ' FIG. 33. Aspirator Made from Two Flasks. 
 
 33. Two large conical flasks 
 
 (I. and II.) are furnished with rubber stoppers and glass tubes 
 arranged (Fig. 33) as for wash bottles, and connected with a rub- 
 ber tube in the manner shown. A pinch-cock is placed at c, and 
 the upper flask filled with water. When the cock is opened and 
 suction applied at a (or the upper flask tipped) till the siphon is 
 filled, the water will flow without interruption from I. into the 
 empty flask II., inducing a uniform and continuous suction of air 
 into b, until I. is empty. By pouring the water back into I. (or 
 reversing the relative position of the flasks) the suction can be 
 recommenced. Knowing the volume of water used, and the 
 time taken to empty one flask into the other, the amount of 
 air sucked into the apparatus during a given time is also 
 known. The rapidity with which the water flows from flask 
 
Bacteriological Technology. 
 
 67 
 
 to flask can by regulated by the pinch-cock, or by the use of 
 glass tubes of various calibre, inserted, somewhere in the 
 rubber tube. A number of such pieces of 
 tubing- are made ready, and the time re- 
 quired for each to permit the passage of 
 a liter of water is experimentally deter- 
 mined and marked on it with a writing dia- 
 mond, so that according to circumstances a 
 larger or smaller tube is employed (Hesse). 
 Another form of aspirator is that shown 
 above in Figure 7. 
 
 c. A drip-aspirator is shown in Figure 
 34 in which the constant stream from a 
 faucet can be used as a means of suction. 
 As the water falls in small portions from 
 the faucet, through the tubes a and &, 
 the little columns of fluid carry along be- 
 tween them larger and smaller columns 
 of air, producing at c a powerful suction, 
 which, however, is not so uniform as in the 
 other two aspirators. [The German deal- 
 ers supply a very simple aspirator essen- 
 tially on this model, consisting of a T- 
 shaped brass tube representing abc, joined 
 by suitable corks and rubber tubing with 
 a flexible lead tube some yards long, which, 
 being non-collapsible, makes a very good 
 connection with the other apparatus em- 
 ployed. W. T.] Nor does the apparatus 
 directly indicate the quantity of air sucked 
 through, like the others, so that if this is 
 to be determined it must be used in connec- 
 tion with a gas-meter. 
 
 I. Collection of Dust for Direct Mi- 
 croscopical Examination. The dust 
 which has accidentally accumulated in dif- 
 ferent places, often in a thick layer, can be 
 simply collected, even if the layer is a 
 slight one, or is easily swept on to a piece 
 of paper with a feather. Small quantities of the dust col- 
 lected in this manner are mingled with a drop of glycerin or 
 
 FIG. 34. Drip Aspirator 
 (Filter Pump). 
 
68 
 
 Bacteriological Technology. 
 
 other fluid on a slide and subjected to microscopic and micro- 
 chemical examination in the usual manner. 
 
 If the dust is to be collected for a given time and place, 
 this is best done by the use of a so-called aeroscope (Ponchet, 
 1859). Of the various models, only Schnauer's will be de- 
 scribed here a form which can easily be put together in any 
 laboratory. It consists (Fig-. 35) of a bell-glass (a) with ground 
 base and short neck. A bottle with the bottom broken off 
 and the lower end ground flat can be made to serve the same 
 purpose. The neck is fitted with a rubber stopper provided 
 with two holes, in one of which a short glass tube (d) is fitted, 
 while a longer tube (e), bent in an arch above and drawn out 
 to a point with an opening of about 1 mm. at the other end, 
 passes through the second. The 
 ground base of the bell-glass is 
 smeared with vaseline and pressed air 
 tight against a glass plate (b). Un- 
 der the bell-glass is placed a slide (c) 
 on which is a small drop of a steril- 
 ized solution of grape sugar 1 -part, 
 and glycerin 2 parts (Miquel). The 
 tube e is then adjusted so that its 
 point is about 1 mm. above the surface 
 of the drop, and the tube d is con- 
 nected with an aspirator. As soon 
 
 FIG. 35. Schoenauer's Aeroscope. 
 
 as the suction begins, the air, and the 
 
 dust suspended in it, flow through the curved tube directly 
 against the drop of glycerin, in which the flow of air causes 
 a slight depression, and where a large part of the dust is 
 stopped. A part settles in the curved tube, which must, 
 nevertheless, have this form in case the apparatus is to be 
 used in the open air in rainy weather. 
 
 When the aspirator has operated long enough, the slide is 
 removed, and the dust uniformly distributed through the 
 glycerin with a sterilized needle ; a cover glass placed on it, 
 and it is ready for microscopic and micro-chemical examina- 
 tion. 
 
 Preparations of this sort give a good notion of many of the 
 inorganic and organic substances which float in the air in 
 enormous quantities, e.g., coal dust, sand, small crystals of 
 various salts, woollen and cotton threads, hair, starch-grains, 
 
Bacteriological Technology. 69 
 
 fragments of vegetable tissue, pollen, etc. . Many mould spores 
 are also recognizable, but the germs of bacteria are too small 
 and too little characteristic to be identified in such prepara- 
 tions. The number and character of such germs present in 
 the air at a given time can only be determined by sowing the 
 dust in suitable culture -vessels. 
 
 II. Collection of Dust for Cultures. Very pretty and 
 useful preparations are obtained by simply letting the germs 
 settle from the air upon the surface of gelatin. Several pairs 
 of shallow glass trays (Fig. 12) are sterilized at 150, each pair 
 wrapped singly in paper. The nutrient gelatin (most com- 
 monly peptonized meat infusion with agar-gelatin) is carefully 
 and rapidly poured in a shallow layer into the smaller tray, 
 which is at once covered with the other (cf. p. 63). When 
 the gelatin has solidified, the trays are wrapped up and taken 
 to the place where they are to be used. Here they are un- 
 packed, the lids (i.e., the larger, upper trays) taken off, and 
 the gelatin is left exposed to the air for a longer or shorter 
 time, varying with the abundance of germs from a few min- 
 utes to an hour. After replacing the lids, the trays are set 
 aside at room temperature, for observation, the germs begin- 
 ning to show evidence of germination after a few days. 
 
 It is best to use the sterilized paper to wrap the lids in 
 while the gelatin is uncovered, and, after they are replaced, 
 to again wrap the pairs of trays in it for their return to the 
 laboratory. Instead of pouring the gelatin directly into trays, 
 it may, .of course, be spread on a glass plate, which is then set 
 within a pair of trays. 
 
 Simple as this method is, it gives, as has been said, very 
 useful results. This may easily be shown by exposing a num- 
 ber of trays of gelatin simultaneously and for the same length 
 of time in two different places. The differences in number and 
 kinds of atmospheric germs in different localities is then very 
 evident. It is to be recommended for the qualitative air anal- 
 yses most frequently called for, but it does not give the num- 
 ber of germs contained in a given quantity of air. A number 
 of methods and appliances for this quantitative analysis of 
 atmospheric air have been indicated, but there is at present a 
 difference of opinion as to which is to be preferred. I have 
 not sufficient experience in this directior. to pronounce very 
 authoritatively on the question, but I have no doubt the air- 
 
7 Bacteriological Technology. 
 
 filters using (insoluble or soluble) powders are to be given the 
 preference. In view of this difference of opinion it must be a 
 satisfaction to know that at present the exact determination 
 of the number of germs in the air is not of very great import- 
 ance. I shall, therefore, merely describe briefly the choice of 
 apparatus representing the various systems, viz., automatic 
 suction balloons, settling apparatus, such in which the air is 
 allowed to bubble through fluid or gelatinized contents, and 
 filters, which may be insoluble or soluble. 
 
 The automatic balloon was first used by Pasteur in 1860. 
 As shown in Fig. 36, it is at once culture vessel, aspirator, and 
 gasometer. It consists of a globular flask with a long neck 
 drawn out into a capillary tube. The tube is 
 hermetically sealed, and the flask sterilized at 150 
 C. When cool, the point is broken off, the air 
 within the balloon is warmed a little over a burner, 
 and the tip dipped into the sterilized culture-fluid. 
 As it cools, a few drops of fluid pass into the flask, 
 this is carefully heated to the boiling point over 
 the burner, and, while the steam is escaping from 
 the opening, this is again clipped into the fluid, a 
 greater quantity of which is sucked in. When the 
 flask is about half full, the point is removed from 
 the fluid, and the contents carefully heated over 
 the flame and allowed to boil gently for a couple Fio 36 _ Aut 
 of minutes. While the steam is escaping with a matic suction- 
 whistling sound from the end of the tube, the ? ulbfor Collect - 
 
 mg Air-germs. 
 
 flame is removed, and the point is at once care- 
 fully fused. The apparatus is now ready for use, for which it is 
 held as far from the experimenter as possible, in the direction 
 from which the wind comes, so that germs shall not be blown 
 from one's body toward it. The point is opened by a pair of 
 pincers or shears that have been flamed, and the air rushes m 
 with a whistle and fills the receptacle. The opening is once 
 more sealed, the flask shaken so as to wash all germs from 
 the walls into the fluid, and the development of micro-organ- 
 isms in the fluid is awaited. [But this gives no quantitative 
 determination, and a satisfactory investigation of even the 
 aerobic forms present in the fluid is only possible when some 
 of the fluid after thorough shaking is at once taken, with the 
 usual precautions, as a basis for some form of plate-culture. 
 W. T.I 
 
Bacteriological Technology. 
 
 Hesse's aeroscope (Fig-. 3?) consists of a glass tube 50 to 
 70 cm. long- and 4 cm. in diameter, one end of which is some- 
 whab flaring and closed by a perforated rubber stopper (d), 
 A sf| through which passes a short glass tube 10 
 
 mm. in diameter, plugged with cotton (b). The 
 flange at the other end is somewhat more prom- 
 inent; over it are tied two thin rubber caps, 
 one outside the other, the inner of which is 
 pierced with a round hole about 10 mm. in 
 diameter, as shown in the section in Figure 37. 
 The tube is used with nutrient gelatin, the sur- 
 face of which must not reach above the lower 
 side of the small glass tube or the hole in the 
 inner cap, when the apparatus lies flat. 
 
 Tlie tube, stopper, and caps are cleansed in 
 1.0 per cent sublimate solution, and finally 
 rinsed with boiled water. The inner cap is 
 tied fast with thread, without being- much 
 stretched. If it is found to be water-tight, by 
 half filling the tube with water, and holding- 
 this end downward, the round hole (f and /') 
 is clipped in its centre with sharp scissors, and 
 the outer cap is tied over it, but in a more 
 tense condition. [Rubber caps, fitting- quite 
 well and easily applied, are also supplied with 
 the apparatus by dealers. W. T.] Its power 
 to hold water is then again tested. The stop- 
 per with its plugged tube in place is now in- 
 serted in the other end, and the apparatus, still 
 containing water, is hung in the steam cylin- 
 der, which needs to be lengthened for this pur- 
 pose (Fig. 4. C). A piece of wire (Fig. 37, c) 
 twisted around the upper end is used for sus- 
 pending the tube to the lid by the means 
 shown in Figure 4 A. After exposure to 
 FIG. 37. Hesse 1 * streaming- steam for a quarter of an hour, it is 
 Aeroscope. removed from the sterilizing- cylinder, and 
 when it is somewhat cooled the water is poured out and 
 replaced with all precautions by melted sterile nutrient g-el- 
 atin, poured from a pipette or f wash-bottle. The stopper be- 
 ing again in place, it is once more hung in the steam-cylinder 
 
72 Bacteriological Technology. 
 
 for ten minutes' sterilization at 100, after which it is placed 
 horizontally while the gelatin cools [or rotated until the gel- 
 atin is nearly hard, when it is allowed to lie flat so that one 
 part of the circumference is more thickly coated than the rest. 
 W. T.] When wanted for use, it is set upon a suitable sup- 
 port, where the air is to be examined. Hesse uses a folding- 
 tripod such as is used by photographers, which ends in a flat 
 surface on which the tube can be fastened, the two flasks of 
 the aspirator (Fig. 33) being hung at different heights on the 
 legs. Simple supports, answering every purpose, are also 
 easily improvised. When the apparatus is set up, it is con- 
 nected with the aspirator, the surface of which is carefully 
 washed with 0.1 per cent sublimate, and the aspirator set in 
 operation, the siphon being sucked full beforehand, to prevent 
 a sudden irregular suction when the pinch-cock is opened. 
 The quantity of air which Hesse advises to be passed through 
 the apparatus is 1 to 5 litres for inhabited rooms, and 10 to 20 
 litres out of doors, but in certain cases much greater or smaller 
 quantities may be advisable. He recommends the aspiration 
 of a litre of air through the apparatus in one to three minutes 
 for inhabited rooms, and the same quantity in three to four 
 minutes for the open air, as a proper rapidity. After the air 
 has been sucked through, the apparatus is closed by replacing 
 the rubber membrane (which has been rinsed in sublimate), 
 and set aside at room temperature. In the course of the next 
 eight or ten days, colonies of moulds and bacteria grow on the 
 surface of the gelatin, from which they can be obtained for 
 examination and the inoculation of cultures by using a long 
 glass rod bearing a platinum needle. If the suction has been 
 effected sufficiently uniformly and with the right rapidity, so 
 that all germs have been allowed to settle from the air passed 
 through, the colonies should be most numerous near the end 
 through which the air enters, gradually decreasing in number 
 toward the other end, the last third or quarter of the gelatin 
 surface being entirely free from colonies. 
 
 Bubbling Apparatus. Of the different forms with fluid 
 contents, only one, long employed by Miquel, is described here. 
 This is a flask closed above with a cap plugged with cotton 
 (a), like a Pasteur flask (c/. Fig. 10). The neck is prolonged 
 as a fine tube to the bottom of the flask. The slightly con- 
 stricted side tube b, is plugged at two points with cotton (not 
 
Bacteriological Technology. 73 
 
 shown in the figure), and is connected with some form of 
 aspirator by the rubber tube e. A capillary tube hermetically 
 sealed at d, -is connected by the same means with the other 
 lateral tube (c), which is slightly bent downward. 30 to 40 
 cm. of distilled water is poured into the llask. Sterilization 
 is effected as usual. The aspirator is connected with e, the 
 plugged cap a is flamed and removed, the observer withdraws 
 from the apparatus, and the suction of air into it is allowed to 
 go on, after which the cap is again flamed and replaced. By 
 blowing through e the water is made to rise and fall through 
 the neck of the flask ten times, to wash off adhering germs. 
 The point d is flamed and broken off and by tipping the flask 
 and blowing through e the contents are divided among 30 or 
 
 d 
 
 FIG. 38.- Miquel's Aeroscope. 
 
 40 culture flasks containing bouillon. Finally, the outermost 
 of the two cotton plugs in the tube b is removed and the inner 
 one pushed into the flask with a sterilized platinum wire, after 
 25 cc. of sterile bouillon has been poured into the flask. The 
 flask, and the 30 culture- vessels are set aside at 30 C., and 
 observed for at least a month. When a small number of the 
 vessels show growth, the total of the germs collected by the 
 aeroscope can be approximately calculated (c/. p. 41, method 
 of dilution). 
 
 Tryde, Hueppe, and v. Sehlen formerly caused air to bubble 
 through melted nutrient gelatin, in their aeroscopic analyses. 
 Quite recently, Straus and Wurtz have used the same method, 
 and for this purpose produced the glass apparatus shown in 
 Figure 39. The top and bottom measure 15 mm. in diameter, 
 while the central portion (A) is a wide cylindrical receptacle 
 bearing the slightly constricted lateral tube (D), with two 
 
74 
 
 Bacteriological TecJmology. 
 
 cotton plug's (f and g). The tube B is drawn out to a fine 
 point below, and closed above with a cotton plug- (e). At c it 
 is widened, and ground to fit the mouth of A air-tight. The 
 apparatus is sterilized at 150 C., after which 10 cc. of 10 per 
 cent gelatin (or preferably agar-gelatin) are poured into it 
 and a drop of sterilized olive-oil added, after which all is ster- 
 ilized in the steam cylinder. The addition of oil prevents the 
 gelatin from foaming, even when air is passed through it very 
 rapidly, e.g., 50 litres in ten minutes. The 
 gelatin is melted, D is joined by rubber-tubing 
 with an aspirator, and the plug e is removed. 
 While air is passing through, the gelatin is 
 kept fluid by the warmth of the hand, or, if 
 agar-gelatin is used, a water-bath is employed. 
 Afterward, the plug e is replaced, and, by blow- 
 ing into D the fluid is forced into B several 
 times to wash out any adhering germs. The 
 plug / is removed so as to allow g to be pushed 
 into the gelatin with a sterile platinum wire, 
 after which it is replaced and g is gently 
 shaken about in the gelatin, which is then al- 
 lowed to solidify as for an Esmarch tube-cul- 
 ture, or poured out in glass trays as described 
 on pages 496, 497. The observation of the de- 
 velopment of the colonies is rendered not a lit- 
 tle difficult by the fact that, while air is bub- 
 bling through, the oil becomes finely emulsified 
 in the gelatin, rendering it cloudy. 
 
 Insoluble Powder Filters. As the result FIG. 39. Aeroscope of 
 of a series of very carefully conducted experi- strausandw rtz - 
 ments, Petri has recommended sand as a filtration medium 
 in preference to glass-wool and asbestos, which have been 
 used by others (Miquel, Moreau, Freudenreich, and Frank- 
 land). The details of his method are as follows: sand is 
 passed through a sieve with meshes 0.5 mm. wide, and what 
 passes this is sifted through a second with meshes 0.25 mm. 
 wide, the portion that remains in this consisting of grains 
 of the right size. This sand is heated to redness in an iron 
 crucible (half or three-quarters of an hour being required 
 for 100 cc.), while it is stirred with a glass rod. While 
 warm, it is filled into sterile test-tubes plugged with cot- 
 
Bacteriological Technology. 
 
 75 
 
 are brought 
 
 ton. Two sand-filters (s\ and s 2 ) 3 cm. long 
 into a glass tube 1.5 to 1.8 cm. wide and 9 cm. long, as shown 
 in Figure 40. The sand is held in place by small caps (n\ to 
 7i 4 ) of fine brass gauze (40 meshes to the linear centimetre), 
 which are easily made over the end of a glass tube or metallic 
 rod. This separation of the sand in two parts enables the ex- 
 perimenter to sow the second sand plug separately, as a con- 
 trol filter. The filters being 1 cm. from each end of the tube, 
 it is plugged at/ and g with cotton, and sterilized at 150 C. 
 Before use, the cotton plugs are removed and laid 
 aside in a sterilized glass box. The filter-tube is 
 joined with an aspirator by means of a rubber 
 stopper (a) sterilized in sublimate, pierced for a 
 cotton-plugged sterile glass tube c, which is con- 
 nected by a short piece of rubber tubing with a 
 lead tube (e) 0.5 cm. wide. The filter tube being 
 arranged vertically, the cotton plug is removed 
 from g and the suction can begin. Petri uses 
 as an aspirator a hand air-pump, each stroke of 
 which draws a litre of air through the filter. 
 When, as in his experiments, 100 litres pass 
 through the filter in 10 to 20 minutes, all germs 
 are stopped by the first filter. The material is 
 sown in pairs of trays (Fig. 12) 9 cm. in diameter, 
 the sand of the first filter being divided between 
 three, and of the second, between two such tra3 r s. 
 The sand is first poured into the trays, and 
 melted gelatin is poured over it from test-tubes 
 (cf. p. 57). After the sand is completely wet by 
 the gelatin, so that all air bubbles have been 
 driven out, it is equally distributed by a series of 
 short but forcible horizontal movements, and the gelatin is 
 allowed to harden, when the three pairs of trays are set away 
 one over the other at the temperature of the room. 
 
 Soluble Filters. Miquel, the most experienced living stu- 
 dent of the air, has very recently recommended 5 the employ- 
 ment of soluble filters, an ingenious method suggested by Pas- 
 teur over twenty-five years ago. Of the various powders 
 which may be selected (cane-sugar, table-salt, sodium phos- 
 phate, magnesium sulphate, etc.), Miquel recommends cane- 
 sugar, which demands no special preparation, may be steril- 
 
 FIG. 40. Petri's 
 Sand- Filter. 
 
7 6 
 
 Bacteriological Technology. 
 
 ized at 150 C., without losing- its high degree of solubility or 
 becoming more hygroscopic, or without having any injurious 
 effect on the bacteria. In very foggy weather it may become 
 impossible to work with either insoluble or soluble filters. In 
 this case a bubbling apparatus may occasionally have to be 
 used. The sugar (if loaf sugar is used) is 
 powdered in a mortar and shaken through 
 two metal sieves, as described for sand, so 
 that the size of the grains used is about half 
 a millimetre. This sugar is placed in an 
 aeroscope (Fig. 41, A) consisting- of a glass 
 tube about 20 cm. long and 5 mm. in diam- 
 eter, narrowed at e, and, according- to the 
 suggestion of Freudenreich, provided at b 
 with a ground cap plugged with cotton, as 
 for the Pasteur- Chamberland flasks. Two 
 plugs of (glass-wool or) cotton are inserted 
 at d and /, and the whole is sterilized at 150 
 C. The cap is removed, and enough well- 
 dried sugar (1 to 2 gm.) is poured into the 
 tube to fill it for a length of 8 to 10 cm., after 
 which all is again sterilized at 150 C. When 
 it is to be used, by a series of light percussions 
 the sugar is packed against the plug d, and 
 the tube held nearly vertically (tipped toward 
 the wind), so that during suction the sugar 
 may not fall away from the walls, thus render- 
 ing it possible for the air to pass unfiltered at 
 any point. 
 
 The air is drawn through the filter Vith 
 different rapidity and for a varying length of 
 
 ..e 
 
 A. 
 
 B, 
 
 time, according to circumstances. The aver- FIG. 41. A, Newer, B, 
 age number of sperms for the day at a given Older Model of Miquers 
 
 J Soluble Powder Filter. 
 
 point is obtained by slowly drawing the air 
 through a filter for 12 or 24 hours. To learn the number 
 at a given time, it is necessary to draw the largest possi- 
 ble quantity of air through the sugar in the shortest time 
 possible. 
 
 After the aspiration, the cotton or glass-wool plug is re- 
 moved and the sugar is poured into sterilized water. When 
 many germs are expected, a large quantity of water (500 to 
 
Bacteriological Technology. 77 
 
 1,000 cc.) is used; otherwise a smaller quality (50 to 100 cc.) 
 is taken. 
 
 In case the sugar does not run out of the tube easily, it 
 must be pushed out by the aid of the cotton plug /, and a 
 sterile glass rod introduced at /, and the tube afterward rinsed 
 out with sterile water. The plug d is also to be sown sepa- 
 rately as a test of the efficiency of the filter, which should 
 remain free from germs after the air has been sucked through. 
 To simplify these manipulations, I advise the use of tubes 
 that are not constricted at e, and to proceed as follows : The 
 following articles are to be made ready beforehand: 1, an 
 Erlenmeyer flask, filled with sterile water; 2, a short test-tube, 
 with a few cc. of water ; 3, a test-tube with nutrient gelatin ; 
 4, a sterile glass box (Fig. 11 or 13); 5, a small rubber tube. 
 The plug / is removed with forceps, and laid in the glass box, 
 after which d is similarly removed and cast quickly into the 
 gelatin, / being immediately replaced. The cap a is then taken 
 off, and the sugar poured out into the Erlenmeyer flask, the 
 rubber tube is slipped over the unground end of the glass (/). 
 the ground end being dipped into the sterile water of the short 
 test-tube, when all remaining germs and sugar are rinsed 
 out by alternately sucking water into the tube and expel- 
 ling it; finally this water is poured into an Erlenmeyer flask. 
 The filter tubes which Miquel used in his earlier investigations 
 were drawn out to a capillary point at the end through w r hich 
 air was admitted (Fig. 41 B, \vhere the constriction between 
 the two cotton plugs is also left out), and were opened and 
 closed by breaking off and remelting the point. The use of 
 the ground cap a renders it far easier to empty the apparatus, 
 but adds materially to its expense ; there is, however, no rea- 
 son why simple glass tubes, closed by my rubber-tubing cap 
 (cf. Fig. 9, III.-VI.) should not be used. The gelatin in which 
 the plug d was placed should remain sterile, all germs being- 
 found in the Erlenmeyer flasks. When the sugar is entirely 
 dissolved and the solution well shaken, the number of germs 
 is determined exactly as described for water analysis, prefer- 
 ably using agar-gelatin, so as to avoid as far as possible the 
 disturbing liquefaction of the gelatin. 
 
 A great advantage of this method is that only a fraction 
 of the sugar solution and the germs it contains need be used 
 for quantitative analysis, while the greater part can be used 
 
yS Bacteriological Technology. 
 
 for various qualitative investigations (e.g., a search for anae- 
 robic forms), which it was not possible to carry out in the 
 earlier methods in which all of the collected germs had to be 
 used for the quantitative analyses. 
 
 Of the aeroscopes and methods of analysis here described, 
 the first two have really only historical interest. The auto- 
 matic suction balloon was the instrument with which Pasteur 
 long since outlined the bacteriological topography of the at- 
 mosphere, which Miquel has since persistently worked upon. 
 Hesse's apparatus marks the first attempt to employ the 
 gelatin cultures of Koch in quantitative analysis of the air. 
 Of the other four, doubtless the solid filters will deserve pref- 
 erence. They are easily transported, and can be " charged " 
 with bacteria at any temperature and at places far from the 
 laboratory, after which they can be kept (at least for several 
 days) until cultures can be started under favorable conditions. 
 They also accomplish what is possible toward collecting in a 
 relatively short time all germs from the air of a very large 
 space. Of these solid filters, the soluble appear to me far 
 superior to the insoluble. 
 
 In this chapter, we have as good as exclusively concerned 
 ourselves with the application of Koch's methods of isolation 
 in gelatin to bacteriological analysis. It must, also, be said 
 unqualifiedly to be the chief method of such analysis. Start- 
 ing plate-cultures in one or other of the described 'forms is the 
 means first to be employed when bacteria are to be found and 
 separately cultivated from a given substance, and they will 
 in most cases lead to the desired result if sufficient attention 
 is given to varying the culture-material, temperature, time of 
 observation, number of germs, etc. The later the colonies are 
 counted, the more probability will there be that all germs 
 have developed into visible colonies. It is not so much the 
 danger of atmospheric contamination which leads to an earlier 
 examination than might be wished, as the presence of rapidly 
 growing, liquefying bacteria and moulds, which quickly spread, 
 so as to cover large portions of the gelatin. But it is self- 
 evident that it is not a universal method in the sense of ren- 
 lering every other method superfluous. On the contrary, we 
 may often do .well to use the various other methods described 
 in Chapter IV., either alone or as complementary; but no 
 
Bacteriological Technology. 79 
 
 general rules can be given as to this, so that one must choose 
 his plan for each respective case. 
 
 Finally, a most important point must be noted with refer- 
 ence to all the methods thus far described, viz. : They are 
 calculated for only aerobic bacteria. An exhaustive analysis 
 of an unknown mixture of bacteria, includes also a determin- 
 ation of the pronouncedly anaerobic forms, which play so im- 
 portant a part in the economy of nature, as well as in the 
 causation of disease. In what precedes, these have been left 
 entirely out of consideration, being- reserved for special and 
 detailed discussion in the next chapter. 
 
CHAPTEE YIII. 
 
 CULTURE OF ANAEROBIC BACTERIA. 
 
 IN 1861, Pasteur made the surprising- discovery that organ- 
 isms exist which can live, nourish themselves, and propagate, 
 without access to free oxygen. His doctrine of anaerobiosis 
 ("la vie sans air") was received with doubt and mistrust; but 
 further investigation not only corroborated his observation, 
 but from this as a starting point, Pasteur succeeded in lead- 
 ing the physiology of respiration into new channels, bringing 
 us nearer to an understanding of many important processes 
 of fermentation. Anaerobiosis showed itself to be a very sig- 
 nificant and wide-spread phenomenon, and a closer investiga- 
 tion of the need for oxygen of different bacteria (as well as 
 yeasts and moulds) revealed an extreme variability in this 
 respect. Some bacteria demand a very abundant supply of 
 oxygen, while others are affected by it as a poison, which not 
 only hinders their development, but quickly destroys their 
 life. Between these extremes the marked aerobiotic and 
 anaerobiotic forms every degree of transition is to be found. 
 Some forms seem to thrive about equally well with or without 
 free oxygen; some grow freely exposed to oxygen of a low 
 tension, others when this is greater a circumstance which 
 occasionally finds expression in a very striking manner in the 
 form and appearance of gelatin cultures. While the growth 
 around the puncture matte in the gelatin occurs for many 
 species very abundantly at and immediately below the surface, 
 and diminishes toward the bottom (Fig. 42, c), other cultures 
 may be found (Fig. 42, a) which show an extremely slight 
 growth near the surface, while the colonies of bacteria increase 
 uniformly in size the further they are removed from the at- 
 mosphere. The former is consequently aerobic, the latter 
 anaerobic. Beside these more or less conical cultures, with 
 the point directed downward or upward, others occur as capil- 
 
Bacteriological Technology. 
 
 81 
 
 lary or thick cylinders along- the needle puncture (Fig. 42, 6). 
 The oxygen of the air has neither helped nor hindered the 
 growth of these. 
 
 The discovery of anaerobiosis has necessitated the intro- 
 duction of new apparatus and methods in the cultivation of 
 bacteria. These must be treated now in detail, especially 
 since we already know several pathogenic forms which are 
 markedly anaerobic (malignant oedema, charbon symptoma^ 
 tique). Very different means have been employed for keep- 
 ing the oxygen of the air from culture vessels and material, 
 
 FIG. 42. Test-tube Cultures, a, B. murisepticus, in agar-gelatin ; 6, a colorless yeast in 
 raisin-gelatin; c, B. Anthracis, in agar-gelatin. The three cultures are of like age, and 
 grown under exactly the same condition? 
 
 or for removing all absorbed oxygen from the latter. Some- 
 times the effort has been limited to covering the culture- 
 medium with solid substances (glass, mica, ag-ar-agar), fluid 
 (oil), or g-as (carbonic acid, hydrogen). If g-ood results are de- 
 sired, the free oxygen held by the culture-medium must also 
 be removed by boiling-, the use of the air-pump, the passage 
 of a g-as which is not a supporter of combustion, but at the 
 same time not poisonous (hydrogen), or by the aid of aerobic 
 bacteria or chemicals with an affinity for oxyg-en. 
 
 Examples of most of these methods are described below, 
 6 
 
82 Bacteriological Technology. 
 
 * 
 
 though I shall confine myself to a discussion of a limited num- 
 ber of kinds of apparatus which have received the recommen- 
 dation of expert investigators. In accordance with the plan 
 of the book, the principal weight will then be laid on such 
 methods as demand the most readily obtainable and cheapest 
 material. Those who wish to go further into the technology 
 of this branch of the subject are referred to the two chief 
 treatises on the subject, by Roux 6 and Siborius. 7 
 
 Even by the best of the methods described, it is not possi- 
 ble to remove the last trace of oxygen from the culture- 
 vessels, but they have shown themselves sufficiently free from 
 oxygen under all circumstances for the cultivation of the most 
 pronouncedly anaerobic forms. As an easily applied test for 
 freedom from oxygen, sufficiently accurate for our purpose, 
 indigotin is to be recommended. Enough of a very dilute 
 solution of the sulphate (1 gm. indigotin to a litre of water) is 
 added to the culture -medium to give it a distinct blue color, a 
 small quantity of grape sugar is added, and the reaction ren- 
 dered distinctly alkaline by the addition of potash. Three 
 drops of a 10-per-cent solution of potash are added to 10 cc. of 
 nutrient gelatin or agar, which in addition to the usual sub- 
 stances also contains 1 per cent of grape sugar (Liborius). 
 When (by means of boiling, the passage of hydrogen, or the 
 use of the air-pump) the oxygen absorbed by the blue culture 
 substance is removed, the latter becomes colorless, since the 
 indigo-blue is reduced by the grape sugar to indigo-white. A 
 return of the blue color shows that oxygen is not certainly 
 enough excluded. 
 
 Trustworthy and adequate apparatus for the cultivation 
 of anaerobic forms can only be prepared when a hydrogen 
 developer or air-pump can be used. For the former, Kipp's 
 apparatus is commonly used, though, as Jorgensen has shown, 
 any one can fit up a sufficiently good generator at a cost of 
 about half a dollar, by using a common student-lamp chimney, 
 fitted by a loose cork (Fig. 43, a) into a cylindrical glass which 
 contains a mixture of one part by weight of sulphuric acid 
 and eight parts by weight of water (prepared with care by 
 adding the acid to the water while it is being stirred, never 
 by adding water to the acid), and a couple of drops of plati- 
 num chloride solution. A perforated lead plate covered with 
 muslin is placed at fr, to support a quantity of granulated 
 
Bacteriological Technology. 
 
 zinc in pieces about as large as peas. The stopper c must 
 be of rubber, and fit perfectly. Hydrogen is evolved as 
 soon as the zinc is brought in contact with the acid; but if 
 the rubber tube at d is closed by a pinch-cock, the hydrogen 
 drives the acid down to below 6, and its formation stops, to 
 recommence when the pinch-cock is again loosened. On the 
 way to the culture-vessels, the gas is passed through an alka- 
 line solution of pyrogallic acid (e) (1 part of a 25-per-cent 
 aqueous solution of pyrogallic acid is thoroughly mingled, in 
 
 a perfectly full bottle, with a 
 60-per-cent solution of potash) 
 to remove a trace of oxygen 
 which may be present. 
 
 The air-pump belongs to the 
 class of expensive instruments 
 which it is not proposed to dis- 
 cuss here in detail. It is, in- 
 deed, a great comfort to have 
 the use of a mercurial air-pump 
 when the cultivation of anaero- 
 bic forms is to be undertaken, 
 but it is not a necessity. A 
 comparatively cheap and good 
 model is the Sprengel pump, as 
 modified by Hiifner. One of 
 the simple water air-pumps 
 cannot be relied on for the re- 
 
 Qf oxyg . en ag completely 
 
 FiG.43.-.Toergensen's Hydrogen-generator. 
 
 as is sometimes necessary; but by also employing hydrogen 
 as indicated by Roux (infra, p. 87), it may be effected with 
 sufficient completeness. 
 
 The vessels and methods for cultivating anaerobic forms 
 will be considered under two principal classes, according as 
 they are to be used for isolating and obtaining pure cultures 
 of anaerobic forms from a mixture of bacteria, or merely for 
 propagating a species already pure, either in culture or in a 
 diseased animal. 
 
 I. ISOLATION FROM A MIXTURE OF SPECIES. 
 
 Pasteur succeeded in getting the first pure cultures of an- 
 aerobic forms in fluid media, by the method of isolation based 
 
84 Bacteriological Technology. 
 
 on physiological differences of species, using- carbonic acid or 
 exhausting- the air. More recently, the attempt has been 
 especially made to apply Koch's methods with gelatinized 
 media, even to the analysis of mixtures containing- anaerobic 
 bacteria. 
 
 A. Simpler (and more incomplete) means, which do not 
 .require the use of either air-pump or hydrogen generator. 
 
 1. Plate Cultures under Mica (Koch). Koch made the at- 
 tempt to effect the plate-culture of anaerobic forms by cover- 
 ing the gelatin, while still soft, with a sterile sheet of mica. 
 This must usually be as thin as a sheet of paper, without 
 flaws, and sterilized by flaming immediately before use (or at 
 150 C., wrapped in paper), and care is to be taken not to allow 
 air-bubbles to slip under it when it is laid on the gelatin. To 
 secure a still surer exclusion of oxygen, the margin of the 
 mica can be covered with melted paraffin (Fraenkel), which 
 quickly hardens, forming a solid border. In this way, the 
 usual method of working is preserved, as well as an easy ac- 
 cessibility for microscopic examination. 
 
 It is easy to convince one's self that the mica plate really 
 hinders the access of oxygen to germs beneath it, to no small 
 degree. If a decidedly aerobic non-liquefying form has been 
 plated out in this manner, the colonies may be seen to appear 
 in numbers in the uncovered part of the gelatin, while beneath 
 the mica they occur sparingly near the margin, and in the 
 beginning may be entirely wanting in the centre. Yet the 
 method is frequently unsatisfactory, because the exclusion of 
 oxygen is not sufficiently complete and lasting, and it is infe- 
 rior to those described below, especially that recommended by 
 Liborius. 
 
 2. Isolation in a Very Deep Solid Medium (Liborius). 
 A small quantity of the mixture to be analyzed is sown in a. 
 test-tube filled to a depth of 10 to 20 cm. with nutrient gelatin 
 or agar, which has been melted and cooled to the lowest tem- 
 perature at which it will remain fluid. The material is distri- 
 buted as uniformly as possible by aid of a thin sterile glass rod 
 carried carefully through a rotary as well as vertical move- 
 ment in the gelatin. If the distribution is successful, and the 
 number of germs right, very pretty results may be obtained, 
 the number of aerobic forms decreasing in size and number 
 from the top, while on the other hand the anaerobic forms 
 
Bacteriological Technology. 
 
 fe 
 
 grow only toward the bottom, and are always wanting- from 
 the uppermost part of the gelatin. In either case, the distri- 
 bution of the colonies always gives a good basis for deciding 
 as to their need of oxygen. 
 
 The colonies of bacteria are naturally far less easily acces- 
 sible for examination in the depth of such a test-tube than in 
 a plate-culture. When there are few of them, 
 and each colony is large and liquefies the gela- 
 tin, material for new cultures or for examina- 
 tion is easily obtained by thrusting a sterile 
 capillary tube, open below but sealed at top, 
 through the gelatin until the colony is reached, 
 when the top is broken off. In other cases, 
 it may be necessary to break the tube and to 
 cut the gelatin up in a sterile tray so as to 
 render the colonies accessible to the needle. 
 If agar is used, this can usually be blown out 
 of the tube by means of a Pasteur pipette (p. 
 44) passed down at one side. The micro- 
 scopic examination of the colonies under mod- 
 erately high powers can likewise often be con- 
 veniently made only after removing the con- 
 tents from the glass and cutting it in thin 
 slices. 
 
 B. Methods in which hydrogen is used to 
 replace the air. 
 3. Isolation in Gelatinized Media. Roux recommends a 
 tube similar in all essentials to that shown below in Figure 5G, 
 but of considerably larger size. After hydrogen has been 
 passed for a sufficiently long time through the liquefied and 
 inoculated gelatin, this is allowed to solidify while the tube is 
 horizontal. When the colonies are developed, the tube is cut 
 lengthwise with a diamond into two boat-shaped halves, the 
 one containing the gelatin being then easily accessible for 
 observation. 
 
 Fraenkel uses the following method : The test-tubes, rather 
 thick -walled, of large diameter, and without spreading rims, 
 are plugged with cotton sterilized in the usual way, and inoc- 
 ulated in three degrees of dilution as described on p. 500, after 
 which the plugs are replaced as rapidly as possible, with ster- 
 ilized fingers, by closely fitting rubber stoppers with double 
 
 FIG. 44. Isolation- 
 Culture of Anaerobic 
 Bacteria in a Deep 
 layer of Gelatin. 
 (Liborius.) 
 
86 Bacteriological Technology. 
 
 perforation, containing- two glass tubes bent at right angles 
 above the stopper. One of these reaches the bottom of the 
 test-tube, while the other ends immediately below the stopper. 
 The horizontal branches are contracted to capillary cavities 
 near the ends, which are plugged with cotton. The stopper 
 and tubing must be carefully sterilized, before the test-tube is 
 infected. This is done exactly as described above (Chamber- 
 land filter, p. 10), i.e., by sterilizing the stopper in 0.1 per cent 
 sublimate, and the tubes at 150 C., both being wrapped in 
 paper and finally sterilized by steam after being set up in an 
 autoclave; the stopper and tubing can, of course, be quickly 
 sterilized together. Just before being used, the rubber stopper 
 is unwrapped, the long tube drawn through the flame, to be 
 sure of its sterilization, and the stopper firmly fixed in the 
 test-tube, after which it is coated with paraffin, especially 
 about the small tubes and where it meets the side of the test- 
 tube. The long tube is now connected with the hydrogen 
 generator, while the test-tube is kept at 37 C. by the use of a 
 water-bath. According to Fraenkel, the rapid passage of 
 hydrogen for four minutes insures the removal of free oxygen, 
 after which the two tubes are sealed by melting at the con- 
 strictions, and the test-tube is rotated under a stream of cold 
 water until the gelatin has solidified upon the walls. By this 
 means a "roll-culture" of anaerobic forms is ultimately ob- 
 tained (Fig. 45). 
 
 4. The Capillary Tube Method. This is obviously applic- 
 able to the isolation of anaerobic species, and has been espe- 
 cially recommended for this purpose by Klebs and Yignal. The 
 latter advises its combination with the passage of hydrogen 
 and the use of solid culture-media. A small quantity of nutri- 
 ent agar or gelatin is brought to the boiling poin t and allowed 
 to cool off while a constant stream of hydrogen is passed 
 through it by means of a bent Pasteur pipette inserted be- 
 tween the wall of the test-tube and its cotton plug (Fig. 46). 
 By use of a water bath the gelatinized contents are prevented 
 from solidifying. The tube is infected while hydrogen is pass- 
 ing through. After this has been continued a few minutes 
 longer, and while the stream is still passing, the gelatin is 
 quickly sucked up into capillary tubes about 50 cm. long, 
 which are sealed at both ends and can be fastened later upon 
 black cardboard. 
 
Bacteriological Technology. 
 
 C. Methods depending- upon the use of the air-pump. 
 
 Where an air-pump can be had, a very simple culture- 
 apparatus (Roux, Fig-. 47) can be used. This is closed by a 
 cotton plug and contains a rather small quantity of nutrient 
 gelatin. Afer the latter is infected, the plug- (a) is crowded 
 down to the constriction c, the tube is softened and slightly 
 constricted at b, and the g-elatin remelted. By means of a 
 piece of rubber tubing-, the tube is connected with the air- 
 pump, and the air exhausted from it, the glass being mean- 
 
 FIG. 45. 
 
 FIG. 46. 
 
 FIG. 45. Roll-culture of Anaerobic Bacteria (Fraenkel). 
 
 FIG. 46. Use of a Bent Pasteur Pipette for Passing Hydrogen through Gelatin Prepara- 
 tory to Making Capillary Tube-cultures 
 
 time carefully warmed by passing a g-as or spirit flame up and 
 down its sides at short intervals. When the air is exhausted, 
 the tube is melted at 6, which hermetically seals it and at the 
 same time separates it from the air-pump. A " roll-culture " 
 is finally obtained by allowing- the gelatin to harden in a thin 
 layer on the inside of the tube. 
 
 According to Roux, a small water air-pump (filter-pump) 
 is sufficient if, by means of a T-tube with two glass stop-cocks 
 (Fig. 48), the culture-tube is likewise joined to a hydrogen 
 
88 
 
 Bacteriological TccJinology. 
 
 generator, and the exhaustion of the air alternated five or six 
 times with the admission of hydrogen. 
 
 Having- now explained how pure material of anaerobic 
 
 V.LJ* 
 
 a 
 
 FIG. 48. 
 
 FIG. 49. 
 
 Fio.47. Culture-vessel for Isolating Anaerobic Bacteria in Gelatin in a Vacuum (Roux). 
 FIG. 48. The Same Apparatus Arranged with T-tube for Alternating Connection with 
 Filter-pump and Hydrogen-generator (Roux). 
 
 FIG. 49. Vessel for Cultivation of Anaerobic Species under Hydrogen (Salomonsen). 
 
 forms is obtained by cultures in solid media, it remains briefvy 
 to explain the use of fluid media for the same purpose. Three 
 
Bacteriological Technology. 
 
 courses are open, so that it is possible to employ the capillary- 
 tube method, as described on p. 86, or the method of dilution, 
 by adding- a drop of the greatly diluted substance to each of a 
 large number of culture-flasks, which may then be used for 
 cultures under hydrogen or in a vacuum. 
 
 For the passage of hydrogen, culture-vessels like that of 
 Fraenkel (Fig. 43) may be used, or my own (Fig. 49), in which 
 the part used for the culture is about 10 cm. long" and 2 cm. 
 in diameter. The contents are inoculated through the verti- 
 cal tube, closed at top by a cotton-plugg-ed rubber tube (a), 
 
 J, 
 
 a 
 
 FIG. 50. FIG. 51. 
 
 FIGS. 50 and 51. Two Culture- vessels after Pasteur. 
 
 and hydrogen is admitted through the bent tube, provided at 
 d with an ordinary cotton plug. Just behind each plug", the 
 tubes are narrowly constricted just before the hydrogen is 
 passed through, and as soon as the stream is discontinued the 
 apparatus is hermetically sealed at these points. If the tubes 
 are made long- enough in the first place, these vessels may be 
 used several times in succession. 
 
 Vacuum-cultures may be made in Roux's tubes (Fig. 47), or 
 in those shown in Figures 50 and 51, which have been con- 
 
Bacteriological Technology. 
 
 stantly used in Pasteur's laboratory for many years. These 
 are filled through the capillary tubes (a), by applying suction 
 at 6, after which the fine tubes are hermetically sealed and 
 the hole sterilized. The contents are inoculated by sucking 
 some of the questionable material in through the tube a, after 
 this has been heated to redness and bent away from the larger 
 tube, and its end flamed and broken off, to be subsequently 
 melted together again. The apparatus is connected with the 
 air-pump by means of rubber tubing. Figure 50 shows a sim- 
 ilar vessel in which two cultures may be carried on simultane- 
 ously, or first in one receiver and later in the other, by care- 
 fully pouring a drop into it from the first. 
 
 II. PRESERVATION OF A GIVEN ANAEROBIC CULTURE. 
 
 For keeping a pure culture upon solid media, all of the ap- 
 pliances recommended above may be used, and isolated-colony 
 cultures obtained, which, as a rule, are attended by no incon- 
 venience. If thrust-cultures are desired, other 
 methods must be employed, such as the follow- 
 ing four : 
 
 1. Use of Oil or a Gelatinizing Plug. 
 The inoculation -thrust is made as usual in a 
 tube of agar or serum, but with a glass needle 
 in preference to platinum (p. 45). Immedi- 
 ately afterward, a layer 5 cm. thick of olive-oil 
 previously sterilized by boiling, or of sterilized 
 2-per-cent agar which has been allowed to 
 cool to about 40 C., is poured into the tube, 
 with the usual precautions. The advantages 
 and disadvantages of each of these substances 
 are self-evident. 
 
 2. Use of Entirely Filled and Hermeti- 
 cally Sealed Tubes (Roux). A tube of the 
 
 form shown in Fig. 53 a, is plugged with cot- FIG. 52. - cuitiva- 
 
 r & t i on O f Anaerobic 
 
 ton at its upper end, and the lower capillary species under on or 
 portion is sealed by fusion, after which it is A s ar - 
 sterilized by hot air at 150 C. Sterile nutrient gelatin or 
 agar is brought to the boiling point, and as soon as it has 
 ceased boiling the opened capillary end of the tube is dipped 
 into it, and the tube is filled up to the constriction, by suction. 
 
Bacteriological Technology. 
 
 By pressing- a finger tightly over the upper end, and quickly 
 raising the tube into an oblique position, the gelatin is pre- 
 vented from running out of the end, which is at once sealed 
 in the flame. The upper end is likewise sealed by fusion at the 
 constriction just above the gelatin. When the 
 contents are to be inoculated, one end is opened, 
 a thrust is made with an infected fine glass 
 needle, and the tube is again sealed. When 
 the developed culture is to be examined, it is 
 best to open the end opposite that from which 
 the thrust was made, otherwise the colonies 
 can easily be forced from the tube by the pres- 
 sure of gas generated, during their growth. 
 
 3. Removal of Oxygen by the aid of Aero- 
 bic Bacteria.^ Roux's method is as follows: 
 A suitable quantity of nutrient agar is boiled 
 in a cotton-plugged test-tube, and quickly 
 cooled in cold water. Immediately after it 
 has become solid, it is inoculated by a glass 
 needle, after which a small quantity of melted 
 but not too hot nutrient jelly is poured into 
 the tube, and, as soon as it has solidified, a 
 couple of drops of bacillus subtilis, or some 
 other decidedly aerobic species, are sown upon 
 its surface. The test-tube is then fused to- 
 gether at top, and set in the brood-oven. B. 
 subtilis grows rapidly upon the surface, using- 
 up the oxygen above it, and so preventing this 
 from reaching the agar, in which the anaerobic 
 form is consequently permitted to grow undis- 
 turbed. To obtain material for transfers from 
 this culture, the tube is opened by breaking off 
 its bottom, to avoid the admixture of B. sub- 
 tilis. 
 
 Cultures can also be made in the appa- 
 ratus shown in Figure 54, where the mixture 
 of the two forms is entirely avoided. The inner tube is filled 
 by means of a Pasteur pipette with agar-g-elatin, and the space 
 about it, with bouillon; the former is inoculated with the 
 anaerobic form, the latter, with the aerobic species, and the 
 outer tube is sealed hermetically at a. 
 
 FIG. 53. Pipette 
 Tubes for Cultivating 
 Anaerobic Species 
 (Roux). 
 
Bacteriological Technology. 
 
 4. Removal of Oxygen by Pyrogallic Acid. Buckner has 
 recently recommended the following- method: The anaerobic 
 form is sown in a small cotton-plugged test-tube containing- 
 agar, immediately after the latter has been boiled and rapidly 
 cooled down. This tube is then supported on a simple wire 
 stand (Fig. 55) [or a piece of spiral spring] in a larger test- 
 tube, the lower part of which contains an alkaline solution of 
 pyrogallic acid (1 g-m. of pyrogallic acid ; 10 cc. of 10-per-cent 
 solution of caustic potash). The larger tube is carefully closed 
 by a tightly fitting rubber stopper, which is slightly moist- 
 
 -O, 
 
 FIG. 54. 
 
 FIG. 55. 
 
 FIG. 56. 
 
 FIG. 54. Method of Cultivating Anaerobic Bacteria by Aid of Others which Exhaust the 
 Oxygen (Salomonsen). 
 
 FIG. 55. Apparatus for use of Pyrogallic Acid (Buchner). 
 
 FIG. 56. Arrangement for Cultivating Anaerobic Species on Potato (Roux). 
 
 ened on the sides, and the culture is usually set in the brood- 
 oven at a little over 30 C. Buckner has satisfied himself that 
 the development of so markedly anaerobic a form as the 
 bacillus of malignant cederna is easity effected in this appara- 
 tus, though a little slowly. Absorption of the oxyg-en is hast- 
 ened by forcibly shaking the tube now and then. 
 
 5. Potato-Cultures in a Vacuum. Roux employs the ap- 
 paratus shown in Figure 56 for this purpose. After the piece 
 of potato has been sterilized in the steam-cylinder, and 'the 
 condensed moisture has collected at the bottom of the tube, 
 
Bacteriological Technology. 93 
 
 below the narrowed portion 6, the potato is inoculated in the 
 usual way, and the test-tube hermetically sealed at a. The 
 small lateral tube is then connected with the air-pump, the 
 exhaustion is kept up for several minutes, so that all air may 
 escape from the interior of the potato, and the tube is sealed 
 by melting- at c. 
 
 The preservation of anaerobic pure cultures in fluid media 
 is exposed to no difficulties. The apparatus adapted to the 
 passage of hydrogen (Fig 1 . 49), or to the use of the air-pump 
 (Figs. 47, 50, 51), is employed. 
 
CHAPTER IX. 
 
 CULTIVATING MICRO-ORGANISMS UNDER THE MICRO- 
 SCOPE. 
 
 IF it is desired to follow the development of a microbe by 
 uninterrupted observation for some hours or days, so that 
 growth, fission, the formation and germination of its spores, 
 etc., may be directly seen, the culture must be made in a 
 " moist chamber," attention being given to the usual precau- 
 tions, such as preliminary sterilization of the several parts of 
 the chamber, rapid inoculation, etc. The chambers must be 
 hermetically sealed by melting, using cement, or in some such 
 manner, to prevent evaporation or infection. They must, 
 therefore, be large enough to contain a sufficient quantity of 
 air in addition to the necessary nutrient substance. Investi- 
 gations of this sort are most frequently attended with some 
 difficulty, especially for beginners, who can best practise them 
 upon larger forms, such as yeasts and moulds, passing from 
 these to the spore formation and germination of the large 
 species of bacillus. 
 
 The following rules are observed in employing the various 
 moist chambers, the most important and useful of which will 
 be described. 
 
 Cover glasses are cleansed in the usual manner with hydro- 
 chloric acid, followed by alcohol and finally by distilled water. 
 After careful drying they are laid in a pair of small glass 
 trays (Fig. 13) which are wrapped in paper and, with their con- 
 tents, sterilized in the dry oven at 150 C. For further cer- 
 tainty, the cover-glasses may be drawn several times through 
 the flame, immediately before use. In the same w r ay, the dif- 
 ferent kinds of slides and chambers are sterilized by dry heat, 
 wrapped in paper, in which they remain until the moment of 
 use. 
 
 In preparing the moist chambers it is well to use a larger 
 
Bacteriological Technology. 95 
 
 piece of sterilized paper to unwrap the slides upon, and to 
 cover them with small bell-glasses,, leaving- them uncovered 
 for only the shortest possible time. Cover-glasses must be 
 treated with the same care. The larger shallow glass trays 
 (Fig. 12) serve well as bell-glasses. 
 
 Several chambers are commonly employed at the same 
 time, since some of them are liable to contamination while 
 being prepared, even though they are sow r n quickly and care- 
 fully. 
 
 Cell-cultures are always started from young and strong" 
 cultures, usually in the same medium that is used in the 
 moist chamber. Care is to be taken that the germs are uni- 
 formly distributed, and in proper number, one to four in each 
 field of the microscope being right. As this depends upon 
 repeated preliminary examinations, three test-tubes are used, 
 
 FIG. 57. Hollow-ground Slide for use with Hanging Drops. 
 
 each containing a few grams of a suitable nutrient medium. 
 A rather large number of germs are suspended in the first, 
 and the effort is made by strong shaking to isolate them and 
 distribute them uniformly. A drop is then transferred by 
 means of a platinum loop to the second tube, and after thor- 
 ough agitation the number of germs in the new mixture is 
 determined under the microscope by examining a drop of it in 
 the sort of moist chamber to be employed for the culture, and 
 with the power to be used for this. If there are too many 
 germs, a little fluid is added from the third test-tube, but if 
 too few are present, their number is increased by adding a 
 drop from the first tube. 
 
 One of the simplest and most frequently used moist cham- 
 bers (in which Koch first observed the growth and spore 
 formation of Bacillus anthracis outside of the animal organ- 
 ism, in 1876) is the hollow-ground slide a glass slip of the form 
 and size of an ordinary microscope slide, with a circular de- 
 pression at the centre, about 15 mm. in diameter (Fig. 57). 
 
96 Bacteriological Technology. 
 
 After placing- a small drop of culture-fluid, containing- the 
 microbe to be studied, on the lower side of a cover-glass, this 
 is laid over the hollow and fastened to the slide in an air-tight 
 manner by smearing with vaselin. Aside from the general 
 rules given above, attention is to be given in the use of this 
 cell, and in examination in hanging- drops to the following: 
 The drop must be spread out in as thin a layer as possible, 
 b'ecause it must usually be capable of examination through its 
 entire depth. Often it is well to place a drop of sterile nutri- 
 ent fluid on the cover-glass, and to infect it at some easily 
 recognized point along- the margin of the drop. 
 
 It is best when the drop has been placed on the cover, to 
 fix this to the slide by smearin t g a little vaselin around the de- 
 pression with a sterile glass rod, after which the slide is lifted 
 and turned over, and pressed against the cover-g-lass so that 
 the edges of the latter are covered by the vaselin, while the 
 culture drop projects into the hollow. Instead of fluid, a 
 small quantity of nutrient gelatin may be placed on the lower 
 side of the cover-glass, making a miniature plate-culture. 
 
 The preparation is first examined with a low power to get 
 a notion of the germs present, and to find a place where they 
 are especially well distributed, when the slide is fastened on 
 the stage of the microscope and the g-erms that have been 
 picked out are found with the higher power it is wished to use 
 for the continuous observation. When a hanging drop is to 
 be found and brought into focus under the microscope, this is 
 greatly facilitated by the fact that moisture at once con- 
 denses upon the cover-glass about the drop, so as to give the 
 cover a roughened appearance. By first finding this, and then 
 moving the slide until it disappears, the margin of the drop 
 is reached. Cultures of this sort can be kept on the stage of 
 the microscope for days without suffering- harm, and the de- 
 velopment of a single germ can thus be followed, step by step. 
 
 If it is desired to hasten the process, or if the temperature 
 of the room is too cold for the development of the bacteria, 
 the moist chamber may be placed on some form of warm 
 stage (such as Schultze's, Ranvier's, Israel's, or Vig-nal's), or 
 the thermostat for the microscope, recommended by Panum 
 (Nord. Med. Arkiv, 1874, VI., No. 7), may be used. Usually, 
 however, it is sufficient to place the moist chamber in the 
 brood-oven, removing- it now and then for microscopic exam- 
 
Bacteriological Technology. 97 
 
 ination, care being- taken to find the same part of the prepar- 
 ation each time. The simplest " finder " for marking- a given 
 point so that it can quickly be found again under the micro- 
 scope, is that indicated by Hofmann, viz., scratching a cross 
 on each side of the opening in the stage, one upright (+), the 
 other oblique ( X ). When the point to be marked is centred 
 in the field, two crosses are marked in ink on the slide exactly 
 over those on the stage. 
 
 Boettcher's moist chamber (Fig. 58) consists of a deep 
 glass ring- fastened to a slide by a cement which is not injured 
 by heating to 150 C. for sterilization. The experimenter can 
 easily fasten the ring's by one of the cements for mending- 
 glass-ware; Hansen recommends for the same purpose a solu- 
 tion of gelatin in glacial acetic acid, to which finely pulverized 
 potassium bichromate is added just before use. 
 
 YY////////97/////////Y^^^ 
 
 FIG. 58. Boettcher's Moist Chamber. 
 
 A little sterilized water is placed in the bottom of the cell, 
 and the upper edge of the ring- is painted with vaselin to 
 fasten the cover-glass. The chamber is used like the hollo w- 
 ground slide, either for examination in hang-ing- drops, or for 
 the observation of small plate-cultures in g-elatin. The latter 
 is the method used by E. Chr. Hansen in obtaining- pure cul- 
 tures of yeast for use in breweries, starting- from a single cell. 
 As a rule he employs rather larg-e ring-s (about 25 rnm. inside 
 diameter), and round cover-glasses of corresponding- size. A 
 drop of nutrient gelatin containing- the yeast is spread upon 
 the latter, with all of the precautions indicated above ("Med- 
 delelser fra Carlsberg- Laboratoriet," ii., 152). 
 
 The use of hanging- drops is attended with certain difficul- 
 ties. The depth of the drop is relatively great, its lower sur- 
 face is not plane, and the g-erms easily chang-e position in it. 
 These difficulties are partly avoided by the use of gelatin, 
 which, nevertheless, can by no means entirely replace fluid 
 7 
 
98 Bacteriological Technology. 
 
 culture media because of its influence upon the movements 
 and mode and rapidity of growth of bacteria. Bref eld's film- 
 cultures, described below, avoid these, but are attended with 
 others. 
 
 A simple means of remedying 1 some of the defects of the 
 hang-ing- drop consists in covering- the lower surface of the 
 drop with a fragment of cover-glass, which adheres by capil- 
 lary attraction, insuring- a flat bottom to the fluid. The same 
 end is reached by the use of Ranvier's moist chamber (Fig. 
 59), consisting of a slide with a deep groove ground in the 
 centre, surrounding a circular disk the surface of which is 
 about 0.1 mm. lower than the rest of the slide. The drop of 
 fluid is placed upon this lower central part, vaselin is painted 
 around the groove, and the cover-glass pressed down upon it. 
 In this way the drop is hermetically inclosed between two 
 
 
 FIG. 59. Ranvier's Moist Chamber. 
 
 parallel flat surfaces, while the groove serves as an air- 
 chamber. 
 
 A very expensive and fragile, but otherwise excellent 
 moist chamber, is the modification of De Bary's and Geissler's 
 model used by Brefeld in his study of Bacillus subtilis, in 
 which he succeeded in following- the entire development from 
 spore to vegetative cells and from these to new spores. This 
 is a glass tube 20 cm. long- (Fig. 60), in the middle of which a 
 transversely cylindrical cell 2 mm. deep is blown, the flat top 
 and bottom of which have the thickness of a cover-glass. To 
 prepare it for use, it is thoroughly cleansed with hydrochloric 
 acid, distilled water, alcohol, and ether, and each end of the 
 tube is plugged with cotton. When dry it is sterilized at 150 
 C. The microbes to be studied are uniformly distributed in a 
 suitable culture fluid, until by examining several drops spread 
 very thinly, it is found that they are present in the right 
 number (about one to four to each microscope field). One plug 
 
Bacteriological Technology. 
 
 99 
 
 is now removed, the open end is quickly flamed and immersed 
 in the fluid, which is drawn into the cell by suction applied to 
 the other plugged end of the tube. When the chamber is 
 quite full, the fluid is allowed to run out again, the wet end of 
 the tube is dried with sterilized filter-paper, 
 and replugged, both ends are sealed with seal- 
 ing wax, and the cell is ready for examination. 
 The inside of the thin-walled chamber is here 
 lined by a very thin film of fluid containing 
 the germs, so thin that the bacteria do not 
 move about in it. A suitable part of the pre- 
 paration is found, and the chamber is fixed 
 upon the stage of the microscope. In search- 
 ing through it, which is done with the lowest 
 power sufficient for recognizing the germs, care 
 is taken not to crowd the objective against 
 the thin and fragile wall of the cells, a mis- 
 hap which is rendered all the easier by the fact 
 that the latter is not perfectly flat. It is most 
 easily fastened on the stage by first placing it 
 upon a thin and well-cleansed slide, to which 
 the cylindrical ends can be attached by paraf- 
 fin. Admitting of the safe and easy trans- 
 mission of gases, this moist chamber can also 
 be used for studying the development of bac- 
 teria in different gases. 
 
 Other forms of moist chambers are easily 
 improvised out of the material and appliances 
 to be found in every laboratory. As it may 
 be important at times to work with a very 
 large number of cell-cultures at the same time, 
 some of these easily extemporized forms de- 
 serve description. 
 
 Buchner studied the germination of spores 
 of Bacillus anthracis in a moist chamber such 
 as is shown in Figure 61. A very small quan- 
 tity of a pure culture containing only the spores of this species 
 is dried upon a cover-glass, and a small drop of culture-fluid 
 added. The cover is then supported on a slide at two edges, 
 by fragments of cover-glass, the entire margin sealed with 
 some cement, and the slide placed on a warm stage. 
 
 FIG. 60. Geissler- 
 chamber for Cultiva- 
 tion in a Film (Bre- 
 feld). 
 
IOO 
 
 Bacteriological Technology. 
 
 A cell, the inventor of which is not known to me, consists 
 of rather thick pasteboard (No. 16), cut into the form shown 
 in Figure 62, and carefully sterilized by boiling- or in the steam 
 cylinder, which is best effected by piling several pieces up be- 
 tween a couple of microscope slides and tying them firmly 
 together. One of the pieces of pasteboard, while still wet from 
 sterilization, is laid on a sterile slide and pressed into contact 
 by a glass rod. A cover-glass, with the hanging drop, is laid 
 over the hole in the pasteboard and carefully pressed down 
 on it, after which sterile water is added at one edge until the 
 paper refuses to absorb more. In this simple chamber, the 
 paper serves both as a source of moisture and a means of at- 
 taching the cover, so that it must be kept from drying out as 
 long as the chamber is in use ; consequently, w r hen not under- 
 going examination, cells of this kind, like plate-cultures, need 
 
 FIGS. 61 and 62. Improvised Moist Chambers, 61, Buchner's form; 62, Cardboard for Cell. 
 
 to be kept in a large moist chamber (cf. p. 60), and from time 
 to time the pasteboard must be resaturated by the addition 
 to the outer edge of a little sterile water or very dilute subli- 
 mate solution. For this reason this form of cell is not well 
 adapted to continuous observation under the microscope for 
 a period of days, though it may often prove very useful as an 
 accessory. Other materials may also be used for the prepa- 
 ration of moist chambers of the same sort. 
 
 In his studies of the life-history of the higher fungi, Brefeld 
 has cultivated them under control of the microscope by the 
 simple use of slides upon which a nutrient fluid was spread in 
 a thin layer. These plate-cultures in fluid are not suited to 
 the study of bacteria. So far as yeasts are concerned, they 
 offer no especial advantage over cell-cultures, but are attended 
 by certain disadvantages. On the other hand, they are of 
 the greatest importance for the study of the development of 
 higher fungi, and for this reason merit brief consideration here. 
 
Bacteriological Technology. 101 
 
 The solutions described on pages 19 and 20 are used, gen- 
 erally after being- boiled down so that they are not too fluid. 
 By means of a glass rod, an elongated drop is placed on a 
 sterile slide, and enough of the spores to be studied are added 
 and distributed with a platinum needle so that only one or 
 two occur in each field of the microscope. It is evident that 
 such cultures cannot be used for prolonged uninterrupted ob- 
 servation. If kept long upon the stage of the microscope, 
 they will be ruined by drying out or contamination. Between 
 the, different examinations, they must be kept in larger moist 
 chambers, the air of which is always saturated with moisture 
 (a common flat porcelain platter filled with water and covered 
 by a bell-glass dipping into the water), and they can scarcely 
 be used successfully except in laboratories where the air is 
 kept as free as possible, by great care, from germs, and espe- 
 cially moulds ; but Bref eld's works show sufficiently how far it 
 is possible to utilize this method. Besides other advantages 
 over similar slide-cultures on solid media, these admit of trans- 
 planting the spores after germination has begun, more favor- 
 able conditions are given .the moulds for growth and develop- 
 ment, and it is possible to renew the supply of food material 
 during weeks or even months, as it becomes exhausted, by 
 adding a fresh drop of culture-fluid at the margin every day 
 or two. 
 
CHAPTEE X. 
 
 INOCULATION OF ANIMALS. 
 
 WE come now to a group of experiments to which, in a 
 certain sense, all of the preceding- chapters point experi- 
 ments calculated to give the proof that a large number of the 
 most familiar contagious diseases are induced by bacteria, 
 and to render possible a more intimate study of the mutual 
 relations between these bacteria and the animal organism. 
 In this connection it must be said that conclusive proof that 
 a given infectious disease is due to a specific bacterian form, 
 is given only when such a form, well characterized morpho- 
 logically, chemically, or physiologically, can always be demon- 
 strated in the organs by the microscope and by cultures, in 
 this disease and only in it; and when, further, these bacteria, 
 after being cultivated pure for several generations outside the 
 animal organism, when reintroduced into an animal of the 
 species they were originally obtained from, produce the same 
 disease in this, and are then demonstrable in its tissues under 
 the microscope and by pure cultures. 
 
 Various kinds of animals have been used for experiment, 
 but usually the smaller rodents (rabbits, Guinea-pigs, rats, 
 and mice), or birds (fowls and pigeons) are employed. It is 
 now well known that inoculations, not of one but of a large 
 series of infectious diseases, may be effected upon one of the 
 most accessible, cheapest, smallest, and most prolific of mam- 
 mals the white mouse, which is more or less receptive for 
 splenic fever, chicken-cholera, malignant oedema, several forms 
 of septicaemia, some pyaemic affections, glanders, tetanus, etc., 
 etc. While the white mouse (an albino of the house mouse, 
 Mus musculus) is very slightly receptive for glanders, ac- 
 cording to Loeffler, the European field mouse (Arvicola ar- 
 volis] is very susceptible to it (Loeffler), as is also the wood 
 mouse (Mus sylvaticus) according to Kitt. On the other 
 
Bacteriological Technology. 103 
 
 hand, the field mouse is not receptive for mouse septicaemia, 
 to which the white mouse shows a great susceptibility (Koch). 
 Both field and wood mice are easily kept in captivity if fed 
 upon oats and moistened bread. The wood mouse is very 
 active, and its bite quite painful ; and it is not advisable to 
 keep several individuals of either of these mice in the same jar 
 after inoculation, because in case one dies the others devour it 
 at once. Where it is only desired to perform the most im- 
 portant fundamental experiments in bacterial infection, it is, 
 therefore, usually sufficient to employ white mice. 
 
 Keeping Mice. After many trials, I can highly recom- 
 mend the use of the cracker-boxes spoken of on p. 2, as cages 
 for mice. A large number of holes about as large as a half 
 dime are made in the cover, e.g., nine rows, of nine each. In 
 case the holes are not large or numerous enough, evaporation 
 from the interior of the box is checked too 
 much, and the mice cannot stand the resulting 
 humidity. The box is filled nearly half full of 
 sawdust, a little cotton is laid on this, and the 
 cage is ready for use. The mice are fed upon 
 white bread softened with water and oats. The 
 sawdust contributes largely to lessening offen- 
 sive odors, and, in connection with the cotton, 
 FIG. 68.-Mousfr.jar. gra i n .} lu n s , and the excrement of the animals, 
 forms a warm, soft, and dry mass, in which the mice tunnel, 
 and thrive and breed well. Ten or fifteen adult mice can 
 easily be kept in such a box, or a still larger number of young 
 ones. Emptying the box and renewing the sawdust and cot- 
 ton is only necessary once a month or even once in two or 
 three months, depending upon the number of animals kept in it. 
 For isolating infected mice, it is best to use common glass 
 pickle-jars, holding two quarts (Fig. 63). ' These jars are 
 readily disinfected after use, and the animals can be easily 
 observed in them. Each is filled for a third of its depth with 
 sawdust, and covered at top by a square piece of close iron 
 gauze, large and flexible enough so that it can easily be slipped 
 over the mouth of the jar and pinched into the groove a. 
 For greater security a heavy object may be laid upon the lid. 
 In this way it is fastened securely enough so that an animal 
 cannot lift it and escape, while it is readily taken off and re- 
 placed, and can be disinfected in the flame without difficulty. 
 
IC4 Bacteriological TccJuwlogy. 
 
 When white mice are constantly used for inoculation, they 
 are usually raised in the laboratory. Starting their propaga- 
 tion is not always easy, since mice are quarrelsome, and males 
 which have not been raised together often fight and injure 
 one another, while they are also apt to kill and eat the young. 
 As a general thing, a few pairs are taken for a beginning, and 
 kept apart. When a female is near the term of pregnancy, 
 she is removed from the male and kept in one of the mouse- 
 jars just described, where she remains with her 3 T oung ones 
 until their eyes are open, when they are all removed to one of 
 the large cages, in which two females and their litters can be 
 safely kept, as a rule, provided the young are of about the 
 same age. The young mice which grow up together in this 
 way generally get along so well that it is unnecessary to re- 
 move the pregnant females as long as there is room for their 
 burrows and young. 
 
 The mode of inoculating an animal, and the point at which 
 this is done, depend upon the object in view. If it is merely 
 desired to infect the animal, introducing the contagium 
 through a wound in the skin suffices in many cases. Not in- 
 frequent^, however, it is wished to imitate the natural infec- 
 tion through the uninjured mucous membrane of the respira- 
 tory or digestive system. In other cases it may be necessary 
 to introduce the virus under the dura mater, by trephining. 
 In short, the most different organs and parts of organs are 
 used for the introduction of cultures of bacteria, or infectious 
 material. Only the most important of the methods of inocu- 
 lation are considered in the following pages. 
 
 In most cases the inoculation of an animal is practically 
 painless, so that it is anaesthetized only when some larger 
 operation is involved, as in trephining for inoculation with 
 rabies. Mice and rats are placed under a bell-glass with a 
 wad of cotton moistened with ether, until, after a short period 
 of excitement, they succumb to the anaesthetic. After they 
 are removed from the bell-glass, and tied, insensibility is easily 
 kept up, as they are very tolerant of ether. It is otherwise 
 with Guinea-pigs and rabbits, the latter, especially, dying 
 easily when anaesthetized with ether, or, particularly, chloro- 
 form. Practice, however, enables the operator to lessen the 
 number of deaths from this cause, and in the Pasteur Insti- 
 tute only one to two per cent of the rabbits die from the an- 
 
Bacteriological Technology. 105 
 
 aesthetic, which is administered by pouring* a teaspoonful of 
 chloroform over a piece of absorbent paper folded as if for a 
 filter, which is then held like a cornucopia over the nose of the 
 animal. After a few seconds the respiratory movements stop, 
 and when they are resumed shortly afterward, insensibility as 
 a rule is complete,, as soon as this occurs the administration 
 of chloroform is discontinued. 
 
 Cutaneous inoculation is best performed on mice by abrad- 
 ing- the ear with a vaccine point dipped in the virus, although 
 it may be effected anywhere on the body after removing- the 
 hair. Scarification of the skin followed by rubbing- the virus 
 in, or simply rubbing- the latter into the uninjured skin, may 
 also be practised. 
 
 Subcutaneous inoculation of mice is best performed under 
 the skin of the back, just above the root of the tail. The 
 mouse being- in a glass jar (Fig. 63), its tail is seized between 
 the thumb and forefinger of the left hand and drawn out over 
 the rim of the glass, while the body hang-s into the jar, which 
 is covered with a small piece of board, held in place by the 
 free fing-ers of the left hand, so that there is just room for the 
 passage of the root of the tail between it and the edge of the 
 jar. [When the usual cylindrical jars of the German labora- 
 tories, with covers similar to those of candy -jars, but the top 
 replaced by coarse iron netting and weighted, are used, these 
 covers need only be tilted a little at one side to serve the same 
 purpose. If gray house-mice are used, they are apt to be far 
 more active than white mice, and it is best to quiet them by 
 holding a handkerchief, moistened with a few drops of ether, 
 over the cover, without carrying the etherization to the sur- 
 gical point. In any such operations, the long " mouse-tongs " 
 of the German dealers are a great convenience for first seizing 
 the tail of an animal. W. T.] The hair just above the tail is 
 clipped off, and by mean's of a lancet-needle or a pair of scis- 
 sors a small opening is made in the skin and a pocket torn in 
 the subcutaneous connective tissue, into which the infectious 
 material is placed. Solid bodies are introduced by means of 
 the forceps; very small quantities of cultures, etc., by the 
 lancet-needle or platinum needle; and slightly greater quanti- 
 ties of fluid, by means of capillary tubes or Pasteur pipettes. 
 
 When a considerable amount of fluid is to be injected into 
 the subcutaneous tissue, a sterilized Pravaz syringe must be 
 
io6 Bacteriological Technology. 
 
 used. The common Pravaz syringe is not adapted to sterili- 
 zation at high temperature, since the piston is packed with 
 leather, and the tip cemented on. Generally, a syringe is used 
 the tip of which screws on to a thread on the glass cylinder, 
 and with an elder-pith plunger, so that it may be sterilized by 
 either moist or dry heat (Straus and Collin). 
 
 Material from cultures in fluid or from vigorous cultures 
 upon solid media, can easily be taken from test-tubes by the 
 pipette or platinum loop. But when the bacteria to be used 
 for inoculation grow in a narrow line along the needle-thrust 
 in a test-tube, and it is desirable to have a relatively large 
 quantity of material, one should rather cut out the entire 
 colony and use it either with or without melting. The process 
 is a little different according as the culture is in gelatin, agar, 
 or serum. 
 
 A gelatin culture is dipped in warm water or warmed over 
 the flame until the outer part melts and the gelatin cylinder 
 becomes free from the test-tube, so that it can be shaken down 
 against the cotton plug, when the latter is carefully removed 
 and the gelatin allowed to fall into a sterilized watch-glass, 
 where, with a sterile and slightly warmed knife, all of the 
 peripheral part containing no bacteria is cut off and removed, 
 leaving only a small prismatic piece of gelatin containing the 
 colonies. 
 
 Agar-agar, which neither adheres to the glass so firmly as 
 gelatin, nor melts at so low a temperature, is removed in a 
 somewhat different manner. The test-tube is warmed some- 
 what, a Pasteur pipette is passed to the bottom of the tube, 
 between it and the agar, and by a strong puff through the 
 pipette, the entire culture can usually be blown out of the 
 glass. If this does not succeed, a warmed glass rod, such as 
 the handle of a platinum needle, is passed down to the bottom 
 through the agar at one side, so as not to come in contact 
 with the colonies of bacteria. As soon as it has cooled, it can 
 be used to draw the agar out by a series of pushes and pulls. 
 The agar being removed into a sterilized watch-glass, it is 
 treated just like gelatin. 
 
 Serum is far harder to manipulate, because it adheres to 
 the glass and cannot be removed by melting, so that it is nec- 
 essary to cut the colonies out of the test-tube as well as possi- 
 ble with a long-handled pointed knife. 
 
Bacteriological 
 
 Technology. 
 
 107 
 
 Intravenous injection cannot be resorted to with mice, 
 because of their small size. In the case of larger animals, it 
 can generally be effected without exposing- the vein by simply 
 thrusting- the needle of the syringe through the skin into the 
 cavity of the vessel. 
 
 With rabbits, it is particularly easy to inject material into 
 the veins of the ear and leg. For the former, the rabbit is 
 wrapped tightry and carefully in a long towel, so that only the 
 head projects. The ear is washed with 2-per-cent carbolic 
 acid, partly to disinfect it, and partly because the vessels are 
 more easily seen when the hair is wet. An assistant holds the 
 animal's head and compresses the base of the ear, so that the 
 
 FIG. 64. Injection of Culture into the Vein of a Rabbit's Ear. 
 
 veins swell, the ear is seized between the thumb and forefinger 
 of the left hand so that' it is slightly tense over the side of the 
 forefinger, and the canula is carefully thrust through the skin 
 into the vein (Fig. 64). The assistant now releases the end of 
 the vein, the operator holds the canula still in the vein with 
 his forefinger and thumb, and injects the fluid slowly. Bleed- 
 ing is easily stopped by brief compression, or by amadou. In 
 case the vein has not been pierced, the fluid soon begins to 
 distend the surrounding connective tissue, when it is almost 
 always useless to withdraw the canula a little way and make 
 another effort to force it into the vein, but another vein is 
 usually at once chosen for injection. Commonly the branch 
 of the vein running along the posterior edge of the ear is 
 selected, but there are many other available points on the ear. 
 
io8 Bacteriological Technology* 
 
 When the ear is used for injection, there is the further advan- 
 tage that one may be sure of really intravenous inoculation 
 without attendant accidental inoculation of the wound, by 
 simply removing- the portion through which the injection was 
 made, by a quick clip. 
 
 By connecting- the needle-shaped canula with a larger 
 syring-e by a rubber tube, large quantities of fluid may be in- 
 troduced into the veins of the animal, in the same simple and 
 painless manner. As the injection must be effected very 
 slowly, and in this case requires a longer time, it is best to 
 fasten the animal upon a "rabbit-board" (the French model, 
 used in Marey's laboratory, is far preferable to Czermak's) so 
 that a sudden movement of its head shall not displace the 
 canula. 
 
 For injection into the leg veins, the rabbit is firmly and 
 carefully wrapped in a long towel so that only one hind leg is 
 left free, the animal being allowed to draw the other up under 
 its body. An assistant sits with the rabbit upon his lap, one 
 hand extending the free leg firmly, while with the other he 
 grasps and compresses the thigh a little above the knee-joint, 
 so as to cause the veins to swell, his fore-arm resting upon 
 the body of the animal. One of the subcutaneous veins in the 
 lower joint of the leg is found, the hair over it is clipped off, 
 the skin washed with 2-per-cent carbolic acid, and while the 
 leg is further steadied by the left hand, the canula is pushed 
 through the skin into the vein, where it is held by the pressure 
 of the left thumb. 
 
 Intraperitoneal inoculation is most conveniently effected 
 with a sterilized Pravaz syringe. The entire thickness of the 
 abdominal wall is pinched up in a longitudinal fold, through 
 which the canula is forced crosswise so as to emerge on the 
 other side. On releasing the fold, the canula is carefully with- 
 drawn enough, so that its point lies within the body cavity, 
 while there is no danger of injuring the intestines. 
 
 In case large quantities of fluid are to be injected into the 
 peritoneal cavity, a graduated glass pipette may be used, one 
 end of which is plugged with cotton, while the other is con- 
 nected by rubber tubing with a canula similar to that of the 
 Pravaz syringe. The canula is inserted in the manner indi- 
 cated, and the fluid blown in. The same kind of inoculation 
 can also be effected with a Pasteur pipette, the perforation 
 
Bacteriological Technology. 109 
 
 being 1 made with a lancet-needle, beside which, as a guide, the 
 pipette is passed into the abdominal cavity. Mice and rats 
 are best etherized before inoculation. 
 
 Inoculation in the peritoneal cavity is especially important 
 in admitting of the introduction of very considerable quanti- 
 ties of either fluid or solid substances into the animal. In the 
 latter case, the body cavity must be opened for a greater dis- 
 tance along the linea alba. By such laparotomy, carefully 
 performed, an entire organ from a larger diseased animal 
 may easily be inserted into a smaller one, e.g., the heart and 
 kidneys of a rabbit, into a rat. 
 
 Inoculation into the anterior chamber of the eye acquired 
 especial importance in the study of tuberculosis, since by in- 
 serting small masses of tuberculous material into the eye Con- 
 heim and Salomonsen succeeded in inducing 1 tubercle of the 
 iris, by which it became possible to directly observe the in- 
 cubation period of miliary tuberculosis, and its independence 
 of a preceding suppurative process. It has also recently been 
 used for rabies inoculations. 
 
 The operation is performed by fastening the rabbit, on its 
 belly, usually on an operating board, where its head can be 
 perfectly fixed. An assistant opens the eyelids, most conveni- 
 ently by sitting so as to face the operator, grasping- the ani- 
 mal's head with both hands. Occasionally the nictitating- 
 membrane is so large that it must be held back. With a pair 
 of forceps, a fold of the conjunctiva is seized, and with a suit- 
 able knife a cut 2 to 3 mm. long is made in the cornea, near 
 its margin, with the usual precautions observed in ophthal- 
 mological operations. If the material to be introduced is a 
 solid substance, it is passed through the opening 1 by means of 
 slender curved forceps, usually with very fine smooth points 
 opening parallel, and by carefully stroking the cornea with a 
 DavieFs spoon, the effort is made to crowd the introduced 
 material into the bottom of the anterior chamber. If this is 
 not easily effected, it is allowed to remain where it lies. Fluids 
 are injected through a (usually) blunt and bent canula, in- 
 serted into the cut. To anaesthetize the eye for the operation, 
 a 2-per-cent cocaine solution is dropped into it, the maximum 
 action being reached after fifteen minutes (Howe). 
 
 Another place, easily accessible for observation, which has 
 often been used for inoculations, is the cornea, in which the 
 
iio Bacteriological Technology. 
 
 conditions are very favorable for observing* the effect of bac- 
 teria upon the connective-tissue cells. By means of a blunt 
 needle, a large number of pricks or scratches are made in the 
 cornea! tissue, without perforating* it, and the bacteria are 
 gently rubbed into the wounds; or the needle may first be 
 dipped into the virulent material, but the first method seems 
 to give surer results. 
 
 Inoculation beneath the arachnoid, as first used in hydro- 
 phobia infections in Pasteur's laboratory (Pasteur and Roux), 
 is effected on rabbits and guinea-pigs in the following- manner: 
 A cut is made through skin and aponeurosis, on the crest be- 
 tween the eyes and ears, and the edges of the wound are held 
 apart by a small tenaculum. The trephine (about 5 to 6 mm. 
 in diameter) is applied behind the orbit, on one side of the 
 middle line. When the circular groove in the bone is deep 
 enough, the centre pin is withdrawn so that it shall not 
 injure the dura mater. As a general thing, the fact that the 
 bone is cut through, is observed in time, but for greater cer- 
 tainty it may be seen, from time to time whether the disk of 
 bone cannot be lifted out. When the dura mater is exposed, 
 it is pierced with a needle-shaped Pravaz canula, bent nearly 
 at right angles, which is drawn slightly toward the operator 
 to avoid injury to the underlying brain, and two or three 
 drops of the diseased spinal cord infusion are injected. The 
 wound is cleansed with 2-per-cent carbolic acid and closed by 
 a couple of sutures. In the case of dogs, the skin is pushed 
 aside after the cut is made, the temporal muscle is loosened, 
 and the skull is trepanned in the fossa temporalis, where the 
 skull is thinner, and there is little bleeding. 
 
 As compared with all other methods employed in the 
 study of rabies, inoculation under the dura mater has the 
 advantage of giving absolutely certain infection, as well as 
 the shortest and constant incubation. But according to the 
 latest investigations of Roux and others, the far simpler inoc- 
 ulation in the anterior chamber of the eye gives as certain 
 infection as the subdural. 
 
 Pure virus is always found in the medulla, etc., of animals 
 dead of hydrophobia, which is best suspended in sterile bouil- 
 lon or 0.7 per cent salt solution, and injected with a Pravaz 
 syringe. The removal and preparation of the material must 
 naturally be effected with the greatest possible cleanliness, to 
 
Bacteriological TecJinology. 1 1 1 
 
 avoid accidental infection. In the Pasteur Institute the 
 material for inoculation is prepared by snipping- out a small 
 piece of brain substance from the bottom of the fourth ventri- 
 cle and placing it in a wine glass holding about 150 cm., which 
 has been covered with paper and sterilized at 150 C. Here it 
 is rubbed to a soft paste by means of a glass rod, and a small 
 quantity of sterile bouillon is gradually added, while it is con- 
 stantly stirred. The glass is then covered with paper and set 
 aside until the coarser particles have fallen to the bottom, the 
 upper, slightly turbid, layer of fluid being used for injection. 
 
 The medulla oblongata retains its virulence unchanged for 
 at least a month when kept in pure neutral glycerin a fact 
 which should be remembered whenever the occasion arises for 
 sending parts of the central nervous system of man or one of 
 the lower animals to a distant laboratory for diagnosis. 
 
 Rabies vaccines are prepared as follows (Pasteur and 
 Roux) : A rabbit weighing two kilos and measuring about 45 to 
 50 cm. from the nose to the root of the tail, is inoculated by 
 trepanning with " virus fixe." Six or seven days later it shows 
 symptoms of rabies, and dies on the tenth day. Before putre- 
 faction sets in, the central nerve system is removed as follows : 
 The skin is split down the back from nose to tail, dissected to 
 one side, the dorsal and cervical muscles are loosened from 
 the spinal column and cranium, and the spinous processes are 
 removed by bent shears. The nose is then seized with a pair 
 of strong bone nippers, and so held fast with the left hand, 
 while the theca cranii is snipped and broken off by means of a 
 pair of Liston's bone scissors. Then the vertebral arches are 
 removed one by one, by snipping them through on the right 
 and left side, as near the body as possible, without injuring 
 the spinal cord, and breaking them off. This is more easily 
 described than done, for the spinal cord is easily crushed, es- 
 pecially in the cervical region; but with practice one gradu- 
 ally learns to expose it for its entire length without injuring 
 it with the coarse instrument used. Then the cord is cut off 
 over the cauda equina, its membrane is seized with forceps 
 just above the cut and it is raised for a distance of 6 to 7 cm., 
 and all adhesions are cut. The loosened piece is cut off, a 
 sterile silk thread tied about one end, and hung up (Fig. 65) 
 in a litre flask with two openings previously plugged with 
 cotton and sterilized at 150 C. Enough caustic soda to cover 
 
112 
 
 Bacteriological TecJinology. 
 
 the bottom is dropped into the flask, the spinal cord is hung 
 from its neck, and the whole set aside at 20 to 25 C. The 
 vaccines are prepared in the manner described above, from 
 such pieces of spinal marrow, hung- up and 
 dried for a long-er or shorter time. 
 
 Infection through the digestive tract is 
 effected by carefully ming-ling- the contagium 
 with some food that the animal is fond of, 
 which is given it in not too larg-e quantity, 
 the animal being 1 previously kept without food 
 for a day or less, if necessary. In case of 
 mice and other small animals, this is the only 
 available method. In the case of larger mam- 
 mals (rabbits and Guinea pigs), simple feed- 
 ing is also used when large quantities of 
 solid material* are to be introduced into the FIG. es.-Drymg-jar for 
 stomach, and it is the best means of inducing Hydrophobia v*ine. 
 infection through the alimentary canal, because it is in this 
 way that natural infection by means of food occurs as a rule. 
 Still it cannot always be employed, and other means must 
 then be resorted to. Fluids (e.g., cultures of bacteria) can 
 likewise be poured or injected through the oesophagus, the 
 rabbit being wrapped in a towel with the exception of its head, 
 and held on the lap of an assistant. The animal is made to 
 open its mouth by slight pressure upon its cheeks over the 
 molar teeth, and a small wooden gag (Fig. 66) is inserted so 
 that its incisors rest in the grooves on the upper and lower 
 surfaces (b). Through the perforation in the gag, a No. 17 
 catheter can easily be introduced into the rabbit's ventricle. 
 
 Adhering bits of food show that 
 it has been inserted right. In case 
 of the Guinea pig, a smaller cathe- 
 ter (usually a bulb catheter) must 
 be employed, and it requires to be 
 passed down with especial caution. 
 Solids are quickly introduced 
 into the crop of doves or fowls by 
 
 Fia. 66. Wooden Gag for Passing a 
 Catheter into the Stomach, in Feeding 
 Experiments. 6, Section of same. 
 
 opening the beak, and placing the food in the mouth, as far back 
 on the tongue as possible, when the bird quickly swallows it. 
 Small meal pellets are formed with cultures and other fluids 
 containing bacteria, and these are introduced in the same way. 
 
Bacteriological Technology^ 113 
 
 The introduction of infectious fluids into the stomach and 
 intestine through the abdominal wall, by means of a Pravaz 
 syringe, is more certain and reliable. If the contagium is to 
 be introduced directly into the intestine, the abdomen must be 
 antiseptically opened, a fold of the intestine found and care- 
 fully fixed, and the canula thrust through its wall. In this 
 way cultures of the cholera spirillum are injected into the 
 duodenum (Nicati and Rietsch), so that they do not pass 
 through the stomach, and the action of the gastric juice upon 
 the microbes is avoided. 
 
 Pathogenic bacteria can be brought in contact with the 
 intact surface of the lungs by injection through an opening 
 made in the trachea. For the sake of entire certainty that 
 the virus is not at the same time introduced through the open 
 wound, this may first be allowed to heal, leaving a small 
 tracheal fistula. In the case of large animals, Arloing, Cor- 
 nevin, and Thomas have reached the same end by passing a 
 drainage tube through a metal canula, and injecting the de- 
 sired material through the former, in small quantities, so as to 
 avoid spasms of coughing. 
 
 If the natural means of infection through the mucous mem- 
 brane of the respiratory organs are to be imitated as closely 
 as possible, the bacteria must be very finely and uniformly 
 suspended in the air inhaled by the animal, so as not to cause 
 mechanical irritation. This is best effected by use of a spray or 
 dry powder. The former method is open to the objection that 
 the spray contains not only extremely fine drops, but likewise 
 some which are a little larger, so that the mucous membrane 
 of the nose, or the entire animal, becomes quite damp. To 
 avoid this, Buckner joins the atomizer to a two-necked Woulf 
 flask holding about three litres, where the coarser drops are 
 allowed to settle, while the finer, as a nearly imperceptible 
 mist, are passed out of one neck of the flask through a tube 
 to the inclosed chamber where the animals are. 
 
 For the second method, a fluid-culture of the bacteria is 
 poured over a rather large quantity of spores of Lycoperdon 
 giganteum, which are then dried off over calcium chloride, 
 and the powder is blown into the closed animal cage by the 
 bellows (Buckner). In his experiments on the entrance of 
 spores of B. anthracis through the lungs, Buckner used 0.25 
 gm. of powder for a space of three litres, and allowed the in- 
 8 
 
ii4 Bacteriological Technology* 
 
 halation to continue ten to fifteen minutes. Other powders 
 (carbon, talc, etc.) can also be used as a vehicle for cultures of 
 bacteria, but the puff-ball spores are superior to all of these 
 in being 1 very small, uniform, and of low specific gravity. In 
 case the bacteria are killed or deprived of their virulence by 
 drying-, this method, of course, cannot be employed. 
 
 In such experiments, the animals must be untranimeled so 
 that their respiratory movements are not checked or altered, 
 and it is best, therefore, to let them remain unfastened. A 
 number of mice can be placed together loose in a tight tin 
 box with a.few breathing holes plugged with cotton. If it is 
 necessary to fasten the animals so as to keep their heads in a 
 given direction, wire gauze can be used, as it may be bent and 
 shaped at will. A single mouse can conveniently be placed in 
 a glass tube 3 to 4 cm. in diameter, with a bored and split 
 cork in one end, which serves to hold its tail. 
 
 When inhalation experiments are performed with bacteria 
 that are pathogenic for man, the operator should remember 
 to take sufficient precautions, such as having tight vessels 
 and tubes, cotton-plugged air-spaces about the cages of the 
 animals, and long tubes for the atomizer, and the work should 
 be done in the open air. 
 
CHAPTEE XL 
 
 CULTURES FROM MAN AND ANIMALS. COLLECTING AND 
 PREPARING PRIMARILY STERILE CULTURE-MEDIA. 
 
 IN attempting- to obtain cultures of the pathogenic micro- 
 organism from the blood or organs of an animal which has 
 died from an infectious disease, the rules given in Chapters I. 
 to III., and V., are to be observed, as well as the following : 
 
 Since the more or less dirty hair or feathers of the animal 
 contain a possible source of contamination, it is best to skin 
 or pluck the body before opening it. All of the parts covered 
 by hair may also be washed with sublimate solution, or cov- 
 ered with filter-paper wet with sublimate. 
 
 All instruments used for preparing the body must be care- 
 fully sterilized. Since repeatedly heating them to a high de- 
 gree in the flame w^orks destructively upon knives, scissors, 
 and forceps, it is best to have two sets of these instruments; 
 one intended for the coarser part of the work, sterilized by 
 flaming, and commonly used so hot as to cause the flesh to 
 hiss while the cut is being made; the other for finer opera- 
 tions, first sterilized at 150 C., in a metal case or wrapped in 
 paper, and merely hastily flamed just before use. 
 
 In addition to the gas or spirit flame, a dish of 0.1 per cent 
 sublimate solution, and of 5-per-cent carbolic acid, must be 
 kept ready for the disinfection of hands and instruments. To 
 avoid contamination from contact, the sterilized instruments 
 are laid upon glass supports (Fig. 18) and covered with a bell- 
 glass, or dropped into a short cylindrical vessel with their 
 points projecting out at top. 
 
 While exposing the organs from which material is to be 
 taken for cultures, use is made of strongly heated instruments, 
 which, as far as possible, are kept from touching the organs. 
 No definite rules can be given, further than this : 
 
 If a culture is to be started from fluid contained in a cavity, 
 
1 1 6 Bacteriological Technology. 
 
 it is best to singe the surface over, before piercing 1 the wall. 
 For example, if a collection of fluid in the pleural cavity is to 
 be used, after exposing the ribs and intercostal muscles in the 
 usual way, a part of one of the intercostal spaces is scorched 
 by means of a glass rod heated in the flame, and then, with a 
 glowed needle or strongly heated small knife, the pleural cav- 
 it}^ is opened, and the small gaping wound made for inserting 
 the inoculation-needle, capillary tube, or pipette, according to 
 the quantity of fluid needed. In the same manner, blood is 
 obtained from one of the anterior chambers of the heart. After 
 cauterizing the surface, as a rule the capillary tube or pipette 
 may be thrust through the thin wall, without the previous 
 use of a knife, which only becomes necessary when a thin and 
 flexible platinum needle is to be inserted. 
 
 To obtain material from the substance of a solid organ, 
 one of the thicker and more rigid needles is used. The organ 
 may be snipped in two with scissors carefully flamed or even 
 glowed, and the needle thrust into its substance from the un- 
 contaminated cut surface, or it may be torn open with sterile 
 fingers or forceps, and in this way a surface exposed which 
 has not been touched at all by the instruments. [Loeffler 
 advises that when parts of cut organs are to be broken in this 
 way, the surface be first disinfected by dropping the whole 
 piece into 0.1 per cent sublimate, and moving it about in this 
 for a minute or two]. By using flamed, but not too strongly 
 heated, curved scissors, small pieces of the organ may be 
 snipped out and placed on or in the culture medium, at least 
 after being crushed between two sterilized glass slides. This 
 method is recommended by Koch in sowing from tubercle. 
 
 Since the white mouse is the animal presumably used in 
 the elementary inoculation experiments, the following descrip- 
 tion is given of the best means of obtaining cultures from its 
 heart blood. As soon as the external examination is com- 
 plete especially about the inoculation wound the body is 
 extended upon its back by a shawl pin through each leg, on a 
 small board, -which it is well to first cover with a sheet of par- 
 affined paper over which a couple of sheets of filter-paper are 
 laid to absorb blood, etc. The head is finally fixed by means 
 of a fifth pin through the nose. The skin is dissected away 
 from the ventral surface as far as possible, and entirely cut 
 off, the occasion being taken to examine the axillary and in- 
 
Bacteriological Technology. 1 1 7 
 
 guinal lymphatics, and, if necessary, to obtain blood from the 
 axillary vessels for microscopic investigation. Seizing- the 
 prominent ensiform process with a pair of sterilized forceps, 
 it is ] if ted strongly, and ~by means of flamed sharp-pointed 
 scissors, one end of which is inserted under the ribs, a suffi- 
 cient part of the thoracic wall is removed to expose the peri- 
 cardium. In this way the intestines are not approached, and 
 the instruments are not exposed to infection from this dan- 
 gerous source. Care is taken to avoid touching the anterior 
 surface of the heart with the scissors. With two pairs of 
 small flamed forceps the pericardium is now torn open, and a 
 small cut is made into the exposed heart. Usually sufficient 
 blood to wet the platinum needle at once bubbles out of the 
 wound; otherwise the needle is pushed through the opening, 
 and the culture started from the fluid obtained. The dissec- 
 tion is now completed in the usual way, but with especial care 
 and cleanliness so as to avoid rendering it difficult or impossi- 
 ble to make cultures from other organs in which morbid 
 changes may subsequently be discovered. When the dissec- 
 tion is finished, and the necessary preparations have been 
 saved, the cadaver is wrapped in the filter-paper it lies on, and 
 burned up. 
 
 The manner of making the dissection naturally varies with 
 the organ chosen for starting a culture, the rule being to pro- 
 ceed directly to the organ from which the chief culture is to 
 be made. If, for instance, it is especially important to ob- 
 tain a culture from the pulp of the spleen, the mouse is laid on 
 its side with the spleen up, so that it can be exposed without 
 disturbing the other organs too much. 
 
 The directions and examples given here may serve as a 
 sufficient introduction to the methods of obtaining cultures 
 from the dead body, as well as to the collection and similar 
 use of fluids and tissues from the living body, where, however, 
 consideration for the patient may necessitate many modifica- 
 tions : e.g., scarification with the heated glass rod must be re- 
 placed by a thorough cleansing with disinfectants, the Pasteur 
 pipette must give place to the sterilized Pravaz syringe, etc. 
 
 Few appliances are needled for starting such cultures with 
 all requisite precautions, even at the sick-bed or operating 
 table, or for collecting the morbid products in a state of com- 
 plete purity, for further elaboration in the laboratory. Even 
 
1 1 8 Bacteriological TccJinology. 
 
 when much is aimed at, practically all that is needed is the 
 usual disinfectants (carbolic acid and corrosive sublimate) ; a 
 little sterile absorbent cotton and filter-paper; scissors, knife, 
 and forceps, wrapped in paper and sterilized at 140 C. ; steril- 
 ized Pravaz (or Straus) syringe; alcohol lamp; platinum wire; 
 capillary tubes; Pasteur pipettes and others with a capillary 
 constriction (Fig-. 53, a) ; small test-tubes containing- various 
 culture media, some of them for the culture of anaerobic 
 forms, e.g., with a considerable depth of nutrient gelatin; 
 labels ; and cover-glasses. Some of these can be prepared on 
 short notice in the sick-room, some are only needed for excep- 
 tional use, while the rest should always be kept "in the hospital 
 wards to prevent the waste of much valuable material. 
 
 The collection of primarily sterile culture-media, which 
 should really have been con- 
 sidered in the third chapter, 
 is treated here, because the 
 precautions to be observed 
 are exactly the same as in 
 obtaining cultures from the 
 dead body, the collecting ap- 
 paratus only being different. 
 Usually it is desired to col- 
 lect the sterile fluids (blood, 
 urine, milk, etc.) either in 
 
 rather large vessels from which they can be transferred to a 
 number of smaller culture-glasses, or they are at once drawn 
 into the latter. 
 
 In the first case, the so-called Pasteur wash-bottle (Fig. 67) 
 is best used. As the Figure shows, this is really only a very 
 large Pasteur pipette, with a reservoir of peculiar form. 
 When the capillary point b has been sealed by fusion, and a 
 cotton plug inserted at a, it is sterilized at 150 C. Just be- 
 fore use, the tip ft is broken off, and the entire capillary part 
 is flamed. Observing the precautions indicated above, the 
 point is inserted, and the receiver sucked full, after which the 
 point b is again fused. As soon as possible afterward, the 
 sterile fluid is transferred to smaller culture-vessels, by blow- 
 ing through a, after once more breaking the point off and in- 
 verting the reservoir. The test-tubes or Chamberland flasks 
 which have been filled in this way are kept on probation at 
 
Bacteriological Technology* 119 
 
 30 to 40 C., for several clays before being used. In this way 
 it is possible, for instance, to collect large quantities of sterile 
 urine from the newly slaughtered animals at any slaughter- 
 house, by passing the capillary tip of the flask through the 
 wall of the bladder, after the abdominal cavity is opened. 
 
 Occasionally it may be convenient to collect the fluid at 
 once in smaller culture vessels. For example, if sterile blood 
 is to be taken from the heart of a newly killed animal, it would 
 coagulate in the larger vessel before it could be transferred 
 to the smaller ones. In such cases, Pasteur pipettes may be 
 used, not closed with simple cotton plugs, as is usual, but with 
 rubber tubes plugged with cotton (Fig. 68), which can be re- 
 moved and again replaced with far less danger of contamina- 
 tion when the contents of the tube are to be inoculated. The 
 tip of the pipette is opened, flamed, and inserted into the heart 
 of a newly killed healthy animal, the tube sucked full of blood, 
 once more hermetically sealed below, and set aside some time 
 
 FIG. 68. Pasteur Pipette with Rubber Cap Plugged with Cotton. 
 
 for observation, after which, if free from germs, it may be 
 used directly as a culture vessel. In the same manner, fresh 
 white of egg, containing no germs, can be collected from hens' 
 eggs (cf. p. 33). 
 
 The collection of sterile fluids from living men or other 
 animals is not always as easy or certain as in these cases, 
 though it is by no means impossible. 
 
 For the means of obtaining sterile blood by phlebotomy, 
 see p. 31. Milk can be drawn into sterile test-tubes, after a 
 very thorough cleansing of the surface of udder and teats; 
 but one should expect to find a portion of the tubes accident- 
 ally contaminated. Urine can be obtained in a sterile condi- 
 tion either by passing it directly into sterile vessels or draw- 
 ing it with a catheter, after a careful disinfection of the 
 urethra by washing it out several times. 
 
 Now and then very good opportunities occur for the collec- 
 tion of a large quantity of sterile culture fluid from living 
 patients. Dropsical and other similar fluids can be collected 
 free from germs without great difficulty. For this purpose, 
 
1 2O Bacteriological Technology. 
 
 a small Southey trocar is used, and the fluid is collected in a 
 large flask furnished with a rubber stopper bored with two 
 holes, one of which is plugged with cotton, while the other 
 admits a short glass tube which is joined by rubber tubing to 
 the trocar. The instrument and glass ware are sterilized at 
 140 C., while the rubber tubing and stopper are steamed be- 
 fore use. 
 
 The rules for preparing primarily sterile infusions are im- 
 plied in the preceding. Meat infusion, for instance, is obtained 
 by cutting a piece of muscle from a recently slaughtered ani- 
 mal, as rapidly as possible, and with all care, and dropping it 
 into sterilized water. Vegetable infusions are generally more 
 easily prepared, if rather large organs, such as fleshy roots 
 and tubers, are employed. The surface of these is cleansed 
 with sublimate solution, after which prismatic pieces are rap- 
 idly cut from the interior with a knife that has been heated 
 in the flame so that it hisses with each cut. From the nature 
 of the case, rules for each particular instance cannot be given, 
 often it is necessary to specially prepare the substance, e.g., 
 in forming the primarily sterile infusion of jequirity as de- 
 scribed by Salomonsen and Christmas ("Hosp. Ticl.,' 1 1884). 
 
 It is always to be expected that a certain number of the 
 fluids and infusions prepared in the ways indicated above have 
 become accidentally contaminated in the process of prepara- 
 tion; hence they must never be made use of until they have 
 been kept several days in the thermostat at about 35 C., re- 
 maining completely clear and unchanged. 
 
CHAPTER XII. 
 
 DISINFECTION EXPERIMENTS. 
 
 WHEN it is desired to learn whether a disinfectant is capa- 
 ble of destroying- a given contagium, the most natural way 
 is to expose this particular germ, if it is known, or if not, sub- 
 stances which carry it, to the influence of the disinfectant, 
 after which it is used for the inoculation of a suitable organ- 
 ism. But the difficulties in the way of such a test of disinfect- 
 ants are evident. Often the contagium is one to which our 
 experimental animals are not susceptible, and even when in- 
 oculations are possible, they are often too complicated to be 
 feasible on a large scale. Hence, long before pathogenic bac- 
 teria could be isolated in cultures, putrefactive bacteria and 
 other micro-organisms were employed as substitutes for the 
 real contagia, in investigations concerning the efficacy of disin- 
 fectants, because of the resemblance of pathological processes 
 to those of putrefaction and fermentation. 
 
 To-day, with greater reason, non -pathogenic bacteria can 
 be employed as tests of the relative disinfecting power of 
 various substances ; for we now know that a large part of the 
 contagia really are bacteria, and a better knowledge of the 
 natural history of the latter has rendered possible the avoid- 
 ance of numerous errors pertaining to earlier investigations 
 and results. For instance, it is now known that a motile 
 species is not to be regarded as dead because its motions 
 cease; and the extremely different resisting power of different 
 species, and the surprising vitality of spores, are now recog- 
 nized, as well as the fact that conclusions cannot be drawn 
 for bacteria in general, and hence for all contagia, from what 
 is learned of casual mixtures of bacteria. E.g., the bacillus 
 of typhoid fever is very resistant toward carbolic acid, a cir- 
 cumstance which, according to Chantemesse and Vidal, facil- 
 itates their recognition in mixtures of bacteria, since they are 
 
122 Bacteriological Technology. 
 
 capable of developing in nutrient gelatin which contains 0.2 per 
 cent of carbolic acid. On the other hand, substances are to 
 be found which are poisonous to a single species, while they 
 are quite harmless for others: e.g., iodoform, on the anti- 
 bacterial influence of which, blind reliance was placed before 
 the investigations of Rovsing and Heyn. But while this sub- 
 stance is not fatal to the greater number of bacteria, such as 
 Micrococcus pyogenes aureus, the tubercle bacillus (Rovsing) 
 and a number of other pathogenic forms, either within the 
 animal organism or in cultures, it is extraordinarily destruc- 
 tive to the cholera spirillum (Buckner). 
 
 Koch's directions for experiments with disinfectants will 
 be followed here very closely, since he has recentty stated the 
 problem clearly, and given the simplest and best means of 
 solving it. It is also well known that the researches of Koch, 
 Gaffky, and Loeffler concerning many points have caused an 
 entire revolution in the methods of disinfection. 
 
 The rules to be followed in testing the germicidal value of 
 a disinfectant, are, briefly, the following : 
 
 Pure cultures of well-known bacteria are used as reagents, 
 not mixtures of unknown composition. For a complete knowl- 
 edge of the rank of the disinfectant, these pure cultures should 
 represent the several groups of micro-organisms which are 
 active in disease and fermentation, i.e., moulds, yeasts, and 
 bacteria. Bacteria which do not form spores should also be 
 represented, as well as the most resistant bacilli (e.g., the 
 hay bacillus, Chapter IV.). But if it is only desired to learn 
 whether a given substance is capable of destroying all organic 
 life, and consequently all living contagia, it is sufficient to use 
 bacillus spores alone, since these are the most resistant living 
 beings known at the present time. 
 
 The forms chosen as reagents oug'ht to be easily recog- 
 nizable by macroscopic characters; consequently chromogenic 
 species are preferable, such as the black Aspergillus niger, 
 the pink yeast [" Saccharomyces glutinis "], the blood-red Mi- 
 crococcus prodigiosus, etc., etc. The persistence or absence 
 of ability to develop after exposure to the action of the disin- 
 fectant is taken as evidence of their life or death. 
 
 According to the nature of the disinfectant, and the cir- 
 cumstances under which it is tested, the cultures employed as 
 reagents are used in various ways. Sometimes they are not 
 
Bacteriological Technology. 123 
 
 prepared in any way; e.g., a tube or flask with the contained 
 bacteria and culture medium can be exposed to a higii tem- 
 perature for a given time, and the power of its contents to 
 develop tested by inoculation in a fresh tube; or a drop of a 
 pure culture can be distributed through a disinfecting- fluid 
 and the mixture sown upon a suitable culture medium. 
 
 But it is generally more convenient to use the cultures 
 dried upon a solid substance, in which condition they are 
 more easily preserved, transported, and made use of. They 
 may be employed in either of the following ways : 
 
 The pure zoogloea is sliced off from a potato culture by 
 means of a flamed knife, with as little of the tissue of the 
 potato as possible, and laid away to dry in a sterilized glass 
 tray which is wrapped in a double layer of filter-paper instead 
 of being covered by the lid. This prevents the access of dust, 
 while it permits evaporation to go on rapidly enough to com- 
 pletely dry the culture in a couple of days. The dried cultures 
 are kept in sterilized test-tubes plugged with cotton, until they 
 are to be used. 
 
 Small pieces of filter-paper sterilized at 150 C. are rubbed 
 upon the potato culture to be used, by the aid of a sterilized 
 glass rod, and dried and preserved as in the preceding case, 
 after being cut into narrow strips with a pair of scissors. 
 These strips are subsequently cut into smaller square pieces 
 when they are to be used. In case of fluid cultures, the paper 
 may be saturated with them, and dried, but it is better to use 
 the next method. 
 
 White silk thread is cut into pieces 8 to 10 mm. long, which 
 are sterilized at 150 C. in test-tubes plugged with cotton. 
 Though they may be rubbed upon solid culture, as in the last 
 case, these are especially suited to fluid cultures, into which a 
 large number of them are dropped. After being well shaken 
 about here for some time, they are taken out and dried as 
 above, care being taken to lay them in the glass well sepa- 
 rated, since, especially when taken from liquefied gelatin cul- 
 tures, they are very apt to cling together. They are preserved 
 as in the preceding cases. 
 
 Frequent use is made of the spores of Bacillus anthracis 
 and B. subtilis, in disinfection experiments. To be sure of 
 abundant formation of spores in the former, it must be sown 
 on the surface of nutrient agar, and kept in the brood-oven at 
 
1 24 Bacteriological Technology. 
 
 30 to 37 C., for a week or longer. When it is found by mi- 
 croscopic observation that fully developed normal spores are 
 abundant, and the vegetative filaments beg-in to disappear, 1 
 to 2 cm. of sterilized water is added to the tube containing the 
 spore bearing culture, which is distributed through it by ener- 
 getic shaking. The pieces of sterile silk are dropped into the 
 turbid fluid, and preserved in the manner indicated. The 
 hay bacillus is always certain to form its spores in a fluid 
 medium such as bouillon. It is sown in a conical flask con- 
 taining bouillon to a depth of 2 cm., and kept at 30 to 37 C. 
 A day or two later it has developed a firm spore-bearing mem- 
 brane upon the surface of the turbid liquid. After some time 
 the culture becomes exhausted and loses its turbidity, the spores 
 sinking to the bottom as a white powder. Three-fourths of 
 the supernatant fluid is poured off, the precipitate of spores 
 is shaken up in the remainder, and silk is saturated with it. 
 
 If spores are wanted which have even more vitality than 
 those of these two species, they may be obtained from garden- 
 earth, etc. 
 
 The spores of moulds are best prepared in a somewhat dif- 
 ferent manner. The mould is sown upon gelatinized beer-wort 
 in a pair of small glass trays (Fig. 13). When it has covered 
 the surface and is in fruit, the entire culture c'an be stripped 
 off in one piece, when it is laid upon a piece of filter-paper in 
 which it is loosely wrapped to prevent the scattering of the 
 spores, and when dry it is clipped into strips. 
 
 Garden earth has also, by the advice of Koch, frequently 
 been used as a bacteriological reagent in disinfection experi- 
 ments. In this case the rule that only pure cultures are to 
 be used is set aside, and in this fact lies the disadvantage of 
 using earth, which contains different bacilli in different places, 
 so that it is impossible to draw general conclusions from the 
 results obtained with a single sample of earth. On the other 
 hand, it has the advantage of being quickly prepared any- 
 where with ease; but in comparative mvestigations it is neces- 
 sary to use small portions of the same bit of earth for the 
 entire series. Pure cultures of the most resistant earth 
 bacilli can easily be obtained by suspending a rather large 
 quantity of dirt in bouillon, which is afterward boiled in a 
 cotton-plugged flask for 15 to 30 minutes, set aside at 30 to 40 
 C. for a couple of days, and then plated out in agar-gelatin. 
 
Bacteriological Technology. 1 2 5 
 
 It must be remembered that these reagents, excepting 1 , 
 perhaps, the bacillus spores, cannot be kept in a useful state 
 for an unlimited time, but that they generally die after a longer 
 or shorter time. It is, therefore, necessary to ascertain their 
 ability to develop before using them in case they are rather 
 old. It is safest to use freshly prepared material. 
 
 The general method of carrying out an investigation is as 
 follows : 1, exposing the bacteria to the action of the disinfec- 
 tant; 2, removing the latter; 3, sowing or inoculating the 
 exposed bacteria on a suitable culture medium or animal; 4, 
 sowing on inoculating control material at the same time ; 5, 
 observing the result of the cultures or inoculations, not merely 
 as to whether or not they grow, but also rapidity of growth, 
 virulence, etc., etc. 
 
 When it has been shown by such experiments that a given 
 disinfectant really destroys one of the pathogenic bacteria 
 quickly and surely, it by no means follows that the substance 
 is practically useful for the destruction of this contagium. 
 Before a disinfectant can be recommended, it is particularly 
 necessary to show that it not only kills the germs on our silk 
 threads, bits of papers, and potatoes, but is also capable of 
 attacking them as they occur in nature in clothing, excre- 
 ment, sputum, etc. The effectiveness of the most powerful 
 disinfecting agents may under such circumstances be neutral- 
 ized by chemical changes or the impermeability of the sub- 
 stance which harbors the contagium, which, for example, pre- 
 vents the successful employment of the corrosive sublimate 
 solution for disinfecting vaults and tuberculous sputum. On 
 the other hand, contagia may occur in nature under circum- 
 stances more favorable for their destruction by a given disin- 
 fectant than is the case when pure cultures of them are ex- 
 posed to its action. [Laplace's solution of sublimate in crude 
 hydrochloric acid, diluted for use to the standard ratio of 1 : 
 1,000, has the advantage over the simpler aqueous solution, of 
 not so readily forming insoluble precipitates with albuminoids. 
 W. T.] How to proceed so as to accomplish the purpose of 
 the investigation, is implied in what has been written above. 
 Either the infected excrement, sputum, or clothing is exposed 
 to the action of the disinfectant and then used for inoculation 
 experiments (e.g., by inoculating guinea-pigs with the tuber- 
 culous sputum that has been treated with carbolic acid or 
 
126 Bacteriological TecJmology. 
 
 corrosive sublimate, or causing- healthy people to wear pieces 
 of clothing disinfected by heat or chemicals) ; or the micro- 
 organisms mentioned above are used as reagents, carefully 
 mingled with excrement or sputum, or placed in the pockets 
 or under the lining of clothing in which they are disinfected, 
 and their power of development subsequently tested in the 
 usual way. 
 
 Finally, a large number of questions must be answered 
 which are of a clinical or technical nature, etc., and hence lie 
 without the scope of this work (e.g., those concerning the dan- 
 gerousness of the substance, its possible injuriousness to the 
 objects to be disinfected, offensive odor, expensiveness, etc.); 
 and an opinion as to its practical utility can only be reached 
 after these are considered. 
 
 The rules given here must be kept in mind in all disinfec- 
 tion experiments, whether referring to the germicidal power 
 of heat, sunlight (Duclaux), sound waves, electricity, fluids, or 
 gases. But the methods must obviously be modified in their 
 details to suit each case. As examples, a detailed account is 
 given here of the best manner of investigating the disinfecting 
 value of a fluid, and of testing a disinfecting oven. 
 
 1. TESTING A FLUID DISINFECTANT. 
 
 A. By the Use of Fluid Cultures. 'Equal quantities of 
 rather dilute and more concentrated solutions of the disinfect- 
 ant are poured into a number of sterilized vessels. With the 
 same Pasteur pipette, an equal number of drops of a well- 
 shaken pure culture is added to each of these tubes, and care- 
 fully mingled with its contents by shaking. The time is noted, 
 and the same number of drops of the culture are then added to 
 a control test-tube containing sterile bouillon or 0.7 per cent 
 solution of table salt. 
 
 A large number of test-tubes, with 5 to 10 cc. of nutrient 
 jelly in each are kept at 25 to 30 C., so that their contents 
 remain fluid. From each of the antiseptic solutions that have 
 received bacteria, as well as from the control tube, a drop is 
 transferred to one of the tubes of jelly, the same platinum 
 loop being used for all, to insure the transfer of about the 
 same quantity in each case. This is repeated at longer or 
 shorter intervals for the various degrees of concentration, and 
 
Bacteriological Technology. 127 
 
 the time for which the disinfectant was allowed to act in each 
 case, as well as its concentration, is marked upon the tube. 
 
 The appearance or absence of bacteria in these isolation- 
 cultures, as well as their abundance if present, give the data 
 for a determination of the time required by the disinfectant in 
 a certain degree of concentration, to destroy a certain kind of 
 bacteria. It is a weak point in this mode of investigation, 
 that a small quantity of antiseptic is always transferred to 
 the nutrient jelly, together with the bacteria; but this can be 
 assumed to be inactive in the degree of dilution caused by 
 mixing it with the jelly, and this source of error can always 
 l>e subjected to the test of control experiments. 
 
 B. By Using Cultures Dried on Silk, etc. The investiga- 
 tions are carried on along the same lines as the last. Sterile 
 vessels are filled with solutions of an antiseptic of various de- 
 grees of concentration. Several pieces of silk, charged with 
 bacteria, are dropped in each. By means of a platinum wire, 
 pieces are fished oat at definite intervals, and, after removal 
 of the disinfectant, sown in a gelatinized medium. The re- 
 moval of the disinfectant is effected by carefully pressing the 
 threads out in sterile filter-paper, washing them in water, and 
 again pressing them between folds of filter-paper. The paper 
 for this use is cut in quadrangular pieces of about 5 sq. cm., 
 folded like sheets of writing paper. These are wrapped in 
 paper in small parcels, and sterilized at 150 C. before use. 
 The control silk is placed in sterile bouillon or 0.7 per cent salt 
 ; solution, and then washed in the same manner as the pieces 
 that have been exposed to the antiseptic solutions. When the 
 disinfectant has been thoroughly removed by washing and 
 the capillary action of the filter-paper, the pieces of silk are 
 sown in melted jelly or on solid jelly. In the first case, test- 
 tubes are used, which are rolled or laid horizontally while 
 cooling, to facilitate the counting of the germs. In the second 
 case, watch-glasses or small uncovered trays (Fig. 13) are 
 used. Six to ten of these are placed in a larger pair of trays 
 (Fig. 12), sterilized in these at 150 C., and only filled immedi- 
 ately before use. This arrangement facilitates the microscopic 
 control of the cultures, but it makes later contamination pos- 
 sible, and requires a more careful washing than the isolation 
 .cultures in test-tubes. 
 
 If the threads are not sufficiently washed out, the disinfect- 
 
128 
 
 Bacteriological Technology. 
 
 ant remaining in them may diffuse through the surrounding* 
 gelatin in sufficient quantity to prevent all growth about the 
 threads, even when these contain living bacteria a fact which 
 can cause (and has caused) mistakes. The thoroughness of 
 the washing may be tested in the following manner : Some of 
 the agar-gelatin to be used is inoculated with the same sort of 
 bacteria as those on the threads, just before being poured in 
 the watch-glasses or trays, where it is then allowed to solidify, 
 and the silk is laid on its surface as usual. Some days later, 
 the colonies appear in it, reaching quite to the thread in case 
 the washing has been sufficient, while a sterile zone surrounds 
 the thread if it has not been thorough enough. 
 Attention should be given to this fact in test- 
 ing the freedom of antiseptic dressings from 
 bacteria by sowing them in nutrient gelatin; 
 control cultures being made by means of a 
 series of thrust-cultures of various bacteria in 
 the immediate vicinity of the pieces of mate- 
 rial in question. 
 
 2. TESTING A DISINFECTING OVEN. 
 
 For this purpose are needed : 
 
 a. A variety of the objects the oven is made 
 for (usually bed-clothes and wearing-apparel) 
 are needed. 
 
 b. Thermometers. Usually it is sufficient 
 to have a few maximum thermometers, which, 
 to avoid injury during the investigations, are 
 
 encased in metal or wood, in the latter case openings being* 
 provided to prevent them from checking the penetration of 
 the heat. But if it is wished to learn easily and quickly when 
 a certain temperature (e.g., 100 C.) is reached at a given point 
 within the oven, an electric contact-thermometer Fig. 69) is 
 employed. This consists of a thermometer in the walls of 
 which two platinum threads are fused, one (pi) entering the 
 mercury in the bulb, the other (p) touching the mercury only 
 when the boiling-point of water (or other desired temperature) 
 is reached. ~By means of screws (s and Si), the thermometer is 
 connected with wires that may be passed through the door 
 of the oven and inserted in an electric circuit including a bell 
 which sounds when the circuit is closed. 
 
 FIG. 69. Electric 
 Thermometer. 
 
Bacteriological Technology. 1 29 
 
 c. Bacteria. The principal of these are resistant spores, 
 like those of Bacillus anthracis, B. subtilis, Tyrothrix scaber 
 (Duclaux), and certain earth-bacilli. The material is prepared 
 as indicated on pages 123-125. For packages, filter-paper is 
 used, folded like the papers used by druggists for " powders," 
 and sterilized at 150 C. The papers are marked and num- 
 bered with a lead -pencil, and those containing the different 
 sorts of spores are collected in small parcels which can event- 
 ually be tied to a thermometer. 
 
 When the material for the test is ready, the oven is packed 
 as it is intended to be in regular use. The thermometers and 
 test-bacteria are distributed through the oven during this pro- 
 cess, not merely between and upon the clothing, but within it, 
 in places where contagia might easily exist, but where the 
 heat penetrates only with difficulty. For instance, mattresses 
 and pillows are ripped open enough to allow of the insertion 
 of thermometer and bacteria into the straw, hair, moss or 
 feathers, and again closed by sewing or the use of safety pins; 
 one or more blankets are rolled tightly about thermometer 
 and bacteria, etc. If the bacteria are destroyed under these 
 circumstances, it is evident a fortiori that as g-ood results 
 are attainable with the looser packing- which may be employed 
 in the daily use of the oven. Care must be taken that the 
 thermometers are not all collected about a single place, but 
 are distributed through the oven. 
 
 After the oven is packed, it is best to make a diagram (Fig-. 
 70) of its contents, that the temperature reached and its in- 
 fluence on the bacteria may be subsequently noted at various 
 points, giving an excellent synopsis of the results of the test. 
 
 At the end of the experiment, as accurate notes as possible 
 are made of the time for which it was continued, the amount 
 of fuel consumed, etc.; the articles are unpacked, and their 
 appearance, degree of dryness and brittleness, etc., observed, 
 and the thermometer readings made. 
 
 The packets of bacteria are wrapped in paper, in which 
 they can be carried to the laboratory without danger of con- 
 tamination. Cultures are made from them, as outlined on p. 
 127, either in test-tubes or trays. But it is to be observed 
 that the latter sort of cultures is not suited to g-arden -earth, 
 because, even when the dirt is poured from the papers very 
 slowly, particles easily fall into adjacent trays. It must, 
 
130 
 
 Bacteriological TccJmology. 
 
 therefore, be poured on the surface of gelatin which has been 
 allowed to harden obliquely in test-tubes. In larger series of 
 comparative tests, it is well to place the cultures in a brood- 
 oven at 20 C., but isolated experiments are as well made at 
 the varying- temperature of the room. Most cultures require 
 to be kept only a week, but those from garden-earth must be 
 watched for a couple of weeks, as they occasionally contain 
 resistant forms which develop very late. It is necessary to 
 note not only whether or not growth occurs, but also whether 
 there is any retardation of the development of the bacteria, 
 and, in isolation cultures, their number. 
 
 The final results of the experiment are most conveniently 
 
 FIG. 70. Diagram of a Packed Disinfection Oven, with Indication of the Results of a Test. 
 
 registered by noting the thermometric and bacteriological ob- 
 servations upon the diagram representing the contents of the 
 oven. In Figure 70, + indicates growth; no development; 
 and the species of bacteria are indicated by the relative posi- 
 tion of the marks, which are always arranged in the same 
 sequence earth, hay, anthrax-bacilli. E.g., within the upper 
 mattress of the Figure, the temperature reached was 102 C., 
 and all three species of bacteria were killed, while the earth- 
 bacilli survived the disinfection within the rolled woollen 
 blankets, where the temperature was between 103 and 104 
 C., etc. 
 
 As has been intimated above, one should be careful not to 
 
Bacteriological Technology. 1 3 1 
 
 confound the power of a disinfectant to check or prevent the 
 growth of the bacteria, with its power of killing them. That a 
 substance, added in a certain quantity to a readily putrefying 
 fluid, prevents decomposition, by no means requires that 
 it should have killed the bacteria in the fluid. The development 
 and dissemination of the germs may, perhaps, have been pre- 
 vented; and it may be that when brought into a new and fa- 
 vorable soil they would prove very well able to develop. It is 
 not superfluous to mention this perfectly obvious fact, because 
 it is often overlooked, giving origin again and again to errors. 
 
 If it is wished to learn the degree of concentration in which 
 a given material begins to check the growth of different 
 micra organisms, and that in which it renders growth entirely 
 impossible, the following method is employed, bacteria and 
 moulds being used, selected as indicated on p. 122. 
 
 A suitable number of test-tubes are filled with nutrient 
 material (generally gelatin) adapted to the organisms selected. 
 Some of these glasses are left untouched, serving for control, 
 while larger or smaller quantities of the substance to be tested 
 are added to the rest, so that all degrees of concentration are 
 obtained. In preliminary experiments, it is best to pour nearly 
 equal quantities of gelatin into all of the tubes, adding to them 
 different quantities of a strong solution of the substance, of 
 known concentration, by means of glass pipettes graduated to 
 0.05 cc. From these data, the percentage of the substance in 
 each culture glass can readily be calculated. 
 
 The species of bacteria on which it is desired to test the 
 disinfectant is sown in the vessels prepared in this way, the 
 material used for inoculating all being taken from a single 
 culture and by means of the same needle or pipette. The 
 quantity used for inoculation, and the manner in which the 
 needle is used should also be, as far as possible, exactly the 
 same for the different glasses, which are then observed daily, 
 the least degree of concentration which permits no growth 
 along the thrust and the least which perceptibly checks the 
 growth of the bacteria, being noted. The latter point is chiefly 
 indicated by a retardation of development, and by a change 
 in the macroscopic habit of the colonies. 
 
 Figure 71 illustrates this. It represents three test-tubes 
 with peptonized meat-infusion gelatin to which 0.05 per cent of 
 carbolic acid was added in the first, 0.4 per cent in the second, 
 
I 3 2 
 
 Bacteriological Technology. 
 
 and 0.7 per cent in the third. The same bacillus was sown in 
 all three glasses, which eight days later presented the appear- 
 ance represented. In the first tube, the appearance of the 
 culture was the same as in the untreated control-tube a large 
 close irregular felt of bacilli floating- on a quantity of melted 
 g-elatin at the very top, and below, above the thrust in the 
 still solid gelatin, a number of dot-like colonies, from some of 
 which, especially the uppermost, fine undulating 1 threads radi- 
 ate in all directions. In the 
 second tube, during the same 
 time, the felt of bacilli next the 
 surface had not reached the 
 glass, liquefaction was limited 
 to a small hemispherical de- 
 pression immediately about 
 the needle-thrust, while no 
 trace was to be seen of fila- 
 ments from the deeper colo- 
 nies, which did not approach 
 the bottom so nearly as in the 
 first glass. The third tube 
 showed little surface growth, 
 no liquefaction, and only a 
 short, firm, inversely conical 
 colony in the solid gelatin. 
 Very similar differences are 
 found in cultures of Koch's 
 bacillus of mouse septicaemia, 
 grown in g-elatin with and 
 
 . . FIG. 71. Three Cultures of B. anthracis after 
 
 Without Carbolic add. The Seven Days 1 Growth in Peptonized Meat Gela- 
 
 peculiar cloud-like fine growth tine ' to which has been Added the Percentage 
 
 of Carbolic Acid Indicated in Each. 
 
 never appears in the cultures 
 
 containing much carbolic acid, in which the bacilli show as 
 
 small, dense, dot-like colonies. 
 
 This prepares the observer for morphological changes in 
 the microscopic appearance of the bacteria so grown; and it 
 must also be remembered that under such conditions a perma- 
 nent physiological transformation of the bacteria has been 
 induced, as in the attenuation of the virulence of B. anthracis 
 by cultivating it in carbolic acid (Toussaint, Chamberland, and 
 Roux) a result which gives especial interest to experiments 
 of this sort. 
 
CHAPTER XIII. 
 
 MICROSCOPIC EXAMINATION, AND STAINING OF BACTERIA. 
 
 ONE side of the microscopic examination of bacteria has 
 already been considered in Chapter IX. (moist chambers), and 
 in sketching the plate-cultures of Koch, where not only the 
 peculiarities of the colonies (whether entire, lobed, fringed, 
 smooth, granular, wrinkled, etc.) can be observed with weaker 
 and medium powers, but which without further contrivances, 
 may also be studied with higher powers, the most superficial 
 colonies being even accessible to the most powerful immersion 
 lenses when covered with a cover-glass. 
 
 If it is wished to study individual cells more closely, the 
 colony need only be touched with a sterile platinum needle, 
 and the adhering material distributed through a small drop 
 of 0.7-per-cent solution of table salt (free from bacteria), spread 
 in a thin layer under a cover-glass. If the bacteria grow in a 
 fluid medium, a small drop of the culture is placed under the 
 cover-glass without further treatment except for final dilation 
 with the standard salt solution. The addition of such a neu- 
 tral fluid may occasionally facilitate the investigation, not 
 only by isolating the germs, but by modifying the differences 
 of refraction. 
 
 If such preparations are to be examined for a longer time 
 or with an immersion lens, it should be sealed with paraffin, 
 which is best applied by means of a glass tube 4 mm. in diam- 
 eter tilled with paraffin and drawn out to a short open point, 
 from which the melted paraffin flows. 
 
 The movements of living- bacteria cannot be observed long 1 
 in such sealed preparations, since they are stopped by want of 
 oxygen. This may be put off for a time by having- a number 
 of rather larg-e air-bubbles under the cover-glass, or, in case 
 the bacteria are contained in water, by introducing one of the 
 filamentous green algae (Engelmann). Impelled by their need 
 
1 34 Bacteriological Technology. 
 
 of oxygen, the bacteria collect about the bubbles or algae (the 
 latter of which continue to set free oxygen, as a result of as- 
 similation), and keep up their motions until the supply is ex- 
 hausted or the activity of the algge ceases. The study of the 
 manner in which the bacteria move can be facilitated by 
 slightly coloring them, as indicated below. The movements 
 of the very active species can be checked by compressing them 
 strongly by pressure on the cover-glass. All motion in a lim- 
 ited part of the preparation may be stopped, by the same 
 means, individuals here and there afterward recommencing 
 their movement, but at first very slowly. 
 
 The best way of studying the movements of bacteria is, 
 naturally, by the use of a moist chamber, Ranvier's moist 
 chamber, and the method indicated on p. 98 is especially 
 advisable, as the difficulties of using the hanging drop are 
 thus avoided. 
 
 The use of staining fluids, especially certain aniline colors, 
 is an indispensible adjunct in the microscopic study of bac- 
 teria. The employment of the methods of staining now used, 
 not only greatly facilitates the demonstration and observation 
 of bacteria in fluids, but the detection of structural peculiari- 
 ties which escape observation in the unstained cells, e.g., the 
 flagella of certain bacteria, the peculiar ends of the cells of 
 B. anthracis, etc. ; and it also discloses chemical differences 
 which are sometimes of extraordinary importance for clinical 
 diagnosis, e.g., in demonstrating the tubercle bacillus, stain- 
 ing has also rendered it possible to obtain handsome perma- 
 nent preparations of free bacteria, and, above all, to demon- 
 strate bacteria within the tissues and to study their distribution 
 in the organs and their relations to the cells. The develop- 
 ment of the technology of staining to the high point it has 
 now reached is due chiefly to three men, Weigert, Koch, and 
 Ehrlich. 
 
 The dyes used in bacteriological investigation nearly all 
 belong to the large class of aniline colors, and especially to 
 the group called basic by Ehrlich, who first called attention 
 to the fact that the aniline colors are divisible into two main 
 groups : the acid colors, in which the staining principle is an 
 acid (e.g., ammonium picrate); and the basic, consisting of a 
 staining base in combination with an acid that does not 
 stain (e.g., acetate of rosaniline). Methylene blue, fuchsin,. 
 
Bacteriological Technology. 1 3 5 
 
 
 
 gentian-violet, vesuvin, etc., belong- to this group of basic 
 dyes (used by liistologists as nuclear stains), which have 
 proved especially. adapted to the staining- of bacteria, free or 
 in situ, so that it has proved possible by their use to stain all 
 known pathogenic bacteria in sections, strongly, permanently, 
 sparingly, and distinct from the invaded tissue. Ehrlich like- 
 wise calls those dyes neutral which are formed by the union 
 of a base and acid both of which are capable of staining (e.g., 
 picrate of rosaniline). 
 
 Naturally, it is not always possible to at once secure all of 
 these desirable results equally well, and especially ,soft stain- 
 ing of the histological elements is often advantageously made 
 to give place to the intense coloring- of all bacteria present. 
 But Baumgarten has, for instance, succeeded by a rather com- 
 plicated method in showing upon the same slide the tubercle- 
 bacilli and the caryokineses resulting- from their invasion. 
 Baumgarten's method is the following: The diseased rabbit is 
 killed, and small pieces of the tuberculous organs are cut out 
 as quickly as possible and at once cast into 0.2-per-cent chromic 
 acid, where they are hardened for forty-eight hours, then 
 washed out thoroughly in running water for as much as 
 twenty-four hours, and rehardened for twenty -four hours in 
 strictly absolute alcohol. The thinnest possible sections are 
 laid in freshly prepared aniline-methyl violet (No. 8) for a long- 
 time (as much as forty-eight hours), washed not to exceed 
 thirty seconds in 1 part of nitric acid to 5 of water, the de- 
 colorization being then completed in 60-per-cent alcohol (No. 
 17), after which they go into a mixture of equal parts of a 
 concentrated alcoholic solution of fuchsin (No. 1) and distilled 
 water for half an hour to an hour, then for five to ten seconds 
 in an aqueous solution of methylene blue (1 : 1,000), and finally 
 for five to ten minutes in absolute alcohol, changed once or 
 twice. The sections are finally mounted in balsam thinned 
 with bergamot oil, but without chloroform. 
 
 Instead of the more complicated method of staining the 
 nuclei with fuchsin and methylene blue, according- to Baum- 
 garten a concentrated solution of vesuvin in 1-per-cent acetic 
 acid may be used. 
 
 For securing- intense and permanent staining, the same 
 means are employed as in dyeing 1 on a larger scale; i.e., by 
 the prolonged action of the staining- fluid; warming- the lat- 
 
1 36 Bacteriological Technology. 
 
 ter, either for a long- time in the thermostat at 40 to 50 C., 
 or more intensely but for a shorter time over the flame; or by 
 the use of mordants, e.g., potash, carbolic acid, aniline oil, or 
 tannin which have a certain tendency to unite with both the 
 dye and the object to be stained, so that they serve as a sort 
 of connecting- link. 
 
 The possibility of staining' bacteria in contrast with the 
 surrounding- tissue is partly due to the different "elective" 
 power of various dyes (Ehrlich), i.e., their different power of 
 staining- certain tissue elements; and partly to the different 
 strength with which they stain various parts of the prepara- 
 tion, i.e., the different stability of the union they form with 
 them. The following- experiment of Ehrlich and Schwarze 
 illustrates in a striking- way the different elective power of 
 three acid aniline colors, aurantia, indulin, and eosin. When 
 a cover-glass preparation of blood is made by distributing it 
 in a thin layer, drying- it, and heating to 120 C., and the pig- 
 ments are tested on it, it is seen that each of the latter pos- 
 sesses the power of coloring- the red corpuscles as well as the 
 nuclei of the white corpuscles and the peculiar granules, found 
 in some of the white cells, which Ehrlich calls a-granules. 
 But if the blood-preparation is treated with a solution of the 
 three dyes in glycerin (made by adding- an excess of eosin and 
 indulin to a mixture of 1 part of saturated solution of aurantia 
 in glycerin, and 2 parts of pure glycerin), the hsemoglobin is 
 colored yellow by the aurantia ; the nuclei, gray or black by 
 the indulin, and the a-granules, red by the eosin. The fact 
 that certain parts of the preparation hold the coloring- matter 
 more tenaciously than others, is used in demonstrating bac- 
 teria in sections, the g-eneral plan being to overstain the entire 
 preparation, subsequently securing- the differentiation by de- 
 colorizing (and finally staining in some contrasting color) cer- 
 tain parts, leaving the others colored as at first. The degree 
 of decolorization can be regulated by the substance chosen to 
 effect it. If, for example, we have a section of an organ con- 
 taining bacteria, which has lain in a strong solution of methyl 
 violet, and wash it in water, microscopic examination will 
 show the bacteria, as well as the protoplasm and nuclei of the 
 cells, colored deep violet (diffuse staining). If it is washed in 
 dilute acetic acid (p. 141, No. 11), the protoplasm becomes de- 
 colorized, while the bacteria and nuclei retain the color 
 
Bacteriological Technology. 137 
 
 (nuclear staining-). If it is washed in potassium carbonate 
 (No. 15), the coloring- matter is also driven from the nuclei, the 
 bacteria alone remaining stained (isolated staining- of the bac- 
 teria). By still more powerful ag-ents, such as 25-per-cenfc 
 nitric acid or hydrochloric acid, most species of bacteria can 
 likewise be decolorized, so that only a very few sorts remain 
 stained (tubercle stain) ; but repeated treatment with strong 1 
 solutions of mineral acids, if continued sufficiently long-, also 
 removes the stain from these species, and it must be remem- 
 bered throughout that the time for which the preparations 
 are exposed to the action of the decolorizing agent is of de- 
 cisive importance for the result, since the power of the differ- 
 ent elements to resist decolorization usually differs only in 
 degree, not in kind. In each case, therefore, it is necessary to 
 avoid decolorizing too little or too much, and as it is not easy 
 to give very exact instructions as to the time needed, this is 
 one of the points on which long- experience plays an import- 
 ant part. 
 
 To further differentiate the bacteria from the tissue ele- 
 ments, and to bring the form and disposition of the latter out 
 more clearly, multiple staining (usually double staining-) can 
 be employed, either by using a new staining- fluid on the partly 
 decolorized preparations (successive staining-, e.g., No. 17), 
 or by treating them with a fluid which at once decolorizes and 
 restains them in part (replacement staining). Treating- the 
 same preparation with two colors is also sometimes resorted to 
 for another purpose, namely to render the staining more re- 
 sistant toward decolorizing substances, either by forming a 
 new compound color, or otherwise, e.g., the gentian-violets 
 iodine method (Gram) and the fuchsin-methylene-blue method 
 described on p. 147. 
 
 Occasionally double staining may be secured by the em- 
 ployment of a single dye, e.g., when methylene blue is used to 
 stain a section which includes bacteria as well as the pecu- 
 liarly granulated connective-tissue cells known as "Mastzel- 
 len," the bacteria are colored blue, and the granules violet. 
 These granules (the ^-granules of Ehrlich) also deserve notice 
 for another reason. Since, like bacteria and nuclei, they are 
 stained by basic aniline colors, and have about the size of 
 micrococci, they may be mistaken for the latter, as has often 
 been the case with inexperienced observers. As an especially 
 
138 Bacteriological Technology. 
 
 favorable object for the comparative study of micrococcus 
 cells and these /'-granules, I can recommend the mesentery of 
 a rather lean mouse, dead of suppurative peritonitis as a result 
 of inoculation with pyogenic staphylococci. The mesentery is 
 spread out on a cover-glass which is slipped beneath it while 
 it is still attached to the intestine, by the weight of which it 
 is stretched over the glass. As soon as it has dried fast, the 
 part projecting around the edge of the glass is cut away, and 
 it is treated as if it were an ordinary cover-glass preparation 
 (by drying, passing through the flame, etc.), and stained by 
 methylene blue. Among the blue cocci will be found small 
 clusters of granules of unequal size, grouped irregularly 
 around an unstained nucleus for the nuclei of these cells, 
 unlike those of other connective-tissue cells, are not colored 
 by this mode of staining with basic aniline colors. In double- 
 staining, colors are naturally chosen which contrast sharply 
 and prettily with one another e.g., fuchsin and methyl green; 
 fuchsin and methyl blue; methyl violet and vesuvin [or 
 methyl violet and eosin]. 
 
 An extremely large number of dyes have gradually come 
 into use in various ways for staining bacteria ; but only a lim- 
 ited number of stains and methods will be considered here, 
 and it is best, for beginners especially, to be content with one 
 or two of the most universal and important methods, practising- 
 them carefully until they are mastered, before going further. 
 It is not possible to limit one's self exclusively to a single dye, 
 for the reason that the behavior of different bacteria toward 
 the various staining fluids, and when different methods are 
 used, is of diagnostic importance, while there is no one dye 
 which stains all bacteria equally well. E.g., the slight power of 
 methylene blue to stain the bacillus of leprosy can be used to 
 distinguish the latter from that of tubercle, which otherwise 
 resembles it closely (c/. p. 155). In other cases, the behavior 
 when the Gram method is employed can be utilized for diag- 
 nosis; e. g., with gonococci, which (like the typhoid bacillus, 
 the cholera spirillum, and the microbes of chicken cholera and 
 the septicaemia of mice) are decolorized when the Gram method 
 is used, while other pyogenic micrococci, which closely resem- 
 ble them, retain the stain. The most nearly universal pig- 
 ment is methylene blue, a substance introduced by Ehrlich, 
 which can well be used as the principal stain. By employing- 
 
Bacteriological Technology. 1 39 
 
 it in the manner indicated below (No. XIII.), Kiihne succeeded 
 in staining in situ all of the bacteria that he investigated. In 
 leprosy and mouse septicaemia, alone, he failed to obtain com- 
 pletely satisfactory results/and for the bacilli of these diseases 
 he employed fuchsin. Consequently, it is best to carefully 
 practise this " universal method with methylene blue ; " and if 
 to this are joined one of the tubercle methods (e.g., No. 
 XVIII.), and the Gram method, one is* well equipped, so far as 
 sections are concerned. It is also best to confine one's self to 
 a few methods for cover-glass preparations. 
 
 The materials employed, in the methods described below, 
 are the following : Distilled water, absolute alcohol, glycerin, 
 acetic acid, hydrochloric acid, nitric acid, sulphuric acid, 
 chromic acid, carbolic acid, aniline oil, potash, potassium car- 
 bonate, potassium acetate, potassium iodide, lithium carbon- 
 ate, iodine, clove oil, bergamot oil, cedar oil, turpentine, xylol, 
 Canada balsam, shellac, paraffin, fuchsin, methylene blue, 
 methyl violet, gentian violet, methyl green, vesuvin, picro-car- 
 mine, and extract of logwood. Different aniline colors bearing 
 the same name may differ to such an extent as not to be 
 equally adapted to histological purposes, so that attention 
 should be given to the source and trade mark of good sorts. 
 
 All of the aniline colors named should be kept in stock in 
 the dry form. Fuchsin, methylene blue, methyl violet, and 
 gentian violet may be further kept in a saturated alcohol 
 solution (No. 1), i.e., 25 gm. of the dye to 100 gm. of absolute 
 alcohol, which always leaves an abundant excess of undis- 
 solved pigment in the bottom of the flask. Vesuvin (or Bis- 
 marck brown) is best kept only in powder, but if a solution is 
 to be kept, this is best made (No. 2) by saturating equal parts 
 of water and glycerin. 
 
 For use, aqueous solutions of the powders (No. 3) may be 
 directly prepared, but these do not keep long, and are there- 
 fore always freshly prepared from water free from bacteria, 
 and are filtered before use. It is very convenient to keep 
 a little of the dry pigment constantly used, in this way, on a 
 little filter in a glass funnel. When it is needed a small 
 quantity of water is poured over it, and the filtrate is collected, 
 the powder soon drying again. Dust is kept from it by cover- 
 ing the funnel with a layer of filter-paper. 
 
 When it is not exceptionally necessary to avoid every trace 
 
140 Bacteriological Technology. 
 
 of alcohol in the staining- fluid (as is, for example, the case 
 when staining- living- bacteria), the aqueous solution is re- 
 placed without disadvantag-e by that (No. 4) made by adding a 
 suitable quantity of the saturated alcoholic solution (No. 1) to 
 water. This diluted alcoholic solution is more durable than 
 the aqueous, but it usually requires renewal once or twice a 
 month, so that it is best to prepare it when needed, by adding 
 five to six drops of No. 1 to a watch-glass full of distilled water. 
 
 The principal mordants used are 0.01-per-cent solution of 
 potash (Koch, Loeffler) : 5-per-cent carbolic acid (Ziehl) ; and 
 aniline-water (No. 5), a concentrated aqueous solution of ani- 
 line oil, prepared by very thoroughly shaking about 1 part of 
 aniline oil and 20 parts of distilled water in a test-tube, allow- 
 ing- it to stand five minutes, and filtering- through a filter 
 moistened with distilled water. (It must be perfectly free 
 from turbidity, or it should be again shaken, and refiltered.) 
 The staining fluids with mordants for which we shall find 
 application, are : 
 
 (No. 6.) Kuehne's carbolic blue. 1.5 parts of methylene 
 blue, and 10 parts of absolute alcohol are triturated lightly in 
 a watch-glass with 100 parts of 5-per-cent carbolic acid which 
 is added little by little. When all is dissolved, it is bottled. 
 To facilitate rapid preparation, several test-tubes with feet 
 may be graduated to 20, 22, and 24.2 cc. (cf. Fig. 72). 
 
 (No. 7.) Ziehl's carbolic fuchsin. 1 part of fuchsin, 10 of 
 alcohol, and 100 of 5-per-cent carbolic acid. 
 
 (No. 8.) Aniline methyl violet (Ehrlich-Weigert). 11 cc. of the 
 saturated alcoholic solution of methyl violet, 10 cc. of absolute 
 alcohol, and 100 cc. of aniline-water. Or the dry powder may 
 be added in excess to aniline water. [In Koch's laboratory it 
 is customary to add the stock alcoholic solution (No. 1) to a 
 watch-glass of aniline-water until the latter is shown to be 
 saturated by the formation of a film at top.] 
 
 (No. 9.) Aniline gentian violet (Ehrlich). 5 cc. of the satu- 
 rated alcoholic solution of gentian violet, to 100 cc, of aniline- 
 water. 
 
 (No. 10.) Loeffler's alkaline blue. 30 cc, of the saturated 
 alcoholic solution of methylene blue to 100 cc. of 0.01-per-cent 
 caustic-potash solution. 
 
 A large part of the chemicals enumerated above are used 
 for washing preparations for decolorization or differentiation. 
 
Bacteriological Technology. 141 
 
 For this purpose, water, alcohol, and glycerin (all perfectly, 
 free from acid) are used, as well as clove-oil and aniline-oil, 
 and acids and salts in the following- forms : 
 
 (No. 11.) Very dilute acetic acid (0.5 per cent to 1 per cent). 
 
 (No. 12.) Very dilute hydrochloric acid (10 drops of the 
 acid to 500 gm. water). 
 
 (No. 13.) 75 parts of water containing- 25 parts of nitric 
 acid (Ehrlich), or the same quantity of hydrochloric or sul- 
 phuric acid. 
 
 (No. 14.) Lithium water (Kuehne). 6 to 8 drops of a con- 
 centrated aqueous solution of lithium carbonate and 10 gm. 
 water. For neutralizing- an acid washing- fluid. 
 
 (No. 15.) Potassium carbonate solution (Koch). Equal 
 parts of a saturated aqueous solution, and water (Koch) ; or 2 
 parts of a 2-per-cent aqueous solution, and 1 part of absolute 
 alcohol (Malassez and Vig-nal). 
 
 (No. 16.) Gram solution. Iodine 1 part; potassic iodide, 2 
 parts; distilled water, 300 parts. 
 
 (No. 17.) Alcohol, 60 parts; water, 40 parts (Koch). For 
 colored wash -alcohol (Kuehne), see p. 151. 
 
 (No. 18.) Aniline-oil blue. Kuehne recommends rubbing as 
 much methylene blue as can be raised on the point of a knife, 
 with 10 gm. clarified aniline oil, and allowing- the excess of un- 
 dissolved pigment to settle in a bottle. A. few drops are added 
 to aniline oil in a watch-g-lass, until the desired concentration 
 is reached. 
 
 The manner of using these staining and decolorizing- ag-ents, 
 as well as the others for hardening-, anhydrating-, clearing, 
 mounting-, and sealing 1 preparations, receives more detailed 
 consideration under the several methods described below; 
 
 In what follows, it is assumed that the common micro- 
 scopic appliances and methods are understood. Staining is 
 usually effected in small glass trays (Fig-. 13), "individual salt 
 cellars" or watch-glasses. Some of the trays must have 
 ground tops so that they can be tightly sealed with a glass 
 plate if they are to kept at an elevated temperature for some 
 time. Watch-glasses, which are inconvenient because of their 
 lack of stability, are necessary if sections are to be stained by 
 heating over the flame. In this case, they are conveniently 
 placed upon the stand shown in Figure 72, which consists sim- 
 ply of a strip of sheet tin with three holes having a somewhat 
 
142 Bacteriological Technology. 
 
 smaller diameter than the watch-glasses. The stand is also 
 useful for filtering, as indicated in the cut. 
 
 STAINING BACTERIA IN FLUIDS. 
 
 A. By the Simple Addition of the Staining Fluid. A lit- 
 tle drop of very dilute aqueous solution (No. 3) of fuchsin or 
 methyl green (Mace) is placed on a slide, a small quantity of 
 the culture, etc., is distributed through it with a platinum 
 needle, and a cover-glass applied. The staining fluid must 
 usually be so dilute that it appears nearly colorless under the 
 microscope. When pure and extremely dilute aqueous solu- 
 
 FIG. 72. Tin Support for Funnels and Watch-glasses, in Staining and Filtering. 
 
 tions of fuchsin are used, the bacteria can remain alive and 
 continue their motions after staining (Salomonsen). 
 
 If a drop of the culture and one of a more concentrated stain- 
 ing fluid than in the last case are placed in proximity. on the 
 slide and covered with a single cover-glass, a series of bacteria 
 may be found in the same preparation, showing all gradations 
 from cells that are unstained to others strongly overstained. 
 
 A rapid microscopic view of a large number of crowded 
 colonies in a plate -culture is obtained by the following method : 
 A well-cleansed cover-glass is laid upon the gelatin over the 
 colonies and pressed firmly into contact with it everywhere, 
 by a glass rod or pair of forceps. Fragments of the super- 
 ficial colonies adhere to the cover, forming an "impression- 
 preparation " of the culture, so that when it is removed from 
 the gelatin and lowered on to a drop of staining fluid on a 
 
Bacteriological Technology. 143 
 
 slide, the opportunity is given to see small samples of the con- 
 tents of all of the colonies touched. Any of these preparations 
 can be sealed with paraffin, as described above. 
 
 B. Staining Preparations Dried on the Cover glass. 
 Weigert was the first, in 1876, to recommend an aniline color 
 (methyl violet) for staining 1 bacteria. Shortly afterward, with- 
 out knowing- of Weigert's work, I introduced fuchsin as par- 
 ticularly good for staining- bacteria, and indicated especially 
 its value as a means of diagnosis in the microscopic analysis 
 of putrefying- blood. By the introduction of the basic aniline 
 colors, the demonstration and examination of bacteria in fluids 
 was greatly facilitated; but a further and decisive advance in 
 methods was made in 1878, when Koch showed how bacteria 
 might be colored after drying them in a very thin layer upon 
 the cover-glass, which, likewise, rendered possible the complex 
 micro-chemical treatment of free bacteria which now plays so 
 important a part. The great advantages of this method have 
 caused it to be almost exclusively used in all chemical examin- 
 ations of blood, pus, urine, and sputum ; consequently the mak- 
 ing and examining stained cover-glass preparations will be 
 sketched in detail in the following pa.ges. Obviously, the use 
 of such preparations does not render superfluous the simple 
 staining- already described, since the latter completely pre- 
 serves the form and turgescence of the cells, while a shrinking 
 always results from drying them. 
 
 New cover-glasses are carefully washed in warm water, 
 dried, and treated with absolute alcohol for the removal of all 
 grease. Those that have been used are first laid in strong- 
 mineral acid (hydrochloric or sulphuric), then washed clear in 
 water and rinsed until every trace of acid is removed, when 
 they are treated as if new. 
 
 The fluid is best spread in a thin layer, by placing- a small 
 drop on a cover-glass, laying another cover upon it, and sepa- 
 rating them by drawing- one over the other, in which way two 
 preparations are obtained. It is also possible to scrape the 
 fluid in as thin a layer as possible by the edge of a second 
 cover-glass. The covers are then laid to dry under a bell- 
 glass, with the smeared side up. If the drying is to be accel- 
 erated by warming, this must be effected at a very low tem- 
 perature. The air-drying must be complete before the next 
 step is taken. 
 
144 Bacteriological Technology. 
 
 When such a dried preparation of blood, pus, sputum, or 
 other fluid which contains albumen, is at once stained, two 
 defects are sometimes observed ; sometimes the dried film sep- 
 arates in part from the glass; sometimes the presence of sol- 
 uble albuminoids causes disturbing precipitates in the prepa- 
 ration. These are avoided when the albuminoids are rendered 
 insoluble by suitable hardening'. The second defect can also be 
 avoided by using 1 aniline brown (Bismarck brown or vesuvin) 
 in glycerin (No. 2), and washing 1 in pure glycerin (Koch). 
 Absolute alcohol (Koch) may be used for this purpose, the 
 cover-glass being- placed in it for some time, but the requisite 
 time varies much for different preparations. Partly for this 
 reason, and partly because of its slowness, this method is far 
 inferior to that of Ehrlich, by heating- up to 120 to 130 C. for 
 two to ten minutes. (In a protracted exposure to this tem- 
 perature, e.g., for an hour, as in Ehrlich's studies of the gran- 
 ules of the white blood-cells, the bacteria have been found to 
 lose their power of staining.) This heating may be effected 
 in the sterilizing oven (Fig. 1), but it is more convenient to 
 follow Koch in passing the cover three times through the 
 flame of a Bunsen burner. The cover-glass is seized with the 
 forceps by one corner, the smeared side up, and passed three 
 times through a vertical circle about a foot in diameter, the 
 clean side of the cover being brought down against the top of 
 the flame each time. The best rapidity for the flaming, upon 
 which success depends, is reached by taking about three sec- 
 onds for describing the three circles. 
 
 Of the various ways in which the staining fluid and the 
 hardened preparation can be brought together, we usually 
 employ that of allowing the cover-glass to float upon the fluid, 
 film downward. In all of the following manipulations, care 
 must be taken to remember which side of the cover bears the 
 bacteria, for when the film is very thin it may be difficult to 
 distinguish it. Sometimes it may be recognized by scratching 
 upon the cover with a sharp pin, the irregularities of the 
 smeared side being felt, or the scratches seen. 
 
 The smeared and dried cover-glasses can be kept, with or 
 without hardening, for an indefinite time before staining, being 
 simply laid in an ordinary cover-glass box, the different sets 
 separated by labels of the size of the covers. [For clinical 
 purposes, it is convenient to use a small oblong' box, a couple 
 
Bacteriological Technology. 145 
 
 of indies long 1 , with a rack inside like a slide-box. Twenty or 
 more clean covers are easily carried in such a box, which is 
 small enoug-h to be slipped into the vest pocket or instrument- 
 case, so that they are always ready for use at the desk or bed- 
 side. W. T.] Two opposite corners are lifted with the thumb 
 and forefing-er of the rig-ht hand, the cover is held horizontal, 
 and allowed to fall upon the fluid from a heig-ht of several 
 centimetres. The time required for the action of the staining* 
 fluid varies according- to circumstances from a few minutes 
 to an entire day. Long- staining- at the temperature of the 
 air can often be replaced by a shorter staining- at a hig-her 
 temperature (Koch, Loeffler), e.g., No. IV., p. 147, infra. 
 This is best effected by setting- the watch-glass of staining- 
 fluid on which the cover floats, upon the tin stand (Fig-. 72), 
 and heating- it with a small flame until vapor rises freely from 
 the surface. The lamp is then removed for a short time, after 
 which the heating- is renewed, and this is repeated several 
 times for a few minutes, or long-er, as may be necessary. 
 Sometimes it may be convenient to heat the fluid until it 
 beg-ins to boil, e.g., in No. IV. 
 
 When the staining-, or, rather, overstating- of the prepara- 
 tion is finished, the latter is washed (partly decolorized or 
 " differentiated "). This is effected according- to circumstances 
 by the use of one or other of the fluids enumerated above (cf. 
 pp. 140 and 141), which is then removed with water either by 
 rinsing- under the faucet or with a wash-bottle, or by moving- 
 the cover back and forth in a larg-e dish of distilled water. 
 Washing- out the excess of color is the most difficult part of 
 staining-, because no rules can be given as to the length of 
 time it oug-ht to be continued in each case: frequently the 
 rig-ht time is only learned by experiment. When it has been 
 finished, the preparation can be examined at once in distilled 
 water or g-lycerin. 
 
 It should also be observed that preparations stained with 
 Bismarck brown or vesuvin (No. 2), can be permanently 
 mounted in g-lycerin, while this substance decolorizes prepara- 
 tions stained with the other aniline colors. On the other hand, 
 a concentrated solution of acetate of potassium (1:2) serves 
 especially well, according- to Koch, for the preservation of bac- 
 teria stained with violet or fuchsin, and, it may be added, of 
 unstained bacteria; but it should not be used for those stained 
 
 brown. 
 
 10 
 
1 46 Bacteriological Technology. 
 
 If the preparation is to be permanently mounted in balsam, 
 it is next dried, by carefully wiping- off the clean side of the 
 cover with a piece of soft linen, and holding- it obliquely with 
 one edge upon a piece of filter-paper, so that as much water 
 as possible will flow off or be absorbed by the paper. Any 
 large drops remaining on the film are carefully removed with 
 filter-paper, and the cover is allowed to become air-dry. To 
 hasten the drying, the cover may be pressed between folds of 
 filter-paper (Ehrlich), waved back and forth through the air, 
 or air may be blown over it by means of a rubber bulb fur- 
 nished with a pointed giass tube (Kuehne). 
 
 The dried preparation is mounted in Canada balsam. It is 
 most convenient to use this greatly thinned with xylol, and 
 kept at hand in collapsible tubes. Turpentine can also be used 
 for this purpose, but chloroform [and benzol] are to be avoided, 
 as they remove the basic aniline colors. Of the ethereal oils, 
 clove-oil is especially apt to decolorize the bacteria, so that 
 sections should rather be cleared with turpentine, oil of berga- 
 mot, or cedar-oil. A drop of this is applied to the middle of 
 the slide, and the cover inverted upon this, spreading- it in a 
 thin layer by its weight. Further treatment is unnecessary, 
 but as it requires a long- time for the xylol-balsam to harden 
 sufficiently to fix the cover immovably, it is convenient to seal 
 the preparation by means of a filtered alcoholic solution of 
 shellac, which may be given a handsome green color by the 
 addition of a little methyl green. Several layers of this 
 cement are painted around the edge of the cover. 
 
 In the preliminary examination of a fluid containing bac- 
 teria, a less complicated method is used. After drying- and 
 flaming the cover-glass preparation, it is stained by a diluted 
 alcoholic solution (No. 3) of methylene blue or fuchsin, rinsed 
 with water, and examined at once in water, or, after drying-, 
 in cedar-oil (c/. No. 1). 
 
 METHODS OF STAINING COVER-GLASS PREPARATIONS. 
 
 (I.) Koch's Original Method (1878). The dried film is 
 stained for a few seconds, or a little longer, with a solution 
 of methyl violet or fuchsin (a few drops of the saturated alco- 
 holic solution to 15 to 20 cc. water), washed with water or 
 acetate of potassium (1:10), dried, and mounted in balsam; 
 
Bacteriological Technology. 147 
 
 or it is stained with vesuvin dissolved in equal parts of gly- 
 cerin and water, and washed and mounted in glycerin. 
 
 (II.) Kuehne's Methylene-Blue Method. The film, after 
 drying- and flaming, is stained for five minutes in carbolic blue 
 (No. 6), rinsed in water, differentiated by laying in dilute hy- 
 drochloric acid (No. 12) for a few seconds or a little longer 
 according to the thickness of the film, rinsed a moment in 
 lithium-water (No. 14), washed under the water-jet for fifteen 
 seconds, dried if necessary by use of the bulb slightly 
 warmed over the flame, cleared in xylol, and mounted in 
 balsam. 
 
 (III.) Gram's Method. The flamed film is treated for one 
 to three minutes with aniline gentian violet (No. 9), laid in the 
 iodine solution (No. 16) for an equal time, washed in alcohol 
 till it appears completely decolorized, dried, and mounted in 
 balsam. 
 
 (IV.) Koch-Ehrlich-Weigert Tubercle Method. The dried 
 and flamed film is heated in a watch-glass of Weigert's ani- 
 line methyl violet (No. 8) until bubbles begin to form beneath 
 the cover-glass, when it is allowed to stand five minutes, de- 
 colorized in a tray of 25-per-cent nitric acid, in which it is 
 moved back and forth for at most five seconds, immediately 
 rinsed in 60-per-cent alcohol (No. 17) until the blue color dis- 
 appears (usually not over one or two seconds), counter-stained 
 for five minutes in a saturated aqueous solution of vesuvin, 
 rinsed in water, dried, and mounted in balsam. 
 
 (Y.) Ziehl-Neelsen Tubercle Method. The flamed film is 
 floated on a watch-glass of carbolic fuchsin (No. 7) three to 
 eight minutes (with heat), decolorized in 25-per-cent nitric or 
 sulphuric acid, treated with 60-per-cent alcohol until only a 
 rosy tint remains, the acid then being completely washed out 
 in a large quantity of water, dried, and mounted in Canada 
 balsam, with or without previous clearing in xylol. 
 
 [For rapidly staining the hardened sputum film, the fol- 
 lowing commonly-used method leaves little to be desired : The 
 cover-glass is held in the forceps by one corner, film up, a large 
 drop of carbolic fuchsin is placed on it so as to entirely cover 
 the upper side, and it is heated directly over the flame until 
 it steams fully or even boils, care being taken not to let any 
 part become dry. If necessary, a second drop of the staining 
 fluid is added as the first evaporates, and the heating is con- 
 
148 Bacteriological Technology. 
 
 tinued for, perhaps, half a minute. After rinsing- most of the 
 staining 1 fluid off under the faucet, the cover is dropped into 
 very dilute nitric acid until the red color changes to a dull 
 olive, or begins to disappear entirely (which usually requires 
 very few seconds), after which it is at once moved about in a 
 dish containing 1 a considerable quantity of alcohol until the 
 dye, which has resumed its original red color, ceases to be 
 given off in clouds. If the film still retains any noticeable 
 color, it is dipped into the acid once more and again washed in 
 alcohol, after which it is counter stained with methylene blue, 
 rinsed in water, and may be at once examined, or dried and 
 mounted in balsam. By this method, a good permanent prep- 
 aration may be had under the microscope within five minutes 
 of the time when the needle is first dipped into the sputum for 
 the transfer of a little to the cover-glass. 
 
 While there are some advantages in floating cover-glass 
 preparations upon the staining fluid, as the author recom- 
 mends, it is more common to keep dilute alcoholic solutions 
 (No. 4) of methylene blue, gentian violet, and fuchsin, as well 
 as the standard alkaline blue (No. 10) and carbolic fuchsin 
 (No. 7) in 2 oz. wide-mouthed bottles closed with bored corks 
 through which dropping-tubes pass into the middle of the bot- 
 tle, so that a drop of the desired staining-fluid may easily be 
 allowed to fall upon the hardened film, heat being applied 
 when necessary by holding the cover above a Bunsen burner 
 until steam begins to rise. W. T.] 
 
 Staining Spores on the Cover-glass. When bacilli which 
 contain spores are stained by the above methods, the spores 
 remain uncolored, so that they appear as colorless spots within 
 the strongly colored rods. This indisposition of the spores to 
 receive coloring matters can be overcome in various ways, 
 and it was simultaneously shown by Buchner and Hueppe 
 that a prolonged exposure to heat, which even unfits the veg- 
 etative cells of bacteria for staining (cf. p. 144, Ehrlich), ex- 
 actly adapts the spores to the reception of basic aniline colors 
 in aqueous or dilute alcoholic solutions. 
 
 ("VI.) If, instead of passing the cover-glass through the 
 flame three times to harden it (p. 144), it is flamed ten times, 
 or heated fifteen to twenty minutes at 120 to 180 C., and 
 then stained with an aqueous solution of one of the usual basic 
 colors, the spores become deeply stained; but the staining of 
 
Bacteriological Technology. 149 
 
 the spores is isolated, because, as has been said, the rods have 
 lost their receptivity for the dye. 
 
 There is, however, a means of obtaining 1 double-stained 
 preparations, in which the spores take one color, the rods, 
 another: namely, by an extremely intense application of the 
 Ehrlich tubercle method. 
 
 (VII.) The cover-glass film is hardened in the flame as 
 usual (three times), stained for an hour floating- on hot aniline 
 fuchsin (p. 144), rinsed in water, decolorized in 25 parts hydro- 
 chloric acid and 75 of alcohol, and counter-stained in a satu- 
 rated aqueous solution of methylene blue. 
 
 There is a great difference in the readiness with which the 
 spores of different bacilli may be stained. [B. Megatherium 
 and B. subtilis are g-ood species to practise with, B. anthra- 
 cis is among the more difficult. The endospores of yeasts 
 may be stained by the same methods, decolorization being 1 
 here effected by alcohol without the addition of an acid. 
 Ziehl's carbolic fuchsin, heated upon the cover, as in tubercle 
 staining, gives excellent results, but it must be renewed sev- 
 eral times and the boiling- correspondingly prolonged. W. T.] 
 
 STAINING THE FLAGELLA OF BACTERIA. 
 
 In the case of certain large bacteria, e.g., the forms of Beg- 
 giatoa roseo-persinica first thoroughly studied by Warming, 
 the flagella can be seen by aid of a good objective without 
 any preparation whatever. In the case of smaller forms, they 
 can only be demonstrated by staining them, or even by means 
 of staining and photography; but none of the methods so far 
 indicated are applicable to this purpose. Koch, however, suc- 
 ceeded in demonstrating them by the use of a saturated aque- 
 ous solution of extract of logwood, added to the fluid contain- 
 ing the bacteria. His permanent preparations were made as 
 follows : 
 
 (VIII.) The bacteria are dried on the cover-glass, stained 
 with an aqueous solution of logwood, laid in dilute chromic 
 acid (which forms a dark brown combination with the dye), 
 dried, and mounted in balsam. 
 
 Neuhaus advises the replacement of chromic acid by neu- 
 tral sodium bichromate, prepared by adding 1 5-per-cent soda 
 solution drop by drop to dilute chromic acid. He recommends 
 
15 Bacteriological Technology. 
 
 the following- as the most certain means of staining- the 
 fl agella : 
 
 (IX.) The cover-glass preparation is boiled for five minutes 
 upon black logwood ink (" Kaisertinte "), from which it is 
 placed for fifteen minutes in dilute neutral bichromate of 
 sodium, this being- repeated several times. 
 
 STAINING BACTERIA IN SECTIONS. 
 
 When it is only necessary to demonstrate the presence of 
 bacteria in the various org-ans, it often suffices to make cover- 
 glass preparations by rubbing- on it a little of the juices from 
 a fresh cut surface; but a more exact examination of the 
 number of bacteria, and of their distribution in the tissues, 
 can be made only by means of sections. Before Weigert pro- 
 vided methods of staining- bacteria in situ, no means of dem- 
 onstrating them existed, except by the use of acids and alka- 
 lies, to which they show great resistance. 
 
 (X.) When a fresh section, or one hardened in alcohol, is 
 treated with strong- acetic acid or dilute (2 per cent) solution 
 of caustic potash or soda, it becomes almost transparent, only 
 the bacteria resisting the acid or alkali, and so becoming evi- 
 dent, especially when collected in masses in or without the 
 vessels; hence such nests of cocci were seen and described 
 long- before their nature was known, e.g., by Beckmann, in the 
 vessels of the kidney. Later, the potash method came to play 
 an important part in the study of pyaemia early in the seven- 
 ties, e.g., in the work of Hj. Heiberg; and quite recently it has 
 been used in isolated cases. It is thus used to render collec- 
 tions of typhoid bacilli evident in the tissues; and, even before 
 knowing- of Koch's investigations Baumgarten had seen 
 tubercle bacilli by the use of the potash method. Still, in gen- 
 eral, this has lost its importance since the introduction of 
 staining- methods. 
 
 One condition of a good staining- of sections is their being- 
 well and completely hardened. The organs are cut into small 
 pieces and hardened in a large quantity of absolute alcohol. 
 
 [If the bottle in which the pieces are to be hardened is 
 filled half full of loose cotton, so that the tissues are kept near 
 the top of the alcohol, the portion of this which becomes more 
 charged with water tends to sink to the bottom because of its 
 
Bacteriological Technology. 1 5 1 
 
 greater specific gravity, and it has been claimed that in such 
 cases more thorough hardening- is effected. W. T.] 
 
 As thin sections as possible are cut from the well-hardened 
 tissues by the use of a razor or microtome. 
 
 [In Koch's laboratory it is now customary to use a drop of 
 melted glycerin -jelly for attaching the organ to a cork, by 
 means of which it clamped in a Schauze or other sledge- 
 microtome, without any sort of imbedding. If perfectly hard- 
 ened material is used, fairly good sections are obtained in this 
 way, the sectioning of course being done under alcohol; and 
 there is no danger of the proper staining- of the bacteria being- 
 interfered with. But in many cases, where the organs to be 
 sectioned are rather large, brittle, etc., and the bacteria will 
 not be injured by the preparatory treatment, the tissue may 
 be imbedded in celloidin for sectioning- in the usual way under 
 alcohol; or it may be impregnated with paraffin melting at 
 about 50 C., and imbedded in this f or> sectioning- dry prefer- 
 ably with some form of rocking--microtome. In the latter 
 case, if the org-an is small, ribbon sections may be obtained 
 and stained on the slide, in the way so generally employed 
 now by histologists, and especially embryologists; or the par- 
 affin may be removed by placing- the sections in turpentine 
 for a few moments, and afterward in alcohol, after which they 
 are stained individually as if cut without imbedding. W. T.] 
 
 The staining of sections is essentially the same as staining 
 cover-glass preparations; but it must be observed that as a 
 rule they require a longer treatment with the staining-fluid, 
 are less tolerant of heating- in the latter, and require more 
 powerful decolorizing media for differentiating the bacteria, 
 while anhydration before they can be mounted in balsam is 
 rarely effected by drying, but by the use of alcohol or anilin 
 oil (Weigert). To prevent these fluids from at the same time 
 partly decolorizing- the preparation, Kuehne has recently 
 adopted the plan of anhydrating- with a fluid tinged with the 
 same dye used in staining the bacteria (No. XIII.). 
 
 Kuehne advises spreading differentiated sections of glanders 
 material upon the cover-glass, blowing- them dry (cf. p. 146), 
 laying- the cover-glass (with the section upward) upon a giass 
 plate, where it is slightly warmed, not to exceed 30 C., over 
 a spirit lamp, until the section becomes transparent, when, 
 after lying on the warm plate for five minutes, it is cleared 
 
152 Bacteriological Technology. 
 
 with ethereal oil and passed through xylol into balsam [so 
 that the use of alcohol is entirely avoided because of its 
 marked decolorizing- action on the bacteria]. 
 
 For the microscopic examination of sections which contain 
 stained bacteria, a sub-stage condenser is used. The Abbe 
 condenser is the most perfect of the various models in use, 
 but the cheaper Dujardin and similar condensers, which are 
 readily used on smaller stands in place of the well-diaphragm, 
 are entirely satisfactory. 
 
 Koch first called attention to the importance of the con- 
 denser in bacteriological microscopy. By means of this in- 
 strument, as he expresses it, it is possible to efface the "struc- 
 ture image " and so to get rid of its disturbing and concealing 
 influence on the "color image." By the former name he 
 designates the image of lines and shadows which reveals to us 
 the structure of the tissue, and which results from diffraction 
 of the rays of light during their passage through the prepara- 
 tion, different parts of which (nuclei, fibres, etc.) differ in re- 
 fractive power from the substratum in which they lie By the 
 " color image," he refers to the image of colored parts, which, 
 if very small, may be completely concealed by the lines and 
 shadows of the structure image. By the use of an Abbe or 
 similar condenser [with full opening], the preparation is illu- 
 minated with so broad a cone of light that the tissue appears 
 as a structureless plane, on which the colored parts, large and 
 small, stand out sharply. It is easy to convince one's self of the 
 great advantage of using such a condenser, for a section which 
 is full of stained bacilli of mouse septicaemia, may appear free 
 from bacteria when examined with ordinary illumination with 
 the concave mirror and diaphragm, while the condenser re- 
 solves a mass of colored rods within it. 
 
 What is here said -of the examination of stained bacteria in 
 sections, is also true of cover-glass preparations, in which the 
 bacteria frequently occur upon or within elements the struc- 
 ture images of which mask them. On the other hand, it must 
 be remembered that the examination of unstained bacteria 
 should always be made without a condenser; with the finest 
 diaphragm which gives sufficient illumination; or, what 
 amounts to the same thing, with the condenser lowered as far 
 as possible. 
 
Bacteriological Technology. 153 
 
 METHODS OF STAINING SECTIONS. 
 
 Several of the numbered methods which follow have already 
 been described for cover-glass preparations, but are repeated 
 here with the changes necessary for sections. 
 
 (XI.) Weigert's Original Method (1876). The section is 
 laid for a rather long- time in a quite strong solution of 
 methyl violet, washed out in dilute acetic acid, anhydrated in 
 alcohol, cleared in oil of cloves, and mounted in balsam. 
 
 Weigert's improved method (1881) employs an aqueous 1- 
 per-cent solution of gentian violet, BR., from which the sec- 
 tion g-oes to absolute alcohol, clove oil, and balsam. (Weigert 
 recommended gentian violet, BR., especially because it is not 
 so easily removed from the bacteria when the section is treated 
 with alcohol and clove oil.) 
 
 (XII.) Loefflers Potash Blue Method. The sections are 
 placed for a few minutes in alkaline methylene blue (No. 10), 
 differentiated for some seconds in 0.5-per-cent acetic acid, an- 
 hydrated in alcohol, cleared with cedar-oil, and mounted in 
 balsam. 
 
 (XIII.) Kuehne Universal Method. 8 The section is laid in 
 carbolic methylene blue (No. 6), as a g-eneral thing- for half an 
 hour (but for leprosy, two hours), rinsed with distilled water, 
 treated with dilute hydrochloric acid (No. 12) until the color 
 is pale blue, rinsed in lithium-water (No. 14), laid in distilled 
 water for some minutes, dipped for anhydration into absolute 
 alcohol (which may be colored with methylene blue, p. 590), 
 before it goes into a salt-cellar of aniline-oil colored with 
 methylene blue, where in a few moments it becomes anhy- 
 drated without being- decolorized, then rinsed in pure aniline 
 oil, cleared in turpentine, and when all aniline oil has been 
 carefully removed by treatment with xylol, usually renewed 
 once, it is mounted in balsam. When the aniline oil is not 
 completely removed, the balsam becomes brown with time. 
 
 The differentiation in hydrochloric acid is the most difficult 
 point in the process. Thin sections need only be dipped into 
 it; and in any case a sharp watch must be kept upon the de- 
 gree to which decolorization has progressed, so that the sec- 
 tion may be rinsed in lithium water at once when the right 
 shade is reached. 
 
154 Bacteriological Technology 
 
 (XIV.) Koch's Isolated Staining Method. After staining 
 in an aqueous solution of fuchsin, methyl violet, or methylene 
 blue, the sections are washed out in a half saturated solution 
 of carbonate of potassium (No. 15 a), then passed through 
 alcohol, xylol, and balsam. Weigert was the first to undertake 
 an isolated staining of bacteria in tissues, since by the use of 
 carmine, followed by washing in glycerin acidulated with hy- 
 drochloric acid, he stained clusters of micrococci in 1871. 
 
 Malassez and Vignal, after staining in methylene blue, 
 wash in No. 15 b, etc. 
 
 (XV.) The Grain Method. The sections are transferred 
 directly from absolute alcohol -into aniline gentian violet, and 
 treated like cover-glass preparations (No. III.), except that 
 they are anhydrated in absolute alcohol and cleared in cedar- 
 oil preparatory to being mounted in balsam. For Weigert's 
 modification of the method, see No. XIX., infra. 
 
 When sections stained by this method are also treated with 
 a nuclear stain like carmine, a very pretty double staining is 
 obtained. Fraenkel recommends the following method, which 
 is also thought to more frequently escape the troublesome 
 granular precipitates which now and then appear in the prep- 
 aration when the original Gram method is used. 
 
 (!XVI.) The sections are transferred from alcohol to strong 
 picro-carmine, where they remain half an hour, are rinsed in 
 50-per-cent alcohol, then go for half an hour into unsaturated 
 aniline gentian violet prepared by dropping a few drops of 
 the saturated alcoholic solution (No. 1) into a watch-glass 
 containing aniline w r ater (No. 5) until the mixture begins to be 
 opaque from which they are placed directly in the iodine 
 water for three minutes, then into alcohol, where the red color 
 reappears and they become anhydrated, when they go through 
 cedar-oil into balsam. 
 
 (XVII.) Koch-Ehrlich-Weigert's Tubercle Method. The 
 sections are laid in aniline gentian violet (No. 9) for twelve 
 hours, moved about for a few seconds in 25-per-cent nitric acid, 
 rinsed in 60-per-cent alcohol until they chow only a slight blue 
 color, counter-stained for some minutes in a saturated aqueous 
 solution of vesuvin, rinsed in 60-per-cent alcohol, anhydrated 
 in absolute alcohol, cleared with cedar-oil, and mounted in 
 xylol balsam. 
 
 The tubercle bacilli are also stained by Kuehne's method 
 
Bacteriological Technology. 155 
 
 (XIII.) and the Gram method, but where a differential diag- 
 nosis is required decolorization must be effected by strong* 
 mineral acids (e.g., as in Nos. XVII. and XVIII.) ; for it is its 
 resistance to decolorization by mineral acids which distin- 
 guishes the tubercle bacillus from all other known species ex- 
 cept the bacillus of leprosy, which, however, differs from it in 
 staining readily with a simple aqueous solution of methyl 
 violet or fuchsin. The so-called smegma-bacilli also show a 
 decided power to resist the action of mineral acids, but they 
 are readily decolorized by subsequent treatment with alcohol, 
 which is not the case with either the tubercle or leprosy bacil- 
 lus. The smegma-bacilli are probably only different species of 
 putrefactive bacteria which, from growing in the smegma> 
 have become so impregnated with oily matters that they have 
 become difficult to stain. This view is supported by the fact 
 that it has proved possible to change the behavior of other 
 species toward staining methods, by growing them in sub- 
 stances containing butter, etc. (Bienstock). On the other 
 hand, the diagnosis of smegma-baciUi may be attended with 
 some difficulty as compared with Lustgarten's syphilis bacil- 
 lus, the etiological relation of which to the disease is still 
 very problematical, although it has been demonstrated in 
 syphilitic secretions and neoplastic growths. Like the syphilis 
 bacillus, after being stained on the cover-glass for twenty -four 
 hours in aniline gentian violet, they remain colored after treat- 
 ment for ten seconds with 1.5-per-cent solution of potassium 
 permanganate, followed by sufficient washing in sulphurous- 
 acid water to entirely decolorize the preparation. The syph- 
 ilis bacillus cannot be confounded with that of tubercle, be- 
 cause it is very easily decolorized by mineral acids. 
 
 (XVIII.) Ziehl-Neelsen Tubercle Method. Sections are 
 more rapidly stained by laying them for fifteen minutes in 
 carbolic fuchsin (No. 7), agitating them a few seconds in 25 -per- 
 cent sulphuric or nitric acid, rinsing in 60-per-cent alcohol 
 until only a light rosy color remains, counter-staining for some 
 minutes in a saturated aqueous solution of methylene blue, and 
 rinsing, anhydrating, and mounting as before. 
 
 For staining bacteria in sections of hardened gelatin-cul- 
 tures, see Neisser, "Centralbl. f. Bakteriologie," 1888, III., 506. 
 
156 Bacteriological Technology. 
 
 MOULDS. 
 
 The following notes on mounting- moulds refer chiefly to 
 the directions of O. Johan-Olsen. 
 
 Temporary preparations are obtained by laying the mould 
 for a few minutes in dilute alcohol, then in weak ammonia, 
 which is removed with filter-paper, the surplus washed out 
 with water, and this replaced by glycerin, in which the exam- 
 ination is made. 
 
 Permanent preparations are made in the same manner, 
 after staining the material in osmic acid, which is kept for use 
 in a 0.5-per-cent solution in a dropping -bottle that has been 
 well cleansed with alcohol and ether and is stopped with glass. 
 Two drops of this are added to eight drops of water in a 
 watch-glass (i.e., about 0.1 per cent), in which the mould is 
 allowed to lie for an entire day; or, if the 0.5-per-cent solution 
 is used directly, a few minutes suffice. The preparation is 
 then washed with alcohol, followed by distilled water. If it is 
 still too black, it is decolorized sufficiently in weak ammonia, 
 followed by water, and mounted in glycerin. Preparations 
 stained with osmic acid may also be further stained by saffra- 
 nin, usually a very dilute solution, in which they are allowed 
 to remain for a long time. 
 
 Preparations of an entire culture can be made upon the 
 slide. Usually the cultures are obtained from fluid media 
 (e.g. } Brefeld's slide-cultures, supra, p. 100), and those which 
 are not too far advanced are chosen. The colony being placed 
 on a slide, a cover-glass is let down upon it carefully espe- 
 cially in case of the erect forms and it is allowed to dry 
 down so that the cover -glass does not move around (usually 
 only a few minutes being needed), when 0.1-per-cent osmic acid 
 is added at one side and drawn under by applying bibulous 
 paper to the other edge of the cover. After a proper time 
 the acid is thoroughly washed out with water that is drawn 
 under the cover in the same way, and, either with or without 
 saffranin staining, mounted by drawing glycerin under and 
 sealing in the usual way: or else dilute alcohol is used instead 
 of the osmic acid, and followed by ammonia, etc., as indicated 
 above for temporary preparations. 
 
 For demonstrating moulds in sections, Hueppe recommends 
 
Bacteriological Technology. 157 
 
 the methylene blue universal method (Nos. XII. and XIII.). 
 Blbbert advises the following 1 modification of the Gram 
 method : 
 
 (XIX.) Weigert's Fibrin Stain. The section hardened in 
 alcohol is stained for a long- time in saturated aniline gentian 
 violet (No. 9), rinsed in 0.7-per-cent solution f table salt, and 
 transferred to the slide, where the other steps are taken. The 
 superfluous water is removed with filter-paper, iodine water 
 (No. 16) is dropped upon it and removed with filter paper, and 
 a couple of drops of aniline oil allowed to fall upon the section 
 for the purpose of at once decolorizing and anhydrating it. 
 
 As the aniline oil soon becomes dark, it must be removed 
 and replaced by fresh oil once or twice, after which it is en- 
 tirely replaced by xylol and the section mounted in balsam. 
 
 ACTINOMYCES. 
 
 When it is only desired to obtain a sure diagnosis of Acti- 
 nomyces cushions in pus or sections, treatment with acetic 
 acid or alkalies and examination in glycerin suffices. For 
 staining cover-glass preparations, Fraenkel employs the Gram 
 method, letting the section stay in aniline gentian violet for 
 twenty -four hours, and in the iodine solution for fifteen min- 
 utes. The Weigert-Gram method (No. XIX.) appears suited 
 to staining sections for Actinomyces. 
 
 AMCEBOIDS. 
 
 The amoebae or amoeboid organism detected by Laveran 
 and subsequently more fully studied by Marchiafava and Oelli, 
 Golgi, Osier, and others, and held to be the cause of malaria, 
 are not adapted to staining by any of the above methods. It 
 is, indeed, possible, when cover-glass preparations of the blood 
 are treated with methylene blue or vesuvin, to find within the 
 red corpuscles larger or smaller irregular spots, colored blue 
 or brown (Marchiafava and Celli). These are the stained 
 amoebae; but a clear view of all the developmental forms of 
 the parasite is obtained only by examination of the fresh 
 blood. Laveran gives the following directions for this : The 
 amoebae are most easily found in patients already anaemic, and 
 who have not taken quinine, at any rate just before the exam- 
 
158 Bacteriological Technology. 
 
 ination. The blood is obtained just before, or :n the beginning 
 of an attack, by a needle puncture in the finger-tip or lobe of 
 the ear, after careful cleansing- with water and alcohol. The 
 surface of the drop which exudes is touched with a well- 
 cleaned slide, and the small drop of blood which adheres is 
 quicidy covered with a cover-glass, and sealed with paraffin 
 when the layer of blood is thin enough for examination. An 
 enlargement of 400 to 500 diameters is sufficient for the inves- 
 tigation, places being chosen where the red corpuscles lie flat 
 and isolated. Few parasites are usually to be seen, rarely as 
 many as eight to fifteen in the same field, so that one must be 
 prepared to spend some time in searching- for them. The pig- 
 ment granules that occur in most of the amoebae give a clue 
 in the search. Richard recommends the addition of a drop of 
 dilute acetic acid for the rapid demonstration of these para- 
 sites, as it removes the annoying- red blood-corpuscles without 
 destroying the parasites. According to Laveran, the addition 
 of water is better, as it does not kill the amoebae so soon as 
 acetic acid does, but allows them to continue their peculiar 
 movements. 
 
 BIBLIOGRAPHY. 
 
 1. Fortschritt. d. Medicin, 1886, iv. 2. Meddelelser f. Carlsberg 
 Lab., ii., 218. 3. C/. Fortsch'r. d. Med., 1888, No. 1. 4. Zeitschr. f. Hy- 
 giene, 1887, 521. 5. Ann. de Micrographie, i., 153. 6. Sur la culture des 
 microbes anae*robies (Annales de 1'Institut Pasteur, 1887, No. 2). 
 7. Beitrage zur Kenntniss des Sauerstoffbediirfnisses der Bacterien 
 <Zeitschr. fur Hygiene, 1886, i., 115). 8. FromKuehne: Praktische An- 
 leitung zum mikroskopischen Nachweis der Bakterien iin thierischen 
 Gewebe, Leipzig. 
 
INDEX. 
 
 ACID, pyrogallic, removal of oxy- 
 gen by, 92. 
 
 Actinomyces, examination of, 157. 
 Aeroscope, Hesse's, 71. 
 Miquel's, 73. 
 Schoenauer's, 68. 
 Straus and Wurtz's, 74. 
 Agar-agar for culture, 25. 
 for plate culture, 62. 
 peptonized, preparation of, 28. 
 Agar-gelatin for culture, 26. 
 for plate culture, 63. 
 peptonized, preparation of, 28. 
 Air, bacteriological analysis of, 66. 
 Air-pump, cultivation of anaerobic 
 
 bacteria by the aid of the, 87. 
 Alg'se, use of, in examination of bac- 
 teria, 133. 
 
 Amoeboids, examination of, 157. 
 Anaerobic bacteria, cultivation by 
 
 the aid of the air-pump, 87. 
 bacteria, culture of, 80. 
 bacteria, culture vessels for, 88. 
 bacteria, isolation by the capilla- 
 ry-tube method, 86. 
 bacteria, isolation in gelatinized 
 
 media, 85. 
 
 bacteria, isolation of, 83. 
 bacteria, roll-culture of, 87. 
 culture by the aid of aerobic 
 
 bacteria, 91. 
 culture in hermetically sealed 
 
 tubes, 90. 
 
 culture, pipettes for, 91. 
 culture, preservation of, 90. 
 culture under a gelatinizing 
 
 plug, 90. 
 
 culture under oil, 90. 
 Analysis, bacteriological, of various 
 
 substances, 55. 
 Aniline staining, 134. 
 Animals, inoculation of, 102. 
 Apparatus, bubbling, for air analy- 
 sis, 72. 
 
 for culture, 14. 
 Arachnoid, inoculation beneath the, 
 
 110. 
 Aspirators for air analysis, 66. 
 
 BACILLUS anthracis, effect of car- 
 bolic acid on, 132. 
 Bacteria, anaerobic, cultivation by 
 
 the aid of the air-pump, 87. 
 anaerobic, culture of, 80. 
 anaerobic, culture vessels for,88. 
 anaerobic, isolation by the capil- 
 lary-tube method, 86. 
 anaerobic, isolation in gelatin- 
 ized media, 85. 
 anaerobic, isolation of, 83. 
 anaerobic, roll-culture of, 87. 
 in sections, staining, 150. 
 microscopic examination of, 133. 
 staining flagella of, 149. 
 staining* of, 133. 
 staining of, in fluids, 142. 
 Bacteriological analysis of various 
 
 substances, 55. 
 Baumgarten, staining method of, 
 
 135. 
 
 Beer-wort for culture, 20, 29. 
 Bench of sheet zinc for supporting 
 
 a plate culture, 60. 
 Blood, collection of sterile, 31, 119. 
 
 serum, sterilization of, 8. 
 Boettcher's moist chamber, 97. 
 Bohr's thermo-regulator, 51. 
 Bottles for culture, 15. 
 Bouillon for culture, 19. 
 Box, glass, for culture, 17. 
 Bread, white, for culture, prepara- 
 tion of, 32. 
 Brood-ovens, 48. 
 
 Bubbling apparatus for air analysis, 
 72. 
 
 CAPILLARY tubes for inoculation, 
 44, 45. 
 
 Capillary -tube method of separa- 
 tion, 39. 
 
 Cement for fastening capillary 
 tubes, 40. 
 
 Chamber, Geissler, 99. 
 moist, Boettcher's, 97. 
 moist, Ranvier's, 98. 
 
 Chamberland flasks, 17. 
 
 Chambeiiand's filter, 9. 
 
i6o 
 
 Index. 
 
 Chamberland's filter, cleaning' of, 12. 
 Chambers, moist, 94. 
 Cibil's extract for culture, 20. 
 gelatin, preparation of, 27. 
 Clarifying of nutrient jelly, 26. 
 
 preparations, 146. 
 
 Cleanliness in sterilization, neces- 
 sity for, 7. 
 
 Cohn's heating- method for obtain- 
 ing pure cultures, 38. 
 Condenser, necessity for use of, in 
 
 examination of sections, 152. 
 Contamination of sterilized ob- 
 jects, guarding against, 4. 
 Cooling apparatus for plate cul- 
 tures, 59. 
 
 Counting germs, 41. 
 Cracker box for sterilization, 2. 
 Cultivation of micro-organisms un- 
 der the microscope, 94. 
 Culture, anaerobic, by the aid of 
 
 aerobic bacteria, 91. 
 anaerobic, in hermetically sealed 
 
 tubes, 90. 
 
 anaerobic, pipettes for, 91. 
 anaerobic, preservation of, 90. 
 anaerobic, under a gelatinizing 
 
 plug, 90. 
 
 anaerobic, under oil, 90. 
 apparatus, 14. 
 bottles for, 15. 
 flasks for, 15. 
 glass box for, 17. 
 glasses, 15. 
 media, and their introduction 
 
 into vessels, 18. 
 
 media, collection of sterile, 118. 
 media, preparation of, 18. 
 obtaining pure material for, 36. 
 of anaerobic bacteria, 80. 
 potato, in a vacuum, 92. 
 test-tubes for, 14. 
 vessels, tilling of, 34. 
 Cultures, collection of dust for, 69. 
 from man and animals, 115. 
 inoculation of, 43. 
 Klebs' fractional, 37. 
 pure, Cohn's heating method 
 for, 38. 
 
 DECOCTION of dried fruits for cul- 
 ture, 20, 29. 
 
 of horse dung for culture, 20. 
 of liver, etc., for culture, 20. 
 of prunes for culture, 20. 
 of wheat, hay, cabbage, for cul- 
 ture, 20. 
 Digestive tract, infection through 
 
 the, 112. 
 
 Dilution, method of, for obtaining 
 pure cultures, 41. 
 
 Discontinuous heating, sterilization 
 
 by, 7. 
 
 Disinfectant, testing a fluid, 126. 
 Disinfectants in sterilization, 13. 
 Disinfecting oven, diagram of, 130. 
 
 oven, testing a, 128. 
 Disinfection experiments, 121. 
 Drip-aspirator, 67. 
 Drying-jar for hydrophobia vaccine, 
 
 Dust, collection of, for cultures, 69. 
 collection of, for microscopical 
 examination, 67. 
 
 EGGS for culture, preparation of, 33. 
 Erlenmeyer flasks, 15, 18. 
 Extracts of meat for culture, 20. 
 Eye, inoculation of the anterior 
 chamber of the, 109. 
 
 FIBRIN stain, Weigert's, 157. 
 Filling culture vessels, 34. 
 Filter, Chamberland's, 9. 
 
 Chamberland's, cleaning of, 12. 
 
 simple, for gelatin, 27. 
 Filtering of nutrient jelly, 26. 
 Filters, insoluble powder, 74. 
 
 porcelain, 9. 
 
 sand, 74. 
 
 soluble, 75. 
 Filtration, 10. 
 Finder, simple, 97. 
 Flagella of bacteria, staining of, 149. 
 Flask, Pasteur-Chamberland, 17. 
 Flasks, conical, in bacteriological 
 analysis, 56. 
 
 for culture, 15. 
 Flesh water for culture, 18, 19. 
 
 GAG for use in feeding experiments, 
 
 112. 
 
 Geissler chamber, 99. 
 Gelatin, Cibil's, preparation of, 27. 
 colored nutrient, for culture, 
 
 preparation of, 33. 
 filter for, 27. 
 for culture, 25. 
 for plate culture, 62. 
 Gelatinized media, separation of 
 
 germs by, 42, 56, 58. 
 Germs, counting, 41. 
 Glass box for culture, 17. 
 
 needles for inoculation, 45. 
 plates in bacteriological analy- 
 sis, 58. 
 
 trays for culture, 24. 
 trays in bacteriological analysis, 
 
 57. 
 
 Glycerin B. P. A. for culture. 28. 
 Glycerin-serum, 32. 
 Gram's staining method, 147, 154. 
 
Index. 
 
 161 
 
 HAY infusion, sterilization of, 7. 
 Heat, sterilization by, 1. 
 Heating, discontinuous, steriliza- 
 tion by, 7. 
 
 Hesse's aeroscope, 71. 
 Hydrogen generator, Joergensen's 
 
 83. 
 Pasteur pipette for passing, 
 
 through gelatin, 87. 
 Hydrophobia vaccine, drying- jar 
 
 for, 112. 
 virus, 110. 
 
 INDIGOTIN test for oxygen, 82. 
 Infection by inhalation, 113. 
 
 through the digestive tract, 112. 
 
 through the trachea, 113. 
 Infusion of wheat, hay, cabbage, for 
 
 culture, 20. 
 
 Inhalation, infection by, 113. 
 Inoculation, capillary tubes for, 44, 
 45. 
 
 glass needles' for, 45. 
 
 of a test-tube, 46. 
 
 of animals, 102. 
 
 of cultures, 43. 
 
 of the anterior chamber of the 
 eye, 109. 
 
 Pasteur pipettes for, 44, 46. 
 Irish -moss for culture, 26. 
 
 JELLY, nutrient, clarifying of, 26. 
 
 nutrient, filtering of, 26. 
 Joergensen's hydrogen generator, 
 
 83. 
 
 KLEBS' fractional cultures, 37. 
 Knife-rests for supporting pipettes, 
 
 35. 
 
 Koch-Ehrlich-Weigert's staining 
 . method, 147, 154. 
 Koch's method of separating germs, 
 42, 56, 58. 
 
 staining method, 143, 146, 154. 
 
 steam sterilizer, 4. 
 Kuehne's staining method, 147, 153. 
 
 LEVELLING tripod, 60. 
 
 Liborius' method of isolation of bac- 
 teria, 84. 
 
 Liebig's extract for culture, 20. 
 
 Liquids, bacteriological analysis of, 
 56. 
 
 Loeffler's staining method, 153. 
 
 MATERIAL for culture, obtaining 
 
 pure, 36. 
 
 Meat broth for culture, 18. 
 Media, fluid, for culture, 18. 
 solid, for culture, 21. 
 
 Mica, plate cultures under, 84. 
 Mice, keeping*, 103. 
 Microscope, cultivation of micro- 
 organisms under the, 94. 
 Microscopic examination of bacte- 
 ria, 133. 
 Miquel's aeroscope, 73. 
 
 soluble powder filter,, 76. 
 Moist chamber, Boettcher's, 97. 
 
 chamber, Ranvier's, 98. 
 
 chambers, 94. 
 Moulds, culture media for, 20, 29. 
 
 mounting, 156. 
 Mounting moulds, 156. 
 
 preparations, 146. 
 Mouse jar, 103. 
 
 OVEN, sterilizing, 2. 
 
 Oxygen, indigotin test for, 82. 
 
 removal of, by pyrogallic acid, 
 92. 
 
 PAPIN digester for sterilization, 2, 
 
 7. 
 Paraffin, application of, to slides, 
 
 133. 
 
 Pasteur culture vessels, 89. 
 flasks, 17. 
 pipette with rubber cap plugged 
 
 with cotton, 119. 
 pipettes for inoculation, 44, 46. 
 wash-bottle, 118. 
 Pasteurization, 8. 
 Petri's sand filter, 75. 
 Physiological differences, utilization 
 of, in obtaining pure cultures, 37. 
 Pipette with cotton plug, 34. 
 Pipettes for anaerobic culture, 91. 
 Pasteur, for inoculation, 44, 46. 
 Pasteur, with rubber cap 
 
 plugged with cotton, 119. 
 Plan tarn our hot- water funnel for fil- 
 tration of gelatin, 28. 
 Plaster, moist, for culture, prepara- 
 tion of, 33. 
 
 Plate culture, agar-agar for, 62. 
 culture, agar-gelatiri for, 63. 
 culture, bench of sheet zinc for 
 
 supporting, 60. 
 culture, gelatin for, 62. 
 culture, serum for, 63. 
 culture, Soyka's method, 61. 
 cultures, cooling apparatus for, 
 
 59. 
 
 cultures, preparation of, 59. 
 cultures under mica, 84. 
 Platinum needle for inoculation, 43, 
 45. 
 
 Plugs, tubular, 16. 
 Porcelain filters, 9. 
 
162 
 
 Index. 
 
 Potato, boiled, for culture, 23. 
 broth for culture, 25. 
 cultures in a vacuum, 92. 
 Powder filters, insoluble, 74. 
 Purification blotches in cylindrical 
 
 glass, 40. 
 
 Pyrogailic acid, removal of oxygen 
 by, 92. 
 
 RABBITS, inoculation of, 107. 
 Rabies, inoculation of, 110. 
 
 vaccines, preparation of, 111. 
 Ranvier's moist chamber, 98. 
 Rei chert's thermo-regulator, 51. 
 Rice milk for culture, preparation 
 
 of, 33. 
 
 Rohrbeck's thermo-regulator, 51. 
 Roll-culture of anaerobic bacteria, 
 
 87. 
 
 SAND filters, 74. 
 Schoenauer's aeroscope, 68. 
 Sections, staining bacteria in, 150. 
 Separation, methods for obtaining 
 
 pure cultures by, 39. 
 Serum for culture, preparation of, 
 29. 
 
 for plate culture, 63. 
 
 of blood, sterilization of, 8. 
 Slide, hollow-ground, 95. 
 Soil, artificial, for culture, prepara- 
 tion of, 33. 
 
 bacteriological analysis of, 64. 
 Solids, bacteriological analysis of, 
 
 64. 
 
 Soluble filters, 75. 
 Soyka's box, 17. 
 
 method of plate culture, 61. 
 Spores, staining, on the cover glass, 
 
 148. 
 
 Sputum, staining, 147. 
 Stain, Weigert's fibrin, 157. 
 Staining bacteria, 133. 
 
 bacteria in fluids, 142. 
 
 bacteria in sections, 150. 
 
 differential, 136. 
 
 double, 137. 
 
 flagella of bacteria, 149. 
 
 materials employed in, 139. 
 
 method, Baumgarten's, 135. 
 
 method, Gram's, 147, 154. 
 
 method, Koch's, 143, 146, 154. 
 
 method, K o c h-E h r 1 i c h-Wei- 
 gert's, 147, 154. 
 
 method, Kuehne's, 147, 153. 
 
 method, Loeffler's, 153. 
 
 method, Weigert's, 153. 
 
 method, Ziehl-Neelsen's, 147, 
 155. 
 
 methods of, 146. 
 
 mordants used in, 140. 
 
 Staining preparations dried on the 
 cover glass, 143. 
 
 sections, methods of, 153. 
 
 spores on the cover glass, 148. 
 
 sputum, 147. 
 
 Steam sterilizer, Koch's, 4. 
 Sterilization, 1. 
 
 by discontinuous heating, 7. 
 
 by heat, 1. 
 
 by steam, 4. 
 
 cracker box for, 2. 
 
 disinfectants in, 13. 
 
 instruments needed for, 2. 
 
 of blood serum, 8. 
 
 of hay infusion, 7. 
 
 Papin digester for, 2, 7. 
 
 water bath for, 8. 
 Sterilizer, Koch's steam, 4. 
 Sterilizing oven, 2. 
 Stoppers, tubular, 16. 
 Straus and Wurtz's aeroscope, 74. 
 Suction-bulb, automatic, for collect- 
 ing air germs, 70. 
 Support for funnels, etc., 142. 
 
 TESTING a disinfecting oven, 128. 
 
 a fluid disinfectant, 126. 
 Test-tube, inoculation of a, 46. 
 Test-tubes for culture, 14. 
 
 in bacteriological analysis, 56. 
 
 plugging of, 14. 
 Thermometer, electric, 128. 
 Thermo-regulators, 48. 
 Thermostat for solidifying serum, 
 
 31 
 
 Thermostats, 49. 
 Tripod, levelling, 60. 
 Tubular plugs, 16. 
 
 VACCINE, hydrophobia, drying-jar 
 
 for, 112. 
 
 Vaccines, rabies, preparation of , 111. 
 Vacuum cultures for bacteria, 89. 
 
 potato culture in a, 92. 
 Virus, hydrophobia, 110. 
 
 WAITING for germination after ster- 
 ilization, 8. 
 
 Wash-bottle, Pasteur, 118. 
 Water, bacteriological analysis of, 
 
 63. 
 
 bath for sterilization, 8. 
 Weigert's fibrin stain, 157. 
 staining method, 153. 
 Wine for culture, 20. 
 
 YEAST, culture media for, 20, 29. 
 water for culture, 20. 
 
 ZIEHL-NEELSEN'S staining method, 
 147, 155. 
 
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