REVIEW OF EXISTING METH- ODS FOR CULTIVATING ANAEROBIC BACTERIA ,^»^ OTTO F. HUNZIKER \.K-'- LABORATORY OF COMPARATIVE PATHOLOGY AND BACTERIOLOGY, NEW YORK STATE VETERINARY COLLEGE, CORNELL UNIVERSITY, ITHACA, N. Y. n\ri\t\7P.(i l^y MinrntnfK^ Mini ^<^Bo»tocn p. knottier Cibrarg THIS BOOK ISTHE GIFTOF A)tAA/vvMA> f^ . TA «yv8- . Digitized by Microsoft® [Reprinted from the JOURNAL OF APPLIED MICROSCOPY AND LABORATORY METHODS, Rochester, N. Y., Vol. V, No. 3.] A Review of the Existing Methods for Cultivating Ana&robic Bacteria. Since the discovery by Pasteur in 1861 of the fact that some species of bac- teria can thrive in the absence of oxygen only, various methods have been intro- duced involving many devices for the study of anaerobic bacteria. Notwith- standing this fact, our knowledge of anaerobes is as yet very limited. While bacteriology has made rapid progress along the line of aerobic species, a compar- atively small number of anaerobic species have been carefully studied and identified. Concerning the character of some of the known anaerobes, such as the bacilli of tetanus, of malignant oedema, and of black leg, it becomes evident that the cause of the slow development in the study of anaerobic species does not lie in the fact that these specips are of less patholdgic and economic importance than their brothers the aerobes. Nor can it be attributed to the assumption that this class of organisms covers a very limited number of species only, for the results of bacteriological research of recent years are rapidly revealing the existence of a liberal distribution of anserobic bacteria in nature. On the other hand, the bacteriologist realizes that the production of anaerobic conditions for bacterial growth involves greater difficulties and meets more fre- quently with failure than the simple cultivation of aerobes. It is, therefore, reasonable to recognize this great«!r difiiculty, complexity and expense of technique as the most potent obstacle in anaerobic research. While, from the above statement, it is obvious that none of the methods for anaerobic cultures are quite so simple and easy as those for aerobes, still, a thorough review of the multitude of methods introduced and devices invented shows that there are some that involve little difficulty, are simple, and can be manipulated in any laboratory. Methods for the cultivation of anaerobic bacteria are referred to in most text-books and manuals on bacteriology ; however, space in such books does not permit a full list of these methods, with directions for practical application ; and a search through the entire bacteriological literature in pursuit of the easiest, simplest and most efficient method is a task that does not appeal to the investigator eager to grow cultures with the greatest economy of time. In order to fill this gap, to afford the investigator easy access to the existing methods, and by so doing encouraging th^ study of anaerobic bacteria, it was thought desirable to carefully review the literature on this subject, and to present the principal methods and r their modi ficakions in this article. For the purpose of being able to treat the diverse methods in logical pr^e^r, the principles upon which they are based are arranged in tl^e fftollowifag classifi- cation, in which order they will be reviewed. The Anaerobic conditions are brought about : 1. By formation of a vacuum. 2. By replacement of air by inert gases. Digitized by Microsoft® [Reprinted from the JOURNAL OF APPLIED MICROSCOPY AND LABORATORY METHODS, Rochester, N. Y., Vol. V, No. 3.] »agc 1B96 3_ By. absorption of oxygen. 4. By reduction of oxygen. 5. By exclusion of atmospheric oxygen by means of various physical principles and mechanical devices. 6. By the combined application of any two or more of the above princi- ples. METHODS FOR CULTIVATING ANAEROBIC BACTERIA IN A VACUUM. In the search for methods that would enable the investigator to obtain cul- tures in the absence of oxygen, the principle of exhaustion was naturally one of the first resorted to. It was much used in early research concerning anaerobic bacteria. While absolute anaerobic conditions can be obtained in a vacuum only, the difficulty of successful evacuation and the fact that a good vacuum pump generally lies beyond the means of small laboratories, explain the reason why the principle of exhaustion has been losing ground in recent years and is gradually being replaced by more simple and less expensive means to produce anaerobic conditions. Pasteur's Method. — Use a small flask with a long neck or test tube ; fill it one-half (in case of flask) or one-third (in case of test tube) with the inoculated medium. Constrict the neck at b (Fig. 1) and connect the end of the neck c with the vacuum pump. > While evacuating place the bulb of the flask or the tube in a water bath at 37° C, caus- ing the liquid to boil. Evacuate for thirty minutes ; then, while still exhausting, seal at b in the flame. According to Fitz this is the oldest and simplest method ; it was introduced by Pasteur and tested by Cohn, Lister, Tyndall, Aitken, and Fitz. Nenki introduced a method identical with the above. Roux, in 1887, modified Pasteur's apparatus as shown in Fig. 2. This type of apparatus has been in constant use in Pasteur's laboratory for many years. The bulbs a and a' hold the medium, which is introduced through the lateral capillary tubes b and V by means of suction at c. Method. — Push a loose cotton plug into tube c. Seal the lateral tubes b and b' in the flame and sterilize the apparatus in the hot air sterilizer. Break .a^ *the tips on the lateral tubes b and V and immerse the tubes in bouillon. Apply suction at c. When the tubes ^a and a' are about one- third full seal the ends of the lateral tubes again and sterilize in steam sterilizer. When cool reopen the lateral tubes 'apd introduce the inoculating mate- rial by suction at c. Melt off the lateral tubes in the flame and connect c with a vacuum pump. When completely evacuated seal tube c at its con- striction and remove the apparatus to the incubator. Digitized by Microsoft® Fig. 1. [Reprinted from the JOURNAL OF APPLIED MICROSCOPY AND LABORATORY METHODS, Rochester, N. Y., Vol. V, No. 8.] Page 1696 [\ In this apparatus two cultures may be grown- simultaneously. Apparatus shown in Fig. 3 works on the same principle with the exception that it can take care of only one culture at a time. The latter can also be used for cultures on solid media and Esmarch roll cultures. Gruder's Method. — Take a long test tube 22 to 25 cm. long. About 15 cm. from the bottom draw it out into a constriction as illustrated in Fig. 4, plug the tube with cotton as usual, and sterilize. By means of a funnel pour about 10 c. c. of the sterile medium and 2 c. c. of sterile water into the tube and sterilize. Then inoculate Fig. 3. the medium as usual. Push the cotton plug well down into the tube and insert the rubber stopper. Connect the glass tube in the rubber stopper with a vacuum pump and immerse the lower part of the test tube in water at 37° C. in case of bouillon or gelatin, and at 42° C. in case of agar. In ten to fifteen minutes the tube is evacuated. To avoid wetting of the cotton plug by the boiling and foaming medium, the con- striction may be slightly and carefully flamed. While still evac- uating, the tube is sealed in the flame. If agar is used, cool the hermetically sealed tube in a water bath to 40° C, then roll it until the medium congeals ; in ease of gelatin cool slowly in the air by constant rolling of the tube in the hand, so that at Fig- 4. room temperature the vacuum is filled with vapor. In case of bouillon cultures the operation is finished as soon as the tube is sealed. Roux's tube for potato cultures in vacuum is operated in the same way, but the potato is introduced into the test tube and- the inoculation is made before the tube is constricted. The same investigator also recommended for potato cultures a tube as shown in Fig. 5. As soon as the potato in the culture is inoculated the tube is sealed at «, and the lateral tube closed with a cotton plug is connected with the vacuum pump. When evacuated the lateral tube is sealed in the flame at c. Novy constructed an apparatus for plate cultures in vacuum (Fig. 6). It consists of a cylinder ending in a firm, broad rim. On the rim is placed a thick rubber ring. The cylinder is closed by a bell jar, the lower rim of which corresponds with the upper rim of the cylinder. The whole apparatus terminates in a stop-cock. Method. — Place the inoculated Petri dishes in the Digitized by Microsoft® Fig. 5. [Reprinted from the JOURNAL OF APPLIED MICROSCOPY AND LABORATORY METHODS, Rochester, N. Y., Vol. V, No. 3.] Page 1697 cylinder, invert the bell jar over it, and close firmly by applying clamps with rubber lined jaws to the union of the cylinder and bell jar. One end of the turn cock (x-y) is connected with a thick-walled rubber tubing which is closed by a screw compressor. The other end is connected with a vacuum pump and when evacuation is complete the turn cock is turned and the vacuum pump is disconnected. Zupinski'xa. 1898 introduced a new and simple method of evacuation. It is based on the principle of Toricelli's vacuum. The apparatus (Fig. 7) consists of a tube which is constricted at each end, each constriction carrying a glass turn cock, a long glass tube holding a column of mercury, and a small beaker filled with mercury. Method. — Fill tube m completely with medium ; inocu- late through c, connect the constriction at c' by means of a piece of stout, elastic rubber tubing with glass rod g. Close both turn cocks. Reverse the whole apparatus, fill the glass tube g with mercury; close the end with the finger and return the apparatus to its original position. Fig. 7, standing the open end of the glass tube ^ in a beaker g^S fn b containing mercury. The column of mercury falls to 750 mm., and above it there is an absolute vacuum, the Toricellian vacuum. Now open cock c', instantly the medium descends and above it there is formed an absolute vacuum. Close cock c' and paraffin it. Remove glass tube g and put the apparatus into the incubator. The diameter and length of glass tube g govern the capacity of the vacuum. The gases produced by bacterial activity can evolve without endangering the apparatus and can be examined without interference by other gases. For the above method any flask or test tube may be used. When filled with inoculated medium it is closed with an air tight, mono-perforated rubber stopper. The perfo- ration carries a snugly fitting glass tube which is connected with a long, heavy glass tube by means of a short piece of rubber tubing, carrying a firm clamp, which serves in the place of the glass turn cock. The manipulation is the same as with the previous apparatus. While the few devices and methods described in this category appear to be the only ones specially designed for vacuum cultures, there exist a large number of apparatus which may be and have been used for this purpose. Thus most of the devices invented for anaerobic cultures in an atmosphere of inert gases may easily be adapted for vacuum cultures. For their description the reader is referred to their respective classes. Fig. 7. 1. I wish to express my appreciation to Dr. V. A. Moore and Dr. E. M. Chamot for valu- able suggestions in this work. 2. The entire bibliography will appear at the end of this seiies. Digitized by Microsoft® IKeprlnted from the JOURNAL OP APPLIED MICROSCOPY AND LABORATORY METHODS, Rochester, N. Y., Vol. V, No. 4.] II. REPLACEMENT OF AIR BY INERT GASES. Page 1741 This principle has been employed by Pasteur in the study of his Vibrion butyricus as early as 1861. He cultivated this organism in an atmosphere o£ hydrogen and carbonic acid. Since then many methods of this type have been introduced and put in practice. Of the various gases that have found applica- tion as a means of replacing, the air, hydrogen proved to be, everything consid- ered, the most satisfactory. Hydrogen is produced most conveniently by means of the Kipp Generator, dilute hydrochloric acid and metallic zinc being used. The hydrogen thus pro- duced is freed from traces of AsHg, SHg and PHj that may be present, by passing the gas through concentrated KMnO^, from acids that may have been carried over from the generator by passing it through concentrated KOH, from traces of oxygen by passing it through an alkaline solution of pyrogallol, and from water by passing it through dry CaCl2 or concentrated H2SO4. If chemically pure zinc is used, washing is not necessary. When a union is to be made between two glass tubes by means of rubber tubing the ends of the glass tubes should meet. This avoids the direct exposure of large surfaces of rubber tubing to the action of hydrogen and prevents the diffusion of hydrogen through the porous walls of the rubber tubing. In any case it is advisable to vaselinate the rubber . tubing. If the apparatus is sealed by means of glass turn-cocks, only cocks with diagonal openings should be used. They afford a perfect seal. High pressure in the apparatus tends to seriously affect the cultures and should therefore' be avoided. Slight over-pressure which will not materially dis- turb growth is desirable as a means of preventing any possible diffusion of gases. Where convenient, it is well, instead of filling the apparatus with hydro- gen and sealing it hermetically, to pass the hydrogen through the apparatus con- tinually during the whole period of cultivation. In this case the hydrogen, upon leaving the culture apparatus, is conducted through a doubly perforated rubber stopper to the bottom of a wash bottle containing distilled water. The water forms a hermetical seal, preventing the air from entering the apparatus in case the gas pressure should diminish. _ The purity of the gas in the culture apparatus is tested by filling a test tube with the escaping hydrogen and applying a match to it. If the gas burns with explosion, the apparatus still contains some atmospheric oxygen, a quiet flame indicates pure or nearly pure hydrogen. A. CULTURES IN TUBES AND FLASKS. Hauser and Liborius (18^6) introduced the apparatus shown in Fig. 8. A short distance above the surface of the medium the test tube carries a lateral Digitized by Microsoft® Fig. 8. [Reprinted from the JOURNAL OF APPLIED MICROSCOPY AND LABORATORY METHODS, Rochester, N. Y., Vol. V, No. 4.] Page 1742 _ j^ljg^ constricted near its union with the test tube, and cotton-plugged at its outer opening. The test tube is also constricted below the lower end of the cotton plug. Method. — Introduce the medium into the test tube by means of the drawn-out funnel (f), replace the cotton plug and sterilize as usual. Inoculate the medium and connect the end (b) of the lateral tube with the Kipp generator. Pass gas through for fifteen to thirty minutes. In case gelatin or agar are used stand the test tube in a water bath at 40°C. while the gas is introduced. Now seal first the test tube and then the lateral at their re- spective constrictions in the flame. Similar ap- paratus have been constructed by Exner, Buchner and Roux. For agar, blood serum, and potato slant cultures Liborius arranges the medium so that the slanted surface is opposite the lateral tube. Hueppe closes the test tube or flask (Fig. 9) containing the inoculated medium with a doubly perforated rubber stopper. In one perforation rests a glass tube (L) which reaches to the bottom of the flask, the other holds the glass tube (A) containing at its lower curve a small amount of mercury. Method. — Introduce hydrogen at (L).; the air is forced out through (A). Hav- ing passed hydrogen through the tube for from ten to twenty minutes, seal the glass tube (L) in the flame. The iriercury in tube A serves as indicator of the changes in pressure, which may take place as the result of gas production by the growing bacteria in the tube or flask. If this indicator is not desired, a short glass tube is used in the place of tube (A) and when all the air is replaced by hydrogen the glass tube is sealed in the flame. Fraenkel's method (1888) : Into a test tube (Fig. 10) containing liquified inoculated gelatin or agar, insert a doubly perforated well fitting rubber stopper carrying two glass tubes ; one reaching to the bottom of the test tube, the other to- the lower surface of the stopper. Cover the top of the rubber stopper and test tube with an air- tight layer of paraffin or sealing wax. In- troduce hydrogen through the long glass tube. When all the air is replaced by the gas, seal first the exit and then the entrance tube over the flame, and roll the test tube until the medium is congealed. For agar stick and stab cultures, Blucher (1890) recommends the follow- ing method : Digitized by Microsoft® Fig. 9. Fig. 10. LKeprlntea trom the JOURNAL OF APPLIED MICROSCOPY AKD LABORATORY METHODS, Rochester, N. Y., Vol. V, No. 4.) Page 1743 Reverse the inoculated test tube (see Fig. 11), remove the cotton plug ; with the open end downward dip the test tube into a beaker containing a solution of diluted glycerin (equal parts glycerin and water). By means of glass tube (g) introduce hydrogen into the test tube. In about five minutes all the air is replaced by the gas, the generator is disconnected, and the beaker containing the test tube is put into the incubator. Hesse (1890) modified Blucher's methods by inverting the inoculated test tube into mercury instead of glycerin. Fuchs (1890) rejects the condensation water from slanted blood serum tubes, inoculates the slanted surface, reverses the tube and introduces hydrogen for about five minutes. Fig. 11. Without changing the position of the test tube he then inserts a tightly fitting, sterilized rubber stopper and seals hermetically by dipping the sealed part of the tube into liquid paraffin. Ogata (1892) used a method very similar to that of Liborius. Instead of in- troducing the gas through the lateral tube as Liborius did, Ogata does away with the lateral tube and conducts the gas down into the medium by means of a capil- lary glass tube running through the cotton plug of the test tube down to the bot- tom of it (see Fig. 12). The air is slowly forced out of the tube in form of gas- bubbles, some of which collapse, others form foam. As soon as the foam has passed up through the narrow part of the tube, the capillary tube is removed and while there is still foam in the upper portion of the tube the constriction is sealed over the flame. Heim (1892), who claims to be the inventor of this method, recommends that, except when Esmarch roll cultures are made, the test tube should be sealed without removing the capillary tube ; that the inoculation should be made be- fore the tube is constricted, and that the constriction should not become wet, as the glass is liable to crack during the operation of sealing in the flame. For liquid medium Roth (1893) used an upright flask (see Fig. 13). In the lower part of the neck rests a cotton plug, which is attached to a wire running out through the flask. Through the cotton plug passes one end of a glass tube, the other being conducted into a cup of glycerin. Method.— ¥\!i\. the flask about half full with liquid me- dium, press the cotton plug (c) well down into the neck, push the glass tube down near the bottom of the flask and connect the other end of it with the gas generator. When all the air is replaced by the gas, and before disconnecting the apparatus from the generator, raise the glass tube out of the medium as shown in Fig. 13, immerse the other end of Fig. 12. the glass tube in glycerin, fill the neck above the cotton Digitized by Microsoft® Fig. 13. [Reprinted from the JOURNAL OP APPLIED MICROSCOPY AND LABORATORY METHODS, Rochester, N. Y., Vol. V, No. 4.] Page 1744 . ^,.=_ — plug with paraffin and disconnect at (e). The flask is opened by heating the neck in the flame and raising the plug by means of the attached wire. In case of very large flasks Roth covers the cotton plug with a piece of rubber containing an opening for the introduction of the glass tube before the apparatus is con- nected with the generator. This will increase the pressure from within and prevent any possible entrance of air during the introduction of gas. Novy (1893) devised an apparatus which allows a large number of tube cultures to be made simultaneously. The apparatus as shown in Fig. 14 con- sists of a cylinder 20 x 10 cm. (not counting the neck). The neck carries two lateral tubes. Into the neck of the cylinder is fitted a glass stopper with ground surface. The glass stopper also carries two perforations on opposite sides corresponding to those in the neck. From the inside of the glass stopper one of the perforations is connected with a glass tube, reaching nearly to the bottom of the cylinder. If a gas heavier than the air is introduced, the glass stopper should be turned so as to conduct the gas through the tube in .the interior to the bottom of the cylinder, thus allowing air to escape on top through the other lateral tube. In case of a gas lighter than air, as for in- stance hydrogen, it should enter the cylinder on top, forcing the air out through the long tube reaching to the bottom of the cylinder. Method. — Inoculate forty to fifty ordinary culture tubes (12 to 15 cm. long). Insert loose cotton plugs and cut them off at the end of the tubes. With a pair of long tongs place the tubes in the cylinder, the bot- tom of which is covered with cotton, cover the surface of the glass stopper with paraffin or vaseline and insert the latter in its place, care being taken that the perforations in the stopper correspond with those in the neck of the cylinder. Connect the apparatus, as above directed, with the gas generator and lead the exit tube into a wash bottle containing water. After passing the gas through for from one to two hours, carefully turn the glass stopper an angle of 90°, disconnect the cylinder and put it aside for the development of the bacteria. If instead of replacement by gas the cylinder is evacuated, it becomes impossible to turn the glass stopper. In this case ^c^^^^^ral^be^c^ges a small glass turn-cock for Fig. 14. Fig. 15. [Reprinted from the JOURNAL OF APPLIED MICROSCOPY AND LABORATORY METHODS, Rochester, N. Y., Vol. V, No. 4.] Page 1746 the purpose of sealing the apparatus when exhaustion is complete. ffewett (1894) recommends for bouillon cultures the use of an yeast flask of 90 c.c. capacity. The flask is closed by a mbnoperf orated, well fitting rubber stopper through which a glass tube passes to the bottom of the flask. The part of the tube extending above the stopper is cotton plugged. A lateral tube projects from the side of the neck as shown in Fig. 15. The lateral tube is also plugged with absorbent cotton. It leads into a cup containing mercury, the latter forming a valve. The surrounding air cannot enter, while the interior air and the gases formed by bacterial activity have free exit. Method. — Fill the flask about two-thirds full with glucose bouillon, sterilize, cool and inoculate the medium. Introduce hydrogen through the glass tube in the rubber stopper for one hour, before disconnecting the generator dip the end of the lateral tube into the cup containing mercury, and seal tube (b) above the • rubber stopper in the flame. Lubinski (1894) constructed two forms of apparatus. One of these resem- bles that of Novy so closely that it need not be described here. The second apparatus is illustrated in Fig. 16. It is closed with a ground glass stopper. Immediately below the neck the cylinder carries at two diametrically opposed places tubes (t) and (t,), ending in glass bulbs. Both bulbs are partly filled with liquid paraffin or vaseline. In order to prevent the liquid in bulb (t) from passing over into the apparatus, bulb (t) is separated from the cylinder by a second bulb. The gas is introduced through bulb (t), ; bulb (t) serves as exit for the air. As in Novy's apparatus the entrance of gas and exit of air take place at different heights. The manipulation is very similar to that of Novy. Jacobitz (1901) successfully made agar slant cultures in nitrogen atmosphere. He used Fraenkel's tube. Into the boiling hot agar he introduced a current of nitrogen purified by running through concentrated sulphuric acid, through alka- line pyrogallol and through potassium hydroxide. He then lays the tube on an ice tray to .slant the agar, continuing the current of nitrogen until the agar is congealed. After inoculating in the usual way more nitrogen is introduced. Finally the entrance and exit gas tubes are sealed hermetically in the flame. B. PLATE CULTURES. Kitasato (1889) used a flattened receptacle about 2 cm. thick (see Fig. 17) for the isolation of tetanus. The manipulations of the apparatus resemble those employed in the case of Liborius tubes. The two openings are plugged with cotton and the apparatus is sterilized. Then the surface tube (b) is constricted in the middle. With a well drawn out funnel about 20 c. c. of the liquified inoculated medium are poured Fig. 16. Digitized by Microsoft® Page 1746 Fig. 17. wire baskets. [Reprinted from the JOURNAL OF APPLIED MICROSCOPY AND LABORATORY METHODS, Roeliester, N. Y., Vol. V, No. i.] through the opening at (a) into the receptacle, then (a) is also constricted. Having passed hydrogen through until all the air has escaped, the two ends, first the exit, then the entrance, are sealed in the flame. JioiA (1893) recommended a similar device. His ap- paratus is illustrated in Fig. 18. It is used for solid medium. Method. — Plug both openings with absorbent cotton and sterilize. The plug at (a) carries a corkscrew, that at (b) is attached to a fine copper wire. Introduce about 8 c. c. of the liquified medium (gelatin or agar) and sterilize on three succes- sive days. For this purpose the flasks may be stood upright in After inoculating in the usual way, let the medium congeal. Then push by means of the cork screw the plug at (a) down far enough to touch the medium ; introduce hydrogen at (b) by connecting (b) by means of rubber tubing with a generator. The rubber tubing carries a clamp. In order to prevent the mixing of air and gas as much as possible, it is best to incline the apparatus so that the neck points downward. When all the air is replaced by the inert gas pour a little melted paraffin on the cotton plug in the neck. When congealed dip tube (b) into liquid paraffin and remove the rubber tubing. " The plug in tube (b) is thus saturated and the tube filled with paraffin. This parslifin seal proved very satis- factory. In order to get access to the grown colonies, warm the neck and pull ^ the cotton plug out by means of the corkscrew. ^^ ^^^^ = - '^^^^~ ^^~^^^^ '^ ^^ For making cultures in the field Roth used a similar apparatus (see Fig. 19). In order to avoid breakage, the small tube (b), shown in Fig. 18, is discarded and the hydrogen is intro- duced in the laboratory. For this purpose a small cotton plugged sterile metal tube is inserted in the apparatus. When the air is all driven out, the neck is filled with paraffin and the metal tube carefully removed by means of a copper wire which had previously been attached to it. Blucher (1890) recommends the apparatus illustrated in Fig. 20. A funnel shaped bell jar with a cotton plugged opening (D) and weighted down with lead (F) rests in a glass bowl (A). The petri dish is kept in its place by means of a spring wire ring with three projections reaching to the walls of the bowl. Method. — Pour the inoculated me- dium into the open petri dish (B). Place the bell jar over it and pour into the glass bowl diluted glycerin (1 part glycerin, 3 parts water) until the in- terior is completely separated from the exterior. Introduce hydrogen through the opening at (D). The air escapes through the glycerin in bubbles Fig. 18. [Reprinted from tlie JOURNAL OF APPLIED MICROSCOPY, AND LABORATORY METHODS, Rochester, N. Y., Vol. V, No. 4.] Page 1747 about 10 minutes, close the rubber tubing at (D) by means of a clamp, cut the tubing about two cm. above the clamp and, fill the end with glycerin. The freedom of the apparatus from oxygen can be tested as follows : Bring a burning match close to the glycerin in the bowl where the bubbles escape. If the latter burn regularly and without explosion, the apparatus may be considered oxygen-free. In order to ob- tain access to the petri dish when the culture has developed, carefully and slowly raise the bell jar on one side, allowing small bubbles of air to enter. This will prevent the glycerin from spattering into the culture. Botkin's apparatus (Fig. 21) is a modification of that of Blucher. It contains a glass dish (D) 20 to 25 cm. in diameter (much the same as those used for potato cultures). In the dish (D) stands a wire support for the petri dishes, which are covered by bell jar (B). The latter has a diameter about three cm. smaller than dish (D). It does not touch the bottom of (D) directly, but it rests on a cross band of lead (L) one cm. in thickness. U tube (U) is a thin rubber tube ; its lumen contains a fine, soft, and flexible copper wire. Opposite tube (U) there is another similarly constructed rubber tube (F) leading from the Fig. 20 Fig. 21. interior of the bell jar into a wash bottle (W) containing water and closed by a doubly perforated rubber stopper ; the second perforation carries a glass tube continuing into a rubber tube and closed by a clamp (C). Method. — Disinfect the interior of the apparatus by washing with a solution of sublimate and drying with alcohol and ether. Sterilize the wire support in the flame. Prepare the culture plates in the ordinary way and place them on the wire stand. Pour a layer, three cm. high, of paraflin liquidum, preferably Buch- ner's mixture (1 part glycerin to 3 parts water) into dish (D). Insert the U tubes Digitized by Microsoft® Fig. 22. [Reprinted from the JOURNAL OF APPLIED MICROSCOPY AND LABORATORY METHODS, Rochester, N. Y., Vol. V, No. 4.] Page 1748 ^ in their respective places and cover with the . bell jar. Introduce hydrogen through tube (U). The air escapes in bubbles through the glycerin in dish (D). In about ten minutes open clamp (C), allowing the air in wash bottle (F) to escape. After two more minutes light the gas escaping at (C). If the apparatus contains pure hydrogen the escaping gas will burn with a quiet, even flame, other- wise with a crackling noise. Being assured of the complete replacement of air by hydrogen, carefully withdraw the U tubes (U) and (F) from the apparatus. In order not to disturb the glycerin seal by transportation, Botkin recommends placing the apparatus in the incubator before hydrogein is introduced. Hesse (1892) introduced the type of apparatus shown in Fig. 22. It con- sists of the following parts : a. A cast iron plate 20 cm. in diameter, with a channel (2 cm. wide and 3 cm. deep) at its periphery ; on one side the channel is 2^ cm. deeper than at the other. It is filled with mercury. The plate is smeared with shellac. b. A bell jar fitting into the channel and floating on the mercury. c. Two U tubes (c) and (c,), with extensions for the entrance and exit of gas and air respectively. Tube (c,) contains at (w) a wire gauze to ensure a safe test of the escaping gas by burning. Method. — Cover the center part of plate (a) with a blotting paper for the pur- pose of absorbing moisture. Upon this place the inoculated, loosely covered plate cultures, invert the bell jar over them and insert the U tubes (c) and (c,) in their proper places. Connect (c) with the Kipp generator. The purity of the escaping gas is tested by applying a burning match to the capillary end of the tube (c,). Baginsiky constructed^an apparatus (Fig. 23) that appears to be simple in construction and easy to manipulate. It consists of a large metal plate, the circumference of which is covered with a thick rubber ring. A bell jar is inverted over the plate and rests on the layer of rubber. Over the bell jar is placed a metal plate similar to that which forms the bot- tom of the apparatus. The bottom part contains four projections in which are hinged n?etal rods, the outer ends of these rods fit into similaJ- projections in the cover plate. By means of these four metal Ejcs Digitized by Microsoft® Fig. 23. Fig. 24. [Reprinted ftom the JOURNAL OP APPLIED MICROSCOPY AND LABORATORY METHODS, Rochester, N. Y., Vol. V, No. 4.] Page 1719 rods the upper and the lower metal plates are tightly pressed against the bell jar, closing the apparatus hermetically. On opposite sides the bell jar contains small lateral tubes by means of which hydrogen is introduced and air driven out. Method. — Place the inoculated petri dishes upon the bottom plate of the apparatus. Invert the bell jar and seal the apparatus by screwing the upper and lower plates firmly against the bell jar. Intro- duce hydrogen at the upper lateral tube, the air will escape through the other. When all the air is driven out, which is determined by the hydrogen tube test, seal first tlie exit, then the entrance of gas and place the apparatus in the incubator. A very satisfactory way is also to run the hydrogen through continuously until the cultures are grown. This apparatus is also well suited for a large number of tube cultures in hydrogen atmosphere. Novy (1893) recommends his apparatus (Fig. 6), designed for plate cultures in a vacuum, also for plate cultures in hydrogen atmosphere. In this case hydrogen is introduced at one end of the glass cock (x-y) and the air escapes at the other. In addition he modified his apparatus for tube cultures (Fig. 14) so that plates can be placed in it (see Fig. 24). Its manipulation is the same as that for the tube cultures. Kedrowski's apparatus (Fig. 25) consists of a deep glass plate (C) with cover (D). On the sides at diametrically opposite points plate and cover are perforated for the entrance of gas and the exit of air. Method. — Into the sterile plate (C) put an open petri dish containing the in- oculated medium. Coat the inside of the rim of cover (D) with vaseline. Place D over C, so that the perforations in plate and cover meet perfectly. Introduce hydrogen, and when the gas has replaced all the air, then turn the cover 90° and put the apparatus into the incubator. Gabritschewsky's plate (Fig. 26) consists of a sub-plate with a rim at its per- iphery. The outer end of the rim is so constructed as to expose a broad horizontal surface for the cover to rest on. This surface is perfor- ated at two diametrically opposite points corresponding to two similar perforations in the cover. Method. — Pour the inoculated medium into th'e central part (c) of the sub-plate. Cover with a paraffined ground glass cover so that the holes in the rim cor- Digitized by Microsoft® [Reprinted fl-om the JOURNAL OF APPLIED MICROSCOPY AND LABORATORY METHODS, Rochester, N. Y., VoL V, No. 4 ] Page 1750 respond with those in the cover. Introduce hydrogen and when all the air is re- placed, carefully turn the cover 90 degrees, sealing hermetically. In addition Gabritschewsky recommends the use of an alkaline solution of pyrogallol which is poured into the rim before the gas is introduced. A similar plate (Fig. 27) was constructed by Kamen. Its method of manipulation is ^limDHmmiimiimimiiiinnmmmmmjg Fig. exactly the same as that of Gabritschewsky. In case of Beckys plate (Fig. 28) a common petri dish is used. It is covered with a plate carrying two lateral tubes for the introduction of gas and exit of aiir. The periphery of the cover is so shaped as to form a small reservoir which may be filled with water in case of cultures that require a long period of incubation. By means of short pieces of stout rubber tubing the laterals are connected with short constricted glass tubes which are sealed in the flame when the plate is oxygen free. Method. — Pour the inoculated medium into the sterile plate and put the cover in its place. When the medium is congealed fill the rim (X) between Fig. 27. Fig. Fig. 28. the plate and the cover with liquified paraffin. When this is solidified replace the air by gas and seal the constricted tubes in the flame. Areris plate (Fig. 29) consists of an ordinary petri dish carrying lateral tubes which open into the interior of the plate. The space between the rim of the cover and that of the plate is filled with liquified paraffin. When the air is expelled by gas the laterals are sealed in the flame. EpsteirCs plate is a modification of that of Aren. Instead of the lateral tubes of glass similar tubes of firm rubber are used. The rubber tubes are a part of a solid rubber band that covers the per- iphery of the plate. When the hydrogen has replaced the air the plate is sealed by pushing well fitting glass rods into the lateral rubber tubes. Fig. 29. Digitized by Microsoft® [Eeprintea from the JOURNAL OF APPLIED MICROSCOPY AND LABORATORY METHODS, Rooliester, N. Y., Vol. V, No. 4.] III. ABSORPTION OF ATMOSPHERIC OXYGEN. Page 1751 Xo this Category belong all those methods, in which advantage is taken of the oxygen-absorbing power of some chemical agents. The adoption of this principle for the purpose of bringing about anaerobic conditions is of more recent date than that of exhaustion and of replacement by inert gases. In 1878 Gunning used a mixture of a ferro salt with an excess of sodium hydroxide. He also recommended a solution of glucose containing indigo car- mine and scTdium hydroxid. In 1880 Nenki found that anaerobic bacteria grow well in an atmosphere from which the oxygen has been absorbed by an alkaline solution of pyrogallic acid. Practical application of this principle, however, •was not made until 1888, when Buchner invented the method which now carries his name. Buchner's Method. — Use two test tubes of different sizes (see Fig. 30), drop into ■ the large tube a small wire support on which the small tube is subsequently placed. The small test tube con- tains the inoculated medium and is closed with a loose cotton plug. Put into the large test tube one gram of dry pyrogallic acid and then ten c. c. of a one-tenth solution of potassium hydroxid (1 part Liquor Kali caust. to 10 parts of water). Then quickly lower the small culture tube and close the latter hermetically with a new, elastic, well fitting, paraffined rubber stopper. §hake well. Buchner observed that the above stated amount of pyro- gallic acid and potassium hydroxid will completely absorb the oxygen in a tube with a cubic content of 100 c. c. in 24 hours at incubator temperature. At lower temperatures, as for instance in the refrigerator, the absorption of oxygen is much slower. Fre- quent shaking hastens the absorption. For plate cultures Buchner incloses the plates filled with the inoculated medium under a hermetically sealed bell jar and uses larger amounts of pyrogallic acid and potassium hydroxid. Babes and Puscarin in 1890 modified Buchner's method by putting the inoc- ulated culture tubes into a Fresinius desiccator containing a large amount of pyrogallic acid and potassium hydroxid. After closing the desiccator hermetic- ally with a vaselined ground glass plate, the apparatus is put into, the incubator. This method enables the experimenter to cultivate numerous tube cultures sim- ultaneously and under identical conditions. The authors obtained the best re- sults when they used media containing 2 per cent, glucose. Blucher (1890) recommends the following method for plate cultures : Put a petri dish 6 cm. in diameter, minus cover, into a glass saucer 10 cm. in diameter and 4 cm. high. The upper edge of the saucer is ground convexly and it fits exactly into a cover with a concave rim. Into the outer saucer pyro- gallic acid and potassium hydroxid are placed and' the cover, the concave panel of which has been well vaselinated, is inverted over the saucer. The decrease Digitized by Microsoft® Fig. 30. Fig. 31. [Reprinted from the JOURNAL OF APPLIED MICROSCOPY AND LABORATORY METHODS, Rochester, N. Y., Vol. V, No. 4.] 1752 of pressure within, due to the absorption of the oxygen in the apparatus, causes the over-pressure from without to force the cover tightly over the saucer, form- ing a hermetical seal. In order to reopen the apparatus the cover must be turned before it can be taken off. TrambusWs Method. — The apparatus used by this investigator is illustrated in Fig. 31. It consists of two parts : a cone-shaped flask (A) containing the medium, and a cylinder (B) which is screwed into (A) by means of a fine thread. In the interior of (B) there is a small tube which opens into (A). Method. — Pour the inoculated medium, gelatin or agar, into flask (A) and distribute it over the surface of the bottom uniformly, the same as in case of a petri dish. Screw (B) into (A), remove the stopper and fill (B) with an alkaline solution of pyrogallol (2 grams of dry pyrogallic acid plus 15 c. c. of 10 per cent, solution of KOH), care being taken that the reagent does not enter the small tube. Replace the stopper which has previously been covered with paraffin or vaseline and remove the apparatus to the incubator. Arens in 1894 used a common small desiccator with a ground glass cover for anaerobic plate cultures. Method. — Fill the desiccator partly with quartz sand, to this add an optional amount of dry pyrogallic acid, leaving just room for one or two small petri dishes. Use deep ■ layer of agar in the plates. Before putting the plates into the apparatus pour a considerable amount of a 10 per cent, solution of caustic potash all over the surface of the sand and acid. Then place the petri dish, minus cover, upon the sand and close the desiccator with a well vaselined ground glass cover by rotary motion. Lubinsky in 1894 modified Buchner's method for tube cultures. Instead of putting the culture tube into a large test tube, Lubinsky placed it in a glass cylinder (Fig. 32) 12-15 cm. high and 3-4 cm. in diameter. Into this cylinder he pours the alkaline solution of pyrogallic acid ; then he pushes a snugly fitting, manifold perforated cork about half way down into the cylinder. In the center of the cork there is a large perforation in which the culture tube is inserted. The cylinder is closed with a glass stopper of ground glass and the edges are sealed with paraffin. When sealed he shakes the apparatus vigorously for 2 to 3 minutes to hasten the process of absorption. Fig. 32. Digitized by Microsoft® [Reprinted from the JOURNAL OP APPLIED MICROSCOPY AND LABORATORY METHODS, Rochester, N. Y., Vol. V, No. 4.] Page 175S Novy recommended his apparatus (Fig. 24) designed for cultivating anaerobes in a hydrogen atmosphere, also for the pyrogallic acid method. Ucke (1898) manipulated Novy's apparatus (Fig. 24) as follows: Put 10 grams of dry pyrogallic acid into a small beaker, float this beaker in a larger one (120 c. c. capacity) containing 50 c. c. of a 20 per cent, solution of potassium hydroxid and stand the two beakers in the apparatus, so that the glass tube reaching from the glass stopper nearly to the bottom extends into the small beaker. At the last moment, when the apparatus is "ready to be put into the incubator, introduce 50 c. c. of water through the long glass tube and turn the glass stopper 90°. This causes the small beaker to sink and the solution of potassium hydroxid mixes with the pyrogallol. By this manipulation the pyrogallic acid does not gel access to the alkali until the apparatus is sealed up, hence it is a mearis of economizing the oxygen absorbing power of the pyrogallol. Wright (1901) recommends the following modification : Into a medium-sized test tube (see Fig. 33) containing the in- oculated culture medium, push a sterile cotton plug well down. By means of a pipette pour J^ c. c. of a saturated solution of pyrogallol and 1 c. c. of a 50 per cent, solution of sodium hydroxid upon the cotton plug. Seal the tube with a well fitting rubber stopper with as little delay as possible. The quantity of the reagents added is so small that there is no danger of its running down along the side of the tube into the culture medium. According to Wright this method can be used successfully for all kinds of flasks and tubes. He lays stress on the fact that the nutrient medium should be freshly boiled and of alkaline reaction. Fig. 33. Slupski (1901) uses the following modification for plate cultures : His appa- ratus (Fig. 34) consists of a bell jar 15 cm. in diameter and 5 cm. high, ending at the bottom in a ground glass rim 1 J^ cm. in width. The bell jar is placed in a glass dish about 10 cm. high. On the bottom of this glass dish rests a double plate. The outer plate (b) contains water and the inner (a) is designed for pyrogallol. Over the double plate rests a tripod which supports the open petri dish. ' Method. — Pour the inoculated culture medium into the open petri dish. Fill plate (b) with water and heap plate (a) with dry pyrogallic acid (25 grams). To this add 50 c. c. of warm distilled water. Replace tripod. Throw two pieces of KOH (14 gms.) into the pyrogallol. Quickly put a blotter and black paper on top of the tripod, upon which place the open petri dish, invert bell jar (X) into glass ^ish (Y) and seal with a thin layer of pafaifin. Then pour hot paraffin three to four cm. deep into dish (Y) and when cooled and congealed pour some liquid paraffin on top. Place the apparatus for about 50 hours in the re- frigerator at a temperature of 5-6°C. in order to prevent any growth while the absorption of oxygen is still in process. Then put it in the incubator. Hammerl (1901) uses a mat of heavy cardboard, felt, or cellulose which he Digitized by Microsoft® [Reprinted from the JOURNAL OP APPLIED MICROSCOPY AND LABORATORY METHODS, Rochester, N. Y., Vol. V, No. 4.] Page 1754 saturates with a concentrated solution of pyrogallic acid. This absorbing mate- rial he fits into the cover of a deep petri dish. Method. — Weigh 4-5 gms. of dry pyrogallic acid into a small beaker, add a 50 per cent, solution of potassium hydroxid, drop by drop, and shake until the Fig. 34. pyrogallol is perfectly wet. In a few minutes it will be dissolved. The less potassium hydroxid used the greater is the absorbing power of the solution. Drop the solution of alkaline pyrogallol upon the mat of cardboard, felt or cel- lulose. Do not saturate the absorbing material completely, otherwise some of the reagent might run down into the medium. Invert the cover containing the absorbed reagent over the culture plate, which has previously been filled with the inoculated medium. Seal the space between the rim of the cover and that of the plate with a mixture of white wax (12 parts) and beef tallow (100 parts) and finally cover the seal with a firm and well fitting rubber band. For single tube cultures Smith* uses the following de- vice : Into one end of a large U tube insert a small test tube containing the inoculated culture medium. Invert the other end over a small slender flask or vial containing a strongly alkaline solution of pyrogallol and standing the ends of the tube in a dish containing oil. The writer modifies Smith's method as shown in Fig. 35. Into one end of a large U tube place about three grams of dry pyrogallic acid and three grams of sodium hydrate. Close this end with a rubber stopper and pour about 15 c. c. of water into the other end (b) holding the U tube so that the water all escapes into the branch (a) containing the reagents. Now insert in the second Ijranch .(b) a small test tube (c) containing the inoculated medium and a loose cotton plug. Close this end of the U tube with a rubber stopper and stand the U tube in a beaker (d) containing mercury or glycerin. Good results were obtained by this method. kQ_0^ Fig. *The method of Dr. Erwin F. Smith, Department of Agriculture, Bureau of Plant Industry, Washington, D. C, has nS;gi/§fzelSb(byt)MW*»fiO/?® [Reprinted from the JOURNAL OP APPLIED MICROSCOPY AND LABORATORY METHODS, Rochester, N. Y., Vol. V, No. 4.] Page 1755 Nikirforoff introduced a method by which anaerobes can be cultivated in a hanging dropj Method. — Use a slide which carries a ground glass ring, just as for making an ordinary hanging drop preparation. Cover the ground edge of the ring which is used for the support of the cover glass with vaseline. Place the cover glass with the hanging drop on the ring, so that one side of the ring is not covered. Insert here a small drop of pyrogallic acid. Move the cover glass into its proper place, that is, so that it covers the ring completely. The drop of pyrogallol now spreads over the whole periphery 'between the cover glass and ring. Now carefully move the cover glass in the opposite direc- tion and put a drop of potassium hydroxid upon the thus un- covered part of the ring. Replace the cover glass and make it fast. The two drops will mix and the absorption of oxygen takes place. Instead of applying the alkaline pyrogallol on the surface of the ring, it may be put on the bottom of the object carrier in the center of the ring. Later, Nikirforoff and Braatz devised special object carriers in which the hanging drop may be examined in a hydrogen atmosphere. Salomonson (1889) suggests the use of aerobic bacteria as the agents for the absorption of oxygen. He uses two test tubes (Fig. 36). The smaller inner tube (a) contains the culture medium inoculated with the anaerobic species. The larger outer tube (b) with constricted neck and cotton plug holds a bouillon culture of one or more strictly aerobic species. As soon as both tubes are inoculated with their respective organisms the outer tube (b) is sealed at (c). Besides the various apparatus described in this category, most of the apparatus for plate cultures referred to under Classes I and II may also be used for the pyrogallol method. Fig. 36. IV. REDUCTION OF OXYGEN. In order to simplify apparatus and manipulation used for cultivating anaero- bic bacteria, and to be able to make cultures of anaerobes in the presence of air, attempts were made to reduce the oxygen in the medium. Various agents have been used to accomplish this end. A. Mixed cultures. One of the simplest though not altogether satisfactory ways to cultivate anae- robes under aerobic conditions is that of making mixed cultures, i. e., the culture medium containing the anaerobic species sought for is inoculated with one or more strictly aerobic species. While many investigators have used this method successfully, there seems to exist some controversy regarding the specific role which the aerobes play in preparing favorable conditions for the anaerobes in mixed cultures. Pasteur holds that the aerobic bacteria are capable of using up Digitized by Microsoft® [Reprinted from the JOURNAL OF APPLIED MICROSCOPY AND LABORATORY METHODS, Rochester, N. Y., Vol. V, No. 4.] Page 1756 all the oxygen present in the culture medium, and that in this way they make it possible for the anaerobes, to thrive in mixed cultures. Kedrowski attributes this fact to another phenomenon. He holds that the conditions favorable for anae- robic development in mixed cultures are not due to the assimilation of the oxygen present in the nutrient medium by the aerobic species, but that the latter produce substances which form a suitable medium for the anaerobes to grow in. Roux recommends the following method for mixed cultures : Boil a conven- ient quantity of nutrient agar in a cotton plugged test tube and cool rapidly in cold water. Immediately after the agar is solidified inoculate it with the anaero- bic organism by means of a glass needle, then pour a small amount of melted but not too hot nutrient gelatin into the tube. When this is congealed pour a few drops of a bouillon culture of Bacillus subtilis upon the surface of the gelatin. Seal the tube in the flame and place it in the incubator. B. subtilis grows very rapidly upon the surface, forming a tough membrane and using up the oxygen in the tube. In order to obtain material for anaerobic subcultures, the bottom of the test tube is broken, avoiding the mixing of the two cultures. Penzo (1891) used a similar method. He cultivated the bacillus of malignant oedema successfully under aerobic conditions in agar and gelatin containing cul- tures of Bacillus prodigiosus or Proteus vulgaris. Scholtz (1898) observed that anaerobes grow much more vigorously in a medium containing a vigorous culture of an aerobic species. While it is evident that mixed cultures are a simple and generally successful means for obtaining vigorous anaerobic growth, it is equally obvious that this method is unsatisfactory where pure cultures of anaerobic bacteria must be obtained. Therefore, the use of mixed cultures can serve only as an indirect means in studying anaerobic bacteria. B. Chemical reducing agents. Glucose. Librius, in 1886, observed that the addition of glucose to nutrient bouillon, gelatin or agar exerted a favorable influence on the development of anaerobic species. This action has been explained by the fact that glucose in an alkaline solution possesses an oxygen-reducing power. Novy (1893) successfully used and recommends media with the following ingredients for the cultivation of anaerobes : 1. Beef broth containing i^ per cent, sodium chloride, 2 per cent, peptone and 2 per cent, glucose. 2. Beef broth as above plus 2 per cent, gelatin. 3. 1-15 per cent, nutrient gelatin with the same constituents as under I. 4. Ij^ to 2 per cent, agar containing i^ per cent, sodium chloride, 2 per cent, peptone and 2 per cent, glucose. Hewletf'S method : Fill a test tube two-thirds with 2 per cent, glucose agar and steam immediately before inoculation to expel any traces of oxygen that may be present in the nutrient medium. When the agar has set, inoculate well into the depth of it. Carefully warm the tube over the flame, melting the superficial layer of agar and filling the puncture made by the inoculation. Now flame the Digitized by Microsoft® [Reprinted from the JOORNAL OF APPLIED MICROSCOPY AND LABORATORY METHODS, Rochester, N. Y,, Vol. V, No. i.] "^' upper portion of the tube to expel the air and slip a well fitting rubber cap over the hot test tube. Babes and Puscarin (1890) use high layers of two per cent, glucose agar in tubes. After inoculation they expose the tubes to 80°C. for a short time, push the plug down nearly to touch the surface of the medium and then pour liquified paraffin on top of the plug. Ucke (1898) recognizes in glucose a two-edged sword, and attributes the frequent failures in the cultivation of anaerobic bacteria to the use of glucose. Although a more vigorous growth is formed on glucose medium, the spores, if formed at all, are less numerous ; degeneration forms are apt to develop, and the virulence of the organism is lessened. He holds that if vigorous growth and spore producing material shall be obtained, media without sugar should be used. Other reducing agents: Kitasato and Weyl (1890) tested the action of a large number of reducing agents in alkaline media on the development of anae- robic bacteria. Their purpose was Xn find a substance of a greater reducing power than glucose, and which at the same time would not exert a harmful influence on the bacterial development. They demonstrate that the addition to the culture medium of Pyrocatechin CgH4(OH)2, the sodium salt of Amidonaph- tol-monosulphonicacidCijHgOHNHjSOgNa, sodium indigo sulphonate CuHg N202(SOgNa)2, Sodum formate HCOjNa, in quantities of 0.1 per cent, exerted a favorable influence on the anaerobic growth, while other reducing reagents, such as Hydroxylaminehydrochloride NH2(OH)Hcl, Resorcin CjH4(OH)2, Hydroquinone Q.^YLj;<:}Yi.^, Pyrogallol CgH3(OH)3, Acetaldehyde CHgCOH, Benzaldehyde C j H 5COH, phenylhydrazinehydrochloride NH 2 — NH(C ^ H gHcl), etc., etc., either did not affect the culture materially, or exerted a strong poison- ous action on the microorganisms. The authors especially recommend the use of sodium sulphonate (use 0.1 per cent.). This agent is valuable, however, as an indicator of the reducing effect of certain bacteria rather than as a reducing agent in itself. Buchner first recommended litmus to indicate changes of reaction caused by bacteria. Later, Cohen proposed its use as a reduction indicator. This is import- ant, as many bacteria, and especially anaerobic fornis, reduce litmus rapidly to a colorless leucosubstance, which, upon access to oxygen, colors red or blue accord- ing to the reaction changes. Novy observed that the addition of litmus favors to a certain extent the growth of the microorganisms, and that it exerts a protecting influence over the anaerobic bacteria. Cultures of the bacilli of tetanus, black leg and malignant oedema retain their vitality even in liquid media exposed to the air for months if they are colored with litmus. Trenkmann (1898) recommends the addition of a few drops of a ten per cent, solution of sodium sulphide NajS to bouillon; take 20 c. c. of nutrient medium and two drops of a ten per cent, solution of NajS. The author cultivated the bacillus of black leg successfully in such a medium in the presence of air. He admits, however, that NajS gradually decomposes by the action of CO 2 in the atmosphere, forming NajCOj and HjS. As soon as this action takes place the Digitized by Microsoft® [Reprinted from the JOUKNAL OF APPLIED MICROSCOPY AND LABORATORY METHODS, Rochester, N. Y., Vol. V, No. 4.] Page 1758 atmospheric oxygen reenters the medium and checks the growth of the anaerobic organisms. Hammer/ (1901) tested the efficiency "of Trenkmann's Na^S, of KjS and of Ammonium sulph-hydrate NH^SH, as reducing agents. Parallel experiments showed that NH4SH was much more efficient than either NajS or K^S. In the ammonium sulph-hydrate he believes he has found a substance which, without checking the bacterial development, reduces the oxygen in the nutrient medium and thus prepares ideal conditions for anaerobic cultures. Unfortunately the ordinary NH^SH as kept in the laboratory is not fit for this purpose. It is nec- essary that it should be fresh and sterile. To obtain sterile NH^SH the follow- ing procedure is recommended : Fill a glass stoppered bottle of 100 to 150 c. c. capacity completely with sterile water. Replace the glass stopper by a cotton plug and sterilize the apparatus in the steam sterilizer. After sterilization cool it to room temperature and introduce by means of a sterile glass tube, reaching to the bottom of the bottle, a vigorous current of washed H^S for about six minutes. The open end of the bottle is loosely plugged with sterile cotton. Now draw from the sulphurated water accurately measured portions of 10 c. c. into each of 6-8 sterile test tubes ; by means of a pipette drop into the first tube 2 drops, into the second 4 drops, etc., etc., of a 1 per cent, solution of ammonium chloride NH4CI. After vigorous shaking add three drops of a concentrated solu- tion of methylene blue to each tube. The latter operation is done most easily by pouring the 10 c. c. into a tube containing the three drops of methylene blue. Mark the time required for complete decolorization. Generally the optimum amount of ammonium salt lies between 4-8 drops and the minimum time for de- colorization from ^ to 1 minute. Upon finding the optimum amount of NH^Cl a corresponding number of drops of a 1 per cent, solution of NH4CI is added to the sulphurated water in the bottle. Then add to ten parts of the nutrient medium one part of the thus prepared solution. If about three drops of con- centrated methylene blue are added to this medium, the latter decolorizes rapidly and completely in 2-3 minutes. From the above review of the various reducing agents and their comparative efficiency as a means for producing conditions favorable for the development of anaerobic bacteria in the presence of air, it may be seen that none of the agents and substances so far in use are entirely satisfactory. If aerobic species are used for this purpose, it is difficult to obtain the anaerobes in pure cultures with- out the aid of some other method for the cultivation of anaerobic bacteria. If we resort to chemical reducing agents we find that while they may favor anaerobic growth, they may do it ^t the expense of spore formation, and that they may cause the development of degeneration forms, as in the case of glucose, or the reducing substance may have a poisonous effect on the microorganisms as in the dase of Hydroxylaminehydrochloride, or the preparation of the reducing agent in sterile form may be too complicated for practical purposes, as in case of Ammoniumsulph-hydrate, and finally that, no matter how great the reducing power of any one of these chemical reducing agents may be, they are not able to make harmless the atmospheric oxygen, which reenters the medium when the Digitized by Microsoft® [Reprinted from the JOURNAL OF APPLIED MICROSCOPY AND LABORATORY METHODS, Rochester, N. Y., Vol. V, No. 6.1 latter is poured into petri dishes. Generally speaking then, in order to use re- ducing agents successfully, they should be used in connection with some other method for cultivating anaerobic bacteria. V. EXCLUSION OF ATMOSPHERIC OXYGEN BY MEANS OF VARIOUS PHYSI- CAL PRINCIPLES AND MECHANICAL DEVICES. Page 1800 This category embraces all those methods and appliances that do not belong to any of the preceding principles. They are arranged as follows : The atmospheric oxygen is excluded : A. By deep layers of solid medium. B. By layer of oil. C. By layer of parafSn. D. By mica or glass plates. E. By boiling the medium to expel the air and sealing the appar- atus. F. By the use of the fermentation tube and its modifications. G. By inoculating into a hen's egg. A. DEEP LAYERS OF SOLID MEDIUM. Hesse (1885) and Liborius (1886) recommended the following method : Pour into a test tube, Erlenmeyer flask, or deep Petri dish a sufficient quantity of nutrient gelatin, nutrient agar, or blood serum to form a layer of from 5 to 20 cm. deep. Boil the medium for at least five minutes to expel the air, cool down to a temperature of about 40°C., inoculate the medium, distributing the inoculat- ing material well through it. Care must be taken not to shake the tube or flask ; after inoculation cool rapidly by standing the apparatus in cold water until the medium is set. In this way a large number of isolated colonies usually develop, which are either distributed all through the medium, or appear only in the higher zone, or inhabit exclusively the lower layers. The sharply marked distribution of the colonies over the different zones, their size and other characteristics per- mit a fairly accurate estimate of their relative want of oxygen. The inoculation is made with a long, firm platinum wire, which has previously been brought into contact with the culture material, or a long capillary pipette may be used, into which the culture has been introduced by suction. This simple method, which is very effective even in the case of the .strictest anaerobes, is being used success- fully in many laboratories. Often a layer of sterile medium is poured on top of the inoculated layer after the latter has solidified. In order to gain access to the colonies the tube is broken. Sanfelice, 1893, warms the bottom of the tube and shakes the column of agar out into a sterile glass plate, where it can be cut into slices, the colonies can be examined and subplanted. Esmarch recommends the following procedure : Make Esmarch roll cultures in the ordinary way. Stand the tubes containing the roll cultures in ice-cold water and pour liquified gelatin into the cavity of the tube. Upon cooling the Digitized by Microsoft® [Reprinted from the JOURNAL OF APPLIED MICROSCOPY AND LABORATORY METHODS, Rochester, N. Y., Vol. V, No. 5.] Page 1801 gelatin, the inoculated medium is almost entirely protected from the access of the atmosphere. SchiU's modification of the above method : Pour from 10 to 15 c. c. of liquiiied gelatin or agar into a large test tube, inoculate it with the anaerobic organism, insert a narrow sterile test tube into the inoculated medium in the large tube and roll the whole apparatus in ice water or on a cake of ice. When the operation is completed the thin layer of solidified medium in the outer tube is protected from the air by the inner, smaller test tube. Both methods have the advantage over the ordinary Esmarch roll culture that they furnish a support for the thin layer of culture medium, and prevent it from sliding down to the bottom fig. 37. of the tube. B. LAYER OF OIL. This method, illustrated in Fig. 37, and used by Pasteur and Hesse, offers to be of little if any advantage over those described under A. Method. — Pour about 7 c. c. (in case of small tube) and 15 c. c. (in case of large tube) of liquified gelatin or agar into a sterile test tube, boil to expel the air, cool to 40°C., inoculate without shaking the tube, cool rapidly in ice water until the medium is set and pour a layer of sterile oil on the surface of the inoculated medium. This method may also be used for stab cultures (see Fig. 38). Cultures prepared in this way have the disadvantage that the oil which ad- heres everywhere renders them somewhat objectionable. 'TWh ] 1 i - 1, ^ ^^3 M J9 - 1 I:: Fig. 38. C. LAYER OF PARAFFIN. Kitasato was the first to use paraffin as a cover over the inoc- ulated medium. He poured liquified paraffin on the inoculated medium. This method has several disadvantages ; with the paraffin foreign germs may be introduced into the medium, the neck of the culture apparatus is smeared with the paraffin, and the heat of the liquified paraffin may be injurious or even detri- mental to the development of the inoculated organisms. Kasparec (1896) introduced the following method: Use a flask with a long tapering neck as illus- trated in Fig. 39. Blow a small lateral tube, terminating in a bulb, into the neck of the flask, about 1 cm. above (c). Fill the sterile flask with bouillon almost to the neck, then add about 3 c. c. of liquid paraffin and sterilize the whole in the steam sterilizer. The heat expands the bouillon and causes the paraffin to rise in the neck of the flask and to over- flow into the lateral tube, filling the bulb (a), so that after sterilization there re- mains only a very thin layer of paraffin Digitized by Microsoft® IReprliuea from the JOURNAL OF APPLIED MICROSCOPY AND LABORATORY METHODS, Rochester, N. Y., Vol. V, No. 5.] 1802 on tjie surface of the bouillon at (c). During the heating a large portion of the air absorbed by the medium is driven out, and its re-absorption, while the flask is cooling, is prevented by the paraihn. When cool inoculate the bouil- lon by piercing the solid film of paraffin with a platinum needle, loop or capillary glass tube charged with inoculating material. Now liquify the paraffin in the lateral bulb by carefully heating it over the flame and pour the liquified paraffin on the thin film of solid paraffin on the surface of the medium by inclining the flask slightly. Upon hardening this additional layer of paraffin constitutes an almost perfectly air tight cover which becomes even more effective by being pressed into the tapering neck when the culture medium is warmed in the incu- bator. This seal is made tighter yet by the pressure of the gases generated in the culture. When the flask is to be emptied, warm the vessel and incline the flask so that the liquified paraffin will flow over into the lateral bulb. This method is likewise advantageous in the preparation of toxins, as the culture can be easily transferred in a very pure condition to the filter after the warmed portion of the neck of the flask has cooled and the paraffin in the lateral bulb has hardened. Park (1901) modified the above method as follows : Use tubes or flasks con- taining sterile nutrient glucose bouillon. If non-spore-bearing anaerobes are to be cultivated, cool the medium quickly, inoculate and cover the bouillon with a layer of very hot, sterile paraffin. Where absolute exclusion of oxygen is desired sterilize the tubes containing the medium and the layer of paraffin in an autoclav ; this renders the bouillon free from oxygen. Spore-bearing bacteria are inocu- lated through the liquid paraffin before the bouillon is cooled down. In case of bacteria without spores the medium is first cooled down and the inoculation is made by breaking through "the solidified film or by heating and melting the paraf- fin in the gas flame. A pipette charged with the culture can then be carried through the hot paraffin into the cool liquid medium. D. MICA OR GLASS PLATE. Koch's method : Prepare a gelatin plate in the usual way. Before the inoc- ulated gelatin is completely congealed cover it with a thin, sterile blade of mica. The mica blade has the thickness of a thin sheet of writing paper. It must be well flamed, and after cooling so placed upon the semi-liquid gelatin that no air bubbles form beneath it. It must cover the gelatin completely and perfectly. Sanfelice modified Koch's method by using a glass plate instead of a mica plate as a cover. Pour the inoculated gelatin or agar into the sterile cover of a Petri dish and when the medium is nearly congealed press the other half of the Petri dish, bottom downward, gently into the medium. E. EXCLUSION OF AIR BY BOILING THE MEDIUM AND SEALING THE APPARATUS IN THE FLAME. Hufner and Rosenbach use a heavy 250 c. c. flask with flat bottom and taper- ing neck as shown in Fig. 40. Immediately below the tapering neck a lateral tube projects horizontally. At its union with the neck it measures 3 mm. in diameter. At (a) the lateral tube is constricted, then blown out into a small bulb Digitized by Microsoft® Fig. 40. [Reprinted from the JOURNAL OF APPLIED MICROSCOPY AND LABORATORY METHODS, Rochester, N. Y., Vol. V, No. 5.] Page 180S ca c which terminates in a capillary tube 8 cm. in length. The bulb (b) constitutes the reservoir for the inoculating material. Method.— Pour about 20 c. c. of medium into the flask by means of a drawnout pipette. Then taper the neck still more so that it can be sealed easily and quickly. After cooling dip the lateral tube into the inoculating ma- terial and by means of suction at (S) draw the latter up, until it reaches (a).. Seal the outer end of the lateral tube in the flame until a powerful stream of vapor evolves from the narrow opening of the neck. After the steam has escaped for about four minutes seal the tapering neck in the flame. When the medium is cooled down to 40°C., "slightly warm the inoculating material in the reservoir of the lateral tube. By this means it is forced into the flask, where it should be well distributed all through the medium by slightly shaking the flask. Now melt oil the lateral tube at (a) and place the flask in the incubator. " VignaPs Method. Taper an open glass tube (Fig. 41) at one end and plug it with absorbent cotton at the other. When sterilized dip the tapered end of the sterile tube into the inoculated medium (liquified agar or gelatin) and fill the tube by suction at (a) ; when filled seal both ends in the flame. In order to make subcultures from the isolated colonies developed in this tube, cut the tube in two. Houx's pipette cultures : (Fig. 42). The pipette consists of a glass tube drawn into a capillary tube at its lower end and constricted at (r). Method. — Seal the capillary end (B) in the flame, insert a loose cotton plug at end (A) and sterilize the apparatus at 150°C. Break off the seal at (B) and dip the "'*^ capillary tube into sterile, liquified nutrient agar or gelatin which has been boiled immediately before. When the tube is filled up to constriction (r), press the finger tightly over the upper opening (A) and quickly raise the tube into an oblique position. This will prevent the medium from running out. Now seal at (B) and then at (r) in the flame. For inoculation open one end, make a thrust with a fine, infected glass needle, and seal again. Nikiforoff (1890) constructed the capillary tube shown in Fig. 43. The apparatus consists, of a reservoir (a), which is constricted and sealed at one end (b) and drawn out into a capillary tube about 25 cm. long at the other end (c). The capillary y tube is U-shape. Heat the reservoir (a) of the Fig. 41. sterile capillary tube, then dip its open end (c) into Fig. 42. Digitized by Microsoft® Fig. 43. [Reprinted trom the JOURNAL OF APPLIED MICROSCOPY AND LABORATORY METHODS, Rochester, N. Y., Vol. V, No. 5.] Page 1804 _«.c£a_ a test tube containing sterile water. By letting the reservoir cool, a little of the sterile water is sucked up into it. Now draw the capillary tube several times through the flame and invert it in a test tube containing the inoculated medium. The end of the capillary tube should be near but should not touch the surface of the medium. Heat the capillary tube and its reservoir, causing the enclosed water to boil, and when most of the water has escaped from it in form of vapor, im- merse the open end of the capillary tube in the medium in the test tube. Upon cooling a va- cuum is formed in the capillary tube and the in- oculated medium is drawn up and fills the entire capillary tube and the reservoir. Now remove the test tube and seal the capillary tube at its curve in the flame. For inoculation break the seal, introduce a fine capillary tube charged with inoculating material, and then seal the tube in the flame. Van Senus (1890) uses a glass tube about 1 m. in length and 6 mm. in diam- eter (Fig. 44). End (a) is drawn out into a narrow opening and wrapped up in cotton ; end (b) is plugged with cotton. Method. — Pour into a sterile test tube about 20 c. c. of liquified, sterile agar or gelatin and inoculate it. Remove the cotton from end (a) of the glass tube and immerse end (a) in the inoculated medium by pushing it through the cotton plug into the test tube. While the U-shape is being turned upward apply suc- tion at end (b) till the medium reaches the curved part. Then turn the latter down, the liquid will now fill the rest of the tube by itself. Seal end (a) in the flame ; the cotton plug at (b) prevents contamination. For reaching the isolated colonies proceed as follows : Mark the place, where a well isolated colony is located, with concentrated H2SO4 by means of a glass rod. Wash off with sterile water, scratch with file, and break the glass tube. The colony is now ready for examination and sub-plantation. Schmidt (1895) uses a test tube into which a monoperf orated rub- ber stopper is well fitted ; the perforation carries a glass tube reaching to the lower surface of the rubber stopper. The glass tube extends upward about 16 cm. and is then bent into a U-shape as shown in Fig. 45. Method. — Fill the sterile test tube with bouillon up to 5 mm. below the upper edge of the tube, carefully insert the rubber stopper carry- ing the glass tube so that the air escapes through the latter. The stopper is pushed down until the medium reaches the upper end of Fig. 45. the glass tube. If possible prevent the flowing-over into the turned Fig. 44. m Digitized by Microsoft® [Reprinted trom the JOURNALOF APPLIED MICROSCOPY AND LABORATORY METHODS, Rochester, N. Y., Vol. V, No. 5.] Page 1805 down part of the glass tube. If gas bubbles have remained in the test tube force them out by lightly striking at the sides of the tube. If during the sub- sequent sterilization so much of the liquid evaporates that the glass tube has become empty, replace the evaporated medium by fresh, sterile medium. The tubes prepared in this way can be stored away indefinitely without becoming contaminated. In order to inoculate remove the stopper, inoculate the medium in the tube, and replace the stopper carefully so that the bouillon rises into the glass tube. An ingenious and simple device has been invented by Wright (1901), (Figs. 46 and 47). The apparatus con- sists of a system of glass and rubber tubes standing in an ordinary test tube. (A), (Fig. 46), is a glass tube some- what constricted at each end ; (B), (C), and (E) are short pieces of rubber tubing; glass tube (D) carries in its upper extremity a small cotton plug. The test tube con- tains some culture fluid as indicated in Fig. 46. Method. — Arrange the apparatus exactly as shown in Fig. 46. To expel the air from the fluid, boil the culture medium immediately before inoculation over the flame without removing the inner system of tubes. Then cool the apparatus by placing it in cold water and inoculate the liquid medium in the test tube in the usual way. Draw the fluid up into the system of glass and rubber tubes to a level above the rubber tube (C) by suction. Compress rubber tube (E) between the fingers to prevent the down-flow of the fluid, now push downward the system of tubes in such a way as to bend rubber tubes (B) and (C) in the manner shown in Fig. 47. If the test tube and the inner tube system are of suitable size the rubber tubes mentioned will remain in this bent position. The fluid in tube (A) is thus contained in a water tight space sealed by the acute angle of the rubber tubes. When it is desirable to transplant some of the culture the tube system is straightened out, this will allow the fluid in them to flow out into the test tube, where it is accessible to the platinum loop in the usual way. F. THE FERMENTATION TUBE AND ITS MODIFICATIONS. The methods and apparatus belonging to this type deserve special mention- ing owing to their great simplicity and efficiency. Previous to the invention of the fermentation tube Pasteur devised an appar- atus (Fig. 48) which operates on a similar principle. It consists of a flask (a) which contains the liquid nutrient medium. Tube (b) is conducted into a porce- lain dish containing the same medium as the flask, tube (c) serves for the pur- pose of introducing the medium and the culture. It carries a glass turn cock (e) above which it is extended into a short rubber stoppered bulb (f ) which forms the reservoir of the inoculating material. Figs. 46 and 47. Digitized by Microsoft® [Reprintea from the JOURNAL OF APPLIED MIOKOSCOPY AND LABOEATGHY METHODS, Rochester, N. Y., Vol. V, No. 5.] P»ee iso« „f, Mei/iod— Boil ,the medium in flask (a) and in porcelain dish (d) for one-half hour simultaneously to drive the atmospheric oxygen from the medium. The evolving vapor forces the medium out of the flask, but the liquid thus freed from air returns back into the flask. The process repeats itself several times during the boiling. When boiled for 'the required length of time cool apparatus and medium. While cooling cover the medium in the porcelain dish with a layer PiQ 48 °^ sterile oil to prevent a sub- sequent absorption of oxygen from the air. Fill (f ) with the culture material and seal with the rubber stop- per. When apparatus and medium are cool introduce a part of the culture material from reservoir (f ) into the medium by quickly opening and closing turn cock (e). The latter must fit perfectly. Now submerge the outer end of tube (b) in sterile mercury for the purpose of collecting the gases formed by the bac- terial activity, and place the apparatus in the incubator. For a control test the author uses a flask as shown in Fig. 49. It is twice as large as that shown in Fig. 48, and filled only one-half with medium so that the culture is freely exposed to atmospheric oxygen. Smith found the fermentation tube to be an ap- paratus of considerable antiquity and of unknown origin. He says: "In Detmer's pflanzenphysiolo- gischem Practicum I find' it figured as Kiihnesches Gahrungsgefass. More recently it has been adapted by Einhorn for the quantitative determination of sugar in urine and by Doremus for that of urea in the same fluid." Smith, in 1889, first conceived the value and made practical application of this tube with reference to anaerobioses and gas-formation among bacteria. The illustration (Fig. 50) represents a model fer- mentation tube. With regard to its construction Smith says : " In the construction of this simple bit of apparatus several points must be borne in mind. The bulb should be large enough to receive all the fluid con- y\q, 49 tained in the closed branch. Moistening the plug im- perils tRe purity of the culture. Jf the bulb is suflnciently large this difficulty will not arise. The connecting tube should not be too small, for then the filling and emptying of the closed branch becomes very tedious. Nor should it be too Digitized by Microsoft® [Reprinted from the JODKNAL OF APPLIED MICROSCOPY AND LABORATORY METHODS, Rochester, N. Y., Vol. V, No. 5.] Page 1807 large, otherwise the anaerobic properties of the fluid in the closed branch may be less efEective. Lastly, the angle formed by the two branches of the tube must not be too acute, otherwise the tube must be tilted so much during the trans- ference of the fluid from the tube to the closed branch that there is danger of its moistening the plug or even running out of the bulb." Method. — Heat the fermentation tube con- taining peptonized, sterile glucose bouillon in the steam sterilizer, cool, and inoculate it with the culture in question. In case of a pure anaerobic culture the growth will take place in the closed bulb and the line of demarcation between the turbid, teeming liquid of the closed branch and that of the bulb and connecting tube is sharply drawn. In 1899 Smith recommended a slight modifi- cation of the above method, using the same fer- mentation tube. Method. — Kill a guinea-pig, rabbit, pigeon or other small animal with chloroform ; tear pieces of the internal organs, more particularly of the spleen, liver, and kidneys, as large as peas or beans from the organs with sterile forceps and quickly introduce them into the fermentation tubes, containing ordinary sterile, peptonized bouillon. The tissue should be eventually forced into the closed branch of the tube with a sterile platinum wire. A series of tubes are prepared at on'e time and placed in the- incubator for several days to reveal any contaminating bacteria from the air or from the introduced tissue. Tubes provided in this way with bits of sterile tissues furnish most favorable conditions for the cultivation of anaerobes. They may be kept indefinitely, and when partly dried out they may be refilled with sterile water. Anaerobes will still multiply freely in them though they have not been reboiled. In fact, boiling would cloud the bouillon by coagulating the albumin from the intro- duced material. It is frequently very de- sirable to have on hand fluid cultures of anaerobic species for the study of morpho- logical and physiological characters, a fact which njakes this method especially valu- able. Fig. 51. Smith constructed two more apparatus Fig. 50. Digitized by Microsoft® [Reprinted from the JOURNAL OF APPLIED MICROSCOPY AND LABORATORY METHODS, Rochester, N. Y., Vol. V, No. 5.] Page 1808 fgj. liquid culture media belonging to the same fermentation tube principle. Being of large capacity they are especially adapted for the cultivation, on a large scale, of the tetanus bacillus for the production of a tetanus toxin. In apparatus illustrated in Fig. 51 there are two bulbs (A) and (B) of nearly equal capacity, connected with a heavy rubber tube (C) which carries a clamp (D) to regulate the communication between them. This apparatus is best manipu- lated in a tin rack (F). The bouillon occupies the whole of (A) and all below the dotted line in (B). It is inoculated by transferring the culture material with a platinum loop or pipette through the cotton plugged opening (E). The growth travels down into bulb (A) within 24 hours. Fig. 52 represents an apparatus consisting of a stout liter flask (A), into Fig. 52. Fig. 53. which is fitted a rubber stopper (c) carrying a 100 c. c. pipette (b) with the lower portion bent as shown in Fig. 52, and the upper shortened and provided with a cotton plug. The bouillon fills the flask completely and extends down the nar- row tube to the dotted line in the bulb. The inoculation takes place through the opening (d), the growth proceeds unaided along the narrow tube and reaches the flask in from 24 to 36 hours. The second form (Fig. 52) cannot be autoclaved when filled, as some of the fluid will be thrown out. To obviate this the flask is only partly filled and the extra bouillon required is autoclaved with it in an ordinary flask. There is no difficulty if the Arnold sterilizer is used. Digitized by Microsoft® [Reprinted from the JOURNAL OF APPLIED MICROSCOPY AND LABORATORY METHODS, Rochester, N. Y., Vol. V, No. 5.] Page 1809 Hill (1899^ constructed the fermentation tube shown in Fig. 53. It differs from that of Smith in that the open bulb has twice the capacity of the closed branch. This does away with the danger of wetting the plug, when the gas pres- sure in the closed branch forces the liquid into the open bulb. The closed branch is sealed by means of a conical ground glass stopper (S). The stopper is made thimble-shape to avoid the danger of cracking under high temperatures, which might affect a solid stopper. This arrangement enables the experimenter to examine the liquid in the closed branch without disturbing the liquid culture. In addition to these advantages it permits a more ready and thorough cleaning, and simplifies the process of filling. G. THE HEN'S EGG AS A CULTURE MEDIUM FOR ANAEROBIC BACTERIA. Hueppe (1891) recommends the following procedure : Use freshly laid eggs. Clean the shell from all foreign matter ; sterilize it by washing it in a solution of sublimate ; rinse in sterile water and dry the shell with sterile cotton. With a flamed instrument make a small opening at the point of the egg. Through this opening inoculate by means of a platinum loop, platinum needle or capillary tube. Then cover the opening with a piece of thin, sterilized paper and seal hermeti- cally by covering the paper with a film of collodion. Pearmain's and Moor's Method. — Wash the newly laid egg in a soda solu- tion ; lay it in a ^Tnrtr solution of bichloride of mercury for a short .time ; then rinse the egg thoroughly in water that has been well boiled, finally rinse it in strong alcohol and ether immediately before inoculation. For inoculation pierce the shell with a strong sterile needle and introduce the inoculating material by means of a glass capillary tube, from which it is blown with great care, close the hole with sterile cotton wool. Mad (1901) recommends shaking of the fresh egg so that the yolk mixes well with the white. Instead of just washing the egg with sublimate for a short time Mac^ lets it soak in the sublimate solution for 24 hours. VI. COMBINED APPLICATION OF TWO OR MORE OF THE ABOVE PRINCIPLES. Little need be said with reference to the apparatus that belong to this cate- gory. It is obvious that the large number of methods introduced permits a great variety of combinations that may be successfully used in cultivating anaerobic bacteria. Thus for instance, where it is desired to cultivate bacteria in hydro- gen atmosphere, instead of forcing the air out by the current of hydrogen, the apparatus may first be partly or wholly evacuated by means of a vacuum pump, then it is connected with the Kipp generator. The exhaustion and filling may be repeated alternately several times. This combination has been used and recommended by Pasteur, Novy and other experimenters. Where large apparatus are used, as those of Novy (Fig. 24), Zubinsky (Fig. 16), Gabinsky (Fig. 23), etc., a vessel containing a concentrated solution of alka- line pyrogallol may be placed in the apparatus immediately before it is sealed. Digitized by Microsoft® |.Reprinte