98 CULTIVATION OF ANAEROBIC MICRO-ORGANISMS 



In the case of media which cannot be heated strongly, Ch. Nicolle recommends 

 the following modification of the method: Pour sufficient sterile vaseline oil into 

 the flask or tube containing the medium to form a thin layer on the surface. Stand 

 the culture vessel in a water bath at 40 C. and connect the mouth of the vessel to 

 a water pump. After exhausting the whole of the dissolved air the culture medium 

 is protected from air by the layer of oil. 



K. Rosenthals method. Method recommended. 1. Distribute the medium 

 (broth, milk, etc.) into tubes. Pour lanolin previously liquefied by heat into 

 each tube so that it forms a layer 15 mm. thick on the surface. Plug the 

 tubes with wool and autoclave at 120 C. After sterilization, cool the tubes 

 rapidly in a vertical position. 



Tubes prepared by this method can be kept for about two months. If kept 

 longer than this, it is well to heat them to 100 C. for 15 minutes before sowing 

 them. It is an advantage to use tubes slightly constricted about the middle ; the 

 medium occupies the lower part of the tube up to the constriction, while the 

 lanolin fills the constricted part. Any gas which may be formed easily escapes 

 by pushing the plug of lanolin into the upper non-constricted portion of the 

 tube. 



2. When required for use, melt the layer of lanolin in the flame (it liquefies 

 at 42 C.), and sow the organism in the ordinary way through the melted 

 lanolin. Cool rapidly to solidify the fat. Growth takes place in what 

 is practically a sealed tube (Rosenthal). 



L. Tarozzi's method. To grow the strictly anaerobic organisms (Bacillus 

 tetani, Bacillus maligni cedematis, etc.) by this method it is only necessary 

 to add to broth contained in ordinary tubes a fragment of tissue freshly 

 removed from a rabbit, mouse or guinea-pig, and to proceed as in the case of 

 aerobic organisms. 



Pieces of liver, spleen, kidney or lymphatic glands may be used with success, 

 but blood, milk, or the connective tissues are useless for the purpose. To 

 tubes of broth add a small piece of one of the above-mentioned internal 

 organs which has recently been excised with the usual aseptic precautions. 

 Incubate the tubes for a day or two at 37 C., and they are then ready for 

 use. They may be heated to 100-107 C. for a minute or two, but if the 

 heating be prolonged for more than 5 minutes growth will fail. Cultures 

 will grow even if the piece of tissue be removed before sowing. 



A number of other substances have a similar action in facilitating the growth 

 of anaerobic organisms. Wrzosek, Ori, for example, were able to obtain cultures 

 under ordinary conditions in broth by simply adding pieces of vegetable tissue 

 (potato, elder pith, mushrooms, etc.) to the medium. Tarozzi used a slightly 

 alkaline glucose-broth, which had been heated under a pressure of two atmospheres 

 in the autoclave, with successful results. Aperlo was able to grow strictly anaerobic 

 organisms in a simple pep tone- broth by sterilizing the medium for half an hour 

 under a pressure of half an atmosphere, and using it within 24 hours of its 

 preparation. 



Kata also succeeded with ordinary broth containing a small piece of agar and 

 0'3-0'7 per cent, of sodium sulphite, and even better with the same amount of 

 sulphite and a little fresh serum. The latter medium would appear to be very 

 useful for toxin production. 



Pf uhl recommends a broth made with liver instead of ordinary meat, and sterilized 

 in the autoclave. Satisfactory results were also obtained with the following 

 technique : To a tube containing 10 c.c. of ordinary broth add 1 gram of spongy 

 platinum, boil for 10 minutes, sow as soon as cool and put in the incubator without 

 shaking the tube. 



The vitality of anaerobic organisms is exhausted much more quickly on 

 media prepared on these principles than on media under anaerobic conditions 

 (Jungano and Distaso). 



