Cultivation 



453 



The methods for excluding the oxygen from the cul- 

 tures and replacing it by hydrogen, as well as other methods 

 suggested for the cultivation of the strictly anaerobic 

 organisms, are given under the appropriate heading (Anae- 

 robic Cultures), and need not be repeated here. 



I have suggested a simple method of cultivating the 

 bacillus in bouillon for the purpose of securing its toxin * in 

 an ordinary bottle which is rilled to the mouth with bouillon, 

 then closed by a perforated rubber stopper containing a 

 glass tube a couple of inches long. Connected with this 

 glass tube, by means of a short piece of rubber tubing 

 (a), is the bulb of a broken 

 pipet (6), the other end of which 

 is plugged with cotton (Fig. 137). 

 The only object of this attach- 

 ment is to provide for the ex- 

 pansion of the contained fluid 

 during steam sterilization. When 

 the steam sterilization takes 

 place, the expanding fluid ascends 

 to the reservoir represented by 

 the pipet bulb (6), descending 

 again as the fluid cools. When 

 the sterilization is completed, 

 the reservoir is detached at a, 

 the inoculation of the bouillon 

 made by passing a fine pipet 

 through the glass tube into the 

 bottle, the projecting glass tube 

 heated in a flame and drawn out 

 to a fine tube. The bottle is 

 then stood in hot water until the 

 expanding fluid ascends to the top of the glass tube, when 

 it is sealed in a flame and the bottle and its contents per- 

 mitted to cool. In cooling, the retracting fluid leaves a 

 partial vacuum which at once draws up any minute bub- 

 bles of air remaining, and allows the tetanus bacillus to 

 grow in a condition of almost perfect anaerobiosis. 



Park,f following the suggestion of Kitasato, covers the 

 surface of the bouillon with a layer of paraffin about 1-2 cm. 



* "Centralbl. f. Bakt. u. Parasitenk.," xix, Nos. 14 and 15, April 

 25, 1896, p. 550. 



f "Jour. Med. Research," N. S., vol. i, No. 1, p. 298. 



Fig T 37- Tetanus bottle: 

 1, During sterilization; 2, 

 after inoculation and sealing. 



