334 HARRIET LESLIE WILCOX 



paraffin and the tubes incubated at 37°C. After one week's 

 incubation the cultures are stored in the ice chest, where they 

 may be kept for six months without affecting their abihty to 

 produce toxin. 



THE BROTH FOR TOXIN AND PRELIMINARY CULTURES 



The following broth is used both for the toxin and for the 

 preliminary cultures for inoculating the toxin broth: 



To every pound of market veal, add 1000 cc. of water and place 

 in ice chest over night. The next morning the infusion is placed 

 over a free flame and raised to 45°C. and held at that temper- 

 ature for one hour; it is then boiled briskly for one-half hour 

 and the broth strained through cheesecloth. The amount of 

 filtrate is measured and the following added: 



Witte's Peptone 1.0 per cent 



Glucose (anhydrous) 1.0 per cent 



NaCl 0.5 per cent 



When the above ingredients are added, the broth is boiled 

 until the peptone, glucose, etc., are melted, and is then titrated 

 with T^ NaOH. After the reaction has been corrected to +1 

 to phenolphthalein, the flame is turned out. The broth is 

 then filtered through two layers of absorbent cotton directly 

 into 2 litre Erlenmeyer flasks, leaving only sufficient space in 

 the flasks for the expansion of the broth during the sterilization 

 in the Arnold. The flasks are sterilized for 1| hours on the 

 first day and one hour on the second day. 



PRELIMINARY CULTIVATION 



Fill potato tubes with about 40 cc. of the glucose broth and 

 sterilize for one and one-half hours on the first day and one 

 hour on the second day. These tubes may be kept for two 

 weeks when they will still give satisfactory growth. To make 

 the first transfer for the preliminary cultivation, add 8 cc. of 

 the melted semi-solid agar stock culture of the B tetani to two 

 tubes of glucose broth from which the air has been previously 



