BACTERIACE.E : THE SPIROGENIC ANAEROBES 279 



Isolation of B. tetani from mixed material or from wounds 

 known to contain it is not always easy. The material should 

 be planted in glucose broth and incubated in hydrogen at 37 C7 

 for 2 to 3 days. Microscopic examination of the sediment may 

 then reveal the drumsticks. Kitasato's procedure should then be 

 followed, employing agar distinctly alkaline to litmus and con- 

 taining 2 per cent of glucose. If many other spore-forming bac- 

 teria are present in the mixture, special procedures are necessary, 

 such as preliminary culture for 8 days at 37 C. in a deep stab in 

 coagulated rabbit's blood with subsequent heating to 80 C. to 

 get rid of B. edematis, or culture for 8 days at 37 C.^ih milk with 

 subsequent heating to get rid of B. welchii. Aerobic spore-formers 

 may be eliminated by successive transfers in animals. 



The spores of B. tetanic esist the temperature of boiling water 

 for 5 to 30 minutes. Biological products to be introduced into 

 the human body need to be sterilized in the autoclave or else 

 carefully examined by anaerobic culture methods to insure their 

 freedom from tetanus spores. The danger of infection from this 

 source has been emphasized by Smith. 1 



The colony in glucose gelatin or glucose agar consists of a 

 compact center with slender, radiating, straight or irregularly 

 curved threads about the periphery. Liquefaction of gelatin 

 becomes evident in stab-culture after about two weeks at 20 C. 

 Milk is sometimes but not always coagulated and the casein is 

 eventually digested. 



The cultures of the tetanus bacillus are extremely poisonous, 

 especially so when they are developed under very strict anaerobic 

 conditions. A nerve poison, tetanospasmin, and a hemolytic 

 poison, tetanolysin, are present. The former is the more impor- 

 tant constituent of the tetanus toxin. Neutral or slightly alka- 

 line plain nutrient broth, incubated in an atmosphere of hydrogen 

 for ten days after inoculation gives the most powerful toxin. 

 The bacteria-free fluid from such a culture has been found to kill 

 a mouse of ic-grams weight in a dose of o.ooo 005 c.c. The toxin is 



1 Journ. A. M. A., Mar. 21, 1908, Vol. L., pp. 929-934. 



