BIOLOGICAL PROPERTIES 541 



Single colonies in a Vignal's tube. Small whitish points appear after incu- 

 bating for about 4 or 6 days : these soon form cloudy spheres from which 

 fine spicules radiate on all sides. Bubbles of gas appear around the colonies. 

 Liquefaction of the gelatin commences about the tenth or fifteenth day 

 and progresses slowly. The colonies appear as whitish flakes floating in the 

 liquefied gelatin. 



Agar. A deep stab culture in agar incubated at 37 C. rapidly gives rise 

 to a cloudy growth which is not very characteristic. The agar is split by 

 numerous gas bubbles. 



Serum. In stab culture in coagulated serum, covered after sowing with a 

 layer of agar, a cloudy growth results. The serum is not liquefied. 



Potato. The tetanus bacillus grows on potato under anaerobic conditions, 

 but poorly and with difficulty. In an experiment of Vaillard and Vincent 

 the bacillus formed a thin, moist, glistening layer rather like that of the 

 typhoid bacillus and microscopically was seen to be made up of long rods 

 without spores. 



Milk. The bacillus grows in milk and does not coagulate the medium. 



SECTION III. BIOLOGICAL PROPERTIES. 

 1. Vitality and virulence. 



The spores of the tetanus bacillus are very difficult to sterilize. In a 

 closed vessel and in a moist atmosphere they withstand a temperature of 

 80 C. for 6 hours, 90 C. for more than 2 hours, and the temperature of 

 boiling water for 3 or 4 minutes ; but they can be destroyed with certainty 

 by boiling for 8 .minutes. 



Spores dried and mixed with soil and kept exposed to the air but in the 

 dark, retain their vitality and virulence for several months (Kitasato), but 

 if dried on paper or silk thread and exposed to air and diffused daylight or 

 direct sunlight they rapidly undergo profound modification. 



These modifications vary with the length of time of exposure. At first, the 

 germination of the spores is slower and their growth less rapid. Later they develop 

 into non-sporing, non-pathogenic bacilli and finally perish. All these changes can 

 be brought about in less than a month. But when exposed to light alone in the 

 absence of air the dried spores are more resistant ; in the experiments of Vaillard 

 and Vincent they were still able to germinate and give rise to spore- bear ing toxi- 

 genic bacilli after more than 2 months, during which they were exposed to sunlight 

 for 59 hours. 



The tetanus bacillus in the superficial layers of the soil is evidently continuously 

 exposed to these destructive and attenuating influences, and it rapidly disappears 

 if by passage through the alimentary canal of herbivora it does not find conditions 

 favourable to life and multiplication (Sanchez Toledo and Veillon). 



If dried in pus or albuminous fluids or on porous substances such as 

 splinters of wood taken from tetanus infected wounds the bacillus retains 

 its virulence and vitality for a long time. 



2. Toxin. 



Faber, and after him Vaillard and Vincent, by filtering broth cultures of 

 the organism obtained a very toxic liquid the inoculation of which into 

 animals gave rise to a typical attack of tetanus. 



Preparation of tetanus toxin. Experience has shown that the composition 

 of the medium has considerable influence on the toxin content of the product. 

 Peptone -beef-broth is the best medium for preparing tetanus toxin. 



Sow the bacillus in fresh peptone-beef-broth and arrange the flask as 



