540 THE TETANUS BACILLUS 



entirely of bacilli with spores. In cultures incubated at 20 C. or 25 C., 

 spore formation is slower and only commences after the cultures have been 

 incubated for about 10 days. 



Spore-bearing bacilli occur as rather short, slender rods with a small sphere 

 situated exactly at one end : the spore is refractile and has a diameter two 

 to four times the width of the bacillus. This is known as the pin form of 

 the bacillus (fig. 260). 



In old cultures, the body of the bacillus breaks off and spores and swollen, 

 irregular, dumb-bell shaped involution forms only are found. 



Staining reactions. The bacillus of tetanus is easily stained with the 

 basic aniline dyes, and is gram-positive. If spore-bearing bacilli be stained 

 in the ordinary way the rods and the outline of the spores alone are stained, 

 leaving the centres of the latter unstained and giving the organism its charac- 

 teristic appearance, which has been compared to a tennis racquet. 



The spores may easily be stained by the methods already described (p. 145). 



2. Cultural characteristics. 



Conditions of growth. The tetanus bacillus is an anaerobic organism 

 though not so strictly anaerobic as the bacillus of malignant oedema. Growth 

 will take place in media containing small quantities of oxygen and the organism 

 can be trained to grow in a slightly rarefied atmosphere. 



Growth takes place at all temperatures between 14 and 43 C. Below 

 20 C. the growth is very poor. Spores form very slowly below 25 C. The 

 optimum temperature is 38 C. At 42 and 43 C. the bacilli multiply 

 rapidly but few of them form spores. 



The tetanus bacillus grows on the ordinary neutral or slightly acid or 

 alkaline media made from broth provided they be made with fresh broth 

 (Kitasato). Ordinary fresh beef broth, Martin's broth or Nicolle's medium 

 (vide infra) are among the best, while such media as white of egg and fresh 

 serum yield very poor growths. 



Debrand has shown that in presence of the Bacillus subtilis the tetanus 

 bacillus can be easily cultivated in broth in contact with air. Under these 

 conditions the tetanus bacillus retains its properties and secretes as powerful 

 a toxin as when grown under strictly anaerobic conditions. 



Characters of growth. Broth. Under anaerobic conditions growth is 

 rapid at a temperature of 37 C. After incubating for about 24 hours the 

 medium is generally cloudy and small bubbles of gas will be seen rising to 

 the surface of the medium. The turbidity increases during the next few 

 days and after incubating for about a fortnight growth begins to slacken 

 and a precipitate falls to the bottom of the tube, the broth becoming clear. 



During cultivation, hydrogen, nitrogen and hydrocarbons are given off in 

 moderate quantity. The culture has a characteristic but most disagreeable 

 odour, which has been compared to that of burnt hoofs. 



The tetanus bacillus forms indol in broth. 



Gelatin. Deep stab culture. A deep stab culture in a tube of gelatin from 

 which the air has been removed gives, after incubating for 4 or 6 days 

 at a temperature of about 20 C., a growth of small cloudy-looking 

 points from which numerous very fine spicules shoot out at right angles to 

 the line of the stab. The cloudiness extends and gradually invades the 

 whole of the gelatin, which commences to liquefy about the tenth day. A 

 flocculent deposit forms at the bottom of the tube and above it the gelatin 

 is clear and liquid. Spores only form when the gelatin has begun to liquefy. 

 Bubbles of gas are also formed. 



