474 ANAEROBIC BACTERIA 



tions made from the material inoculated each time, and the process 

 is repeated until microscopical examination reveals a sufficient number 

 of bacilli of characteristic appearance. Finally, the enriched culture, 

 after a final heating to 80 C. for thirty minutes, is plated out anaerob- 

 ically upon blood agar plates. 



Tetanus bacilli characteristically produce a wide zone of hemolysis 

 around the colonies on blood agar, and the colonies themselves tend 

 to spread rapidly. 1 



Growth in Media. The tetanus bacillus is typically an obligate 

 anaerobe, although various successful attempts to induce aerobic 

 development have been recorded. 2 The characteristic reactions and 

 products of the organism, however, are detected only in anaerobic 

 cultures. On anaerobic agar plates the colonies are filamentous; 

 under the lower powers of the microscope they resemble densely matted 

 strands of cotton fiber. Gelatin colonies are quite similar, except 

 that in sugar-free gelatin liquefaction takes place after three to five 

 days' incubation. The growth in deep stab cultures is distinctive; 

 the organisms grow away from the line of inoculation at right angles, 

 producing an appearance which has been likened to an inverted pine 

 tree. The growth fails to reach the surface of the medium, however, 

 indicating the anaerobic nature of the bacteria. Milk appears to be 

 a favorable medium for their development. A slight acidity is pro- 

 duced, but no coagulation or peptonization. Slightly alkaline, sugar- 

 free broth overlaid with a layer of paraffin or paraffin oil 3 is a favorable 

 medium; the organisms produce a well-defined turbidity after twenty- 

 four to forty-eight hours' incubation at 37 C. which increases in 

 intensity for about fourteen days, at the end of which time the growth 

 begins to settle to the bottom of the flask. Cultures of tetanus bacilli 

 usually possess a very disagreeable odor. 



Conditions of Growth. Bacillus tetani is an obligate anaerobe, but 

 strains may be gradually accustomed to oxygen so that eventually 

 they will grow slowly even in the presence of air. They lose their 

 toxin-producing powers, however, under these conditions. 4 The 



1 Boulton and Fisch, Trans. Am. Phys., 1902, 463. It is occasionally possible to 

 isolate tetanus bacilli directly from mixtures by inoculating dextrose agar with dilute 

 suspensions of the suspected material, which has previously been heated to 80 C. for 

 thirty minutes. The agar is drawn up into long, sterile glass tubes of approximately 

 5 mm. bore, and the ends sealed by heating. After forty-eight hours' incubation at 

 37 C. characteristic colonies are visible through the glass. The outside of the tube 

 is carefully sterilized and cut with a file close to the desired organisms, which may be 

 removed by a sterile capillary pipette. 



2 Ferran, Centralbl. f. Bakt., 1898, xxiv, 28. 



3 Park, Centralb. f. Bakt., 1901, xxix, 445. 4 Ferran, loc. cit. 



