512 THE ANAEROBIC BACTERIA 
therefore, inchuiing; tetanus bacilli (and free from proteus bacilli and 
ordinary "spreaders") is inoculated into the condensation water of an 
agar slant, which is subsequently incubated in an upright position in 
an anaerobic agar. The tetanus growth soon reaches the upper levels 
of the slanted surface, and often may be obtained in a pure state. 
Tetanus bacilli usually produce a clear zone of hemolysis around 
colonies on blood agar. The most trustworthy method for obtaining 
cultures of B. tetani in a state of undoubted purity is to isolate single 
spores from an enrichment culture, as described above, by the modified 
method of Barber.^ 
Growth in Media.— Bacillus tetani is an obligate anaerobe, although 
various successful attempts to induce aerobic development have been 
recorded.^ The characteristic reactions and products of the organism, 
however, are detected only in anaerobic cultures. Colonies upon anae- 
robic plates are rather delicate, thin, filmy growths with somewhat radi- 
FiG. 70.— Bacillus tetani, spore formation. X 1000. (Glinther.) 
ating edges. Submerged colonies are small, flocculent, gray masses 
which resemble somewhat small (1 to 2 mm.) wisps of cotton, denser 
in the center. Gelatin colonies are similar, but develop more slowly. In 
stab cultures the growth of the organism away from the line of inocu- 
lation results in an arborescent formation which is quite characteristic. 
This is sometimes referred to as "an inverted fir tree. ' ' The inoculation 
growth extends upward to about 1 cm. below the surface of the medium, 
indicating thereby the anaerobic nature of the organism. SUghtly 
alkaline (pH 7.5) nutrient broth, in which anaerobic conditions are 
created by heating to 100° C. and rapidly cooling to 40° C, just prior 
to inoculation, and kept oxygen-free by suitable procedures, is an 
advantageous medium for the growth of B. tetani. Within forty- 
eight hours, as a rule, the broth becomes turbid and the turbidity 
increases for several days. After a few days or more, the growth settles 
• See Daee 240. * Ferran: Centralbl. f. Bakteriol., 1898, 24, 28, 
