440 BACTERIOLOGICAL ANALYSES 



3. Incubate aerobically at 37 C. for twenty-four 

 hours. Examine the tubes carefully for visible growth, 

 especially delicate pellicle formation, which if present 

 should be examined microscopically for vibrios, both 

 by stained preparations or by fresh specimens with dark 

 ground illumination. 



4. Inoculate fresh tubes of peptone water from such of 

 the tubes as exhibit pellicle formation from the pel- 

 licle itself and incubate at 3 7 C. for twenty-four hours. 



5. Test the peptone water itself for the presence of 

 indol and nitrite by the addition of pure concentrated 

 H 2 S0 4 . 



5. Prepare gelatine and agar plates in the usual way 

 from such of these tubes as show pellicle formation. 



6. Pick off from the plates any colonies resembling 

 those of the Vibrio cholerae and subcultivate upon all 

 the ordinary laboratory media. 



7. Test the vibrio isolated against the serum of an ani- 

 mal immunised to the Vibrio choleras for agglutination. 



B. Anthracis. 



1. Transfer 5 c.c. of the emulsion from the filter 

 candle to a sterile test-tube and plug carefully. 



2. Place the test-tube in the interior of the benzole 

 bath employed in separating out spore- bearing organ- 

 isms (vide page 257), and expose to a temperature 

 of 80 C. for twenty minutes. 



3. Inoculate a young white rat subcutaneously (on 

 the inner aspect of one of the hind legs) with i c.c. of the 

 emulsion. Observe during life, and, if the animal 

 succumbs, make a complete post-mortem examination. 



4. Melt three tubes of nutrient agar in boiling water 

 and cool to 42 C. 



5. Number the tubes i, 2, and 3. To No. i add 

 0.2 c.c., to No. 2 add 0.3 c.c., and to No. 3 add 0.5 c.c. 

 of the suspension, and pour plates therefrom. 



6. Incubate at 37 C. for twenty-four or forty -eight 

 hours. 



