or the presence of indol and nitrite
by the addition of pure concentrated H_{2}SO_{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 animal immunised to
the Vibrio cholerae 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 organisms (_vide_ page 257), and expose to
a temperature of 80 deg. C. for twenty minutes.

3. Inoculate a _young_ white rat subcutaneously (on the inner aspect of
one of the hind legs) with 1 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
deg. C.

5. Number the tubes 1, 2, and 3. To No. 1 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 deg. C. for twenty-four or forty-eight hours.

7. Pick off any colonies resembling those of anthrax and subcultivate on
all the ordinary laboratory media.

8. Inoculate another young white rat as in 3, using two loopfuls of the
agar subcultivation emulsified with 1 c.c. sterile bouillon. Observe
during life, and if the animal succumbs, make a complete post-mortem
examination.

~B. Tetani.~--

1. Proceed as detailed above in steps 1 and 2 for the isolation of the
B. anthracis.

2. Add 1 c.c. of the suspension to each of three tubes of glucose
formate broth, and incubate anaerobically in Buchner's tubes at 37 deg. C.

3. From such of the tubes as show visible growth (with or without the
production of gas) after twenty-four hours' incubation inoculate
guinea-pigs, subcutaneously (under the skin of the abdomen), using 0.1
c.c. of the bouillon cultivation as a dose. Observe carefully during
life, and, if death occurs, make a complete post-mortem examination.

4. From the same tubes pour agar plates and incubate anaerobically in
Bulloch's apparatus, at 37 deg. C.

5. Subcultivate suspicious colonies on the various media, incubate
anaerobically, making control cultivations on glucose formate agar, stab