of 80 deg. C. for twenty minutes.

3. Number ten tubes of litmus milk consecutively from 1 to 10.

4. Remove the test-tube from the benzole bath and shake well to
distribute the spores evenly through the fluid.

5. To each tube of litmus milk add a measured quantity of the suspension
corresponding to the amounts employed in isolating the coli group
(_vide_ page 437).

6. Incubate each tube anaerobically at 37 deg. C. Anaerobic conditions
can be obtained by putting the cultures up in Buchner's tubes or in
Bulloch's apparatus. If, however, whole milk has been used in making the
litmus milk the layer of cream that rises to the surface will be
sufficient to ensure anaerobiosis; whilst if separated milk has been
employed it will be sufficient to pour a layer of sterile vaseline or
liquid paraffin on the surface of the fluid.

7. Examine after twenty-four hours' incubation. Note (if B. enteritidis
sporogenes is present)--

(a) Acid reaction of the medium as indicated by the colour of the
litmus or its complete decolourisation.

(b) Presence of clotting, and the separation of clear whey.

(c) Presence of gas, as indicated by fissures and bubbles in the
coagulum, and possibly masses of coagulum driven up the tube almost to
the plug.

8. Replace the tubes which show no signs of growth in the incubator for
a further period of twenty-four hours and again examine with reference
to the same points.

9. Remove those tubes which give evidence of growth from the Buchner's
tubes and carefully pipette off the whey; examine the whey
microscopically.

10. Inoculate two guinea-pigs each subcutaneously with 0.5 c.c. of the
whey and observe the result.

~Vibrio Cholerae.~--

1. Number ten tubes of peptone water consecutively from 1 to 10.

2. To each of the tubes of peptone water add a measured quantity of the
suspension, corresponding to those amounts employed in isolating the
members of the coli group (_vide_ page 437).

3. Incubate aerobically at 37 deg. 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 pellicle itself--and incubate at
37 deg. C. for twenty-four hours.

5. Test the peptone water itself f