d
120 deg. C.

4. Remove each capsule from the oven with crucible tongs immediately
after the ten minutes are completed; remove the cover-glass from its
interior with a sterile pair of forceps.

5. Deposit the film in a flask containing 200 c.c. nutrient bouillon.

6. Prepare subcultivations from such flasks as show evidence of growth,
to determine that no accidental contamination has taken place but that
the organism originally spread on the film is responsible for the
growth.

7. Control the result of these experiments.

Dry--Spores: The thermal death-point in this case is that ~temperature~
which with certainty kills the spores of the organism in question when
present in a thin film after a time exposure of ~10 minutes~.

_Apparatus Required:_

As for vegetative forms.

METHOD.--

1. Prepare a sloped agar tube cultivation and incubate under optimum
conditions as to spore formations.

2. Pipette 5 c.c. sterile normal saline into the culture tube and
emulsify the entire surface growth in it. Examine microscopically to
determine the presence of spores in large numbers.

3. Spread thin even films on twelve sterile cover-slips and place each
cover-slip in a separate sterile capsule.

4. Expose each capsule in turn for ten minutes to a different fixed
temperature, varying 5 deg. C, between 100 deg. C. and 160 deg. C.

5. Complete the examination as for vegetative forms.

~III. Reaction of Medium.~

(A) _Range._--

1. Prepare a bouillon culture of the organism and incubate, under
optimum conditions as to temperature and atmosphere, for twenty-four
hours.

2. Pipette 0.1 c.c. of the cultivation into a sterile capsule; add 9.9
c.c. sterile bouillon and mix thoroughly.

3. Prepare a series of tubes of nutrient bouillon of varying reactions,
from +25 to -30 (_vide_ page 155), viz.: +25, +20, +15, +10, +5,
neutral, -5, -10, -15, -20, -25, -30.

4. Inoculate each of the bouillon tubes with 0.1 c.c. of the diluted
cultivation by means of a sterile graduated pipette and incubate under
optimum conditions.

5. Observe the cultures at half-hourly intervals from the third to the
twelfth hours. Note the reaction of the tube or tubes in which growth is
first visible macroscopically (probably optimum reaction).

6. Continue the incubation until the completion, if necessary, of seven
days. Note the extremes of acidity and alkalinity in which macroscopical
growth has developed (Range of reaction).