e flat culture bottles--_vide_ page 5) of nutrient agar and
incubate under the optimum conditions (previously determined), for the
formation of spores.

Examine preparations from the cultures microscopically to determine the
presence of spores.

2. Pipette 5 c.c. sterile normal saline into each culture tube or 30
c.c. into each bottle and by means of a sterile platinum spatula
emulsify the entire surface growth with the solution.

3. Add the 60 c.c. emulsion to 140 c.c. normal saline contained in the
fitted Erlenmeyer flask.

4. Place the flask in the water-bath of boiling water.

5. Connect up the straight tube, after removing the cotton-wool plug,
with the delivery tube of the steam can; remove the plug from the vent
tube.

6. When the thermometer reaches 100 deg. C., open the spring clip on the
_syphon_, discard the first cubic centimeter of suspension that syphons
over (i. e., the contents of the syphon tube); collect the next 5 c.c.
of the suspension in the sterile graduated test-tube and pour plates and
prepare flask cultures therefrom as in the previous experiments.

7. Repeat this process at intervals of twenty-five minutes' steaming.

8. Observe the inoculated plates and flasks up to the completion, if
necessary, of seven days' incubation.

9. Control these experiments, but in this instance syphon off portions
of the suspension at intervals of one-half to one minute during the five
or ten minutes preceding the previously determined death-point.

_Thermal Death-point._--

Dry--Vegetative Forms: The thermal death-point in this case is that
~temperature~ which with certainty kills a thin film of the organism in
question after a time exposure of ~ten minutes~.

_Apparatus Required:_

Hot-air oven, provided with thermo-regulator.

Sterile cover-slips.

Flask containing 250 c.c. sterile normal saline solution.

Case of sterile pipettes, 10 c.c. (in tenths of a cubic
centimetre).

Case of sterile capsules.

Crucible tongs.

METHOD.--

1. Prepare an emulsion with three loopfuls from an optimum cultivation
in 5 c.c. normal saline in a sterile capsule and examine microscopically
to determine the absence of spore forms.

2. Make twelve cover-slip films on sterile cover-slips; place each in a
sterile capsule to dry.

3. Expose each capsule in turn in the hot-air oven for ten minutes to a
different fixed temperature, varying 5 deg. C. between 60 deg. C. an