4. Remove each capsule from the oven with crucible tongs immediately after the ten minutes are completed; remove the cover-gla.s.s 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 5C, between 100 C. and 160C.

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).

7. Control the result of these observations.

(B) _Optimum Reaction._--The optimum reaction has already been roughly determined whilst observing the range. It can be fixed within narrower limits by inoculating in a similar manner a series of tubes of bouillon which represent smaller variations in reaction than those previously employed (say, 1 instead of 5) for five points on either side of the previously observed optimum. For example, the optimum reaction observed in the set of experiments to determine the range was +10. Now plant tubes having reactions of +15, +14, +13, +12, +11, +10, +9, +8, +7, + 6, +5, and observe as before.

~IV. Resistance to Lethal Agents.~--

(A) _Desiccation._--

_Apparatus Required:_

Mueller's desiccator. This consists of a bell gla.s.s fitted with an exhaust tube and stop-c.o.c.k (d), which can be secured to a plate-gla.s.s base (c) by means of wax or grease. It contains a cylindrical vessel of porous clay (a) into the top of which pure sulphuric acid is poured whilst the material to be dried is placed within its walls on a gla.s.s shelf (b). The air is exhausted from the interior and the acid rapidly converts the clay vessel into a large absorbing surface (Fig. 157).

Exhaust pump.

Pure concentrated sulphuric acid.

Sterile cover-slips.

Sterile forceps.

Culture flask containing 200 c.c. nutrient bouillon.

Sterile ventilated Petri dish. This is prepared by bending three short pieces of aluminium wire into V shape and hanging these on the edge of the lower dish and resting the lid upon them (Fig. 158).

METHOD.--

1. Prepare a surface cultivation on nutrient agar in a culture bottle and incubate under optimum conditions for forty-eight hours.

2. Examine preparations from the cultivation, microscopically, to determine the absence of spores.

3. Pipette 5 c.c. sterile normal saline solution into the flask and suspend the entire growth in it.

4. Spread the suspension in thin, even films on sterile cover-slips and deposit inside sterile "plates" to dry.

5. As soon as dry, transfer the cover-slip films to the ventilated Petri dish by means of sterile forceps.

[Ill.u.s.tration: FIG. 157.--Mueller's desiccator.]

6. Place the Petri dish inside the Mueller's desiccator; fill the upper chamber with pure sulphuric acid, cover with the bell jar, and exhaust the air from its interior. Ten minutes later connect up the desiccator to a sulphuric acid wash-bottle interposing an air filter so that only dry sterile air enters.

[Ill.u.s.tration: FIG. 158.--Petri dish for drying cultivations.]

7. At intervals of five hours open the apparatus, remove one of the cover-slip films from the Petri dish, and transfer it to the interior of a culture flask, with every precaution against contamination. Reseal the desiccator and again exhaust, and subsequently admit dry sterile air as before.

8. Incubate the culture flask under optimum conditions until the completion of seven days, if necessary; and determine the time exposure at which death occurs.

9. Pour plates from those culture flasks which grow, to determine the absence of contamination.

10. Repeat these observations at hourly intervals for the five hours preceding and succeeding the death time, as determined in the first set of experiments.

(B) _Light._--

(a) Diffuse Daylight:

1. Prepare a tube cultivation in nutrient bouillon, and incubate under optimum conditions, for forty-eight hours.