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 glass fitted
with an exhaust tube and stop-cock (d), which can be
secured to a plate-glass 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 glass 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.

[Illustration: 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.