_Apparatus Required:_

Bulloch's apparatus.

Sterile gas filter (_vide_ page 40).

Gasometer containing the gas it is desired to test (SO_{2}, N_{2}O, NO, CO_{2}, etc.) or gas generator for its production.

METHOD.--

1. Prepare at least seven tube cultivations upon solid media and deposit them in Bulloch's apparatus.

2. Connect up the inlet tube of the Bulloch's jar with the sterile gas filter, and this again with the delivery tube of the gasometer or gas generator.

3. Open both stop-c.o.c.ks of the Bulloch's apparatus and pa.s.s the gas through until it has completely replaced the air in the bell jar as shown by the result of a.n.a.lyses of samples collected from the exit tube.

4. Incubate under optimum conditions as to temperature.

5. Examine the cultivations at intervals of twenty-four hours, until the completion of seven days.

6. Remove one tube from the interior of the apparatus each day. If no growth is visible, incubate the tube under optimum conditions as to temperature _and_ atmosphere, and in this way determine the length of exposure to the action of the gas necessary to kill the organisms under observation.

7. Control these results.

~II. Temperature.~--

(A) _Range._--

1. Prepare a series of ten tube cultivations, in fluid media, of optimum reaction.

2. Arrange a series of incubators at fixed temperatures, varying 5 C.

and including temperatures between 5 C. and 50 C.

(In the absence of a sufficient number of incubators utilise the water-bath employed in testing the thermal death-point of vegetative forms.)

3. Incubate one tube cultivation of the organism aerobically or anaerobically, as may be necessary, in each incubator, and examine at half-hour intervals for from five to eighteen hours.

4. Note that temperature at which growth is first observed macroscopically (Optimum temperature).

5. Continue the incubation until the completion of seven days. Note the extremes of temperature at which growth takes place (Range of temperature).

6. Control these results--if considered necessary arranging the series of incubators to include each degree centigrade for five degrees beyond each of the extremes previously noted.

(B) _Optimum._--

1. Prepare a second series of ten tube cultivations under similar conditions as to reaction of medium.

2. Incubate in a series of incubators in which the temperature is regulated at intervals of 1 C. for five degrees on either side of optimum temperature observed in the previous experiment (A, step 4).

3. Observe again at half-hour intervals and note that temperature at which growth is first visible to the naked eye = Optimum temperature.

(C) _Thermal Death-point (t. d. p.)_--

Moist--Vegetative Forms:

The _t. d. p._ here is that ~temperature~ which with certainty kills a watery suspension of the organisms in question after an exposure of ~10 minutes~.

[Ill.u.s.tration: FIG. 155.--Hearson's water-bath.]

_Apparatus Required:_

Water-bath. For the purpose of observing the thermal death-point a special water-bath is necessary. The temperature of this piece of apparatus is controlled by means of a capsule regulator that can be adjusted for intervals of half a degree centigrade through a range of 30, from 50 C. to 80 C. by means of a spring, actuated by the handle a, which increases the pressure in the interior of the capsule. A hole is provided for the reception of the nozzle of a blast pump, so that a current of air may be blown through the water while the bath is in use, and thus ensure a uniform temperature of its contents. Through a second hole is suspended a certified centigrade thermometer, the bulb of which although completely immersed in the water is raised at least 2 cm. above the floor of the bath.

Sterile gla.s.s capsules.

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

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

Special platinum loop.

Test-tubes, 18 by 1.5 cm., of thin German gla.s.s.

Case of sterile petri dishes.

Tubes of agar or gelatine.

METHOD.--

1. Prepare tube cultivations on solid media of optimum reaction; incubate forty-eight hours under optimum conditions as to temperature and atmosphere.

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

3. Pipette 5 c.c. salt solution into each of twelve capsules.

4. Suspend three loopfuls of the surface growth (using a special platinum loop, _vide_ page 316) in the normal saline solution by emulcifying evenly against the moist walls of each capsule.

5. Transfer emulsion from each capsule to sterile 250 c.c. flask, and mix.

6. Pipette 5 c.c. emulsion into each of twelve sterile test-tubes numbered consecutively.

7. Adjust the first tube in the water-bath, regulated at 40 C, by means of two rubber rings around the tube, one above and the other below the perforated top of the bath, so that the upper level of the fluid in the tube is about 4 cm. below the surface of the water in the bath, and the bottom of the tube is a similar distance above the bottom of the bath.

8. Arrange a control test-tube containing 5 c.c. sterile saline solution under similar conditions. Plug the tube with cotton-wool and pa.s.s a thermometer through the plug so that its bulb is immersed in the water.

9. Close the unoccupied perforations in the lid of the water-bath by means of gla.s.s b.a.l.l.s.

10. Watch the thermometer in the test-tube until it records a temperature of 40 C. Note the time. Ten minutes later remove the tube containing the suspension, and cool rapidly by immersing its lower end in a stream of running water.

11. Pour three gelatine (or agar) plates containing respectively 0.2, 0.3, and 0.5 c.c. of the suspension, and incubate.