The Elements of Bacteriological Technique Part 83

12. Pipette the remaining 4 c.c. of the suspension into a culture flask containing 250 c.c. of nutrient bouillon, and incubate.

13. Observe these cultivations from day to day. "No growth" must not be recorded as final until after the completion of seven days' incubation.

14. Extend these observations to the remaining tubes of the series, but varying the conditions so that each tube is exposed to a temperature 2 C. higher than the immediately preceding one--i. e., 42 C., 44 C., 46 C., and so on.

15. Note that temperature, after exposure to which no growth takes place up to the end of seven days' incubation, = the thermal death-point.

16. If greater accuracy is desired, a second series of tubes may be prepared and exposed for ten minutes to fixed temperatures varying only 0.5 C., through a range of 5 C. on either side of the previously observed death-point.

Moist--Spores: The thermal death-point in the case of spores is that ~time exposure~ to a ~fixed temperature of 100 C.~ necessary to effect the death of all the spores present in a suspension.

NOTE.--If it is desired to retain the ~time constant 10 minutes~ and investigate the temperature necessary to destroy the spores, varying amounts of calcium chloride must be added to the water in the bath, when the boiling-point will be raised above 100 C. according to the percentage of calcium in solution. In such case use the bath figured on page 227; the bath figured on page 299 can only be used if the capsule is first removed.

It is determined in the following manner

_Apparatus Required:_

Steam-can fitted with a delivery tube and a large bore safety-valve tube.

Water-bath at 100 C.

Erlenmeyer flask, 500 c.c. capacity, containing 140 c.c.

sterile normal saline solution and fitted with rubber stopper perforated with four holes.

The rubber stopper is fitted as follows:

(a) Thermometer to 120 C., its bulb immersed in the normal saline.

(b) Straight entry tube, reaching to the bottom of the flask, the upper end plugged with cotton-wool.

(c) Bent syphon tube, with pipette nozzle attached by means of rubber tubing and fitted with pinch-c.o.c.k.

The nozzle is protected from accidental contamination by pa.s.sing it through the cotton-wool plug of a small test-tube.

(d) A sickle-shaped piece of gla.s.s tubing pa.s.sing just through the stopper, plugged with cotton-wool, to act as a vent for the steam.

Sterile plates.

Sterile pipettes.

Sterile test-tubes graduated to contain 5 c.c.

_Media Required:_

Gelatine or agar.

Culture flasks containing 200 c.c. nutrient bouillon.

[Ill.u.s.tration: FIG. 156.--Apparatus arranged for the determination of the death-point of spores.]

METHOD.--

1. Prepare twelve tube cultivations upon the surface (or two cultures in large 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 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 C. between 60 C. and 120 C.