a cubic
centimetre).

Special platinum loop.

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

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 deg. 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 pass 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 glass balls.

10. Watch the thermometer in the test-tube until it records a
temperature of 40 deg. 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.

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
deg. C. higher than the immediately preceding one--i. e., 42 deg. C.,
44 deg. C., 46 deg. C., and so on.

15. Note that tempe