rature, 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 deg. C., through a range of 5 deg. 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 deg. 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 deg. 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 deg. 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 deg. 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-cock.

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

(d) A sickle-shaped piece of glass tubing passing 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.

[Illustration: 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
larg