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-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.--