TEMPERATURE 303 



vide page 5) of nutrient agar and incubate under the 

 optimum conditions (previously determined), for the 

 formation of spores. 



Examine preparations from the cultures micro- 

 scopically to determine the presence of spores. 



2. Pipette 5 c.c. sterile normal saline into each cul- 

 ture 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 centi- 

 meter of suspension that syphons over (i.e., the con- 

 tents 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. 



