304 METHODS OF IDENTIFICATION AND STUDY 



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 ster- 

 ile 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, vary- 

 ing 5 C. between 60 C. and 120 C. 



4. Remove each capsule from the oven with crucible 

 tongs immediately the ten minutes are completed; 

 remove the cover-glass from its interior with a sterile 

 pair of forceps. 



5. Deposit the film in a flask containing 200 c.c. 

 nutrient bouillon. 



6. Prepare subcultivations from such flasks as show 

 evidence of growth, to determine that no accidental 

 contamination has taken place but that the organism 

 originally spread on the film is responsible for the 

 growth. 



7. Control the result of these experiments. 



Dry Spores : The thermal death-point in this case 

 is that temperature which with certainty kills the spores 

 of the organism in question when present in a thin film 

 after a time exposure of 10 minutes. 



Apparatus Required: 

 As for vegetative forms. 



METHOD. 



i. Prepare a sloped agar tube cultivation and incu- 

 bate under optimum conditions as to spore formations. 



