24 MICRO-ORGANISMS AND DISEASE. [CH. iv. 



holder I hold them above a very small flame until the fluid 

 boils, and keep it so boiling for from two to five minutes. 

 During this process of boiling the cotton- wool is only slightly 

 pulled up, and immediately before ceasing to boil the plug is 

 again replaced, and pushed down with a clean glass rod. 

 Then the test-tube is placed (of course upright) in a beaker at 

 the bottom of which a layer of cotton-wool a sort of cushion 

 has been placed. When finished, the test-tubes in the 

 beaker are all transferred to the incubator and kept there for 

 from twelve to twenty-four hours at a temperature of 

 32 38 C. Then the boiling is repeated once more. After 

 this they are kept in the incubator for several days to several 

 weeks. I generally keep them there for two to three weeks, 

 and all those in which the fluid has remained limpid and 

 clear are considered sterile and ready for use. As a rule, 

 starting with sterile stock fluid, and using thoroughly sterile 

 test-tubes and cotton-wool plugs, after once or twice boiling 

 after decanting, there ought to be no loss of tubes through 

 accidental contamination with air-organisms (during de- 

 canting). Sometimes, however, I have had loss to the 

 amount of 5 per cent, or more, but then there was always a 

 hitch of some kind traceable. To decant under carbolic acid 

 spray is not practicable and possesses many unpleasant draw- 

 backs, besides, in some instances when I used it, there was 

 really a greater percentage of contaminated tubes than without 

 it. I therefore do not use the spray. 



Test-tubes containing solid nourishing material are generally 

 kept sufficiently inclined during solidification of the material 

 (see a former chapter) to allow the material to spread into a 

 layer of large area, although this is not essential. 



