36 MICRO-ORGANISMS AND DISEASE. [CHAP. 



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 to 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 from 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 with- 

 out it. I therefore do not use the spray. 



A simple method is to subject the whole number of test- 

 tubes into which the nutritive material had been decanted 

 (broth, peptone broth, nutritive gelatine, Agar-Agar mixture,) 

 to a steamer (see Fig. 7). The test-tubes are placed into the 

 wire net (see figure), the top of the group of test-tubes is 

 covered with tinfoil, so as to protect the plugs from becoming 

 wet, and then the wire net is placed into the steamer the 

 water at the bottom of which has been previously heated to 

 boiling, the lid is put on and the steaming is kept up for 

 from fifteen to twenty minutes. This is repeated on one or 

 two successive days. I have not seen any tubes go bad, 

 after they have thus been steamed on three successive days 

 each time for twenty minutes. Placed in the incubator and 

 kept at a temperature of 35 to 38 C., from some days to one 



