Iv] PREPARATIOisr OF CULTURE-MEDIA 49 



is again replaced, and pushed down with sterile forceps. 

 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 one to three days, 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 decanting). 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 necessary or practic- 

 able, and possesses many unpleasant drawbacks, besides, in 

 some instances when I used it there was really a greater 

 percentage of contaminated tubes than without it. 



A simple method and now generally used is to subject 

 the whole number of test-tubes or flasks into which the 

 nutritive material had been decanted (broth, peptone broth, 

 potato, milk, alkaline serum agar, 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° 



E 



