22 MICRO-ORGANISMS AND DISEASE. [CHAP. 



little care is required to obtain sterile material ready for 

 inoculation. To start with a stock of nourishing material, 

 however well sterilised, and to decant it into test-tubes with 

 cotton-wool plugs not absolutely sterile must lead to failure. 

 I have seen this happen over and over again, and all the 

 material decanted became consequently contaminated and 

 thereby useless for inoculations. The test-tubes and flasks 

 must be well cleaned, then dried, placed in the air-chamber, 

 and kept there exposed for several hours to a temperature of 

 from 130 150 0. on several successive days, or they may be 

 thoroughly heated in all parts over the open flame of a gas- 

 burner. The same applies to the cotton-wool, as mentioned 

 in a former chapter. The test-tubes and flasks are plugged 

 by means of clean forceps with the cotton-wool which is just 

 brown, and then replaced in the air-chamber and again 

 heated for several hours on two or three occasions up to a 

 temperature of 130 150 C., or they may be well heated 

 over the open flame of the burner. To decant sterile stock 

 fluid into these test-tubes and flasks, I proceed thus : A clean 

 beaker with spout, covered with a clean glass plate, is placed 

 on a wire net on a tripod over the flame of a Bunsen burner, 

 and thoroughly heated for half an hour or so; then it is 

 allowed to cool, and when cool, the plug of the stock flask is 

 lifted with forceps, and some of the sterile fluid quickly 

 poured from the flask into the beaker. The plug is replaced 

 in the neck of the stock flask and the beaker covered with 

 the glass plate. Of course the quantity poured into the 

 beaker should be large enough to supply the required number 

 of test-tubes or small flasks. The stock flask containing still 

 some fluid, having been opened for however short a time, has 

 of course been exposed to air-contamination, and therefore 

 must be treated accordingly, if the fluid left in it is to serve 

 as sterile nourishing material on a future occasion. Con- 

 sequently it is subjected to boiling for from fifteen to thirty 

 minutes, then placed in the incubator, boiled again the next 

 day and put back in the incubator, where it is left at a tem- 

 perature -of from 32 38 C. for several days. If after a week 

 or so the fluid remains limpid, it is of course to be considered 

 sterile. 



Next, the fluid that has been poured into the beaker (covered 

 with the glass plate) is poured as quickly as possible into the 

 test-tubes, one after the other, by lifting with clean forceps 

 the plug and pouring in the fluid to a depth of one and a half 

 to two and a half inches, and the plug replaced. 



During this procedure, contamination with air-organisms, if 



