PREPARATION OF CULTURE MEDIA II 



soda, drop by drop, testing after each addition, until the reaction 

 of the fluid is slightly alkaline.* Boil the fluid for half an hour 

 to coagulate any albumen which may be present. 



Next filter the broth into a sterile flask, passing it through a 

 double thickness of white filter or blotting paper, and plug the 

 flask firmly with sterilized cotton-wool. 



If the broth is to be used for the manufacture of gelatin or 

 agar, it is next sterilized in the flask ; while if it is to be used 

 as it is as a culture medium, it is decanted into tubes and then 

 sterilized. 



In decanting media into tubes be very careful not to get the 

 plug wet, and not to let any of the medium get on to the upper 

 part of the tube ; otherwise the plug will stick to the tube, and 

 there will be some danger of bacteria from the air " growing 

 through" the fluid contained in the interstices of the plug and 

 contaminating the culture. Ordinary non-absorbent (brown) wool 

 is better than the white absorbent wool, as it is less easily 

 wetted. 



The broth (and solid culture media after being melted) may 

 be poured into the tubes in the following way : A sterilized funnel 

 is united by a short length of indiarubber tubing to a piece of glass 

 tubing drawn out to a point ; the rubber tube is clipped by a 

 spring clip or a pair of pressure forceps. The funnel is now 

 mounted on a retort-stand, filled with the medium, and covered 

 over with a piece of glass. The cotton-wool plug is removed from 

 a test-tube, and the latter placed so that the glass tube attached to 

 the funnel reaches nearly to the bottom. The clip is released; and 

 the requisite quantity of broth (enough to fill the tube to the depth 

 of i| or 2 inches) is allowed to run in ; the clip is then reapplied 

 and the tube removed and plugged. This process is repeated 

 until enough tubes have been filled. 



The tubes and the broth which remains over (after having been 

 poured back into the flask and the latter plugged with cotton-wool) 

 are now sterilized. The vessels are placed in the steam sterilizer 

 and exposed to steam for half an hour on thvee successive days ; this 

 process is called intermittent sterilization, and its rationale is very 

 simple. The first steaming destroys all developed bacteria, and 

 would sterilize the fluid entirely if no spores were present. In 



* If during the neutralizing process too much alkali is added, then it is 

 necessary to reacidify with dilute hydrochloric acid and reneutralize. The 

 sodium chloride formed makes no practical difference in the medium. 



