126 A MANUAL OF BACTERIOLOGY 



3. Gelatin and agar media. Media which contain gelatin should 

 not be heated at high temperatures in the autoclave nor for longer 

 periods than absolutely necessary in the Arnold sterilizer. An excess 

 of heat breaks down the gelatin so that it will not harden when cool. 

 Flasks and tubes of gelatin media should be chilled as quickly as 

 possible after removal from the stetilizer. After the gelatin has 

 hardened, the vessels may be put into the locker at room temperature, 

 to await the next sterilization. The temperature at which it is kept 

 should be high enough to permit the germination of such resistant 

 spores as may have survived heating. When three successive steriliza- 

 tions are given at twenty-four-hour intervals, the first heating may be 

 twenty minutes, the second fifteen minutes, and the third ten minutes. 

 After the last heating the media may be put into the locker and held 

 for a few days, during which time they are closely watched for colo- 

 nies. Such tubes as show colonies should then be discarded. Gelatin 

 media may be sterilized in the autoclave for fifteen minutes, provided 

 the pressure does not rise higher than five pounds and the vessels 

 containing them, when removed, are plunged at once into cold water. 



One source of difficulty in sterilizing media containing gelatin 

 comes from the spores of microorganisms which may have developed 

 during the preparation of the gelatin itself while in the vats or dry- 

 ing house of the manufacturer. Only the best grade of gelatin 

 should be used for bacteriological work. Another frequent cause of 

 failure in sterilizing media % is due to the presence of resistant spores 

 in beef extract. These spores develop in the meat juice during the 

 process of preparation and evaporation. It is better, when making 

 gelatin media, to make the meat extract yourself, according to direc- 

 tions given in Exercise 8. If, however, it is necessary to use com- 

 mercial beef extract, make the bouillon first and sterilize it in the 

 autoclave ; then add the gelatin and carry on subsequent operations 

 in the Arnold sterilizer. 



Agar media are not likely to present much difficulty in the way of 

 sterilization so far as the agar itself is concerned, although it is not 

 advisable to use more heat than necessary. If agar media fail to 

 harden after cooling, it is probably due to an excess of sugar or of 

 acid. In making media from fruit decoctions this is an important 

 precaution. If it is found that the acidity of the agar medium is 

 excessive, the medium should be divided into two equal parts. Bring 

 one portion to approximate neutrality with sodium-hydrate solution ; 

 then recombine the two portions and heat for a short time, with 



