266 APPENDIX 



prepared in the usual way shall not soften when kept at 

 a temperature of 25 C. 



Commercial agar in threads shall be of as high a grade 

 as can be obtained. Agar may be purified by washing. 



The various sugars, such as dextr se, lactose, and sac- 

 charose, shall be as nearly as possible the chemically pure 

 compounds designated. Unusual effort to obtain such 

 sugars is considered to be necessary. 



Glycerine shall be double distilled. 



In place of litmus, a i per cent aqueous solution of 

 Kahlbaum's azolitmin may be used. 



Of the various other ingredients used, nearly all of which 

 are of a mineral nature, special effort shall be made to 

 see that they are chemically pure products within the 

 full meaning of this expression. 



STERILIZATION 



Sterilization in the autoclave seems to be preferable to 

 that in flowing steam. Both in the lowering of the melting 

 point of gelatin and in the breaking down of sugar media 

 the time of sterilization has a greater effect than does the 

 temperature within the standard limits. It is, therefore, 

 suggested that small containers be used and that media 

 be sterilized in the autoclave at 120 C. (15 pounds pressure) 

 for 15 minutes. A shorter period than this, in practice, 

 sometimes results in incomplete sterilization, and a longer 

 time results in the inversion of sugars or the lowering of 

 the melting point of gelatin. Agar media should be melted 

 before placing in the autoclave. 



An important point in the sterilization of gelatin and 

 sugar media is to have the sterilizer hot when the media 

 are introduced, so that heating to the point of sterilization 

 will be accomplished as quickly as possible. Also when 

 sterilization is complete the media should be cooled rapidly. 

 This not only reduces the time of heating, thus preserving 



