434 S. HENRY AYERS AND PHILIP RUPP 



Solution II 

 Filtered solution of agar in distilled water 3.0 



Mix solution I and II in equal proportions while hot and put 

 up in definite amounts of 100 cc, or more, in flasks or bottles 

 and then steriHze. The 3 per cent agar solution is made up 

 separately and kept in stock merely for convenience. Agar 

 can be added directly to solution I at the time of preparation 

 if desired, using 1.5 per cent. A slight precipitate may appear 

 upon sterilization, but this does not interfere with the count 

 and may not appear on the plate. 



INDICATOR 



To indicate the acid colonies basic fuchsin is employed and 

 decolorized by sodium sulfite using the following solutions: 



Solution A. One per cent alcoholic solution of basic fuchsin. 



Solution B. Five per cent aqueous solution sodium sulfite, 

 freshly prepared. 



At the time of plating, after the agar medium is melted and while 

 at its highest temperature add for each 100 cc. of medium 0.5 

 cc. of a 1 per cent alcoholic basic fuchsin solution and follow 

 at once with 0.5 cc. of a freshly prepared 5 per cent sodium sulfite 

 solution. Mix thoroughly and allow to cool, then pour into 

 plates as usual. The red color is not entirely destroyed by the 

 sulfite and at time of plating the medium is pink. 



Purified litmus or brom cresol purple can be used as indica- 

 tors, but if more than about thirty colonies appear on the plate 

 the entire medium changes to the acid color of the indicator and 

 it then becomes impossible to detect acid colonies. 



With decolorized basic fuchsin there is a red coloration or at 

 least a deep red colony which is probably caused by the combined 

 action of acid and aldehyde as has been shown by De Bord (1917). 

 This indicator is preferred, therefore, to the two previously 

 mentioned, because the acid-forming organisms of the colon- 

 a,erogenes group produce deep red colonies which can be detected 

 when present in any countable number even when the medium 

 has become entirely red. 



