100 GUY W. CLARK 



dicator. The titration tube and comparator of Hurwitz, Meyer and 

 Ostenberg (1915, 1916) have been found very convenient, using 3 to 5 

 cc. of media and 0.05 normal NaOH. From the results of this titration 

 is calculated the amount of 2 normal NaOH necessary to give the 

 desired pH to the entire lot of medium. After heating to boiling the 

 reaction is again determined and more precisely adjusted. 



Now add 25 to 30 grams of agar which has been melted in 250 cc. of 

 distilled water (soak the finely chopped agar in the water and auto- 

 clave just long enough to melt it before adding to the infusion). Fi- 

 nally add 5 grams of glucose, mix thoroughly and distribute into desired 

 containers. 



Autoclave tubes at 15 pounds for twenty minutes, being sure to dis- 

 place the air in the autoclave. Before slanting, rotate the tubes to 

 make the contents uniform. 



The procedure just outlined differs essentially from that of 

 Hall (1916) in the following points: 



1. The medium is titrated and adjusted to a definite hydrogen 

 ion concentration instead of the old method of titrating to "de- 

 grees of acidity" with phenolphthalein as an indicator. Several 

 writers (Hurwitz, Meyer and Ostenberg, 1915, 1916; Clark, 1915) 

 have recently showm that the last method of adjusting the reac- 

 tion of culture media is not a reliable procedure. For a detailed 

 discussion of this subject reference is made to the article by 

 Clark (1915). 



2. The glucose is added last. This is done in order to mini- 

 mize the caramelizing effects of a hot, alkaline solution. Even 

 though the final concentration of hydroxyl ions is small, consid- 

 erable caramelization may result when strong NaOH is added in 

 adjusting the reaction. 



3. Reduction of the time of sterilization which is of general 

 value to all media and especially those containing carbohydrates. 

 Twenty minutes was found to be the minimum for different sized 

 tubes of this medium. Now and then a lot came through grossly 

 contaminated which is to be attributed to infected testicles. On 

 one occasion a sporulating organism v/as encountered which 

 resisted two of the twenty minute periods in the autoclave. Since 

 it is not easy to detect these infected testicles it seems better to 

 make up small lots (not more than 5 to 7 liters) of this medium. 



