118 



(K 2 HP0 4 ). A dilute solution of the K 2 HP0 4 should give a distinct 

 pink with phenolphthalein. 



2. Heat with occasional stirring over steam for 20 minutes. 



3. Filter through folded filter paper, cool to 20 C. and dilute to 

 1,000 c. c. with distilled water. 



4. Distribute 10 c. c. portions in sterilized test tubes. 



5. Sterilize by the intermittent method for 20 minutes on three suc- 

 cessive days. 



B. Synthetic Medium. (After Clark and Lubs). (Standard 

 Methods A. P. H. A., 1920). 



1. Na 2 HP0 4 (anhydrous) 7 grams 



or 



Na 2 HP0 4 . 2H,0 8.8 grams 



KHphthalate 2 grams 



Asparatic acid 1 gram 



Dextrose 4 grams 



Warm distilled water 800 c. c. 



2. When solution is complete, cool and make up to 1 liter at room 

 temperature. 



3. Heat in an autoclave for 15 minutes after the pressure has reached 

 15 pounds, provided the total time of exposure to heat is not more than 

 one-half hour. 



4. The hydrogen-ion concentration of the medium is fixed by the 

 composition. It should be very close to PH 7.0, slightly red with phenol 

 red. All materials should be re-crystallized or if used from stock fur- 

 nished by manufacturers, should be carefully examined. The di-sodium 

 hydrogen phosphate may be used either as the anhydrous salt obtained by 

 dessication in vacuo at 100 C. or else as the salt containing two mole- 

 cules of water of crystallization. This is obtained by exposing the re- 

 crystallized Na,HP0 4 12H,0 for two weeks. Use 0.88 percent of Na,HP0 4 - 

 2H 2 0. 



C. Uric Acid Medium (Koser) 



Distilled amrnonia-free water 1,000 c. c. 



NaCl 5.0 gm. 



MgS04 0.2 gm. 



CaCL, 0.1 gm. 



K 2 HP0 4 1.0 gm. 



Glycerol 30.1 gm. 



Uric acid 0.5 gm. 



This combination gives a colorless and clear medium. It is filled into 

 ordinary test tubes and sterilized in the autoclave at 13 to 15 pounds extra 

 pressure for 15 minutes. A slight turbidity may be apparent after auto- 

 claving, due, presumably, to a finely divided precipitate of calcium sul- 

 phate. On cooling, the solution becomes clear. 



On the addition of 1.5 percent of washed shred agar to the solution 

 mentioned in the foregoing an agar medium was obtained on which the 

 same distinction between the two types may be brought out. 



