113 



6. Sterilize in the autoclave at 15 pounds (120 C.) for 15 minutes 

 after the pressure reaches 15 pounds. 



7. Prepare a 10 percent solution of basic fuchsin in 95 percent 

 alcohol, allow to stand 20 hours, decant and filter the supernatant fluid. 

 This is a stock solution. 



8. When ready to make plates melt 100 c. c. of agar in streaming 

 steam or on a waterbath. Dissolve 1 gram of lactose in 15 c. c. of dis- 

 tilled water, using heat if necessary. Dissolve 0.25 gram anhydrous sodium 

 sulphite in 10 c. c. water. To the sulphite solution add 0.5 c. c. of the 

 fuchsin stock solution. Add the fuchsin-sulphite solution to the lactose 

 solution and then add the resulting solution to the melted agar. The lac- 

 tose used must be chemically pure and the sulphite solution must be made 

 up fresh. 



9. Pour plates and allow to harden thoroughly in the incubator be- 

 fore use. 



(b) Endo's Medium (Hygienic Laboratory Modification). 1. The 

 Hygienic Laboratory-Endo medium consists of a 3 percent agar which is 

 titrated and corrected to +0.5 to phenolphthalein, to which is added 3.7 

 cubic centimeters of a 10 percent solution of anhydrous sodium carbonate 

 per liter. For convenience it is flasked, sterilized, and stored in 200 cubic 

 centimeter quantities. When ready to use, the following ingredients are 

 added to 200 cubic centimeters of agar as follows: 



2. Dissolve 2 grams C. P. lactose in 25 to 30 cubic centimeters of 

 distilled water, with the aid of gentle heat. 



3. Dissolve 0.5 gram of anhydrous sodium sulphite in 10 to 15 cubic 

 centimeters of distilled water. 



4. To the sulphite solution add 1 cubic centimeter of saturated so- 

 lution of basic fuchsin in 95 percent alcohol. 



5. Add the fuchsin-sulphite solution to the lactose solution, and 

 then add the whole to the agar. Pour plates at once and, after hardening, 

 dry for 15 minutes in the incubator. 



(c) Endo's Medium (Med. Dept. U. S. Army Modification). 



1. Into a container put 1 liter of tap water, marking the level of the 

 fluid. Add 30 grams of thread agar, 10 grams of peptone, 5 grams of NaCl, 5 

 grams of beef extract. Cook until dissolved it is best to autoclave thirty 

 minutes at 15 pounds; filter through sterile gauze or cotton. If necessary 

 clear with egg. For this purpose, for each liter beat up the white of one 

 egg with 10 c. c. of warm water until the egg is well mixed. Add this to 

 agar cooled to 55 C., mix thoroughly, heat for 30 minutes or autoclave and 

 filter through cotton. 



2. This stock agar is kept on hand in quarter-liter flasks or bottles. 

 Agar is standardized just before use and reaction adjusted to 0.2 percent 

 acid to phenolphthalein. Before use, fuchsin and sodium sulphite are 

 added. A filtered, saturated solution of basic fuchsin in 95 percent alcohol 

 is kept on hand. A 10 percent solution of dry sodium sulphite crystals 

 in sterile water is freshly made. 



