, 114 



3. Teague has shown that a 10 percent solution of crystalline sodium 

 sulphite can be heated for twenty minutes at 15 pounds pressure with prac- 

 tically no change, and that the 10 percent sodium sulphite solution covered 

 with a layer of liquid petroleum about one cm. thick and sterilized in the 

 autoclave can be kept at room temperature for three weeks and probably 

 much longer with but very slight change. 



4. One and eight-tenths c. c. of fuchsin solution is added per liter 

 to the agar. After this has been done the sodium sulphite solution is 

 added gradually until the hot agar is almost decolorized usually about 

 25 c. c. to the liter. A pale rose color should be present in the hot agar, 

 which fades to a very faint pink on cooling; 10 grams of lactose is dis- 

 solved in a little water, filtered and added to each liter. 



Various fuchsin solutions may differ and the absolute quantities given 

 above may not be exactly the proper balance in separate lots. These are 

 approximate, however, and the proper balance can easily be attained by a 

 little preliminary testing in which sodium sulphite solution is added to 

 small quantities of fuchsin solution in a test-tube. 



The finished product is poured into large sterile Petri dishes. The 

 cover is left off until the agar is hard. Smears are made on these plates. 



It is helpful to lay a piece of filter paper into the lid of the petri plate 

 in order to absorb liquid evaporating from the agar in the incubator. If 

 there is not enough filter paper for this, the plate should be placed up- 

 side down in the incubator. 



(d) Endo's Medium (Kendal's Modification). (1) Preparation 

 of Agar. (a) Prepare plain, sugar-free nutrient agar, using 15 grams of 

 agar per liter. 



(b) Adjust the reaction to a point just alkaline to litmus. 



(c) Flask the agar, 100 c. c. to a flask, and sterilize in the autoclave. 



(2) Preparation of Indicator (a) Prepare a 10 percent solu- 

 tion of basic fuchsin in 96 percent alcohol. This solution is fairly stable 

 if kept away from light. 



(b) Prepare a 10 percent aqueous solution of chemically pure anhy- 

 drous sodium sulphite (1 gram in 10 c. c. water). This solution does not 

 keep. 



(c) Add 1 c. c. of "2, a" to 10 c. c. of "2, b" and heat in the 

 Arnold sterilizer for 20 minutes. The color of the fuchsin is nearly dis- 

 charged if the solutions are of proper strength. This solution must be 

 prepared each day it does not keep. 



(3.) Preparation and Use of Endo medium (a) Add 1 gram 

 of C. P. lactose (free from dextrose) to 100 c. c. of agar and place in the 

 autoclave until melted and the lactose is thoroughly dissolved. 



(b) Add a sufficient volume of "2, c" (about 1 c. c.) to impart a 

 faint pink color to the medium. 



(c) Pour into sterile Petri dishes and allow to harden in a dark place 

 with the covers partly removed. When cool the medium should be color- 

 less when viewed from above and a very faint pink when viewed from the 



