BACILLUS TYPHOSUS 523 



a. Preparation of agar: 1500 grams of finely chopped beef 

 are placed in two liters of water and set aside for twenty- 

 four hours. This meat infusion is then boiled for one hour, 

 filtered, and 20 grams of Witte's peptone, 20 grams of 

 nutrose, and 10 grams of sodium chloride are added and 

 again boiled for an hour, filtered, and 60 grams of agar-agar 

 are added, boiled for three hours (or one hour in the auto- 

 clave), rendered slightly alkaline to litmus paper, filtered, 

 and boiled for one-half hour. 



b. Litmus solution: (Litmus solution according to Kubel 

 and Tiemann) 260 c.c., boil ten minutes, add 30 grams 

 chemically pure lactose, boil fifteen minutes. 



c. The hot litmus-lactose solution is added to the hot 

 nutritive agar, thoroughly mixed, and the alkaline reaction 

 is again restored. To this medium is then added 4 c.c. of 

 a hot sterile solution of 10 per cent, water-free sodium 

 carbonate, 20 c.c. of freshly prepared solution of 0.1 gram 

 crystal violet (Hochst) in 100 c.c. of warm sterile distilled 

 water. 



One now has a meat-infusion-peptone-nutrose-agar with 

 13 per cent, of litmus solution and 0.01 per thousand crys- 

 tal violet. It becomes very hard on solidifying, without 

 becoming too dry. Plates are poured of this material and 

 held in readiness for some time, and the remainder of the 

 medium is preserved in flasks in portions of 200 c.c. each. 



If the lactose is boiled for a longer time than directed 

 it is reduced, with an acid reaction of the culture medium, 

 and the content in lactose falls below the required quantity, 

 and the alteration in the color of the colon colonies appears 

 too early. For this reason it is also necessary to liquefy 

 the agar as quickly as possible in pouring plates from the 

 agar medium stored in flasks. 



