METHODS FOR THE MICROSCOPIC STUDY OF BACTERIA 201 



Ten per cent, aqueous solutions of various sugars are prepared and 

 sterilized, and a sufficient amount of the desired sugar to make a 

 final concentration of 1 per cent, is added to the sterile serum solution. 

 Sufficient sterile 5 per cent, litmus solution is added for an indicator. 

 Fermentation of the carbohydrate is shown by the development of an 

 acid reaction, and frequently by a well-defined coagulation of the 

 medium as well. 



Endo Medium for the Isolation of Typhoid, Paratyphoid, and Dysentery 

 Bacilli. I. Preparation of Agar. (a) Prepare plain, sugar-free nutri- 

 ent agar as described on page 197, 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. 



II. Preparation of Indicator. (a) Prepare a 10 per cent, solution 

 of basic fuchsin in 96 per cent, alcohol. This solution is fairly stabile 

 if kept away from the light. 



(6) Prepare a 10 per cent, aqueous solution of chemically pure 

 anhydrous sodium sulphite (1 gram in 10 c.c. water). This solution 

 does not keep. 



(c) Add 1 c.c. of "II, a" to 10 c.c. of "II, 6" and heat in the Arnold 

 sterilizer for twenty minutes. The color of the fuchsin is nearly 

 discharged if the solutions are of proper strength. This solution must 

 be prepared each day it does not keep. 



III. Preparation and Use of Endomedium. (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. 



(6) Add a sufficient volume of "II, 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 colorless when viewed from above and a very faint pink when viewed 

 from the edge. The medium must be kept in a dark place because 

 the color is restored by the action of daylight. 



Those bacteria which ferment lactose as Bacillus coli form lactic 

 acid which restores the color of the medium in the immediate neigh- 

 borhood of the colony; the colony therefore is colored red. Some 

 aldehydes also restore the color, but it is not very probable that alde- 

 hyde production is commonly observed among the lactose-fermenting 

 organisms. Non-lactose fermenting bacteria grow as colorless colonies. 



If the plates are to be incubated two or three days it may be 

 advisable to increase the agar to 2.5 per cent, to limit the diffusion of 



