230 MICROSCOPIC AND CULTURAL STUDY OF BACTERIA 
(6) Adjust the reaction to a point just alkaline to litmus. 
(c) Flask the agar, 100 cc. 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. 
(b) Prepare a 10 per cent aqueous solution of chemically pure 
anhydrous sodium sulphite (1 gm. in 10 cc. water). This solution 
does not keep. 
(c) Add 1 cc. of "II, a" to 10 cc. of "11, 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 Endo Medium. — (a) Add 1 gm. of 
C. P. lactose (free from glucose) to 100 cc. of agar and place in the 
autoclave until melted and the lactose is thoroughly dissolved. 
(b) Add a sufficient volume of 'TI, c" (about 1 cc.) 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 should be remembered that 
aldehyde is bound by the sulphite. Non-lactose fermenting bacteria 
grow as colorless colonies. 
If the plates are to be incubated two or three days it may be advis- 
able to increase the agar to 2.5 per cent to limit the diffusion of color 
from the acid colonies. For rapid isolations the medium with the 
normal percentage of agar is preferable.^ 
The technique of inoculation of Endo media is described on page 262. 
Andrade Indicator.^— The Andrade indicator, prepared from acid 
fuchsin exactly decolorized with XaOH, is a better indicator than the 
Endo indicator. The latter turns red upon exposure to light and it 
slowly turns red even when kept in the dark. Andrade indicator is 
adA'antageously substituted for the Endo indicator for all work involv- 
ing the formation of acids of the fermentation type by bacteria. 
Preparation of Andrade Indicator.— An 0.5 per cent aqueous solu- 
tion of acid fuchsin is carefully treated with caustic soda solution until 
the color is just discharged. One per cent of this solution is added to 
the medium just before using. The indicator may be sterilized with 
Kendall and Day: Jour. Med. Res., 1911, 25, 95. 
Jour. Med. Res., 1906, 14, 551. 
