1 82 MILK AND THE PUBLIC HEALTH 



CHAP. 



L.B.A. plate. Distribute with a sterile bent glass rod 

 uniformly over the plate. The L.B.A. plate will be rather 

 wet from condensed water on the surface. To dry, incubate 

 it for 1^ to 2 hours uncovered in the 37 C. incubator. 

 Then cover and invert. Incubate for 24 hours at 37 C. 



B. coli and other lactose fermenters grow as red colonies, 

 colour the surrounding medium red, and many of them pro- 

 duce a haze round the colony. Several of the typical red 

 colonies are picked off, subcultivated, and their characters 

 determined as described below. 



Another procedure for the isolation of B. coli and its allies 

 from milk samples is to directly plate the diluted milk on solid 

 media. Lactose litmus agar, L.B.A., and aesculin agar may be 

 used for this purpose. Aesculin media were introduced by 

 Harrison and Leek. 1 They prepared an aesculin broth and 

 an aesculin agar. 



The aesculin agar is made as follows : 



Ten grammes Witte's peptone, 5 grammes sodium taurocholate 

 (commercial), 1 gramme aesculin, 0'5 gramme ferric citrate, 15 

 grammes agar, tap-water 1 litre. The agar and other ingredients 

 are dissolved in the ordinary way, boiled, filtered, tubed, and 

 sterilised. 



Colonies of B. coli in this medium are black with a black 

 halo, and can be readily counted against a suitable back- 

 ground. The aesculin (a glucoside) combines with the iron 

 citrate and forms a dark-brown salt, the reaction only taking 

 place in sugar-free media. 



The colonies of some other organisms give the reac- 

 tion, notably B. lactis aerogenes. Harrison and Leek say that 

 with care the characters of the colonies of this organism 

 can be distinguished from those of B. coli, being larger, 

 moister, and more raised. These investigators recommend 

 that the diluted milk (dilutions 1:100 and 1:500 being used), 

 and the aesculin agar be mixed together in the Petri dish, 

 allowed to set and incubated at 37 C. The plates are 

 counted after 24 hours, using a white background. To 

 accurately differentiate B. coli and B. aerogenes, they prefer to 

 leave the plates longer in the incubator. By this method 



1 CentrallL f. Bakt. Abt. II., 1909, xxii. p. 55 ; and Amer. Journ. of Public 

 Hygiene, 1908, p. 431. 



