APPENDIX 165 



The presence of fermenting organisms, including B. coli, 

 is shown when the medium turns red (due to acid production) 

 and gas is formed in the inner tube. 



Aescidin Agar (Harrison and Leek 1 ): 10 grammes Witte's 

 peptone, 5 grammes sodium taurocholate (commercial), I gramme 

 aesculin, 0*5 gramme ferric citrate, 15 grammes agar, tap-water 

 I litre. The agar and other ingredients are dissolved in the 

 ordinary way, boiled, filtered, tubed, and sterilized. 



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

 and can be readily counted against a suitable background. 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 reaction, 

 notably B. lactis aerogenes. 



Neutral Red Glucose Broth. To ordinary broth made from 

 Liebig's Extract, peptone and sodium chloride, and made faintly 

 alkaline to litmus, 0*5 per cent, of glucose and 12 c.c. per litre 

 of a O'5 per cent, freshly-made watery solution of neutral red 

 (Grubler's) are added. The solution, after preliminary steaming, 

 is filtered and tubed ordinary or double tubes and sterilized 

 for three successive days for thirty minutes each day. 



Malachite Green Agar (as prepared by Lentz and Tietz). 

 Three pounds of fat-free ox flesh are finely cut up and macerated 

 with 2 litres of water for sixteen hours. The extract is expressed 

 boiled for half an hour, filtered, 3 per cent, agar added, and the 

 mixture boiled for three hours. Then are added I per cent, 

 peptone, 0*5 per cent, sodium chloride, and I per cent, nutrose in 

 240 c.c. of cold water (the nutrose may be omitted). This 

 mixture is brought to the litmus neutral point by soda solution 

 with duplitest paper, then boiled for one hour and filtered through 

 linen. The reaction of the finished agar is sometimes distinctly 

 acid. It is filtered into small flasks of 100 to 200 c.c., and 

 sterilized three times before use in the usual way. Before the 

 addition of the malachite green, the hot agar is tested by 



1 Centralbl. f. Bakt. n. Abt. 1909, xxn, p. 55. 



