50 METHODS OF CULTIVATION OF BACTERIA 



constituents, and is boiled, filtered, and rendered neutral. It is then 

 made alkaline by the addition of 10 c.c. of a 10 per cent, solution of 

 sodium hydrate, and there are added 10 grammes of chemically pure milk 

 sugar (free from cane sugar) and 5 c.c. of a filtered saturated alcoholic 

 solution of basic fuchsin. After thorough mixing there is added 25 c.c. 

 of a freshly prepared 10 per cent, solution of sodium sulphite, the effect 

 of this step being to remove the colour of the fuchsin so that the finished 

 medium when cool is quite colourless. Of the medium 15 c.c. are placed 

 in each of a number of tubes, these are steamed for fifteen minutes and 

 must then be kept in the dark. For use the contents of a tube are 

 poured into a sterile Petri capsule, allowed to set in a still, dustless 

 atmosphere, and are then inoculated as in the other methods described. 

 After twenty-four hours' growth colonies of b. coli appear red, while those 

 of b. typhosus are colourless. Endo also claims for his medium that 

 typhoid bacilli isolated by its means are agglutinable by a typhoid 

 serum. The rationale of the colour reaction appears to be that fuchsin, 

 which is rosanilin hydrochlorate (C 20 H 19 "N3HC1), is reduced to rosanilin 

 (a colourless substance) by the sodium sulphite. This colourless base 

 produces a red colour with acids, such as the lactic acid formed by the 

 b. coli in its fermentation of lactose. 



MacConkey's Bile-Salt Media. These media were introduced for the 

 purpose of differentiating the intestinal bacteria, and have been exten- 

 sively used for the study of the b. coli, b. typhosus, b. dysenteric, etc. 

 The characteristic ingredients are bile salts and various sugars. The 

 stock solution is the following: Commercial sodium taurocholate, '5 

 gramme ; Witte's peptone, 2'0 grammes; tap water, 100 c.c. (if distilled 

 water be used, '03 per cent, of calcium chloride should be added). The 

 solution is steamed for two hours, filtered when hot, allowed to stand for 

 twenty-four hours or till sedimentation has occurred, and filtered again. For 

 a liquid medium there is added to this "25 per cent, of a freshly prepared 

 1 per cent, solution of neutral red J and the sugar, when glucose, dulcite, 

 or adonite is used, '5 per cent, is added, in the case of other sugars 

 1 per cent. The fluid is distributed in Durham's fermentation tubes and 

 sterilised in the steamer for ten minutes on two successive days, care 

 being taken not to overheat the medium. 



For bile-salt agar 1*5 to 2 per cent, agar is dissolved in the stock 

 solution in the autoclave, if necessary cleared with white of egg and 

 filtered. Neutral red and a sugar are added, as in the case of the liquid 

 medium. As with Drigalski's medium, it is well to sterilise it in flasks 

 containing 80 c.c., this being an amount sufficient for three Petri 

 capsules. When this medium is used for examining urine or faeces, 

 plates are inoculated as with Drigalski's medium (supra} ; for its use in 

 water examinations, see p. 157. 



With reference to their behaviour in MacConkey's fluid medium with 

 glucose, organisms are divided into (1) those which produce both acid and 

 gas ; (2) those producing acid only ; (3) those growing but not producing 

 either acid or gas ; (4) those incapable of growing. B. coli belongs to the 

 first group and b. typhosus to the second, and to these groups also belong 

 most ordinary organisms growing in faeces, practically none of which are 

 found in the third and fourth classes. Further, a number of ordinary non- 



1 The use of neutral red in a bile-salt medium was first introduced by 

 Griinbaum and Hume. It gives a deep crimson with acids and a yellow-brown 

 with alkalies. 



