ISOLATION OF BACILLUS COLI 591 



constituents are dissolved by heating ; the mixture is filtered, and 

 after filtration sufficient neutral litmus solution is added to give a 

 distinct colour. The medium is then distributed into Durham's 

 fermentation-tubes and sterilised by steaming for twenty minutes 

 on three successive days. The medium may be put up in various 

 sized tubes, a measured volume in each e.g. 10 c.c., 20 c.c., 25 c.c., 

 etc., according to the quantity of water which is to be added. For 

 the larger quantities the medium may be made double the above 

 strength. The inoculated tubes are incubated at 42 C. for forty- 

 eight hours. The B. coli reddens and ferments both the glucose 

 and lactose media, so that gas collects in the fermentation tube. 



(3) Neutral-red broth (Hunter, Makgill, Savage). The dye known 

 as neutral-red (Griibler's) is reduced by the action of the B. coli, 

 the colour changing to a canary yellow, accompanied by a green 

 fluorescence. The B. enteritidis (Gartner) also reduces neutral- 

 red, but the B. typhosus does not do so, nor do streptococci, 

 B. pyocyaneus, and Vibrio cholerce. Some anaerobes also possess 

 a reducing action. Glucose agar or broth (0-5 per cent, of glucose) 

 is employed, and to every 10 c.c. of the medium 0-1 c.c. of a 0-5 

 per cent, aqueous solution of neutral-red is added. Savage recom- 

 mends the following procedure : 10 c.c. of the water are added to 

 a 10 c.c. tube of neutral-red broth ; also to 40 c.c. of the water 

 contained in a bottle or flask a 10 c.c. tube of the broth of quadruple 

 strength is added. Both are incubated at 37 C., and examined 

 daily up to eight days. If reduction occurs, B. coli is almost 

 certainly present in the water ; if reduction does not occur its 

 presence is highly improbable. 



(4) Glucose formate broth (Pakes). To ordinary meat infusion 

 1 per cent, peptone, 0-5 per cent, sodium chloride, 2 per cent, glucose, 

 and 0-4 per cent, sodium formate are added. When these have 

 been dissolved by heating, the medium is neutralised (indicator, 

 litmus), and after neutralisation 2 c.c. of normal caustic soda solu- 

 tion per litre are added ; the broth is then steamed for twenty 

 minutes, filtered, and distributed into test-tubes, 10 c.c. in each, 

 which are steamed for twenty minutes on each of three successive 

 days. These tubes are inoculated with the water, and incubated 

 anaerobically at 42 C. for twenty-four to seventy-two hours. Tubes 

 showing any growth at the end of twenty-four, forty-eight, or 

 seventy-two hours are removed and examined microscopically and 

 by plating. 



(5) Bile-salt lactose agar (MacConkey). This medium is prepared 

 by adding to 1000 c.c. of tap-water in a flask 2 per cent, of peptone, 

 0-5 per cent, of sodium taurocholate, and 1-5 per cent, of agar. 



