860 THE BACTERIOLOGICAL EXAMINATION OF WATER 



2. Sow 10 c.c. into 10 c.c. of MacConkey's double strength fluid and a 

 second 10 c.c. into 50 c.c. of milk being careful that the water passes beneath 

 the cream. 



3. Sow 1 c.c. into a tube of MacConkey's medium (ordinary strength) ; a 

 second 1 c.c. into 10 c.c. of milk ; a third 1 c.c. into broth and a fourth into 

 9 c.c. of sterile water. 



4. Thoroughly mix the last tube in which the water under examination 

 has been added to sterile water. Sow 1 c.c. of this mixture representing 

 0*1 c.c. of the water into MacConkey's fluid, into gelatin and into agar 

 (melted and cooled to 40 C.) and a further 1 c.c. into a second tube of sterile 

 water (9 c.c.). 



5. After mixing use 1 c.c. of the diluted water equivalent to O'Ol c.c. of 

 the water for sowing MacConkey's medium, gelatin and agar as before ; 

 and if necessary proceed to further dilution. 



6. Pour the agar and gelatin into Petri dishes. Incubate the cultures at 

 37 C. with the exception of the gelatin plates which must be grown at 22 C. 

 The milk tubes must be heated first at 80 C. for 15 minutes and then be 

 grown anaerobically in a Bulloch's apparatus (p. 96). 



The plates must be examined daily and the colonies counted as described 

 above. The other cultures are examined after 48 hours' incubation : 



i. The broth tubes for streptococci by examining the deposit micro- 

 scopically. 



ii. The milk tubes for the " enteritidis change" considerable formation 

 of gas, an odour of butyric acid, separation of the curd from the whey and 

 tearing up of the curd by the gas evolved. 



iii. The MacConkey's tubes for the presence of gas. 



If gas is formed a loopful of the culture is diluted in sterile water and one 

 or two loopsful of the dilution used for sowing a surface culture of MacConkey's 

 taurocholate-lactose-agar for the purpose of isolation. The agar is incubated 

 at 37 C. and after 24 or 48 hours is examined for colon-like colonies. If 

 such colonies be found one or more is sown in a tube or tubes of liquefied 

 glucose-gelatin and the tubes incubated at 20 C. If gas be formed the 

 gelatin is liquefied and the culture used for sowing the following media : 



1. Neutral-red broth. 



1 per cent, solution of neutral-red, 2 c.c. 



Broth, ... . 1000 



2. Peptone water. 



3. Litmus milk. 



These tubes are incubated at 37 C. and then examined respectively for 



i. Fluorescence in the neutral-red-broth. 



ii. Indol in peptone water (Ehrlich's test, p. 374 e). 



iii. Acid and clot in milk. 



If an organism which gives all the reactions described has been recovered 

 from any of the MacConkey tubes the water is said to contain " typical colon 

 bacilli " l in that amount. Thus if an organism having these characteristics 

 be isolated from the 100 c.c. tube but not from the 10 c.c., 1 c.c. or O'l c.c. tubes 

 the water is said to contain typical colon bacilli in 100 c.c, but not in less. 

 Similarly a water from 1 c.c. of which a typical colon bacillus was isolated 

 but not from O'l c.c. is said to contain at least 1 but not 10 colon bacilli 

 per c.c. 



1 Such an organism is described by Houston as a " Flaginac " colon bacillus: Fl, 

 fluorescence in neutral-red-broth ; ag, acid and gas in a lactose medium ; in, indol in 

 peptone water ; ac, acid and clot in milk. 



