158 BACTERIA IN WATER 



2-ouuce cylindrical mediciue bottles, 4 in. high by 1 in. in diameter. 

 The medium, along with the inverted test-tube, is placed in these ; 

 rubber stoppers are inserted in the mouths, and they are sterilised. It is 

 customary to test for the presence of the organisms in any sample by 

 adding to a series of such tubes the following quantities of the, water 

 50 c.c., 20c. c., 10 c.c., 5 c.c., 1 c.c., and, it may be, in specially sus- 

 picious waters, '5 c.c., -1 c.c., and even -01 c.c. The result is estimated 

 in terms of the smallest amount of water with which the occurrence of 

 acid and gas formation is observed. By starting with a. concentrated 

 MacConkey's mixture, it is arranged that, when the sample is added, the 

 resulting fluid shall be of the concentration of MacConkey's medium as 

 ordinarily prepared. Thus, in the bottle to which the 50 c.c. sample is 

 to be added, there are placed 10 c.c. of a sixfold concentration of 

 MacConkey's medium. In the 20 c.c. tube, there are present 20 c.c. of a 

 medium of double strength ; in the 10 c.c. tube, 10 c.c. of a mixture 

 of double strength ; and in the 5 c.c. tube, 5 c.c. of a mixture of double 

 strength. With smaller samples, we simply use the ordinary MacConkey's 

 medium. 



For the taking of the samples, sterile 8-ounce stoppered bottles are 

 convenient, and for each sample it is necessary to have sterile 25 c.c., 

 10 c.c. (graduated to tenths), and 1 c.c. (graduated to hundredths) 

 pipettes. The armamentarium being thus simple, there is. no difficulty 

 in carrying out the necessary manipulations at the spot where the sample 

 is collected. 



The tubes are incubated for forty-eight hours, and it is well to read the 

 results at the end of the first twenty-four hours also. The formation of 

 acid and gas in the tube is usually recognised as " presumptive evidence " 

 of the presence of members of the b. coli group, but it is necessary to 

 further investigate the bacteria giving rise to this change to determine 

 whether they are "typical" or "atypical" b. coli. With this end in 

 view, each bottle in which acid and gas is present is well shaken up, two 

 or three loopfuls are placed on a plate of MacConkey's neutral-red bile-salt 

 lactose agar. These loopfuls are spread over the surface by means of a 

 sterile spreader, made by taking a piece of glass rod and turning a portion 

 about 2 inches long at right angles to the shaft. The plates are incubated 

 for twenty-four hours. As typical b. coli produces acid in lactose, any 

 colonies of such an organism are of a rosy red colour. These are then 

 picked off, sloped agar tubes are inoculated and used for the further 

 investigation of the properties of the bacterium isolated. 



The media inoculated should be gelatin stab, litmus milk, neutral-red 

 lactose bouillon, glucose broth, peptone water, dulcite peptone water, 

 adonite peptone water, inuline peptone water, saccharose peptone water, 

 and potato. 



It is well in dealing with the neutral-red lactose agar plates to inoculate 

 a lactose peptone water tube from all the kinds of colonies present, 

 whether these are red or not, as MacConkey rightly points out that some- 

 times an organism which is really a lactose fermenter does not produce 

 a red colour on the solid medium. There is another point to be noted 

 here, namely, that the naked-eye appearances of colonies on lactose agar 

 are not of value in identifying the kind of organism present. 



The object of growing suspicious colonies on a range of media such as 

 that given, is to enable typical b. coli to be recognised when present. At 

 the present time it cannot be said that bacteriologists are in agreement 

 as to what characters determine the type of organism most frequently 

 found in the human intestine this, of course, being the important point 



