136 . BACTERIA IN WATER 



stopper with forceps. Care must be taken not to touch the water-bed, 

 as the vegetable matter covering it contains a large number of organisms. 

 The bottles ought to be packed in ice and sawdust, and plates must be 

 prepared from the samples as soon as possible. When the object in 

 view is to determine the number of bacteria per cubic centimetre, it is 

 important to note that water bacteria grow at very varied rates, and 

 therefore it is well that the same time should always elapse before the 

 colonies are counted. The period of growing usually allowed is forty- 

 eight hours at 20 C. 



Several points may be here noted. It has been found, for instance, 

 that slight variations in the reaction of the medium affect the number 

 of colonies which develop. A slightly greater degree of alkalinity than 

 peptone gelatin, as ordinarily prepared, possesses such an increased 

 degree as that caused by the addition of '01 grm. Na 2 C0 3 to 10 c.c. 

 peptone gelatin will give a greater yield of colonies than the ordinary 

 gelatin. Again, the natural temperature of the growth of water bacteria 

 in temperate climates is comparatively low, being not often above 18 C., 

 and, on account of this, gelatin suggests itself as the most suitable 

 medium. This can be seen by comparing the growth on an agar plate 

 inoculated with a given quantity of water, and incubated at 37 C., with 

 the growth on a precisely similar gelatin plate incubated at 20 C. , as it 

 will be found that many more colonies have developed on the latter. 

 This fact may be taken advantage of when pathogenic bacteria are being 

 sought for in a water. The latter usually grow well at 37 C., and thus 

 if agar plates be used the search may be facilitated. Apart from the 

 difference of incubation temperatures, however, in such a case as that 

 cited, it is probable that agar is a less suitable medium than gelatin for 

 the growth of water bacteria, for in plates incubated at the same 

 temperature the colonies which grow on the agar are often fewer than 

 those on the gelatin. Probably no one medium will support the growth 

 of all the organisms present in a given sample of water, and under 

 certain circumstances special media must therefore be used. Thus 

 Hansen found that in testing waters to be used in brewing it was 

 advisable to have in the medium employed some sterile wort or beer, so 

 that the organisms in the test experiments should be provided with the 

 food materials which would be present in the commercial use of the water. 

 Manifestly this principle applies generally in the bacteriological examina- 

 tion of waters to be used for industrial purposes. 



In ordinary public health work it may be taken that the most 

 frequent and important inquiry is directed towards the presence or 

 absence of the b. coli and its congeners. Many methods are here used 

 but we consider that in which MacConkey's bile-salt media are employed 

 the most convenient. For small quantities of water, up to 1 c.c., the 

 sample is simply added to a Durham's tube of bile-salt glucose neutral- 

 red broth and incubated for 48 hours. When it is necessary to examine 

 larger samples it is convenient, as Savage recommends, to have the bile- 

 salt broth made of double, treble, or quadruple its usual strength. 

 The water to be examined is used as the diluent by which the medium 

 is brought down to the ordinary concentration. If gas forms, some of 

 the coli group are almost certainly present. The organisms may be 

 plated out by smearing a little of the broth on bile-salt agar for further 

 isolation and examination. 



With regard to the objects with which the bacteriological 



