ISOLATION OF INDICATOR ORGANISMS 23 



equivalent amounts, are added to tubes of sterile whole milk, 

 recently boiled. The destruction of all but spores is ensured 

 by heating to 80 C. and maintaining at that temperature for 

 ten minutes. The milk tubes are then cooled and incubated under 

 anaerobic conditions. The most convenient method of doing 

 this is to employ the pyrogallic acid and caustic potash method. 

 The milk tubes are placed in jars or larger tubes containing 

 pyrogallic acid in powder to which is added, when all is in place, 

 a little 10 per cent, caustic potash solution. The tubes or jars 

 must then be immediately closed by tightly fitting stoppers. The 

 alkaline mixture absorbs the oxygen. The numerical estimation 

 is arrived at by recording the tubes, which show after incubation 

 the characteristic " enteritidis change," as containing the spores 

 of this bacillus while those which do not show it are recorded as 

 negative. 



As a rule no attempt is made by workers to isolate the 

 bacillus in pure culture or to definitely prove its presence by 

 animal inoculation. The estimation is therefore not really one 

 of a definite bacillus but that of a group of closely allied bacilli 

 which, as far as is known, have a very similar distribution in 

 nature and a very similar significance. 



The estimation as carried out is a fairly simple one and 

 involves no great variations in the hands of individual workers. 

 There are two points in connection with the test which require 

 further consideration. Both deal with the question of the 

 amounts to examine. 



It is easy to handle O'l, I and 10 c.c. of the liquid under 

 examination (water, milk, etc.), but when larger amounts have 

 to be examined, as is necessary with water, some method of 

 concentration has to be practised. These concentration of 

 bacteria methods have been mentioned under Streptococci 

 and B, coli, but are more conveniently considered here. 



To obtain the bacteria in a convenient bulk of fluid it is 

 possible to evaporate off the water at a low temperature under 

 reduced pressure, as in the method for B. typJiosus in water 

 described in Chapter III, but this is a procedure very rarely 

 employed. The usual plan is to filter the water through a 

 sterile porcelain filter, the water passing through and leaving 



