1 8 ISOLATION OF INDICATOR ORGANISMS 



The use of tubes of liquid media involves two possibilities of 

 considerable error. One is that the estimations obtained only 

 give very widely spaced results. For example, with water samples, 

 O'l, I, 10 and 100 c.c. are the amounts usually selected for exami- 

 nation, and according to the positive findings the number of B. coll 

 organisms will be 10,000, 1000, 100, 10, etc. to the litre. There is 

 obviously a considerable spacing between these results. This 

 wide spacing can be largely obviated by the use of more 

 tubes but this greatly increases the labour and therefore in 

 practice this method of diminishing the possible error is limited. 

 The other objection is that bacteria are by no means uniformly 

 distributed and if the bacilli under examination happen to be 

 present in the smaller amounts used for examination, misleading 

 results may be obtained and a much greater prevalence recorded 

 than is warranted by the facts. 



This second objection also largely applies to solid media 

 enumeration methods, while these methods have several other 

 decided drawbacks of their own. One of the chief of them 

 is that they enable small quantities (i.e. O'l to i c.c.) only of 

 the material under examination to be dealt with. It is not 

 very convenient to concentrate the bacteria into a smaller bulk, 

 by filtration or other method, so that this method is not suitable 

 for many substances. 



A further decided objection to their use is that they give 

 unreliable colony differentiation. Theoretically the medium 

 used should clearly differentiate the B. coli group colonies from 

 the rest of the bacteria by their being coloured red or otherwise 

 distinguished ; in practice there are many intermediate colonies 

 which it is not possible to say, without further cultural differ- 

 entiation, whether they belong to the B. coli group or not. In 

 particular if the plates are crowded true B. coli group bacilli will 

 frequently not develope into characteristic colonies until after 

 several days so that there is great danger of their being un- 

 enumerated as B. coli colonies. The presence of these indeter- 

 minate colonies seriously reduces the accuracy and value of the 

 direct plating method and makes the results obtained dependent 

 to some extent upon the recorder. From extended comparative 

 work with the examination of milk and other substances the 



