BACTERIA AND RURAL WATER SUPPLIES 7 



water sample is to inoculate a series of tubes with one milliliter of water each 

 and another series with ten milliliters each (1,000 milliliters = about one quart). 

 Gas formation from the one-milliliter inoculations is considered an indication of a 

 more serious degree of pollution than that in tubes receiving ten milliliters each. 

 If a water is suspected of being very badly polluted, a series of tubes may be 

 inoculated with one-tenth of a milliliter each. 



The appearance of gas in the fermentation tubes is termed a "presumptive 

 test," and in itself is not sufficient evidence that the water contains bacteria of 

 the coliform group. There are some other species of bacteria, not indicative of 

 sewage pollution, that may form gas from lactose; and it sometimes happens 

 that two species of bacteria acting together (symbiosis) may produce gas from 

 lactose where neither alone could do it. If gas is produced under either of these 

 conditions, the result is termed a false presumptive test. Consequently, a posi- 

 tive gas test must be confirmed. 



The ne.xt step is to remove, with a sterilized wire loop, some of the fluid from 

 fermentation tubes showing gas and streak it on a differential solid culture 

 medium on which coliform bacteria will give characteristic growth. The two 

 media most used for that purpose in this country are Endo's agar and Eosin- 

 methylene blue agar. The agar in these media gives them a solid base, and both 

 contain proper food materials to support the growth of bacteria. These media 

 also contain dyes that prevent the growth of unimportant bacteria and cause the 

 growth of coliform bacteria to assume a characteristic appearance. One or the 

 other of these media is used, not both at the same time. 



If no growth occurs on the differential solid medium, it can be assumed that 

 gas in the lactose-broth tube was not caused by coliform bacteria and no pollution 

 is indicated. If, however, there is growth on the solid medium, the test must be 

 further confirmed. A sterilized wire loop is employed again, and some of the 

 bacterial growth is taken from the differential medium and put into fermentation 

 tubes identical with those used for the preliminary test. It sometimes happens 

 that the solid medium will produce growth that appears to be coliform bacteria, 

 and yet no gas will be produced when it is put again into lactose broth. Usually, 

 however, it is the non-characteristic growth that will fail to produce gas in the 

 refermentation tests. On the other hand, non-characteristic growth not In- 

 frequently is confirmed as coliform bacteria. 



In the e.xamination of municipal water supplies, the "Standard Methods" pro- 

 cedure does not require confirmation if gas appears within the first 24 hours of 

 incubation. However, experience in our laboratory in testing water from privately 

 owned rural supplies has indicated the desirability of confirming all positive pre- 

 sumptive tests from such waters. 



Differentiation of Coliform Bacteria 



The coliform group of bacteria is made up of a number of similar and closely 

 related species. We may think of them, perhaps, as being arranged in a line. 

 At one end of the line is the colon bacillus {Escherichia coli) and at the other end 

 is the aerogenes bacillus (Aerobacter aerogenes). In between is a miscellaneous 

 lot of species known as intermediates of the group. Some of these will closely re- 

 semble Escherichia coli, some the Aerohacter aerogenes, and some will seem to be 

 somewhere between these two. All of these species behave alike with bacteriologi- 

 cal stains, and they are indistinguishable from one another when viewed under 

 the microscope. 



