8 MASS. EXPERIMENT STATION BULLETIN 414 



If all of these species had the same sanitary significance, it would be unnecessary 

 to go further than to identify them as coliform bacteria. It is the opinion of 

 many who have worked with these bacteria that the Escherichia coli is the pri- 

 mary indicator of sewage contamination of water, and that Aerohacler aerogenes 

 usually comes from soil and indicates pollution of water from the ground's sur- 

 face. The meaning of the intermediates is not clear and is the subject of much 

 controversy and discussion. It is natural, however, to suppose that the sanitary 

 significance of these intermediates depends upon the closeness of their relation- 

 ship to either Escherichia coli or Aerohacter aerogenes. 



To begin with, all of the coliform bacteria ferment lactose and produce gas. 

 Then, they all look alike when viewed through a microscope. When cultures of 

 these bacteria are made with media containing dyes (Endo's or eosin-methylene 

 blue agar), it is possible to make some differentiation of them. From that point 

 on, it is necessary to inoculate the cultures into media designed for the purpose 

 of making possible the separation of the species. A number of tests have been 

 devised and employed for the purpose, and four of them are now in common use. 



These four tests employed for differentiation of the group are designated by 

 the collective term of IMViC test. Each of the four capital letters indicates a 

 test and the small "i" is put in to supph' a necessary vowel for the second syllable. 

 The word is generally written "Imvic." The tests are as follows: 



I, indole test. This depends on the fact that certain of the coliform bacteria 

 will produce a chemical substance, indol, from an amino acid, tryptophane, 

 incorporated in a fluid medium. Indol can be detected by a specific color reac- 

 tion when the proper reagents are employed. 



M, methyl-red test. When certain species of bacteria are grown in a broth 

 containing dextrose (glucose), they will produce acid which is detected by adding 

 an indicator, methyl red, to the broth after incubation is completed. The indi- 

 cator range of the methyl red is such that a positive test indicates rather strong 

 acid production. This is commonly designated as the M. R. test. 



V, Voges-Proskauer test. This test depends on the ability of certain bacterial 

 species, growing in dextrose broth, to produce a chemical compound known as 

 acetyl-methyl-carbinol. This can be detected by a color reaction when the 

 proper reagents are employed. This test is termed the V. P. test. 



C, sodium citrate test. In this test a fluid medium is prepared in which sodium 

 citrate is the sole source of carbon. If bacteria inoculated into the medium can 

 utilize the carbon, growth is evident and the test is termed positive. 



The reactions of Escherichia coli and Aerohacter aerogenes to these tests are 

 shown in the following table: 



Sodium 

 Indole M.R. V.P. citrate 



Escherichia coli + + ~ "~ 



Aerohacter aerogenes — '— + -f 



AU possible combinations of these reactions may be encountered among the 

 intermediates of the coliform group of bacteria. All may be positive; all may 

 be negative; or any combination of positives and negatives may occur. One 

 group, other than E. coli and A. aerogenes, recognized by some investigators, 

 gives a negative V. P. test and is positive for the other three tests. The name 

 Citrobacter has been proposed for this group, and a close relationship is thought 



