Traditionally, the identification of E. coli in environmental samples has involved the use of selective and differential media such as MacConkey, VRB and EMB agars and EC, BGLB, Lactose or other broths.  Confirmation could be through various additional biochemical tests on cultures isolated from the primary media. 

One set of biochemical tests which has been used is the IMViC series, where I=indole, M=methyl red, Vi= voges proskaur, C=citrate and the typical E. coli culture results in ++-- for these four tests, while the typical Enterobacter aerogenes gives --++ for the series.  Of the four tests in this series, it is generally conceded that the production of indole is a very useful test to confirm E. coli identification.  There are numerous published methods for the performance of the indole test.  The most commonly used is probably the isolation of the colony in question by inoculating a tube of tryptone broth, allowing growth for 48 hrs, and then adding several drops of Kovac’s reagent to the broth, agitating slightly and checking for color in the reagent that collects at the top of the broth.  A cherry red color is positive for indole, and a yellow color is negative.  There are also spot tests described in the literature.

The use of Coliscan® MF or Coliscan® Easygel eliminates the necessity of doing all of the above procedures to confirm E. coli in a sample.  Blue/purple colonies on these media are confirmatory for E. coli and indicate the production of glucuronidase and galactosidase.  If the investigator has a problem being sure whether a colony is teal green, pink (magenta) or blue/purple, we recommend they use our Confirmation Media to clear up their confusion of interpretation.  We only recommend going to the additional trouble of the indole test for persons who feel that they need reassurance of their final identification. 

However, for persons using the Coliscan® MF method, it is extremely easy to check individual colonies growing on the membrane for indole production.  This provides an additional verification and confirmation that a blue/purple colony is E. coli.  Following are the instructions for doing this test.

1.  Choose a colony growing on the membrane filter and add a small drop of Kovac’s reagent on or at the edge of the colony.  (Use a small wire or plastic loop, or a new toothpick to pick up the Kovac’s reagent and transfer it.)

2.  If the colony is indole positive, a bright red zone will develop within 5 seconds where the Kovac’s has spread.  (The reagent will likely kill the colony, so any transfer of the colony should be made before testing.)