Confirmed Test

Once it has been established that gas-producing lactose fermenters are present in the water, it is presumed to be unsafe. However, gas formation may be due to noncoliform bacteria. Some to these organisms, such as Clostridium perfringens, are gram-positive. To confirm the presence of gram-negative lactose fermenters, the next step is to inoculate media such as Levine eosin-methylene blue agar or Endo agar from positive presumptive tubes.

Levine EMB agar contains methylene blue, which inhibits gram-positive bacteria. Gram-negative lactose fermenters (coliforms) which grow on this medium will produce "nucleated colonies" (dark centers). Colonies of E. coli and E. aerogenes can be differentiated on the basis of size and the presence of a greenish metallic sheen. E. coli colonies on this medium are small and have this metallic sheen, whereas E. aerogenes colonies usually lack the sheen and are larger. Differentiation in this manner is not completely reliable, however. It should be remembered that E. coli is the more reliable sewage indicator since it is not normally present in soil, while E. aerogenes has been isolated from soil and grains.

Endo agar contains a fuchsin sulfite indicator which makes identification of lactose fermenters relatively easy. Coliform colonies and the surrounding medium appear red on Endo agar. Non-fermenters of lactose, on the other hand, are colorless and does not affect the color of the medium.

In addition to these two media, there are several other media that can be used for the confirmed test. Brilliant green bile lactose broth, Eijkman's medium, and EC medium are just a few examples that can be used.


1 Petri plate of Levine EMB agar or any other testing medium you wish to choose.

1. Select on positive lactose broth tube from the presumptive test and streak a plate of medium. Use a streak method which will produce good isolation of colonies.

2. Incubate the plate for 24 hours at 35C.

3. Look for typical coliform colonies on the petri plate. If no coliforms colonies are present, the water is considered bacteriologically safe to drink.

Note: In actual practice, confirmation of all presumptive tubes would be necessary to ensure accuracy of results.


If your plate contained coliforms then double click on the BACK icon and click on the COMPLETED TEST to provide further test of your sample.

If your plate did not contained coliforms then you can stop the test now.