Multiple Tube Fermentation Method (MTFM):

This method is comprised of three stages: 1) the presumptive test (today), 2) the confirmed test (next lab) and 3) the completed test (Lab 25).  In the presumptive test, 9 different tubes of lactose broth with 10 fold variations of the water sample are inoculated.  If any lactose-fermenting bacteria grow and produce gas, the medium will turn yellow and bubbles will be present in the Durham tub.  It is thus "presumed" that coliforms are present.  This test is also used to determine the most probable number (MPN) of coliforms present per 100 mL of water.  The samples should be incubated for 48 h at 35˚C.  If the test is positive, the confirmed test is performed.

The confirmed test involves taking a sample from a positive lactose broth tube and streaking it onto Eosin Methylene Blue (EMB) agar.  The appearance of dark colonies, often with a metallic sheen, indicate a coliform.  EMB is a selective and differential medium.  It selects for Gram-negative bacteria.  The dyes Eosin & Methylene Blue inhibit the growth of Gram-positive bacteria.  It is differential because lactose fermenters form colonies that are dark in color and sometimes have a green metallic sheen (typical for Escherichia coli); the colonies of lactose non-fermenters appear pale pink.

In the completed test an EMB positive colony (dark, metallic color) is used to inoculate a tube of lactose broth to verify acid and gas production.  A nutrient agar slant is also generally inoculated and serves as a source for stains to confirm the Gram-negative, non-spore-forming nature of coliforms.

In today's lab, the presumptive test will be performed on the same sample of water that was used for the P-A and Membrane Filtration tests.

Rachel Watson, M.S.
AG 5010
766-3524
Cell: 307-760-2942
rwatson@uwyo.edu