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. 

Procedure (Presumptive Test)

Students will work in groups of 4
From the side bench, collect 3 tubes of double-strength lactose broth, also known as DSLB; and 6 tubes of single-strength lactose broth, also known as SSLB.

1. Mix the water sample well.

2. Inoculate the 3 DSLB tubes with 10 mL of the water sample. Label these tubes with your initials, section number, water sample identity, and volume of the water used (10 mL).

3. Inoculate 3 SSLB tubes with 1 mL of the water sample. Label these tubes with your initials, section number, water sample identity, and volume of the water used (1 mL).

4. Inoculate 3 SSLB tubes with 0.1 mL of the water sample. Label these tubes with your initials, section number, water sample identity, and volume of the water used (0.1 mL).

5. Place all 9 tubes in the tray on the side bench to be incubated at 37°C for 48 hours.

Results (Presumptive Test)

Most probable number tables
multiple tube fermentation profile of 3-1-0

Procedure: Confirmed Test (to be performed in the next session) 

For this test select one lactose positive tube from your MTFM (presumptive Test) and use a loopful to t-streak a McConkey/MUG plate. 

Confirmed Test Results

Observe the MacConkey MUG plate for pink lactose fermenting colonies. Coliforms will also fluoresce under UV light.

MacConkey MUG plate with pink coloniesMacConkey MUG plates with pink colonies under UV light

Procedured: Completed Test (to be completed in the third lab session) 

For this procedure select a lactose positive/fluorescent isolated colony to innoculate a lactose broth tube. If no colonies are lactose positive/fluorescent then simply pick your favorite colony! 

Completed Test Results

lactose broth negative and positive results

Background & Introduction

Not all coliforms are of fecal origin; nonfecal coliforms like Enterobacter aerogenes are also commonly found in the environment. Thus, coliforms may or may not signify fecal contamination of waters. If coliforms are detected by using one or more of the tests that have been performed (membrane filtration, P-A, MTFM), one can determine using the IMViC tests whether they are fecal or non-fecal coliforms. 

1. IMViC stands for Indole, Methyl Red, Voges Proskauer and Citrate. 

2. Fecal coliforms like Escherichia coli are + + - - in the IMViC tests. 

3. Nonfecal coliforms like Enterobacter aerogenes are - - + + in these tests 



Methyl Red 



Escherichia coli 





Enterobacter aerogenes 





IMViC Procedure: 

For this procedure use a single isolated colony to inoculate three biochemical tests: 

For the Indole test inoculate a SIM Tube (Stab inoculation Review) 

For the Methyl Red, Voges-Proskauer test inoculate a MRVP Tube (MR-VP Procedure Review) 

For the Citrate test a Citrate Tube (Basic Slant Inoculation Procedure Review) 

IMViC ResultsIMViC Results for fecal and non-fecal coliforms