Coliform Lab

Coliform analysis

Completed by the Public Health Department & Waste Water Treatment Plant

Test water samples for the presence of facial coliform bacteria (drinking water, frequently used beaches, etc.)

Coliform

E. Coli like characteristics

Gram -

Non-spore forming

Bacillus shaped

Able to ferment lactose → formation of acid & gas

E. coli is an indicator for

Fecal contamination in water

Presumptive test

1st test in coliform test

Quick and easy

Method: water samples inoculated into tubes of lactose broth

Positive Presumptive test

Presence of gas in Durham tube after 48 hours of incubation

Gas bubble in Durham tube

Negative Presumptive test

No bacterial growth or bacterial growth but no gas in Durham tube

Confirmed test

Verify that coliform bacteria are present in water sample following positive Presumptive test

Method: bacteria from presumptive test inoculated into tubes of brilliant green lactose bile broth (BGLBB)

Gas production is monitored in Durham tube

Brilliant Green Lactose Bile Broth (BGLBB)

Used in Confirmed coliform test

Selective for Gram - bacteria

Inhibit Gram + growth by generally inhibiting peptidoglycan synthesis

Positive Confirmed coliform test

Gas from fermentation collects in Durham tube and bacterial growth (G-) = confirmed coliform presence

Negative Confirmed coliform test

No bacterial growth or bacterial growth but no gas in Durham tube

Completed coliform test

Confirms the presence of the indicator fecal coliform bacteria E. coli

Method: bacteria from Confirmed test are streaked on to eosin methylene blue agar (EMB)

Eosin methylene blue agar plates

Selective and differential

2 dyes (eosin and methylene blue) suppress growth of Gram + bacteria

The color of the colonies help to identify the bacterium present

Positive results for Completed test

Presence of E. coli

Metallic green colonies = fermentation of sugars creates a lot of acidic byproducts = lower pH of agar = changing colonies’ color

Negative results for Completed test of coliform test

No E. coli colonies or no growth

Completed test - colonies are colorless

Negative results

No fermentation of sugars to lower pH of agar

Gram - present but no E. coli or fecal coliform bacteria

Completed test - colonies are colorless

Negative result

Fermentation of sugars creates some acidic byproducts = lower pH of agar = slightly changes color

Likely coliform bacteria other than E. coli such as E. aerogenes

Completed test can differentiate between

E. Coli and E. Aerogenes

Purpose of lactose broth

Primary medium for Presumptive test that provides nutrients as a selective media for Gram - bacteria favoring bacteria that can utilize lactose as a source of energy through fermentation

Purpose of Durham tube

Small inverted glass tube placed inside the larger tube

As coliform ferments lactose they produce gas

Gas gets trapped in the Durham tube which confirms a positive result for coliform