Multiple Technique Fermentation Process

This method is widely used for detecting and estimating coliform bacteria (like Escherichia coli) in water, food, and environmental samples. It’s a quantitative technique based on microbial fermentation, and it provides an estimate of bacterial density through the Most Probable Number (MPN) method.

Principle:

Coliform bacteria can ferment lactose and produce acid and gas. The Multiple Tube Fermentation Technique uses differential media to observe this fermentation. Through a series of tests, we confirm the presence of coliforms and estimate their population using MPN tables.

Materials Required:

• Lactose broth (or MacConkey broth)
• Brilliant Green Lactose Bile Broth (BGLBB)
• Eosin Methylene Blue (EMB) Agar or Endo Agar
• Durham tubes (inverted small tubes for gas collection)
• Sterile test tubes
• Sterile pipettes
• Incubator (35–37°C)
• Sample (like water)

Procedure:

1. Presumptive Test:

   This is the first screening step to detect possible coliforms.

   • Prepare lactose broth tubes: Use different dilutions of the sample (like 10 mL, 1 mL, and 0.1 mL) in sets of 3, 5, or 10 tubes each.
   • Inoculate the sample: Add measured amounts of the sample into the tubes. Place a Durham tube in each to trap gas.
   • Incubate: Place the tubes at 35–37°C for 24–48 hours.
   • Observe results:
     • Positive result: Gas formation in the Durham tube and turbidity in the broth indicate possible coliform presence.
     • Negative result: No gas or turbidity means no coliforms detected.

2. Confirmed Test:

   This step confirms that the gas-producing organisms from the presumptive test are indeed coliforms.

   • Take positive tubes from the presumptive test and transfer a loopful of broth into Brilliant Green Lactose Bile Broth (BGLBB) tubes.
   • Incubate: Keep at 35–37°C for 24–48 hours.
   • Observe: Gas production in the Durham tube is a positive result, confirming the presence of coliforms.

3. Completed Test:

   This step identifies the specific coliform species (like E. coli).

   • Streak a loopful from positive BGLBB tubes onto EMB agar or Endo agar plates.
   • Incubate: At 35–37°C for 24 hours.
   • Observe colonies:
     • E. coli typically shows green metallic sheen on EMB agar.
     • Other coliforms form pinkish, mucoid colonies.
   • Perform Gram staining and biochemical tests (like IMViC tests) for further identification.

4. Most Probable Number (MPN) Calculation:

   This is a statistical estimation of the bacterial population in the original sample.

   • Record the number of positive tubes at each dilution level (like 10 mL, 1 mL, and 0.1 mL).
   • Compare the results with an MPN table.
   • The table gives an MPN value, which estimates the number of coliforms per 100 mL of the sample.