Enumeration of Bacteria in Drinking Water by Membrane Filter Method

The membrane filter method is a widely used microbiological technique for detecting and quantifying bacteria in drinking water, especially coliforms and E. coli, which are indicators of water contamination. The method involves filtering a water sample through a membrane filter, which retains bacteria, and then incubating the filter on a nutrient medium to allow bacterial growth and enumeration. Below is a detailed process:

Materials Required:

Membrane filter – typically 0.45 µm pore size, made of cellulose acetate or nitrocellulose.
Vacuum filtration apparatus.
Sterile filtration unit (e.g., a funnel, filter holder).
Petri dishes.
Selective growth media – for example, m-Endo agar (for coliforms) or EMB agar (for E. coli).
Sterile pipettes.
Sterile tweezers.
Incubator (set to the required temperature, usually 35-37°C).
Sterile gloves and other appropriate lab safety gear.

Procedure:

1. Sample Collection:

Collect water samples in sterile containers.
If testing multiple samples, ensure proper labeling and handling to avoid contamination.

2. Preparation of Equipment:

Ensure the filtration setup is sterile by cleaning all equipment with 70% ethanol or autoclaving before use.
Prepare Petri dishes with the appropriate agar medium.

3. Filtration Process:

Place the membrane filter onto the filtration apparatus (funnel).
Pour the desired volume of the water sample (typically 100 mL or 1 L, depending on contamination level) into the funnel.
Apply vacuum pressure to pull the water through the filter. Bacteria in the water will be trapped on the membrane.

4. Transferring the Membrane Filter:

Once filtration is complete, use sterile tweezers to carefully remove the membrane filter from the funnel.
Place the filter onto the surface of the prepared agar medium in a Petri dish.

5. Incubation:

Incubate the Petri dishes in an incubator at the appropriate temperature (usually 35-37°C) for 24 hours (for coliforms) or 48 hours (for E. coli) based on the bacterial species being tested.

6. Examination of Results:

After incubation, examine the membrane filter for bacterial colonies.
Colony appearance varies depending on the growth medium used:
- For coliforms, the colonies are typically red or pink on m-Endo agar.
- For E. coli, colonies may be blue or metallic green on EMB agar.
Count the colonies that appear on the membrane filter.

7. Calculating Results:

The number of colonies observed on the membrane filter corresponds to the number of bacteria per unit volume of the water sample.
Colony-forming units (CFUs) can be calculated using the formula:
$CFU/100 mL = \frac{\text{Number of colonies counted}}{\text{Volume of sample filtered (mL)}} \times 100$

Advantages of the Membrane Filter Method:

High Sensitivity: Capable of detecting low concentrations of bacteria.
Selectivity: Using different agar media allows the detection of specific bacterial types, like coliforms or E. coli.
Quantitative: Provides a precise count of bacterial colonies in the sample.

Limitations:

Filtration of large volumes can sometimes be time-consuming.
Clogging of the filter with particulates or high turbidity can interfere with results.
Not suitable for samples with extremely high bacterial loads (may need dilution).

Conclusion:

The membrane filter method is a reliable, standardized approach to enumerate bacteria in drinking water. It is widely used for water quality testing, particularly for detecting indicators of contamination that could pose public health risks.

Enumeration of Bacteria in Drinking Water by Membrane Filter Method