Understanding Indicator Organisms and the Multiple Tube Fermentation Technique for Determining MPN in Water Samples

 

Indicator Organism

An indicator organism is a microorganism whose presence in water indicates the potential presence of pathogenic (disease-causing) organisms. These organisms are used as a proxy for assessing the microbiological quality of water, as direct testing for all pathogens would be complex and expensive. Common indicator organisms include:

• Escherichia coli (E. coli): Indicates fecal contamination.
• Total Coliforms: Indicates general water quality and possible contamination.
• Enterococci: Also used to indicate fecal contamination, particularly in marine waters.

Multiple Tube Fermentation Technique

The Multiple Tube Fermentation (MTF) technique, also known as the Most Probable Number (MPN) method, is a statistical method used to estimate the concentration of viable microorganisms in a water sample. It involves a series of steps to detect and enumerate coliform bacteria.

Steps in the Multiple Tube Fermentation Technique

1. Sample Dilution: Prepare serial dilutions of the water sample to cover a range that likely includes the number of target organisms.

2. Inoculation: Inoculate multiple tubes of a selective liquid growth medium (such as lactose broth) with each dilution of the water sample. Usually, three or five tubes per dilution are used.

3. Incubation: Incubate the inoculated tubes at a specified temperature (typically 35°C for coliforms) for 24-48 hours.

4. Observation: After incubation, observe the tubes for signs of fermentation, indicated by gas production or a color change (depending on the medium used).

5. Confirmation: For tubes showing positive results, further confirmatory tests (such as using selective agars like EMB or MacConkey agar) may be performed to verify the presence of coliforms.

Determining MPN (Most Probable Number)

The MPN is calculated based on the number of positive tubes at each dilution level. The results are compared to statistical MPN tables, which provide an estimate of the number of bacteria per unit volume of the original sample.

Steps to Determine MPN

1. Count Positive Tubes: Record the number of positive tubes (those showing gas production or color change) for each dilution series.

2. Refer to MPN Table: Use a standard MPN table, which corresponds to the number of positive tubes across the dilutions, to determine the MPN value.

3. Calculate: The MPN value from the table gives an estimate of the concentration of target organisms (e.g., coliforms) per 100 mL of the water sample.

Example Calculation

1. If you have three sets of dilutions with 5 tubes each:

   • 10 mL dilution: 4 positive tubes
   • 1 mL dilution: 2 positive tubes
   • 0.1 mL dilution: 1 positive tube

2. Look up the combination 4-2-1 in an MPN table.

3. The table might indicate an MPN of, for example, 15 per 100 mL for this combination of positive results.

Summary

The Multiple Tube Fermentation technique is a reliable method for estimating the microbial quality of water. By identifying the presence and concentration of indicator organisms like coliforms, it helps ensure that water is safe for consumption and use.