Indicator organisms and membrane tube fermentation

Indicator organisms

Indicator organisms are micro-organisms whose presence in water shows faecal pollution and so the possible presence of pathogens. Testing for individual pathogens is difficult, so an easily detected indicator is used. The best-known are coliform group bacteria and E. coli (faecal coliform); others are faecal streptococci and Clostridium perfringens.

An ideal indicator is always present when pathogens are present and absent in safe water, is more abundant and lives longer than pathogens, does not multiply in water, and is easy to detect.

Multiple tube fermentation (MPN) technique

The MTF technique estimates the Most Probable Number of coliform per 100 ml based on gas production from lactose.

Stage 1: Presumptive test

1. Prepare a series of tubes of lactose broth with inverted Durham tubes. Inoculate 5 tubes each with 10 ml, 1 ml and 0.1 ml of sample (use higher dilutions for polluted water).
2. Incubate at 35 degrees C for 24 hours; if there is no gas, incubate for another 24 hours.
3. Gas and acid in the tubes show a positive presumptive result.

Stage 2: Confirmed test

1. Transfer a loopful from each positive tube to brilliant green lactose bile broth, and incubate at 35 degrees C for 48 hours. Gas shows coliform.
2. For faecal coliform / E. coli, transfer to EC broth and incubate at 44.5 degrees C for 24 hours; gas shows E. coli.

Stage 3: Completed test Streak on EMB agar and incubate at 35 degrees C for 24 hours. E. coli forms dark colonies with a metallic green sheen. Gram stain should show Gram-negative, non-spore forming rods, and growth in lactose broth should again produce gas.

Calculation of MPN The number of tubes giving a positive result in each dilution is noted (for example 5-1-0), and the MPN per 100 ml is read from the standard MPN table (5-1-0 gives about 33 per 100 ml). For other combinations Thomas' formula is used:

$$\text{MPN}/100\ \text{ml} = \frac{\text{positive tubes}\times100}{\sqrt{(\text{ml in negative tubes})\times(\text{ml in all tubes})}}$$

Interpretation: drinking water must have 0 MPN of E. coli per 100 ml.