Multiple tube fermentation (MPN) technique

The multiple tube fermentation (MTF) technique estimates the number of coliform bacteria in a water sample as the Most Probable Number (MPN) per 100 ml, using a statistical table. It uses the ability of coliforms to ferment lactose with acid and gas at 35 degrees C within 48 hours. It is done in three stages.

Apparatus and media

Test tubes with inverted Durham tubes, pipettes, incubator at 35 +/- 0.5 degrees C, water bath at 44.5 degrees C, lactose broth (or lauryl tryptose broth), brilliant green lactose bile (BGLB) broth, EC broth, EMB agar, sterile dilution water.

1. Presumptive test

1. Choose sample volumes by the expected pollution. For example, for drinking water: 5 tubes each of 10 ml, 1 ml and 0.1 ml (a 15-tube series).
2. Add the volumes to tubes of lactose broth (double strength for the 10 ml portions) and mix.
3. Incubate at 35 degrees C for 24 hours; if no gas, continue to 48 hours.
4. Gas in the Durham tube plus acid (colour change) is a positive presumptive result. No gas after 48 hours is negative.

2. Confirmed test

1. Transfer a loopful from each positive tube to BGLB broth (for coliform), and to EC broth for faecal coliform / E. coli.
2. Incubate BGLB at 35 degrees C for 48 hours; incubate EC broth at 44.5 degrees C for 24 hours.
3. Gas formation confirms coliform (BGLB) or faecal coliform / E. coli (EC).

3. Completed test

Streak positive confirmed cultures on EMB agar and incubate for 24 hours. Typical E. coli colonies are dark with a green metallic sheen. A Gram stain shows Gram-negative non-spore forming rods, and lactose broth again produces gas.

Calculation of MPN

Count the number of positive tubes in each dilution and read the MPN per 100 ml from the standard MPN table for the combination, for example 5-2-1 (10 ml, 1 ml, 0.1 ml) gives an MPN of about 70 per 100 ml (the 95% confidence limits are also given). For other dilutions, use Thomas' formula:

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

Drinking water should show an MPN of 0 per 100 ml.