Membrane filter technique for coliforms

The membrane filter (MF) technique is a direct way of counting coliform bacteria in a water sample. It gives the count of colonies (CFU) per 100 ml and is faster than the MPN method (results in about 24 hours), uses less media and gives a direct count.

Principle

A measured volume of water passes through a sterile cellulose membrane with 0.45 micron pores. Bacteria are retained on the surface. The membrane is incubated on a selective nutrient medium, where each bacterium grows into a visible colony.

Apparatus and media

Filtration unit (funnel and base), vacuum source, sterile membrane filters, forceps, petri dishes with absorbent pads, m-Endo broth (total coliform) or m-FC broth (faecal coliform), incubator (35 degrees C) or water bath (44.5 degrees C), sterile buffered dilution water, a counting lens.

Procedure

1. Sterilise the unit and place the membrane on the base with sterile forceps.
2. Filter 100 ml of sample (or a dilution) under vacuum, then rinse the funnel.
3. Place the membrane on the medium-soaked pad without air bubbles.
4. Incubate upside down: 35 degrees C for 24 hours for total coliform; 44.5 degrees C for 24 hours for faecal coliform.
5. Count the typical colonies (golden-green sheen with m-Endo; blue colonies with m-FC).

$$\text{Coliform}/100\ \text{ml} = \frac{\text{colonies counted}}{\text{ml filtered}}\times100$$

Merits and limits

It is good for large volumes with low counts (clean water). It is less suitable for turbid water that clogs the filter, and for water with many non-coliform bacteria.