ENUMERATION OF MICROBES FROM WATER USING MEMBRANE FILTER

 ENUMERATION OF MICROBES FROM WATER USING MEMBRANE FILTER 

AIM: 

To determine the quality of water samples using the membrane filter method. 

INTRODUCTION: 

The Membrane Filter (MF) Technique was introduced in the late 1950s as an alternative to the Most Probable Number (MPN) procedure for microbiological analysis of water samples. It involves the use of membrane filters, which are thin porous sheet structures composed of cellulose esters or similar polymeric materials. They act essentially as two-dimensional screens and as such, all particles, both biological and non-biological, which exceed the pore size, are retained upon the surface of the filter from fluids passing through. Bacteria-tight membrane filters capable of retaining microorganisms larger than 0.45 micrometer (μm) are frequently used for analysis of water. 

PRINCIPLE OF MEMBRANE FILTER TECHNIQUE: 

Membrane filters have a known uniform porosity of predetermined size (generally 0.45 µm) sufficiently small to trap microorganisms. A water sample is passed through a sterile membrane filter that is housed in a special filter apparatus contained in a suction flask. Following filtration, the filter disc that contains the trapped microorganisms is aseptically transferred to a sterile Petri dish containing a pad saturated with the appropriate medium. The passage of nutrients through the filter during incubation facilitates the growth of organisms in the form of colonies, on the upper surface of the membrane. Membrane filtration and colony count techniques assume that each bacterium, clump of bacteria, or particle with bacteria attached, will give rise to a single visible colony. Each of these clumps or particles is, therefore, a colony forming unit (cfu) and the results are expressed as colony forming units per unit volume. Discrete colonies thus formed can be easily transferred to confirmation media. Following incubation, the colonies present on the filter are counted with the aid of a microscope.

PROCEDURE OF MEMBRANE FILTER TECHNIQUE: 

1. Collect the sample and make any necessary dilutions.

 2. Select the appropriate nutrient or culture medium. Dispense the broth into a sterile Petri dish, evenly saturating the absorbent pad. 

3. Flame the forceps, and remove the membrane from the sterile package. 

4. Place the membrane filter into the funnel assembly. 

5. Flame the pouring lip of the sample container and pour the sample into the funnel. 

6. Turn on the vacuum and allow the sample to draw completely through the filter. 

7. Rinse funnel with sterile buffered water. Turn on vacuum and allow the liquid to draw completely through the filter. 

8. Flame the forceps and remove the membrane filter from the funnel.

 9. Place the membrane filter into the prepared Petri dish. 

10. Incubate at the proper temperature and for the appropriate time period. 

11. Count the colonies under 10 – 15 X magnification. 

Result:

  The coliform bacteria produce colonies with characteristic "metallic green sheen"