Membrane filtration method is an alternative to the MPN method for the microbiological analysis of water. Sometimes, it also refers as “Millipore filter technique” or “MF technique”. The Millipore filter technique was named by the two scientists Goetz and Tsuneishi in 1951. Membrane filtration method makes the use of membrane filter, or we can say Millipore filter.
A Millipore filter possesses a uniform porosity or a predetermined pore size of 0.45µm. The pores in the membrane filter are sufficiently small enough to trap the different types of microorganisms. A membrane filter is composed of polymeric substances.
Membrane filtration method requires an apparatus refers to as “Membrane filtration assembly” to carry out the water analysis. A filtration unit comprises of many components like a funnel, Vacumn pump, filter flask, filter pump etc.
Content: Membrane Filtration Method
- Definition of Membrane Filtration Method
- Membrane Filter
- Absorbent Pad
- Membrane Filtration Assembly
- Method of Membrane Filtration
Definition of Membrane Filtration Method
Membrane filtration method can define as the process of microbiological analysis of water by making the use of a special filter like membrane filter to trap the microorganisms.
It is a very effective method for the isolation and enumeration of microorganisms in the test water sample. By knowing the quantity of microbial mass, the quality of water can also determine. Therefore, the membrane filtration method can check both the quality of water and the quantity of microorganisms present in the water.
Membrane filtration method was introduced in the late 1950s and widely accepted by the U.S. EPA (Environmental Protection Agency). According to U.S. EPA (Environmental Protection Agency), Membrane filtration is the best technique for the water analysis as it allows the testing of a large volume of water sample in less time.
The main objective of the Membrane filtration method is to determine the presence or absence of particular coliform group that is commonly present in wastewater and groundwater.
Membrane filter can define as the molecular or biological filter which forms by the biologically inert cellulose esters. It is having a uniform or a predetermined pore size, which traps the different types of microorganisms whose size ranges from 0.01-0.10µm.
The membrane filters are widely used in laboratories for the isolation and separation methods. In industries like pharmaceuticals, cosmetics, food and beverages, membrane filters are used to monitor the presence of bacteria and sterilize the heat labile fluids.
An absorbent pad consists of filter paper disks which are free of sulphites and other compounds which suppress the growth of bacteria. The MF technique makes the use of absorbent paper of diameter 48mm with a thickness of 0.8mm. The absorbent paper absorbs 1.8-2.2ml of the nutrient medium.
The absorbent pad saturates with the appropriate liquid broth medium to which 1.5% of agar may be added. Over the saturated absorbent medium, a membrane filter is placed and incubated for the growth of bacterial colonies.
Membrane Filtration Assembly
To perform membrane filtration, there is a filtration unit which commonly refers to as “Membrane filtration assembly”. A filtration unit comprises of the funnel, locking ring, carbon disc, stainless base, rubber stopper, filter flask and Vacumn pump.
First, the water sample passes through a funnel. A stainless funnel is present at the top below which, a locking ring or clamp is present that controls the flow of the test water sample. A membrane filter is placed over the carbon disk.
The stainless base holds the carbon disc and the membrane filter. At the base, a rubber stopper controls the flow of water to the filter flask. On the one end of filter flask, there is a connector which connects the Vacumn motor with the filtration unit. A Vacumn provides a negative pressure which permits the suction of filtrate through the membrane filter.
Through the MF technique, 100ml of the sample can easily pass from the filter membrane of diameter 47mm by means of filtration assembly. The organisms confine on the surface of the membrane filter.
Transfer the organisms retain on the surface of the membrane filter to the Petri dish containing a nutrient medium. There is a passage of nutrients from the Petri plate to the organisms trapped, through the membrane filter.
The passage of nutrients allows the growth of microorganisms and results in the formation of discrete colonies. The culture plate can further transfer to the confirmation media or selective media to confirm the type of organism present in water.
Method of Membrane Filtration
A membrane filtration method involves the following steps like:
Sample collection: Collect water samples like groundwater (high in Cu and Zn) and wastewater (high in heavy metals) in the sterilized sample bottles. Then dilute the water sample making a volume up to 100 ml.
Selection of appropriate nutrient medium: For the isolation and enumeration of different pathogenic microorganisms in water, use a suitable nutrient medium.
|Microorganisms in water||Nutrient medium used|
|Total coliforms||MF- Endo|
|Fecal coliforms||M- FC|
|Yeast and Molds||M- Green YM|
|Total bacteria||M- TGE / Trypticase Soy USP|
|Fecal streptococci||KF- Streptococcal|
|Lactobacillus acid resistant bacteria||Orange serum|
After selecting an appropriate nutrient medium, transfer it to the sterilized Petri plate containing absorbent pad at the bottom. Soak the absorbent pad with the liquid broth. The saturation of the absorbent pad with the nutrient medium will promote microbial growth.
Membrane filtration: For the membrane filtration of water, place a membrane filter over the carbon disk by the help of forcep sterilized with the flame. Then flame the opening of the sample bottles and pour the test water sample, through the funnel.
A Vacumn system allows the complete separation of filtrate through the membrane filter. After complete separation of filtrate, rinse the funnel with the buffered water.
Placing of membrane filter: Flame the forcep and take out the membrane filter from the filtration assembly. Then place a membrane filter over the prepared petri dish containing absorbent pad saturated with liquid nutrient medium.
Incubation: Incubate the Petri plate for 24-48 hours at 35-37 degrees Celsius temperature.
Quantitative analysis: Count the number of colonies directly on the colony counter.
Qualitative analysis: To test the water quality, isolate and identify the type of microorganisms by subculturing the isolated colonies. Then perform staining methods, microscopic observations and further a biochemical test to identify the specific type.
- MF technique is an alternative to the most probable number method.
- One can test a large volume of a water sample by the Millipore filtration method within less time.
- Membrane filtration consumes less time than the MPN method as it only involves four to five steps to get the results.
- MF technique gives relevant and reliable results than the MPN method.
- The number of bacteria present in the water sample can count directly by the colony counter.
- Membrane filtration method is not applicable for the testing of turbid water
- There may be a chance of bacterial overgrowth, as the water contains many microorganisms.
Therefore membrane filtration method is an efficient method to isolate and enumerate the number of microorganisms from the microbiologically contaminated fluid sample.
Membrane filtration method determines the microbial population by counting the number of discrete colonies, which is an advantage over the MPN method. In contrast to membrane filtration method, the MPN method determines the microbial density based on the turbidity of the culture broth.