DETERMINATION OF MICROBIOLOGICAL CONTAMINATION USING MF TECHNIQUE

Theory:
Membrane filters or “membranes” are microporous plastic films with specific pore size ratings. Also known as
screen, sieve or microporous filters, membranes retain particles or microorganisms larger than their pore size
primarily by surface capture. Some particles smaller than the stated pore size may be retained by other mechanisms.
Advantec membranes are produced by three different processes. Mixed Cellulose Esters, Cellulose Acetate, and
Nylon are reverse phase solvent cast membranes, where controlled evaporation or removal of the complex solvent
system forms the porous structure. Both hydrophilic and hydrophobic PTFE are made by a patented process where
the membranes are stretched biaxially to form the porous structure. PCTE membranes are track etched..
Membrane filters have a known uniform porosity of predetermined size (generally 0.45 µm ) sufficiently small to
trap microorganisms.Using the membrane filter technique, sample is passed through the membrane using a filter
funnel and vacuum system. Any organisms in the sample are concentrated on the surface of the membrane. The
membrane, with its trapped bacteria, is then placed in a special plate 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. Discrete colonies thus formed can be easily transferred to
confirmation media.
Membrane filter technique is an effective, accepted technique for testing fluid samples for microbiological
contamination. It involves less preparation than many traditional methods, and is one of a few methods that will
allow the isolation and enumeration of microorganisms. Membrane filters are used extensively in the laboratory and
in industry to sterilize fluid materials.

Advantage of Membrane Filter Technique

 Permits testing of large sample volumes. Theoretically almost any volumes of nontubrid water could be filtered
through the disk, the organisms from any given volume being deposited in the disk.
 The membrane can be transferred from one medium to another for purposes of selection or differentiation of
organisms thus allowing isolation and enumeration of discrete colonies of bacteria.
 Results can be obtained more rapidly than by the conventional MPN standard methods. It provides presence or
absence information within 24 hours.

Uses of Memberane filters

1. Membrane filters are used extensively in the laboratory and in industry to sterilize heat labile fluid materials.
2. Effective and acceptable technique to monitor drinking water.
3. Useful for bacterial monitoring in the pharmaceutical, cosmetics, electronics, and food and beverage industries.
4. Allows for removal of bacteriostatic or cidal agents that would not be removed in pour plate, spread plate, or
MPN techniques.

Microbiological Contamination
Exposure to microorganisms, such as fungi, bacteria, viruses, and their biological by-products, may cause disease
and allergic responses in building occupants. Human exposure to pathogenic microorganisms and their by-products
in an indoor environment usually occurs by inhalation and contact with the mucous mem-branes. In order to achieve
acceptable indoor air quality, airborne exposure to fungi and other pathogens should be minimized.

For airborne exposures to microorganisms to occur, several events must happen. First, there must be a reservoir (i.e.,
a loca-tion where an unusually high concentration of microorganisms is present). Second, the microorganisms must
be allowed to repro-duce. Favorable conditions are needed for reproduction to occur. For example, fungal growth is
usually optimized when moisture levels are high. Last, the microorganisms must be released into the air. For
example, Legionella is released into the environment when cooling tower fans blow contaminated water mist into
the air. Since all three steps are needed for exposure to occur, prevention of one or more of the steps from occurring
will minimize airborne expo-sures to microorganisms.
Certain conditions contribute to microbial contamination in an indoor environment:
Location of fresh air intakes adjacent to outdoor microbial reservoirs
Excessive indoor relative humidity levels (greater than 60 percent)
Stagnant water in air-handling units or other HVAC components
Wet building materials such as carpet, gypsum board, insulation, or ceiling tiles
Wet furniture
Recent flooding within a building
Inadequate building vapor barriers that allow entry of moisture into the building
Voids in exterior insulation or cracks in buildings that allow cold outdoor air to enter the building and cool interior
surfaces, which create condensation and promote microbial growth
Several controls are needed to minimize microbial contamination within a building. The HVAC system should be
properly sized and installed. The system should be capable of handling the cool-ing and heating loads within the
building. The building should be positively pressurized with respect to the outdoors to prevent the uncontrolled
infiltration of moisture and airborne contaminants into the building. A preventive maintenance program should be
implemented that provides regular inspections of HVAC compo-nents and prompt response to faulty equipment. All
moisture leaks should be repaired immediately. Moisture should not be allowed to accumulate within wall cavities.
The EPA recommends that water-damaged items should be discarded or dried within 24 to 48 hours to prevent mold
growth.

Step-by-step Procedures
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 and confirm the colonies and report the result

References

https://microbeonline.com/membrane-filter-technique/

http://www.advantecmfs.com/catalog/filt/membrane.pdf

http://www.ashe.org/compliance/ec_02_05_01/01/microbiological.shtml