The membrane filtration method is one of the most widely used laboratory techniques for enumerating indicator bacteria in water, providing a direct, quantitative measurement of total coliforms, Escherichia coli, thermotolerant (fecal) coliforms, and enterococci within 24 to 48 hours. This article explains the principle of the water microbiology membrane filter experiment, the relevant international and national standards, sample types, the step-by-step analytical procedure, and quality control requirements for laboratories performing routine water quality assessment.
Technical Overview
The membrane filter (MF) method is a culture-based enumeration technique in which a measured volume of water is drawn by vacuum through a sterile membrane filter with a nominal pore size of 0.45 micrometers. Bacteria present in the sample are retained on the membrane surface, which is then transferred onto a selective and differential culture medium and incubated under defined conditions. After incubation, characteristic colonies developing on the membrane are counted, and the result is reported as colony-forming units per 100 milliliters (CFU/100 mL).
Unlike the multiple-tube fermentation technique, which estimates bacterial density statistically through the most probable number (MPN), the membrane filter method is a direct plating procedure that yields a quantitative colony count. Its principal advantages include higher analytical sensitivity for low-turbidity, low-count waters (because large sample volumes of 100 mL or more can be filtered), shorter turnaround time, lower media consumption, and easier colony isolation for confirmation. The main limitation is interference from water turbidity and suspended solids, which can clog the membrane, reduce the filtered volume, or mask target colonies with heavy background growth.
The membrane filter method is standardized by major standards organizations worldwide, including ISO 9308-1 for coliforms and E. coli, ISO 7899-2 for intestinal enterococci, ISO 6461-2 for sulfite-reducing anaerobic spores, ASTM D5392 for fecal coliforms, GB/T 5750.12 and related GB standards for drinking water examination in China, and the procedures published in Standard Methods for the Examination of Water and Wastewater (Methods 9222 and 9230) together with US EPA membrane filter methods such as Method 1600 and Method 1603.
Testing Samples
Drinking Water and Packaged Water
Treated tap water, bottled water, mineral water, and water from vending machines are typically low in turbidity and bacterial density, making them ideal matrices for membrane filtration. Testing focuses on demonstrating compliance with microbiological limits of zero or near-zero indicator organisms in 100 mL samples.
Surface Water and Groundwater
Rivers, lakes, reservoirs, springs, and wells are monitored to assess natural water quality and detect fecal contamination from sewage discharge, agricultural runoff, or livestock activity. Depending on expected bacterial density, smaller filtered volumes or serial dilutions may be required.
Recreational Water
Swimming pools, spa pools, water parks, and bathing beaches are routinely tested for indicator bacteria to protect public health. Enterococci and E. coli are the primary indicators for natural bathing waters, while Pseudomonas aeruginosa is of particular concern in pool and spa water.
Wastewater and Treated Effluent
Raw influent, secondary effluent, and disinfected final effluent from municipal and industrial wastewater treatment plants are analyzed to verify treatment performance and compliance with discharge permit limits. High bacterial loads require significant dilution before filtration.
Industrial and Process Water
Cooling tower water, ultrapure water systems, and food and beverage process water are monitored for heterotrophic plate counts and specific opportunistic pathogens to control biofouling and protect product quality.
Testing Items
The membrane filter method is applicable to a defined set of culturable indicator and hygiene-relevant organisms. Typical testing items and their reported units are listed below.
- Total coliforms — enumerated on m-Endo or chromogenic coliform agar, reported as CFU/100 mL
- Thermotolerant (fecal) coliforms — enumerated on m-FC agar at 44.5 °C, reported as CFU/100 mL
- Escherichia coli — enumerated on m-TEC, TBX, or chromogenic media, reported as CFU/100 mL
- Intestinal enterococci (fecal streptococci) — enumerated on Slanetz-Bartley (m-Enterococcus) agar, reported as CFU/100 mL
- Pseudomonas aeruginosa — enumerated on selective media such as m-PA or CN agar, reported as CFU/100 mL
- Sulfite-reducing anaerobic spores (Clostridium perfringens) — enumerated after heat shock pretreatment, reported as CFU/100 mL
- Heterotrophic plate count (HPC) — enumerated by membrane filtration on R2A or nutrient media, reported as CFU/mL
Testing Methods
Apparatus and Materials
The experiment requires a sterile membrane filtration unit consisting of a borosilicate or stainless-steel funnel, a fritted filter support, a side-arm filtration flask, and a vacuum source; sterile gridded white membrane filters of 47 mm diameter and 0.45 micrometer pore size; sterile absorbent pads; pre-poured selective media or membrane media in ampoules; sterile forceps; phosphate-buffered dilution water; sterile Petri dishes; and calibrated incubators or circulating water baths capable of maintaining 35 to 37 °C and 44.5 ± 0.2 °C. All components that contact the sample must be sterile, either by autoclaving or by using pre-sterilized disposable units.
Sample Collection and Preservation
Samples are collected in sterile, neutral glass or plastic bottles using aseptic technique. For chlorinated or otherwise disinfected water, sodium thiosulfate is added to the container beforehand to neutralize residual disinfectant and prevent continued bacterial die-off between collection and analysis. Samples are transported in the dark at 2 to 8 °C and should be analyzed as soon as practicable, ideally within 6 hours of collection and no later than 24 hours, in accordance with most national and international standards.
Filtration Procedure
- Assemble the sterilized filtration unit on the manifold and moisten the filter support with sterile dilution water.
- Using sterile forceps, place a sterile membrane filter grid-side up on the support and seat the funnel securely.
- Filter a measured volume of sample, typically 100 mL for treated water or an appropriate dilution for polluted water; select volumes that will yield between 20 and 80 (ideally 20 to 60) target colonies on the membrane.
- Rinse the inner walls of the funnel with 20 to 30 mL of sterile dilution water at least three times to ensure complete transfer of organisms onto the membrane.
- Lift the funnel, remove the membrane with sterile forceps, and roll it onto the surface of the prepared medium plate to avoid trapping air bubbles between the membrane and the agar or pad.
- Invert the plate and incubate under the conditions specified for the target organism.
Incubation Conditions by Target Organism
- Total coliforms: m-Endo agar, 35 to 37 °C for 22 to 24 hours; typical colonies show a dark center with a metallic green sheen.
- Fecal coliforms: m-FC agar, 44.5 ± 0.2 °C for 22 to 24 hours, preferably in a circulating water bath for precise temperature control; typical colonies are blue.
- E. coli: m-TEC agar (35 °C for 2 hours of resuscitation followed by 44.5 °C for 22 hours) or chromogenic and fluorogenic media incubated at 35 to 37 °C or 41.5 °C per ISO 9308-1; typical colonies are colored or fluorescent according to the medium.
- Intestinal enterococci: Slanetz-Bartley agar, 36 ± 2 °C for 44 ± 4 hours; typical colonies are red, pink, or burgundy, with confirmation on bile esculin azide agar at 44 °C.
- Pseudomonas aeruginosa: selective CN or m-PA agar at 35 to 37 °C for 24 to 48 hours, with confirmation of pigment production and biochemical reactions.
Colony Counting and Confirmation
After incubation, membranes are examined under a dissecting microscope or illuminated colony counter with daylight-quality lighting. Colonies matching the typical morphology, color, or sheen for the target organism are counted; crowded plates exceeding approximately 200 colonies or plates dominated by spreading growth are rejected. Depending on the standard and the regulatory context, representative typical colonies may require confirmation using subsequent biochemical tests such as gas production in lauryl tryptose or brilliant green lactose bile broth, cytochrome oxidase, indole production from tryptophan, beta-glucuronidase activity, bile esculin hydrolysis, and catalase reaction. Atypical colonies may also be verified when non-coliform background growth is heavy.
Calculation and Reporting
The density of the target organism is calculated as the number of confirmed typical colonies multiplied by 100 and divided by the volume of sample filtered in milliliters, and is reported as CFU/100 mL. When multiple volumes or dilutions are filtered, the count is preferably taken from membranes yielding 20 to 80 typical colonies, and results from duplicate membranes may be averaged. Filters with no target colonies are reported as fewer than 1 CFU per 100 mL when 100 mL was filtered, or below the corresponding detection limit for the actual volume analyzed.
Quality Control
Each analytical batch must include sterility controls for the medium, dilution water, and membrane filtration apparatus, typically by filtering sterile dilution water at the start and end of the session. Positive and negative control strains, such as E. coli, Enterococcus faecalis, and Pseudomonas aeruginosa reference cultures, are analyzed periodically to verify medium selectivity and incubation performance. Incubators and water baths are calibrated and temperature-mapped, forceps are flame-sterilized between membranes, and colony counts, sample volumes, incubation times, and any deviations are fully documented in accordance with laboratory quality management systems such as ISO/IEC 17025.
Applications
The membrane filter method supports a wide range of regulatory, industrial, and research applications in water quality management.
- Compliance monitoring of drinking water against national and international microbiological limits in GB, ISO, and EN standards
- Verification of disinfection performance in water treatment plants, including chlorination, ozonation, and UV systems
- Wastewater effluent monitoring and discharge permit verification for municipal and industrial facilities
- Recreational water quality assessment at beaches, lakes, and swimming pools for public health protection
- Bottled water and beverage industry quality control from source to finished product
- Environmental surveillance, pollution source tracing, and watershed management studies
- Emergency response and outbreak investigation following suspected waterborne disease events
- Validation of water treatment devices, filters, and purification units by accredited third-party laboratories
FAQ
What is the detection limit of the membrane filter method?
When 100 mL of sample is filtered, the theoretical detection limit is 1 CFU/100 mL, because a single viable target organism in the filtered volume will produce one countable colony. Filtering smaller volumes or dilutions raises the detection limit proportionally.
How does the membrane filter method differ from the multiple-tube fermentation (MPN) method?
Membrane filtration is a direct colony-count technique that is faster, uses less media, and provides an actual quantitative result, while the multiple-tube fermentation method estimates density statistically from positive tubes and requires longer confirmation steps. Membrane filtration is preferred for low-turbidity waters, whereas MPN methods are often more suitable for turbid or heavily polluted samples that clog membranes.
Can turbid water samples be tested by membrane filtration?
High turbidity and suspended solids can clog the membrane, reduce the effective filtered volume, and suppress or mask target colonies. For such samples, laboratories reduce the filtered volume, perform serial dilutions, or switch to MPN, enzyme substrate, or chromogenic liquid methods as allowed by the applicable standard.
Why is a 0.45 micrometer pore size used?
A pore size of 0.45 micrometers provides quantitative retention of indicator bacteria such as coliforms and enterococci while maintaining an acceptable filtration flow rate. It has been validated as the standard pore size in ISO, ASTM, and GB methods for the enumeration of these organisms.
Why is sodium thiosulfate added to sample containers?
Sodium thiosulfate immediately neutralizes residual chlorine, chlorine dioxide, or ozone in disinfected water at the moment of sampling. This prevents continued bactericidal action during transport, which would otherwise underestimate the true bacterial density in the sample.
How quickly are results available?
Primary results are typically available after 22 to 48 hours of incubation depending on the target organism and medium. Confirmatory biochemical testing of representative colonies may require an additional 24 to 48 hours.
Are confirmatory tests always required?
Requirements depend on the applicable standard and the intended use of the data. Some modern chromogenic and fluorogenic media achieve high specificity without confirmation, whereas classical media such as m-Endo and m-FC generally require verification of representative colonies through subculture and biochemical testing, particularly for regulatory reporting.
What does a positive result indicate?
A positive result indicates that fecal indicator bacteria are present in the water, signaling possible fecal contamination and the potential presence of pathogenic organisms. Positive findings typically trigger immediate resampling, investigation of the contamination source, and corrective actions such as re-disinfection or system flushing.