Microbial Enumeration in Water: Membrane Filter Technique

Principle of Membrane Filter Technique

The membrane filter technique is based on the principle of using a porous membrane filter to physically trap microorganisms from a liquid sample. By passing an unknown volume of the sample through the membrane, microorganisms are retained on the filter surface. After incubation on a suitable agar medium, microbial colonies develop, allowing for the quantification of viable microorganisms in the original sample.

Introduction to Equipment

Before performing the membrane filter technique, it is important to have the necessary equipment. This includes:

• Membrane filters: sterile filters made of materials like cellulose acetate, nitrocellulose, or other suitable materials with defined pore sizes.
• Filtration apparatus: a device designed to hold the membrane filter securely and facilitate the passage of liquid through the filter. It includes a funnel, filter support, and a receiver for the filtrate.
• Vacuum pump or pressure system: provides the necessary force to pass the liquid sample through the membrane filter. The type of pump or system used depends on the specific filtration setup.
• Sterile forceps or membrane filter dispenser: tools for aseptic handling and transfer of the sterile membrane filter from the filtration apparatus to a petri dish.
• Petri dishes: sterile containers for holding the membrane filter during incubation and colony counting.
• Culture media: sterile agar medium suitable for the growth of the target microorganisms. Nutrient agar is commonly used.
• Incubator: maintains a controlled temperature conducive to the growth of microorganisms. It is typically set to 35 to 37°C for bacterial analysis.
• Disinfectants: solutions such as ethanol or bleach for decontaminating equipment and surfaces to prevent cross-contamination.
• Safety equipment: personal protective equipment (PPE) including gloves and safety glasses to ensure the safety of the personnel handling the samples.

Procedure of Water Testing

Makoni Agar Plate Method

Materials required for the Makoni agar plate method:

• Makoni agar plates
• Sterile syringe
• Incubator set to 37°C

Steps:

1. Ensure the Makoni agar plates are sterile and ready for use.
2. Sample collection: Collect a water sample in a sterile container and label it with the source of water and the date. Also, label the plates with the date and water sample name.
3. Inoculation: Using a sterile syringe, aseptically take 1 mL of water sample. Remove any air bubbles from the syringe and spread the water evenly on the surface of the Makoni agar plate.
4. Incubation: Close the lid of the Makoni agar plate without tilting it and allow the water to be absorbed for a few minutes. Incubate the plates at 37°C for 24 to 48 hours.
5. Observation: After incubation, check for the growth of characteristic colonies. Makoni agar is selected for gram-negative bacteria, and lactose fermenting clones will appear pink or red.

Membrane Filtration Technique

Materials and equipment required for membrane filtration technique:

• Membrane filtration unit: includes funnel, filter flask, and vacuum pump
• Membrane filters
• Nutrient agar plates
• Sterile forceps
• Incubator set to 37°C

Steps:

1. Prepare the filtration unit: Assemble the membrane filtration unit and connect the filter flask to the vacuum pump, ensuring a tight seal for proper filtration.
2. Filter preparation: In aseptic conditions, take a sterile membrane filter out of the box using sterile forceps. Lift the filter from the corner without touching the membrane and remove the covering. Carefully place the exposed membrane filter onto the filter funnel and place the filter funnel on top of the filter membrane.
3. Sample filtration: Pour a small volume of water through the funnel and switch on the vacuum pump. The water will pass through the membrane filter. When all the water has been passed, aseptically remove the filter funnel and the membrane filter from the funnel using sterile forceps.
4. Plate preparation: Place the membrane filter onto the center of a sterile nutrient agar plate, making sure the wide part of the membrane filter is placed on top of the nutrient agar.
5. Incubation: Incubate the nutrient agar plates at 37°C for 72 hours. Check for microbial growth after 24 hours and at regular intervals.
6. Observation: Observe the nutrient agar plates for the presence of colonies. Count and identify colonies as needed. Record observations and report the results, including the source of water and the date.

Precautions

When performing the membrane filter technique for water analysis or other microbial applications, it is essential to follow specific precautions to ensure accurate and reliable results and maintain a safe working environment. Some important precautions to consider are:

1. Personal protective equipment (PPE): Use appropriate PPE, including gloves and safety glasses, to protect yourself from potential exposure to microorganisms and chemicals.
2. Aseptic techniques: Practice strict aseptic techniques to prevent contamination of samples and equipment. Sterilize all equipment before use.
3. Sample handling: Handle samples carefully to avoid spills or splashes. Use sterile containers for sample collection and seal them securely.
4. Decontamination: Decontaminate work surfaces and equipment with appropriate disinfectants before and after use to minimize the risk of cross-contamination.
5. Filter handling: Use sterile forceps or a membrane filter dispenser when handling filters to avoid contamination. Touch only the edges of the filter.
6. Membrane filter placement: Ensure proper placement of the membrane filter in the filter holder to prevent leaks or uneven filtration.
7. Avoid air bubbles: Minimize the introduction of air bubbles during sample filtration as they can impact the accuracy of colony counts.
8. Proper priming (if required): If using a vacuum filtration system, ensure proper priming to eliminate air from the system. Follow manufacturers' guidelines.
9. Quality control: Include positive and negative controls in each analysis to validate the accuracy of the procedure and reliability of the results.
10. Incubation conditions: Maintain consistent and appropriate incubation conditions, including temperature and humidity, for accurate colony development.
11. Record keeping: Keep detailed and accurate records of the procedure, including sample information, filtration parameters, and incubation conditions.
12. Regular calibration: Calibrate equipment regularly to ensure accurate measurement of sample volumes and flow rates.
13. Emergency procedures: Be familiar with emergency procedures in case of equipment malfunction, spills, or other unforeseen incidents.
14. Proper waste disposal: Dispose of contaminated materials properly according to local regulations and guidelines.
15. Training: Ensure the personnel performing the membrane filter technique are adequately trained in microbiological techniques and safety protocols.
16. Follow manufacturer's instructions: Adhere to the manufacturer's instructions for all equipment and agents used in the procedure.
17. Regular maintenance: Perform routine maintenance on equipment to ensure its proper functioning and prevent potential issues.
18. Consult guidelines: Consult prevalent guidelines, standards, and regulatory requirements for water quality testing in your specific region or industry.

By implementing these precautions, you can help minimize the risk of contamination, ensure the accuracy of results, and maintain a safe laboratory environment during the membrane filter technique. Always follow established protocols and guidelines for water quality testing in your specific application.

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