Pour Plate Method- Definition, Principle, Procedure, Uses

The pour plate method is a microbiological laboratory technique that is used to isolate and count the viable microorganisms present in a liquid sample or a suspension of a solid sample. The sample is mixed with a molten agar medium and poured into a Petri dish, where it solidifies and forms a solid culture medium. The microorganisms in the sample grow into visible colonies on the surface or within the medium, which can be counted and identified.

The pour plate method is one of the oldest and simplest methods for isolating microorganisms and estimating their numbers in a given sample. It was developed in the laboratory of Robert Koch, the father of bacteriology, and is still widely used today. The pour plate method can be used for various purposes, such as:

• To determine the number of colony-forming units (CFUs) per milliliter of a liquid sample or per gram of a solid sample
• To isolate pure cultures of microorganisms from a mixed population
• To study the colony morphology and characteristics of different microorganisms
• To test the quality and safety of food, water, pharmaceuticals, and other products by detecting microbial contamination

The pour plate method is suitable for isolating facultative, microaerophilic, and anaerobic microorganisms, as well as some obligate aerobes . However, it has some limitations, such as:

• The need for serial dilution of the sample to obtain an optimal number of colonies (between 20 and 300 CFU/mL) for accurate counting
• The possibility of heat damage to some heat-sensitive microorganisms by the molten agar medium
• The difficulty in distinguishing between surface and subsurface colonies
• The slow growth and small size of some colonies within the medium

In this article, we will discuss the objectives, principle, requirements, procedure, results, precautions, applications, advantages, and limitations of the pour plate method in detail. We will also provide some examples and tips for performing this technique successfully.

The pour plate technique has several objectives that make it a useful and versatile method for microbiological analysis. Some of the main objectives are:

• To isolate the microorganisms from the liquid specimen (or suspension). The pour plate technique allows the separation of individual microorganisms from a mixed population by diluting and dispersing them in a solid medium. This can help in identifying and characterizing the microbial diversity and composition of a sample.

• To calculate viable microbial load by counting colony formation unit (CFU) per mL. The pour plate technique enables the estimation of the number of living microorganisms in a sample by counting the visible colonies that develop after incubation. Each colony is assumed to arise from a single viable cell or a group of cells that act as a unit. The CFU per mL can be calculated by multiplying the number of colonies by the dilution factor.

• To isolate the pure culture of microorganisms from a mixed population. The pour plate technique can also be used to obtain pure cultures of microorganisms by picking isolated colonies from the plates and subculturing them on fresh media. A pure culture is a population of microorganisms that belong to the same species and have identical genetic characteristics.

• To isolate microorganisms in discrete colonies in order to study colony characters. The pour plate technique can also provide information about the morphology, color, size, shape, and texture of the colonies formed by different microorganisms. These colony characters can be used as preliminary clues for identification and classification of microorganisms.

The pour plate method is based on the principle of counting viable colonies of microorganisms using serial dilution. A serially diluted sample (usually 1 ml) is poured into the petri dish, and molten agar at 45-50℃ is added to the dish and swirled. After solidification, the plate is incubated at an optimal temperature.

The microorganisms in the sample will grow and form visible colonies on the surface and within the agar medium. Each colony represents a single viable cell or a colony-forming unit (CFU) that was present in the original sample. The number of CFUs can be calculated by multiplying the number of colonies by the dilution factor and dividing by the volume of the sample.

The pour plate method can be used to isolate and enumerate different types of microorganisms, such as bacteria, fungi, actinobacteria, and yeasts. It can also help identify microorganisms as aerobes, anaerobes, or facultative aerobes based on their location and appearance on the agar medium. Aerobic microorganisms will grow on the surface of the agar, while anaerobic microorganisms will grow within the agar. Facultative microorganisms can grow both on the surface and within the agar.

The pour plate method is an economical and simple technique for laboratories to perform tests focused on a specific number of microorganisms. It can also be used to isolate pure cultures of microorganisms from a mixed population and study their colony characteristics.

To perform the pour plate method, you will need the following materials and equipment:

• Liquid specimen (or suspension of the solid sample): For the pour plate, the sample must be either liquid or in suspension form. The solid sample must be suspended in a suitable solvent that doesn`t influence the growth of any microorganisms or react with any material of the media .
• Suitable solid culture media: Specific culture media is used for the isolation and differentiation of suspected (or specific) bacteria. The culture medium is a solid agar medium that is melted and is at a temperature of 40 – 45°C.
• Petri plates: Mostly 10 cm Petri plates are used. They must be sterile and must be labeled before pouring samples or media .
• Test-tubes: Test tubes are needed for serial dilution during sample preparation. The tubes must be sterile .
• Sterile distilled water (or sterile broth): Either distilled water or broth can be used for serial dilution. They are also used for dissolving the solid or semisolid sample.
• Micropipette (or graduated pipette): A micropipette of 0.1 mL or 1 mL capacity is required for measuring the sample during serial dilution and sample inoculation .
• Other laboratory facilities: You will also need a hot water bath, a flame, a colony counter with a magnifying glass, and an incubator .

The general procedure for performing the pour plate method can be summarized as follows:

• Arrange all the requirements, put on the personal protective equipment (PPE), sterilize the work surface, and set up the laboratory equipment.
• Sample preparation: If the sample is in liquid form, serially dilute it to make the microbial load to the range of 20 – 300 CFU/mL. (Prior pilot test may give exact value. You can prepare serial dilution up to 10^-10^ and use different dilutions.) If the sample is in solid or semisolid form, dissolve it in sterile distilled water or sterile broth, or any other solvent. (Generally, 1 gm sample is mixed with 9 ml of solvent to get the concentration of 10^-1^ gm/mL.)
• Media preparation: Suitable media (general-purpose media like Nutrient Agar and Plate Count Agar for bacteria, and Potato Dextrose Agar or Sabouraud Dextrose Agar for fungi) are prepared and autoclaved. The media is allowed to cool to about 40 – 45°C (maximum up to 55°C), but don’t let it solidify. If the media is prepared already and solidified, melt it by placing it over a water bath or other heat source. If you want to mix the sample in media prior to pouring it into the Petri plate, you can either add approximately 15 mL of media in one test tube or beaker and autoclave it. Alternatively, a fixed volume of media can be prepared in a large beaker or bottle and a sample can be added later by calculating the volume which will be equivalent to 1 mL sample per about 15 mL of media.
• Arrange sterile Petri plates. Label at the edge of the bottom of the plate with the dilution factor, date, name, sample ID, and other required information.

• Inoculation: There are two methods for inoculating the sample and media in the Petri plate.

  • Method I: Dispense 1 ml of diluted sample in the center of the Petri plate using a sterile micropipette or calibrated pipette. Open the lid of the bottle and flame its mouth. Pour about 15 mL of sterilized molten media at the appropriate temperature above the sample. Close the lid of the plate then mix the sample in the media properly by gently swirling the plate. The plate is generally swirled in an “S” or “8” shape. (Put sample at specific dilution in the plate labeled with the specific dilution factor.)
  • Method II: In a tube with about 15 mL of molten media at a suitable temperature, add 1 mL of sample. Mix the sample properly in the media. Pour the media into a sterile Petri plate.

• Close the lid of the Petri plate. Allow the media to completely solidify.

• Incubate the plate in an inverted position under suitable incubation conditions (mostly for 24 hours at 37°C).

Following the incubation, developed colonies are observed. Each colony will account for one viable microbial cell or one colony-forming unit (CFU).

• If all the colonies are of the same type, then we can predict that the sample contained only one type of microbial genera. However, there may be different genera or species producing similar types of colonies. Hence, further tests are required for their identification.
• If the colonies are of different morphology, then we can conclude that the sample contained a mixed population. They can be purified by sub-culturing each colony on a separate culture media plate using the streak plate method.

Count the colonies and calculate CFU/mL by using the formula:

$$CFU/mL = \frac{Number\ of\ colonies}{Volume\ of\ sample\ (mL) \times Dilution\ factor}$$

This will give the total number of viable microbial cells present in the given sample.

For optimum count, the number of colonies must be between 20 – 300 CFU/mL. Beyond this limit, the whole procedure must be repeated.

• If the number of colonies is less than 20, it is suggested to use the sample of lower dilution, whereas, if the total number of colonies exceeds 300, it is suggested to use the sample of higher dilution on successive repeats.
• If the colonies are fused or the whole plate is covered with a single colony, then report as “too numerous to count” (TNTC) and repeat the process of taking the sample at a higher dilution.

• The pour plate technique should be performed under aseptic conditions to avoid any contamination of the sample or the medium. A laminar air flow cabinet or a safety cabinet can be used for this purpose .
• The quantity and accuracy of the sample and the medium should be measured carefully while preparing the serial dilutions and pouring the plates. Sterile pipettes or micropipettes should be used for transferring the sample and the medium .
• The temperature of the molten medium should be between 40°C to 45°C when it is mixed with the sample or poured over it. Higher temperatures can kill or damage the heat-sensitive microorganisms, while lower temperatures can cause the medium to solidify or form clumps .
• The sample and the medium should be mixed properly by swirling the plate gently in an "S" or "8" shape. This ensures a uniform distribution of the microorganisms throughout the medium .
• The incubation conditions should be suitable for the growth of the microorganisms present in the sample. The temperature, time, and atmosphere should be adjusted according to the type of microorganisms being isolated .
• The colony count should be within the range of 20 to 300 CFU/mL for an optimum result. If the colony count is below 20 or above 300 CFU/mL, the procedure should be repeated with a different dilution factor of the sample .
• The colony morphology should be observed carefully to identify different types of microorganisms present in the sample. If the colonies are fused or too numerous to count (TNTC), the procedure should be repeated with a higher dilution factor of the sample .

The pour plate technique has various applications in microbiology and related fields. Some of the common applications are:

• It is used to isolate and enumerate viable bacteria and fungi (calculate CFU/ml) from suspensions or liquid samples. This can help in assessing the microbial quality and safety of food, water, beverages, pharmaceuticals, cosmetics, and other products.
• It is used to isolate and enumerate microorganisms from soil to study soil microflora and their role in nutrient cycling, biodegradation, and bioremediation.
• It is used to generate growth curves while studying microbial metabolisms and biochemical features, and the effects of environmental factors on microbial growth.
• It is used to isolate pure culture of microorganisms from a mixed population. The isolated colonies can be further subcultured and identified by various biochemical and molecular tests.
• It is used to isolate microorganisms in discrete colonies in order to study colony characters such as shape, size, color, texture, elevation, margin, hemolysis, etc.
• It is used to separate pure culture from mixed cultures. This can help in isolating pathogens from clinical specimens or contaminants from industrial samples.
• It is suitable for isolating facultative and anaerobic microorganisms. Aerobes can also be isolated by this method.

The pour plate technique has several advantages over other methods of isolating and enumerating microorganisms, such as:

• It is simple and easy to perform, and does not require extra tools or materials for inoculation.
• It does not require previously solidified agar plates, which can save time and resources.
• It can detect very low loads of bacterial counts as well, as a larger volume of sample can be used than the spread plate method .
• It can also be used for clinical and environmental samples, such as water, beverages, foodstuff, tissue samples, etc .
• It can isolate and enumerate both aerobic and anaerobic microorganisms, as well as facultative and microaerophilic ones .
• It can generate discrete colonies for obtaining pure cultures and studying colony characters .
• It can use any type of specimen, liquid or solid (it can be dissolved), as long as it is properly diluted .

Limitations of Pour Plate Technique

• The pour plate technique has some limitations that may affect the accuracy and reliability of the results. Some of the limitations are:

• Solid or semisolid samples must be suspended prior to inoculation. It is very difficult if the sample is not easily soluble or if it contains large particles that may interfere with the mixing of the sample and the agar .

• Heat-sensitive organisms may be killed or injured by the molten agar. The temperature of the agar medium must be between 40-45°C to prevent solidification and clumping, but some microorganisms may not survive or grow well at this temperature .

• Colonies may be smaller and less visible than in other methods. The colonies that grow within the medium are usually smaller and less distinct than those that grow on the surface. They may also be confluent or overlapping, making them difficult to count or identify .

• Obligate aerobes may have reduced growth in the depth of the agar. The oxygen supply may be limited for the microorganisms that grow at the bottom of the plates, especially if the agar is too thick or dense. This may affect their growth rate and colony size .

• The method is time-consuming and requires more materials than other methods. The pour plate technique involves dissolving or suspending the solid samples, serial dilutions, melting and cooling of the agar medium, and pouring and mixing of the sample and the agar. It also requires more Petri plates, pipettes, test tubes, and media than other methods .

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