Pour Plate Method: Principle, Procedure, Results & Uses

The pour plate method is a microbiological technique used for the isolation and enumeration of culturable microorganisms in a sample.

• In this method, a measured volume of diluted microbial suspension (inoculum) is mixed with molten agar medium in a sterile Petri dish.
• The agar medium is allowed to solidify and then incubated under suitable growth conditions.
• After incubation, microbial colonies are formed both on the surface and within the solidified agar medium.
• The pour plate technique is commonly used for determining the number of viable microorganisms present in water, milk, food and other samples.
• The microbial sample is usually subjected to serial dilution to reduce the number of microorganisms so that separate colonies can be obtained. These colonies are counted to estimate the microbial population in the original sample.
• The count is expressed in colony-forming units (CFU), as a colony may develop from a single viable cell or a group of cells. It is one of the methods used for viable plate counting.
• The isolated colonies can also be selected and transferred to fresh culture medium for further study.

Objectives of Pour Plate Method

The following are the objectives of pour plate method–

• To estimate the number of viable or culturable microorganisms present in a sample.
• To determine the microbial load in water, milk, food, soil and other samples.
• To prepare countable plates from a sample by using proper serial dilution.
• To distribute the microbial cells throughout the molten agar medium. Colonies are formed on the surface and inside the agar.
• To calculate the microbial count as colony-forming units (CFU) per mL or per g of sample.
• To obtain separated colonies from a diluted microbial sample for further study.
• To study the growth of microorganisms in solid agar medium after incubation.
• To compare microbial count between different samples or different dilutions.
• To check the microbiological quality of food, water and other tested materials.
• To detect the presence of culturable microorganisms in a sample under the selected medium and incubation condition.
• To provide colonies which can be selected and transferred to fresh medium for purification or identification tests.

Principle of Pour Plate Method

The pour plate method is based on the growth of culturable microorganisms into separate colonies in a solidified agar medium. In this method, the microbial inoculum is mixed with molten agar which distributes the microorganisms throughout the medium. On solidification, they remain at different positions. During incubation under suitable conditions, these microorganisms multiply at their respective positions and give rise to visible colonies.

The colonies can be formed both on the surface and inside the solidified agar. Oxygen availability may be different at these locations, and the growth of microorganisms depends on their oxygen requirements and other culture conditions.

The serial dilution of microbial sample is done to reduce the number of microorganisms before plating. When a sample contains a large number of microorganisms, the colonies may grow very close to each other or overlap, which makes counting difficult. The diluted sample produces more separated colonies. These can be counted individually.

Each visible colony is considered as one colony-forming unit (CFU). It may be formed from a single viable microbial cell or a group of cells present together as a clump or chain. The number of colonies obtained is used for estimating the culturable microorganisms present in the original sample, considering the dilution of sample and the volume used for plating.

Materials Required for Pour Plate Method

The following are the materials and equipment required for performing the pour plate method. The basic laboratory equipment remains the same, whereas the culture medium, diluent and incubation conditions may vary depending on the sample and type of microbiological test.

• Sterile Petri dishes- These are used to hold the microbial sample mixed with molten agar medium. The Petri dishes must be sterile to avoid contamination during cultivation.
• Molten agar medium- A suitable sterile culture medium such as nutrient agar or plate count agar is required for the growth of microorganisms. The medium is selected depending on the type of microorganisms and the purpose of the test.
• Microbial sample or suspension- The sample containing microorganisms to be examined. It may be a liquid sample or a suspension prepared from other materials.
• Sterile diluent- Sterile water, saline solution or buffered diluent is used for preparing serial dilutions of the microbial sample. The type of diluent depends on the test requirements.
• Sterile dilution tubes or bottles- Different dilutions of the microbial sample are prepared and kept in these tubes or bottles.
• Sterile pipettes- Graduated pipettes or micropipettes with sterile tips are used to transfer a measured volume of the original or diluted microbial sample into the Petri dishes.
• Temperature-controlled water bath- The water bath maintains the agar medium in a molten condition at a suitable temperature before it is mixed with the microbial sample.
• Incubator- The inoculated Petri dishes are placed in an incubator for microbial growth. The incubation temperature and period vary according to the microorganisms and the method followed.
• Colony counter– A manual or illuminated colony counter may be used for counting the microbial colonies after incubation. It is not always required. The colonies can also be counted directly from the plates.

Procedure of Pour Plate Method

The pour plate method involves the inoculation of a diluted microbial sample into sterile molten agar medium, followed by solidification and incubation. The following steps are involved in performing the pour plate technique-

Preparation of Sample and Serial Dilution

1. Clean the working area and arrange all the required materials, such as sterile Petri dishes, pipettes, dilution tubes, sterile diluent and agar medium. All the materials coming in contact with the microbial sample must be sterile to prevent contamination.
2. Take the microbial sample in a sterile container and mix it properly to distribute the microorganisms. In case of a solid or semi-solid sample, a measured quantity is mixed with a suitable sterile diluent to prepare a uniform suspension.
3. Take a series of sterile test tubes, each containing 9 mL of sterile diluent. Using a sterile pipette, transfer 1 mL of the liquid microbial sample into the first tube containing 9 mL of diluent. Mix the contents properly. This gives a 10⁻¹ dilution of the original liquid sample.
4. From the first diluted tube, transfer 1 mL of the suspension to another tube containing 9 mL of sterile diluent. The second tube contains a 10⁻² dilution. Repeat the process in the same way to prepare 10⁻³, 10⁻⁴, 10⁻⁵ and other required dilutions. Use a fresh sterile pipette or tip for each transfer. In case of a sample suspension already prepared at a known dilution, the subsequent dilutions are calculated from that initial dilution.
5. Select two or more suitable dilutions from the prepared series for plating. The selection is made according to the expected microbial population in the sample, so that plates with separately countable colonies can be obtained after incubation.

Agar Preparation and Plate Inoculation

1. Prepare a suitable sterile agar medium, such as nutrient agar or plate count agar, depending on the test. The agar is heated until completely melted and then kept in a water bath at about 45-50°C. At higher temperature, the microbial cells may get damaged, whereas at lower temperatures the agar may begin to solidify before mixing with the sample.
2. Take sterile Petri dishes and label their bottom surface with the sample name, dilution, date and other necessary identification. Separate plates are used for each dilution. Duplicate plates may also be prepared according to the method followed.
3. Using a sterile pipette, transfer 1 mL of the selected diluted microbial sample into the centre of an empty sterile Petri dish. During the transfer, lift the lid slightly to introduce the pipette and close it immediately after transferring the sample. Do not touch the inner surface of the Petri dish with the pipette.
4. To the Petri dish containing 1 mL of microbial suspension, pour about 15-20 mL of sterile molten agar medium which has been cooled to 45-50°C. The agar is added aseptically by lifting the lid only enough for pouring. The medium should be added without delay so that the agar remains in liquid condition during mixing.
5. After adding the agar, gently rotate the covered Petri dish in clockwise and anticlockwise directions. The plate may also be moved slowly back and forth on a level surface for mixing the microbial sample with the molten agar. Avoid vigorous shaking, splashing of medium and formation of air bubbles.
6. Repeat the same procedure with the other selected dilutions, using separate sterile Petri dishes. Uninoculated medium and diluent control plates may also be prepared when required by the test to check for contamination.

Solidification and Incubation

1. The inoculated Petri dishes are placed on a flat horizontal surface and left undisturbed until the agar solidifies completely. During this period, the microbial cells remain distributed within the agar medium. Do not move or invert the plates while the agar is still in liquid condition.
2. Once the agar becomes solid, the Petri dishes are inverted with their bottom facing upwards and placed in an incubator. Inverting the plates prevents condensed water from dripping onto the agar surface. The incubation temperature and period are selected according to the type of microorganisms, culture medium and test method. For example, some standard aerobic plate count procedures use 35 ± 1°C for 48 ± 2 hours.
3. After the required incubation period, remove the Petri dishes from the incubator and examine them for the development of microbial colonies. The colonies may develop on the surface of agar as well as inside the solidified medium. Surface colonies are generally more spread out, while the colonies present within the agar may appear smaller.
4. Select the plates containing well-separated and countable colonies. Count the colonies present on and within the agar using a colony counter or by direct observation. The overlapping or confluent growth should not be counted as separate individual colonies.
5. Record the number of colonies obtained from each plate along with the corresponding dilution and volume of sample inoculated. For microbial enumeration, the colony count is used to determine the colony-forming units (CFU) in the original sample by taking into account the dilution factor and the volume plated. The result is expressed as CFU/mL for liquid samples or CFU/g for samples measured by mass.

Results and Interpretation of Pour Plate Method

After incubation, the pour plates are examined for the development of microbial colonies. The colonies may be found on the agar surface as well as inside the solidified medium. Their number is not always same. It varies with the dilution of sample, microorganisms present and the incubation conditions. The colonies are counted to estimate the microbial population in the original sample.

Colony Appearance and Distribution

The colonies formed in the pour plate method may differ in their morphological characteristics. Some colonies are circular with smooth margins. Others may have irregular edges or rough surfaces, and there may be differences in their size and colour. These characteristics depend on the type of microorganisms and their growth conditions.

There are two types of colony growth based on their position in the agar medium-

• Surface colonies- These are formed on the upper surface of the solidified agar medium. The colonies are usually larger in size and some may spread over the agar surface. Their shape and size vary with the microorganisms and culture conditions.
• Subsurface colonies- The colonies which are formed within the agar medium are referred to as subsurface colonies. They are generally smaller and more compact compared to the surface colonies. Some appear as small dots or lens-shaped growth within the agar. The oxygen and nutrient availability at different depths of the medium may influence their growth.

During counting, both surface and subsurface colonies are included if they are clearly visible and separated from each other.

The presence of colonies with different morphological characteristics may indicate that different types of microorganisms are present in the sample. However, a particular microbial species cannot be identified only by its colony morphology. Further examination and identification tests are required.

Calculation of CFU/mL

The number of microbial colonies obtained from the pour plates is used for estimating the colony-forming units (CFU) in the original sample. For liquid samples, the result is expressed as CFU per millilitre (CFU/mL).

The plates having separate and countable colonies are selected for calculating CFU/mL. In many conventional plate counting methods, plates containing 30-300 colonies are considered suitable for counting. The acceptable colony count range is not same for all the methods. For example, the U.S. Food and Drug Administration (FDA) aerobic plate count method uses 15-300 colonies per plate.

The CFU/mL is calculated from the number of colonies, dilution factor and volume of sample used for plating.

The formula is given below-

CFU/mL = (Number of colonies × Dilution factor) / Volume plated (mL)

Where,

• Number of colonies- Total countable colonies obtained from the selected agar plate.
• Dilution factor- The reciprocal of the sample dilution used for plating.
• Volume plated- The amount of diluted microbial sample inoculated into the Petri dish, expressed in millilitres.

Example-

Suppose 1 mL of microbial sample from a 10⁻⁴ dilution is used for preparing a pour plate. After incubation, 86 separate colonies are observed on the plate.

The calculation is as follows-

CFU/mL = (86 × 10⁴) / 1

CFU/mL = 8.6 × 10⁵ CFU/mL

The estimated culturable microbial population of the original liquid sample is 8.6 × 10⁵ CFU/mL under the cultivation conditions used.

When duplicate plates are prepared from the same dilution, the average colony count may be used for the calculation. In case of countable plates obtained from different dilutions, the counts are combined according to the particular method followed.

A single CFU does not always arise from one microbial cell. It may also be formed from a group of viable cells present together as a clump or chain. Microorganisms which are unable to grow on the selected culture medium and incubation conditions are not included in the colony count.

Interpretation of Uncountable or Unreliable Plates

In the pour plate method, some plates may not be suitable for counting the colonies. The plates may contain very high or low colony numbers. In certain cases, irregular growth, contamination or other changes in the agar medium can make the counting difficult.

The different results observed in such plates are described below-

• Too Numerous to Count (TNTC)- In a sample containing a large number of culturable microorganisms, numerous colonies develop close to each other on the agar medium. The neighbouring colonies may join together, forming confluent growth. Individual colonies cannot be counted properly. Such plates are recorded as TNTC. A plate prepared from a higher dilution is generally selected for counting.
• Too few colonies- The plates prepared from highly diluted samples or samples having low microbial population may contain only a few colonies. These counts have greater statistical variation and may not be reliable under the selected counting standard. If available, a plate from a lower dilution may be selected.
• No colony growth- In some plates, no visible colonies are formed even after the required incubation period. It is recorded as no colonies detected in the plated volume. This does not always indicate the absence of microorganisms in the original sample. The microbial population may be below the detection limit of the method. Some microorganisms may also fail to grow on the selected culture medium or under the incubation conditions used.
• Overlapping and spreading colonies- Certain colonies may spread over a large area of the agar medium or overlap with the surrounding colonies, making individual counting difficult. Such plates are examined according to the counting rules of the method followed. Plates which are unsuitable for counting are not used for an ordinary colony count.
• Contaminated plates- The growth of unexpected microorganisms due to contamination during inoculation or incubation may affect the colony count. Sometimes microbial colonies may also appear on the uninoculated control plate. This indicates possible contamination of the culture medium or during handling. The affected plates should not be used for a reliable microbial count without investigating the contamination.
• Air bubbles and other irregularities- During mixing of the microbial sample with molten agar, small air bubbles may be formed within the medium. They may sometimes look similar to the small colonies present inside the agar. These air bubbles are not included in the colony count. The plates are examined carefully using suitable illumination or a colony counter to distinguish the embedded microbial colonies from the bubbles.

Precautions of Pour Plate Method

The following precautions are followed during the pour plate method–

• Use only sterile Petri dishes, pipettes, dilution tubes and culture media. The working area is disinfected before starting the work.
• Do not keep the Petri dish open for long time. Open the lid only during inoculation and pouring of agar.
• Microbial samples are handled under proper biosafety requirements. Use laboratory coat, gloves and eye protection when required. Mouth pipetting is avoided. Unknown or pathogenic cultures are handled under suitable containment.
• Contaminated pipettes, plates and other materials are decontaminated before disposal.
• The microbial sample is mixed properly before dilution. Unevenly mixed sample may give uneven colony count.
• During serial dilution, correct volume of sample and diluent is used. Each dilution tube is mixed before the next transfer.
• Use a fresh sterile pipette or tip for every transfer. This prevents carry over of sample from the previous dilution.
• The agar medium must be suitable for the microorganisms to be tested. It should be sterile. Unsuitable medium may not allow the growth of some microorganisms.
• The molten agar is maintained at about 45-50°C or at the temperature given in the method. A water bath is used for this purpose.
• Very hot agar may kill or damage microbial cells. Cold agar may start solidifying before proper mixing.
• After adding agar, rotate the covered Petri dish gently. The inoculum should mix uniformly with the molten agar.
• Vigorous shaking is avoided. It may cause splashing, uneven distribution and air bubbles.
• The inoculated plates are kept on a level surface until agar becomes solid. Do not invert the plates before solidification.
• After solidification, the plates are inverted and incubated at required temperature, time and atmospheric condition. Wrong incubation condition may affect the microbial growth.
• Petri dishes and dilution tubes are labelled with sample name, dilution and other details. The volume plated is also recorded for correct colony-forming units (CFU) calculation.
• Uninoculated agar plate and sterile diluent control are used when required. No growth should appear in these controls. Growth in control plate indicates contamination.
• Duplicate plates may be prepared to check variation in colony count.
• Select plates having separate and countable colonies. Surface colonies and embedded colonies both are counted.
• Air bubbles are not counted as colonies. Plates with excessive, spreading or overlapping colonies are not suitable for accurate counting.
• Some errors can be prevented by proper technique, but some are part of the method itself. Heat-sensitive microorganisms may not survive molten agar. Some microorganisms may not grow in the selected medium and incubation condition.
• A colony may arise from one viable cell or a group of cells present together. This cannot be completely avoided by careful plating.

Applications of Pour Plate Method

The following are some of the important applications of pour plate method–

• Viable plate count- It is used to estimate the number of culturable microorganisms present in a sample. The result is expressed as colony-forming units (CFU).
• Enumeration of bacteria in food samples- The pour plate method is commonly used for counting aerobic bacteria in food materials. Examples include milk, meat products, processed foods and other edible samples.
• Water microbiology- It is used for estimating microbial load in drinking water, wastewater and other water samples. The count helps to know the number of culturable heterotrophic bacteria present in the sample.
• Environmental microbiology- Soil, air-settled dust, sewage and other environmental samples can be examined by this method after proper dilution. It gives an idea about the microbial population present in the environment.
• Pharmaceutical quality control- The method is used for microbial count of pharmaceutical products, raw materials and water used in production. It helps to detect excess microbial contamination.
• Testing of milk and dairy products- Pour plate method is used for counting viable bacteria in milk, curd, cream and other dairy products. High count indicates poor handling, storage or contamination.
• Recovery of discrete colonies- Diluted samples give separated colonies after incubation. These colonies can be picked and transferred to fresh medium for further study.
• Study of surface and embedded colonies- The method allows growth of microorganisms on the surface as well as inside the agar medium. It is useful where both aerobic and some less oxygen-dependent organisms may grow.
• Comparison of microbial load between samples- Different samples or different dilutions can be compared by their colony count. It is useful in routine microbiological analysis.
• Checking effectiveness of sanitation and processing- The pour plate method can be used before and after cleaning, treatment or processing. A reduced colony count indicates lower number of culturable microorganisms.
• Microbial monitoring in laboratories and industries- It is used in food industry, water testing laboratories, pharmaceutical laboratories and teaching laboratories for routine microbial enumeration.
• The pour plate method is mainly used for counting culturable microorganisms. It is not used for direct identification of a microbial species without further biochemical, microscopic or molecular tests.

Advantages of Pour Plate Method

The following are the advantages of pour plate method–

• Viable count- It is used to count living or culturable microorganisms present in the sample. Dead cells are not counted because they do not form colonies.
• Useful for microbial enumeration- The diluted sample produces separate colonies after incubation. These colonies are counted and expressed as colony-forming units (CFU).
• Larger sample volume- A measured volume of sample, commonly 1 mL, can be used in this method. It is useful when the microbial number is low.
• Growth throughout the medium- Microorganisms are mixed with the molten agar. Colonies are formed on the surface and also inside the agar medium.
• Recovery of discrete colonies- Well-separated colonies can be picked from the plate and transferred to fresh medium for further study.
• Useful for food and water testing- The method is used for estimating microbial load in milk, food, drinking water and wastewater samples.
• Suitable for routine laboratory work- It does not require highly complex instruments. Sterile Petri dishes, pipettes, dilution tubes, agar medium and incubator are generally enough.
• Reduces overcrowding by dilution- Serial dilution reduces the number of microorganisms before plating. It gives better chance for separated colonies.
• Can detect embedded growth- Some colonies develop within the agar medium. This is useful for observing microorganisms that can grow below the agar surface.
• Quantitative result- The number of colonies gives an estimate of microbial population in the original sample. The result can be calculated with dilution factor and volume plated.
• Comparison between samples- Different samples can be compared by their colony count. It is used in routine microbiological analysis.
• Simple observation of colony characters- Colony size, shape, colour and distribution can be observed after incubation. But species identification requires further tests.

Limitations of Pour Plate Method

The following are the limitations of pour plate method–

• In this method, the microbial sample comes in contact with molten agar. Heat-sensitive microorganisms may be damaged or killed.
• Only culturable microorganisms are counted. Those organisms which do not grow in the selected medium are not detected.
• The incubation condition also affects the count. Some microorganisms may be present in the sample but fail to grow.
• One colony-forming unit (CFU) may not represent one cell. It may arise from one viable cell or from a clump of cells.
• Colonies formed inside the agar are usually small. These embedded colonies are sometimes difficult to see. Counting of embedded colonies needs careful observation. Some colonies may be missed.
• The method is time taking. Dilution, agar preparation, pouring, incubation and counting are needed before result.
• Agar temperature must be controlled. Very hot agar damages the cells. Cold agar may solidify before proper mixing.
• Air bubbles can form during mixing of the sample with agar. These bubbles may look like small colonies in the medium.
• Highly concentrated samples give crowded growth. The colonies may overlap or become confluent. Such plates are not suitable for accurate counting.
• Small errors can change the final count. Error may occur during sterilization, dilution, pipetting or aseptic handling.
• It does not identify the microorganism directly. The method gives colony number and colony appearance only. For identification, biochemical, microscopic or molecular tests are required.
• Fast-growing microorganisms may cover the slow-growing microorganisms. This makes the slow-growing forms difficult to detect on the same plate.
• The result depends on the medium and incubation condition used. Unsuitable medium may not support all microorganisms present in the sample.

Comparison with Other Plating Methods

The pour plate method, spread plate method and streak plate method are used for different purposes in microbiology. The method is selected according to the aim of work, type of sample and need of colony counting.

Pour Plate vs Spread Plate Method

Pour Plate vs Streak Plate Method

References

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