Pour Plate Method

The pour plate method is a technique used in microbiology to isolate and count viable organisms in liquid samples. Developed in the laboratory of Robert Koch, the technique is especially suitable for facultative, microaerophilic, and anaerobic microorganisms. It is widely used due to it's simplicity, cost-effectiveness and the requirement of minimal resources.

Pour plate method

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The technique is based on the principle that each viable organism will grow into a microbial colony when mixed with molten agar and incubated. Usually, a 1 ml of serially diluted sample is mixed with approximately 15 ml of molten agar at 40-45°C in a Petri dish. Once the solution solidifies and is incubated, colonies start to develop on the surface and within the medium. This allows the researcher to perform colony counting and calculate the colony-forming units (CFU) per ml.

Objectives

Isolate microorganisms from a liquid specimen or suspension 

Calculate viable microbial load by counting CFU per ml

Obtain a pure culture of microorganisms from mixed populations

Isolate microorganisms in discrete colonies to study colony morphology

Materials and methods

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Materials required

• Liquid specimen or suspension of the solid sample
• Molten agar at 40-45°C
• Sterile Petri dishes (10 cm diameter)
• Sterile test tubes for serial dilution
• Sterile distilled water or broth
• Micropipette

Step-by-step method

Pour plate method

1. Preparation: arrange all the required materials, put on your PPE and sterilize the work surface.
2. Sample preparation:
   1. If you are using a liquid sample, dilute them serially to achieve 20-300 CFU/ml.
   2. If you are using a solid sample, emulsify it in a sterile solvent before dilution.
3. Media preparation: Prepare the molten agar media and sterilize it by autoclaving. Allow it to cool to 40-45°C, but do not let it solidify.
4. Inoculation: There are two methods that can be used for this step.
   1. Add 1 ml of sample to the Petri dish, pour molten agar on top and gently swirl the dish to mix them together.
   2. Mix 1 ml of the sample with molten agar in a test tube and pour into a Petri dish, sidestepping the swirling step.
5. Incubation: Once the medium solidifies, incubate the samples under appropriate conditions. For example, this could be at 37°C for 24-48 hours.
6. Counting colonies: Following the incubation, you can count the colonies using a colony counter and calculate the CFU/ml using this formula:

Results interpretation

After the incubation, each developed colony represents a viable microbial cell or one CFU. If all the colonies appear the same, it is very likely that only one species was present in the sample. To confirm this, further testing is required as some species can produce the similar colony types. Difference in colony morphologies within the plate suggests that the sample contained mixed population. In this case, it is recommended to sub-culture individual colonies in separate Petri dishes using the streak plate method. It is preferred that the colony count is between 20-300 CFU/ml. If there are less than 20 colonies, it is recommended that a lower dilution be used. In case the count is above 300, a higher dilution should be used. If the colonies are fused together or the entirety of the surface is covered with one colony, the result should be recorded as "too numerous to count" and the process needs to be repeated with higher dilution. 

Precautions

When using the pour plate method, it is important to follow the proper safety protocol. Firstly, it is necessary to treat all the unknown samples as potentially hazardous. As such, it is crucial to keep sterility throughout the whole process, including using sterile Petri dishes, culture media as well as the working environment. The media and water used for the serial dilution should also be kept sterile. Secondly, it is important that the solvent for the emulsification of solid samples does not inhibit or support the microbial growth. Thirdly, the sample needs to be appropriately diluted to maintain viable microbial growth. Precise measurements are necessary and require the use of a micropipette when transferring the samples into a Petri dish or the media. Lastly, all the Petri dishes need to be labelled prior to the media addition, including the specimen name and dilution factor.

Applications of pour plate method

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The pour plate method is often used for the isolation and enumeration of viable bacteria and fungi from liquid or solid samples. It contributes to the control of quality and product safety in the food and pharmaceutical industries by isolating microorganisms and colony counting. The method is integral for generating growth curves for studying microbial metabolism and biochemical characteristics. It is also effective in separating pure cultures from mixed microbial populations.

Advantages

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• Simple and cost-effective, requiring minimal tools and resources.
• Capable of detecting low microbial loads.
• Suitable for isolating facultative and anaerobic microorganisms.
• Produces isolated colonies that can be used for further studies.

Limitations

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• Not suitable for heat-sensitive organisms due to the temperature of molten agar.
• Time-consuming, requiring preparation steps like serial dilution and media melting.
• Difficulties may arise with solid samples that are not easily emulsified.
• May not support the optimal growth of obligate aerobes at the bottom of the plate.

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