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What is the Pour Plate Method?
The pour plate method is a plating technique that is commonly used for obligate and anaerobic bacteria. This technique is used to isolate microbial colonies by serial dilution and then counting the colony forming units (CFUs). In this method, the liquid sample is poured into the petri dish before the solidification of the agar medium. After solidification, colonies grow both inside and on the surface of the medium. However, the colonies growing inside the medium are confluent; those on the surface are used for viable counting.
Pour Plate Method Principle
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. Viable microbial colonies can be observed on the plate after incubation that can be counted. The CFU/ml can be obtained by the following formula:
Pour Plate Method Procedure
- Sterilise all the instruments, flasks, and media that are required for the streaking procedure.
- Clean your work area using a disinfectant to minimise any contamination.
- Set up the bunsen burner in your work area carefully.
- Wash your hands with an antiseptic solution before handling any microbial solution.
- Label the petri dish with all important information, such as your name, date, media used, and the culture being inoculated.
- Sample Preparation: If the sample is in semisolid or solid form, suspend it in sterile water or broth to prepare a liquid solution. If the sample is already in liquid form, prepare serial dilutions of the sample to lessen the load of microbial colonies in the range of 20-300 CFU/ml. You can prepare dilutions up to 10-10.
- For inoculation, open the lids of the Petri dishes and pour 1 ml of the diluted sample. Take the molten agar, heat it a little and pour around 15-18 ml of it onto the sample. Keep in mind that the agar should not be either too hot or too cold. Close the lid of the dish and swirl it slowly.
- Another method for inoculation is to mix the diluted sample in the agar medium, mix it gently, and then pour it into the petri dish. However, this method is less commonly used.
- Let the plate solidify.
- Invert the plate and incubate it at an optimal temperature (usually 37℃) for 24-48 hours.
Read: Morphology and Different Shapes of Bacterial Cell
Interpretation
After incubation, the plates are observed for viable colony counts. If all the colonies look alike, it indicates that only one bacterial species is present on the plate. However, if different colonies are seen, it is either different species that have grown on the plate or there is some kind of contamination.
For obtaining the CFU/ml, count the colonies on the plate, and use the above formula to get the value. The optimum count of colonies obtained must be between 20-300 CFU/ml. If it is more or less than this range, the whole process needs to be repeated.
Applications of the Pour Plate Method
- It is used by scientists to obtain microbial growth curves and in the calculation of the concentration of cells in a particular sample.
- It is also used to check the effect of various growth factors and environmental factors on the growth rate of the bacteria.
Advantages of the Pour Plate Method
- It is useful for counting viable colonies.
- It can detect very low loads of bacterial counts as well.
- It does not require previously solidified agar plates.
- It can also be used for clinical and environmental samples.
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