Pour Plate Method: Procedure, Uses, (Dis) Advantages

By Acharya Tankeshwar •  Updated: 06/05/22 •  4 min read

Pour plate method is usually the method of choice for counting the number of colony-forming bacteria present in a liquid specimen. Because the sample is mixed with the molten agar medium, a larger volume can be used than with the spread plate.

Principle

In the pour plate method, a fixed amount of inoculum (generally 1 ml) from a broth/sample is placed in the center of a sterile Petri dish using a sterile pipette. Molten cooled agar (approx. 15mL) is then poured into the Petri dish containing the inoculum and mixed well. After the solidification of the agar, the plate is inverted and incubated at 37°C for 24-48 hours.

Microorganisms will grow both on the surface and within the medium. Colonies that grow within the medium generally are small in size and maybe confluent; the few that grow on the agar surface are of the same size and appear like those on a streak plate. Each (both large and small) colony is carefully counted (using magnifying colony counter if needed). Each colony represents a “colony-forming unit” (CFU).

The number of microorganisms present in the particular test sample is determined using the formula:

CFU/mL= CFU * dilution factor * 1/aliquot

Pour plate technique

For accurate counts, the optimum count should be within the range of 30-300 colonies/plate. To ensure a countable plate a series of dilutions should be plated. The pour plate method of counting bacteria is more precise than the streak plate method, but, on average, it will give a lower count as heat-sensitive microorganisms may die when they come in contact with a hot molten agar medium.

Uses of Pour plate method

The pour plate technique can be used to determine the number of microbes/mL in a specimen. It has the advantage of not requiring previously prepared plates and is often used to assay bacterial contamination of foodstuffs.

Materials and Equipment

  1. Test sample
  2. Plate count agar (PCA) or nutrient agar
  3. Hot water bath 45°C
  4. Sterile Petri dishes
  5. Flame
  6. Colony counter with a magnifying glass
  7. Sterile capped 16*150 mm test tubes
  8. Pipettes of various sizes (e.g. 01, 1.0 and 2.0 mL)

Procedure of Pour plate technique

  1. Prepare the dilution of the test sample expected to contain between 30-300 CFU/mL. (Follow serial dilution technique)
  2. Inoculate labeled empty Petri dish with specified mL (0.1 or 1.0 mL) of diluted specimen

Note: for the detailed description regarding the use of pipette, inoculation of the sample, dilution technique, etc, follow reference 1.

Pouring the molten agar and incubation

Pouring the moten agar medium
Pouring the molten agar medium
  1. Collect one bottle of sterile molten agar (containing 15 mL of melted Plate Count Agar or any other standard culture media) from the water bath (45°C).
  2. Hold the bottle in the right hand; remove the cap with the little finger of the left hand.
  3. Flame the neck of the bottle.
  4. Lift the lid of the  Petri dish slightly with the left hand and pour the sterile molten agar into the Petri dish and replace the lid.
  5. Flame the neck of the bottle and replace the cap.
  6. Gently swirl the plate on the benchtop to mix the culture and the medium thoroughly. Ensure that the medium covers the plate evenly and do not slip the agar over the edge of the Petri dish.
  7. Allow the agar to completely gel without disturbing it, it will take approximately 10 minutes.
  8. Seal and incubate the plate in an inverted position at 37°C for 24-48 hours.
Overview of Pour plate method and spread plate method
Overview of Pour plate method and spread plate method

Results

After 24-48 hours, count all the colonies (note that the embedded colonies will be much smaller than those which happen to form on the surface). A magnifying colony counter can aid in counting small embedded colonies.

Calculate CFU/mL using the formula: = (number of colonies x dilution factor) / volume of culture plated*

Suppose the plate of the 10^4 dilution yielded a count of 32 colonies. Then, the total number of colony-forming units in 1 ml of the original sample is (32) x (104 ) x1*= 3.2 × 105

*We have used a 1 mL sample in this pour plate technique.

Disadvantages of Pour plate method

  1. Preparation for the pour plate method is time-consuming compared with the streak plate/and or spread plate technique.
  2. Loss of viability of heat-sensitive organisms coming into contact with hot agar. The organism to be counted must be able to withstand brief exposure to the temperature of molten agar (∼45°C to 50°C)
  3. Embedded colonies are much smaller than those which happen to be on the surface. Thus, one must be careful to count these so that none are overlooked.
  4. The reduced growth rate of obligate aerobes in the depth of the agar.

References and further readings

  1. Basic Practical Microbiology A Manual by Society for General Microbiology (SGM)

Acharya Tankeshwar

Hello, thank you for visiting my blog. I am Tankeshwar Acharya. Blogging is my passion. As an asst. professor, I am teaching microbiology and immunology to medical and nursing students at PAHS, Nepal. I have been working as a microbiologist at Patan hospital for more than 10 years.

15 responses to “Pour Plate Method: Procedure, Uses, (Dis) Advantages”

  1. Egwuatu says:

    quite good

  2. michael says:

    Would anaerobic bacteria grow in a pour plate as inoculated and incubated above? reason?

  3. Prachi says:

    Why use 250 ml sample for water analysis through membrane filter method

  4. karthick says:

    Why giemsa stain is used for viewing blood cells??

  5. Afreen Shaikh says:

    Quite helpful…!!!👍
    Can have approach for these…??
    M in fy level now…will b helpful to me?

  6. ashokreddy says:

    nice information

  7. Ingrid says:

    pour plate usually give lower count than membrane filtration, why?, how do you ensure that the membrane filtration does not get contaminated especially when testing more samples of different types.

  8. jasmeet says:

    why embedded colonies are smaller in size as compared to surface colonies?

  9. Mayank Kaushik says:

    Can you please tell me , why there is white patches occcur in plate count agar even when no microbial growth white patches still form on the plates. Why this happen with plate count agar?

  10. Deeksha Sharma says:

    explain why pour plate method is known as minimal counts

  11. situ says:

    how to count colonies on agar plates which are merged with each other especially fungal colonies??

  12. Montu says:

    During pour plate method, spreader colony found during total viable count then how to overcome this problem and which precautions take care during analysis.

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