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 the spread plate.
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
For accurate counts, the optimum count should be within 30-300 colonies/plate. To ensure a countable plate, plate a series of dilutions. The pour plate method of counting bacteria is more precise than the streak plate method. 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 the Pour plate method
The pour plate technique can 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
- Test sample
- Plate count agar (PCA) or nutrient agar
- Hot water bath 45°C
- Sterile Petri dishes
- Colony counter with a magnifying glass
- Sterile capped 16*150 mm test tubes
- Pipettes of various sizes (e.g. 01, 1.0 and 2.0 mL)
Procedure of Pour plate technique
- Prepare the dilution of the test sample expected to contain between 30-300 CFU/mL. (Follow serial dilution technique)
- 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
- 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).
- Hold the bottle in the right hand; remove the cap with the little finger of the left hand.
- Flame the neck of the bottle.
- 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.
- Flame the neck of the bottle and replace the cap.
- Gently swirl the plate on the benchtop to thoroughly mix the culture and medium. Ensure that the medium covers the plate evenly and do not slip the agar over the edge of the Petri dish.
- Allow the agar to gel completely without disturbing it. It will take approximately 10 minutes.
- Seal and incubate the plate in an inverted position at 37°C for 24-48 hours.
After 24-48 hours, count all the colonies (note that the embedded colonies will be much smaller than those 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
- Preparation for the pour plate method is time-consuming compared with the streak plate/and or spread plate technique.
- 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)
- 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.
- The reduced growth rate of obligate aerobes in the depth of the agar.
References and further readings
- Basic Practical Microbiology A Manual by Society for General Microbiology (SGM)