Pour Plate Technique For the Isolation of Microorganism

Pour plate technique for the isolation of microorganism is often used to determine the quantity of colony-forming bacteria available in a liquid sample. When compared to the spread plate method, this method allows for the use of a greater amount of material due to the fact that the sample is combined with the liquid agar media.

Principle of Pour Plate Method

The pour plate technique involves using a sterile pipette to deposit a predetermined volume of inoculum (often 1 milliliter) from a broth or sample into the middle of a sterile Petri dish. Once the inoculum has been added, 15mL of cooled agar (about) is placed into the Petri plate and stirred well. After the agar has had time to set, the plate will be turned upside down and placed in an incubator set to 37 degrees Celsius for anywhere between 24 and 48 hours.

Both the surface and the inside of the medium will support the growth of microorganisms. Colonies that develop inside the medium are often of a small size and may be confluent. The few colonies that grow on the top of the agar are all of the same size and seem to be in the same pattern as those on a streak plate. Every colony, no matter how big or how little, is counted individually. The term “colony-forming unit” (CFU) refers to each individual colony. The following formula is used to figure out how many microorganisms are in a particular test sample:

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

Pour Plate Method

The ideal colony count is between 30 and 300 colonies per plate. Dilutions should be plated to guarantee that the plate is countable. The Pour Plating Technique is more accurate than the streak plate method for counting bacteria; nevertheless, the average count will be lower due to the fact that heat-sensitive microorganisms are more likely to perish when they come into contact with a hot molten agar media while using this pouring agar plates method.

Requirements for Pour Plate Method

Materials

1. Test sample

2. Plate count agar (PCA)

Equipment’s

1. Water bath 45°C

2. Petri dishes

3. Flame

4. Colony counter

5. Sterile tubes

6. Pipettes

Procedure of Pour Plate Method

1. Make the dilution of the test sample, which is anticipated to contain between 30 and 300 CFU per milliliter. (Follow serial dilution procedure for pour plate inoculation).

2. Inoculate an empty, labelled Petri dish with the required amount (0.1 or 1.0 milliliters) of the diluted specimen.

Pour Plate Method Steps

3. Retrieve one bottle of sterile molten agar from the water bath at 45 degrees Celsius. The bottle should contain 15 ml of melted Plate Count Agar or any other standard culture medium.

4. Keep the bottle steady with your right hand as you unscrew the cap with the smallest finger on your left hand. Put some flame on the bottle’s neck.

5. Pour the sterilised molten agar into the Petri dish by lifting the lid slightly with your left hand and then replacing the cover. Put some flame on the bottle’s neck, and then reinstall the top.

Pour Plate Method Steps 2

6. Stir the culture and the media completely by gently swiping the plate on the worktop. Make sure the medium covers the plate evenly, and don’t let the agar spill out of the Petri dish.

7. Allow the agar to harden for about 10 minutes without tampering with it. For 24-48 hours, incubate the plate at 37°C in an inverted position with the lid on.

Results for Pour Plate Method

Count all colonies after 24-48 hours (Keep in mind that the embedded colonies will be significantly smaller than the ones that spontaneously develop on the surface). Counting tiny embedded colonies is made easier using a magnifying colony counter.

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

Significance of Pour Plate Method

1. This method is utilised in the process of doing viable plate counts, which involves the enumeration of the total number of colony-forming units present on a single plate, both within the agar and on the surface of the agar.

2. Viable plate counts give researchers a standardised method for generating growth curves, calculating the concentration of cells in the tube from which the sample was plated, and investigating the effect of different environments or growth conditions on the rate at which bacterial cells survive or multiply.

Advantages of Pour Plate Method

1. Pour Plating Technique will detect lower concentrations than surface spread method because of the larger sample volume. Pour Plating Technique requires no pre-drying of the agar surface.

2. The pour-plate method is the one that is utilised the vast majority of the time for calculating the total viable count.

3. The Pour Plating Technique can be used to find out how many microbes/mL are in a sample.

4. Pour Plating Technique is frequently used to test for bacterial contamination of foods and has the benefit of not requiring previously prepared plates.

Disadvantages of Pour Plate Method

1. When compared to the streak plate/and or spread plate approach, the pour plate technique requires significantly more time for preparation.

2. When heat-sensitive organisms come into touch with hot agar, their viability is significantly reduced. The organism that is going to be counted has to be able to survive a brief exposure to the temperature of molten agar, which can range anywhere from 45°C to 50°C.

3. Colonies that are embedded are often significantly smaller than those that are found on the surface. Consequently, one must use extreme caution when counting them to ensure that none are missed.

4. In the event that a solid sample dissolves in water, there is a possibility that certain species will suffer a degree of loss of viability if the solution is diluted quickly in cold water. As a result, an isotonic buffer (such as phosphate-buffered saline, for example) or peptone water is utilised as a solvent or diluent in this Pour Plating Technique.