Introduction
Pour plate method is usually the method of choice for counting the number of colony-forming bacteria present in a liquid specimen. In this method, fixed amount of inoculum (generally 1 ml) from a broth/sample is placed in the center of 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 may be confluent; the few that grow on the agar surface are of the same size and appearance as 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 the range of 30-300 colonies/plate. To insure 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 the average, it will give a lower count as heat sensitive microorganisms may die when they come contact with hot, molten agar medium.
Principle
The modern pour plate culture method was initially developed in the laboratory of the famous bacteriologist and the father of bacteriology, Dr. Robert Koch. In Pour Plate technique, successive dilutions of the inoculum (serially diluting the original specimen of old broth culture) is added to the sterile Petri plates containing the melted and cooled (40-45 °C) agar medium & thoroughly mixed by rotating the plates which are then allowed to solidify. After incubation, the plates are examined for the presence of individual colonies growing throughout the medium.
The pure colonies which are of different size, shape, and color may be isolated or transferred into test tubes containing liquid culture media (broth) or directly inoculated on the solid agar media by streak plate method for making pure cultures.Pour Plate culture technique is also used as a means of determining the numbers of viable organisms in a liquid such as water, milk, Urine, or Broth cultures as well as to determine the hemolytic activity of deep colonies of some bacteria, such as the Streptococci, by using an agar medium containing blood.
Experiment
Material Required
- 24 hours old nutrient broth culture of two or more bacteria (Mixed Culture) or Sample/Specimen.
- Nutrient Agar Medium
- Six 9 ml Sterile Water Blanks
- Sterile Petri plates
- Marker
- Graduated pipette (1ml)
Procedure Of Pour Plate Method
- Melt the nutrient agar medium and keep it in the water bath set at 45 °C.
Serial Dilutions of the Specimen / Sample
- Label the 6 Sterile Water blanks (9ml sterile water in each tube) as number 1 to 6 with the help of Marker. Also, label the Sterile Petri plates as number 1 to 6.
- Place the labeled tubes in the test tube rack.
- Mix well the 24 hours old broth culture to equally distribute the bacterial cells in the tube.
- After mixing, Remove the Cotton plug and aseptically transfer the 1 ml of the bacterial suspension from the tube of culture to sterile water blank tube no. 1 using a graduated pipette.
- Shake the tube no. 1 to mix well the content to uniformly distribute the bacterial cells. Transfer the 1 ml of this to the water blank tube no. 2 by using the graduated pipette.
Note: Use the separate sterile pipette each time to transfer the contents from one tube to another.
- In this way, make serial dilutions till the six water blanks (no. 1 to no. 6).
Inoculating the Specimen / Plating of Specimen
- Transfer 1 ml of the bacterial suspension each from the tube no. 1 to 6 to Petri Plates labeled as 1 to 6 by using separate sterile pipette each time.
- Now, take out the Molten Nutrient Agar Medium (at 45 °C) from the water bath and pour the medium into the Plates no. 1-6 containing the specimen at different dilutions.
- Rotate the plate gently to ensure the uniform distribution of cells in the media plates.
- Allow the medium to solidify at room temperature.
- Incubate the inoculated media plates for 24-48 hours at 37 °C in an inverted position.
Results Interpretation
The colonies in the culture media plates inoculated by the serial dilutions of the specimen will show the lesser and lesser no. of the colonies as the dilution factor increased, which will be distributed more or less sparsely in the entire plate. These colonies may be transferred i.e. sub-cultured to the fresh media plates by streaking to obtain the pure culture of the bacterial cells for further study.
Limitations of Pour plate method
- Preparation for pour plate method is time consuming compared with streak plate/and or spread plate technique.
- Loss of viability of heat-sensitive organisms coming into contact with hot agar.
- Embedded colonies are much smaller than those which happen to be on the surface. Thus, one must be careful to score these so that none are overlooked.
- Reduced growth rate of obligate aerobes in the depth of the agar.
Precautions Of Pour Plate Method
- The protocol should be followed under all aseptic conditions preferably in Laminar Air Flow (Safety cabinet) to avoid any contamination.
- Accurately measure the quantity while preparing the serial dilutions of the specimen.
- Use the sterile pipettes each time to avoid any contamination and errors in the result.
- Accurately measure the quantity of Diluted specimen while inoculating onto the Solidified Media Plates.
- Uniformly spread the specimen in the Media plate to get the discrete & well-developed colonies.
Use
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 food stuffs.