The forerunner of the present pour-plate method was developed in the laboratory of the famous bacteriologist, Robert Koch. In normally microbial population exists as a mixture of many other cell types, this laboratory technique is used to produce pure culture from these population.
This method also used to count the number of viable organisms in a liquid such as water, milk, urine or broth culture 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.
Aim of Pour Plate Method
The main purpose of the pour plate method is to isolate the pure culture from a mixture of different populations and demonstrate the cultural characteristics of the bacteria such as color, texture, size, elevation etc.
Principle of Pour Plate Method
In this Method, serial dilutions of the inoculum (serially diluting the primary specimen) are added within sterile Petri plates to which is poured melted and cooled (42-45°C) agar medium and completely mixed by revolving the plates which are then left to solidify.
After incubation, the plates are observed for the appearance of individual colonies growing everywhere in the medium. The pure colonies which are of varying size, shape and colour may be isolated/transported into test tube culture media to prepare pure cultures.
Requirement for Pour Plate Method
- 24 hours old nutrient broth culture of two different organisms.
- Nutrient Agar Medium
- Six 9 ml Sterile Water Blanks
- Sterile Petri plates
- Graduated pipette (1ml)
Pour plate method procedure
- Place the 7 agar deep tubes within a boiling water bath for melting of agar.
- Allow them to cool to 48°C.
- Label the empty sterile culture tube as number 1 and the six water blanks (containing 9ml of water) as numbers 2 through 7 with a wax marking pencil. Also, label the Petri plates 1 through 7.
- Now Place the labeled tubes in a 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 1 mL of the bacterial suspension from tube number 1 to water blank tube number 2 and return the pipette to tube number 1.
- Shake the tube number 2 and with a fresh sterile pipette transfer 1 mL to tube number 3. Return the pipette to tube number 2.
- Prepare serial dilutions till the 6 water blanks (number 2-number 7) are used by using fresh sterile pipettes each time by repeating step 7.
- Transfer 1 mL of the bacterial suspension each from tube numbers 1 to 7 to Petri plates numbers 1 to 7 using respective pipettes.
- Remove a nutrient agar tube from the water bath (at 45°C) and pour the medium into plate 1 and rotate the plate gently to ensure uniform distribution of cells in the medium.
- Repeat step 10 for the addition of medium to plates 2, 3, 4, 5, 6, and 7.
- Allow the medium to solidify.
- Incubate the inoculated plates for 24-48 hours at 37°C in an inverted position (lid on bottom).
Observation and Result
Study the plates for the appearance of individual colonies growing throughout the agar medium. It will be observed that progressively poured plates will have a lesser and lesser number of colonies which will be distributed more or less sparsely in the plates which may be transferred (subcultured) to other media (fresh plates) or agar slants for further study.
Importance of Pour Plate Method
- The spread plate method is used for viable plate counts. In this method, the total number of colony-forming units ( CFU) on the surface of an agar medium is enumerated. The Calculation of CFU/mL is done by using the formula: CFU/mL= CFU * dilution factor * 1/aliquot (the volume of diluted specimen (aliquot) is either 0.1 or 1.0 mL).
- The viable plate counts help to generate growth curves of an individual organism. From this growth curve, we can calculate the concentration of cells.
Advantages of Pour Plate Method
- Can detect the lower concentration of microorganism as compared to spread plate method.
- Requires no pre-drying of the agar surface.
- Easy to perform.
- A useful method to determining the total viable count.
- It can detect the number of microbes/ mL.
Limitation of Pour Plate Method
- The hot agar medium may kill some sensitive bacteria.
- Small colonies may be overlooked.
- It is a time-consuming method as compared to the spread plate or streak plate method.
- If a solid sample dissolves in water, some species may undergo a degree of viability end if diluted immediately in ice-cold water; consequently, isotonic buffer (phosphate-buffered saline for example) or peptone water are accepted as solvents or diluents.
- Difficulties may occur in the detection of contaminants if the Colonies of distinct species within the agar look similar.
- In the depth of the agar, the growth rate of obligate aerobes is reduced.
- MICROBIOLOGY Laboratory Theory & Application by Michael J. Leboffe and Burton E. Pierce
- Benson, H. J. (2005). Benson’s microbiological applications: Laboratory manual in general microbiology. Boston: McGraw-Hill Higher Education.
- James G. Cappuccino, Chad T. Welsh (2017). Microbiology: A Laboratory Manual, 11th Edition. Pearson Publications.