Pour Plate Technique- Procedure, Advantages, Limitations
- In nature, microbial populations do not segregate themselves by species but exist with a mixture of many other cell types.
- In the laboratory, these populations can be separated into pure cultures.
- These cultures contain only one type of organism and are suitable for the study of their cultural, morphological, and biochemical properties.
- At times also the determination of viable cells is very crucial in many microbiological procedures.
- To accomplish this, the serial dilution–agar plate technique is used.
- Briefly, this method involves serial dilution of a bacterial suspension in sterile water blanks, which serve as a diluent of known volume.
- Once diluted, the suspensions are placed on suitable nutrient media.
- The pour-plate technique is the procedure usually employed.
The pour-plate technique
- The pour-plate technique requires a serial dilution of the mixed culture by means of a loop or pipette.
- Molten agar cooled to 45°C, is poured into a Petri dish containing a specified amount of the diluted sample.
- Following the addition of the molten-then cooled agar, the cover is replaced, and the plates gently rotated in a circular motion to achieve uniform distribution of microorganisms.
- This procedure is repeated for all dilutions to be plated.
- Dilutions should be plated in duplicate for greater accuracy, incubated overnight, and counted on a Quebec colony counter either by hand or by an electronically modified version of this instrument.
- If the material to be tested is an aqueous liquid, a known volume is simply placed in the base of the Petri dish and 10–25 ml of molten culture medium (typically tryptone soy agar or plate count agar at 45o C) is poured onto it and quickly mixed by gentle swirling, then the plate is placed on one side for the agar to set.
- If the sample is a solid that is soluble in water that solid would normally be dissolved, but if it were insoluble then a suspension would be used. The problem in the last case would be to ensure that the suspension remained uniformly dispersed during pipetting and any dilution steps.
- Because the sample is dispersed through the medium before the gel sets, the colonies that grow are similarly dispersed throughout the agar.
- Consequently, the colonies are usually of different sizes because there is less oxygen available within the gel than at its surface and most organisms are either strict aerobes or facultative anaerobes which will grow best where the oxygen concentration is highest.
The procedure of Pour Plate Technique
- Label around the edge of the bottom (not the lid) of a sterile but empty Petri dish with at least your name, the date, the type of growth medium, and the type of organism to be added to the melted agar medium.
- Include the dilution factor if plating serial dilutions, or a series of repeated dilutions, which results in a systematic reduction in the concentration of cells in the sample. Preparing serial dilutions is necessary if the number of cells in the sample exceeds the capacity of the agar plate, in which the statistically significant range is 30 to 300 CFU. If there are more than 300 CFU on a plate, then the colonies will be crowded and overlapping.
- Obtain a tube containing 18 ml of melted agar medium.
- The agar medium should be dispensed into test tubes and pre-sterilized in an autoclave. On the same day, it is needed for an experiment, the agar should be melted in a steamer for 30 minutes then transferred to a 55 °C water bath. Only as much agar as is needed for the experiment should be melted as it cannot be re-used.
- Ten minutes before pouring plates, the tubes of melted agar should be transferred from the 55 °C water bath to a heat block on the laboratory bench set at 48 °C. Once the agar reaches this temperature, it is ready to pour. If the agar is too hot, the bacteria in the sample may be killed. If the agar is too cool, the medium may be lumpy once solidified.
- Obtain your sample, which should be either a broth culture or a suspension of cells produced by mixing cells from a colony into buffer or saline.
- The samples may be derived from a dilution series of a single sample.
- The sample volume to be plated should be between 0.1 and 1.0 ml.
- Open the lid of the empty Petri dish, and dispense your sample into the middle of the plate. Close the lid.
- Use aseptic technique throughout this procedure.
- Use either a serological pipette or micropipette to transfer your sample to the plate. Control the flow of the sample so it does not splash out of the plate.
- Remove the cap from the tube of the melted agar, and pass the rim of the open tube through the flame of the Bunsen burner.
- Open the lid of the Petri dish containing your sample and pour the agar in carefully. Close the lid then mix the sample with the agar by gently swirling the plate.
- Allow the agar to thoroughly solidify before inverting the plate for incubation.
Significance of Pour Plate Technique
- This technique is used to perform viable plate counts, in which the total number of colony-forming units within the agar and on the surface of the agar on a single plate is enumerated.
- Viable plate counts provide scientists a standardized means to generate growth curves, to calculate the concentration of cells in the tube from which the sample was plated, and to investigate the effect of various environments or growth conditions on bacterial cell survival or growth rate.
Advantages of Pour Plate Technique
- Easy to undertake.
- Will detect lower concentrations than surface spread method because of the larger sample volume.
- It requires no pre-drying of the agar surface.
- The most common method for determining the total viable count is the 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.
Disadvantages of Pour Plate Technique
- The use of relatively hot agar carries the risk of killing some sensitive contaminants, so giving a low result.
- Small colonies may be overlooked.
- In the case of solid sample dissolving in water, some species may suffer a degree of viability loss if diluted quickly in cold water; consequently, isotonic buffer (phosphate-buffered saline for example) or peptone water are used as solvents or diluents.
- Colonies of different species within the agar appear similar — so it is difficult to detect contaminants.
- The reduced growth rate of obligate aerobes in the depth of the agar.
- Preparation for the pour plate method is time consuming compared with streak plate/and or spread plate technique.
- 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.
- Stephen P. Denyer, Norman A. Hodges, Sean P. Gorman. (2004). Hugo and Russell’s pharmaceutical microbiology. Malden, Mass. :Blackwell Science,
- Sanders E. R. (2012). Aseptic laboratory techniques: plating methods. Journal of visualized experiments : JoVE, (63), e3064. doi:10.3791/3064