Pour Plate Technique: Method, Benefits, and Limitations
In nature, microbial populations coexist alongside a variety of different cell types rather than grouping themselves according to species.
In the laboratory, certain populations may be separated into pure cultures.
These cultures are appropriate for the research of their cultural, morphological, and biochemical characteristics since they only contain one kind of organism.
The identification of live cells may sometimes be highly important in several microbiological techniques.
To do this, the serial dilution-agar plate approach is employed.
A bacterial suspension is serially diluted using this approach in sterile water blanks that operate like a known-volume diluent.
The suspensions are put on an appropriate nutritional medium after being diluted.
The method most often used is the pour-plate method.
The pour-plate technique
The mixed culture must be serially diluted using a loop or pipette in order to use the pour-plate method.
A Petri plate filled with a certain quantity of the diluted sample is loaded with molten agar cooled to 45 degrees Celsius.
To ensure a uniform distribution of microorganisms, the cover is replaced after the agar has been added, melted, and cooled. The plates are then slowly rotated in a circular motion.
This process is continued for each dilution that has to be plated.
For improved accuracy, dilutions should be plated in triplicate, allowed to incubate for an extended period of time, and then the colonies should be counted manually or using an electronic version of a Quebec colony counter.
If the material being evaluated is an aqueous liquid, the Petri dish’s base is simply filled with a known volume, which is then quickly mixed by gentle swirling with 10–25 ml of molten culture medium (usually tryptone soy agar or plate count agar at 45 °C). The plate is then placed on its side to enable the agar to solidify.
Typically, a sample consisting of a material that is soluble in water would be dissolved. However, if the material were insoluble, it would be employed as a suspension. In the final scenario, the challenge would be to make sure that the suspension was evenly distributed throughout the pipetting and diluting processes.
The colonies that develop are equally scattered across the agar because the material is spread throughout the medium before the gel hardens.
Since there is less oxygen available within the gel than at its top, and because the majority of organisms are either strict aerobes or facultative anaerobes that grow effectively in conditions in which the oxygen concentration is the highest, the gel does not support microbial growth. The colonies are often various sizes as a result.
The procedure of the Pour Plate Technique
Label the border of the bottom (not the lid) of a hygienic but unfilled Petri dish with at least your title, the date, the type of growth media, and the organism to be put into the melted agar medium.
If you are plating serial dilutions—a series of successive dilutions—which cause a systematically lower cell concentration in the sample, include the dilution factor. If the sample contains more cells than the agar plate can hold—in which case the statistically meaningful range is between 30 and 300 CFU—serial dilutions must be prepared. The colonies will be dense and overlapping if there are more than 300 CFU on a plate.
Acquire a tube holding 18 ml of agar media that has melted.
The agar medium has to be pre-sterilized in an autoclave before being distributed into test tubes. On the 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. Agar cannot be reused, thus just the needed quantity for the experiment must be melted.
Ten minutes prior to pouring the plates, the tubes of melted agar must be transferred from the 55 °C water bath to a 48 °C heat block on the laboratory bench.
When it reaches this temperature, the agar is ready for pouring. As the agar is cooked excessively, the bacteria in the specimen may be killed. When the agar solidifies, it could be lumpy if it’s too cold.
Collect your specimen, which should consist of either a broth culture or a cell suspension created by mixing colony cells with buffer or saltwater.
The specimens might have originated from a series of dilutions of a single specimen.
The volume of the sample for plating should vary between 0.1 and 1 mL.
Remove the lid from the Petri dish, then pour the sample onto the centre of the plate. Put the lid on.
Always use an aseptic approach during this procedure.
Transfer your material to the plate using a micropipette or a serological pipette. Control the sample’s flow to prevent it splashing onto the plate.
Detach the top from the agar tube and pass the tube’s open rim over the flame of the Bunsen burner.
The Petri dish containing your specimen must have an open cover, so gently pour the agar inside. After gently spinning the plate, cover the sample and blend it with agar.
Before turning the dish over for incubation, let the agar solidify completely.
Significance of Pour Plate Technique
Viable plate counts are carried out using this method. In a single plate, all colony-forming organisms inside the agar and on its surface are counted.
Researchers may use viable plate counts to generate growth curves, quantify the number of cells in the sample tube from which they plated the sample, and assess the effect of varying habitats or growing conditions on the survival or growth rate of bacteria.
Advantages of the Pour Plate Technique
Simple to complete.
Owing to the increased sample volume, will be able to identify concentrations at a lower level than the surface spread method.
There is no need to dry the agar surface in advance.
Pour-plate counting is the most common method for determining the total healthy population.
The pour plate technique may be used to determine the number of germs per millilitre of a sample.
It is often used to test for bacterial contamination of foods and has the benefit of not needing previously prepared plates.
Disadvantages of the Pour Plate Technique
When using somewhat hot agar, there is a chance that it can kill any delicate impurities, producing a subpar outcome.
Small colonies can go unnoticed.
When a solid sample dissolves in water, certain species may lose some viability if the solution is rapidly diluted in cold water; for this reason, isotonic buffers (such as phosphate-buffered saline or peptone water) or these liquids are used as solvents or diluents.
It is challenging to identify contaminants in the agar, since colonies of various species there seem identical.
The depth of the agar, where required aerobes develop more slowly,
In comparison to the streak plate, spread plate, or pour plate techniques, the pour plate approach requires more preparation time.
References
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,
