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Translate PDF. Introduction Microorganism is an organism that is microscopic or submicroscopic, which is too small to be seen under naked eyes.
However, the numbers of microorganisms in a given sample are required to know in certain aspect such as dairy industries, diseases investigation, and so on.
Because of this, a variety of methods have been developed for the enumeration of microorganisms like direct microscopic counts, filtration and viable plate counts Jacquelyn G.
Black Among the methods of enumeration, viable plate counts are being used most frequently to measure bacterial populations. In viable plate counts, we are measuring the number of viable cells, unlike the microscopic counts which cannot distinguish live from dead cells.
However, it takes some time for the visible colonies to grow Gerard J. Tortora, Before doing plate counts, serial dilutions are required. This is because it is hard to count more than colonies on an agar plate if we inoculated directly from the original bacterial suspension or sample without serial dilutions Kathleen Talaro, To complete plate counts, we could either use pour plate method or spread plate method and each of them have their advantages and limitations.
All the visible colonies are calculated and represented as colony forming units CFU. Then, the CFU is multiplied with the corresponding dilution factor. As a result, the population of original sample is known. The objectives of this experiment are to learn the process of enumeration to determine the number of microorganisms in a sample and utilize two methods in enumeration which are pour plate method and spread plate method.
Pour Plate Method The pour plate, like other viable plate count methods, involves adding a sample to a solid medium that will support microbial growth incubating the plates so that each bacterial cell multiplies to form a colony, and counting the number of colonies that develop. Generally we have no idea of the number of bacteria in a sample, so it is almost always necessary to prepare a dilution series to ensure that you will obtain a dilution containing a reasonable number of bacteria to count.
Six nutrient agar pours are melted in a boiling water bath. Three 99 mL dilution blanks are labeled as 10 -2, , and respectively.
Six Petri plates are labeled through The unknown sample is shaken to ensure an even distribution of microorganisms generally shaking side to side for 25 times. The dilution blank is shaken vigorously to distribute the bacteria evenly. Using a new sterile pipette, 0. With the same pipette, an additional 1. The original 1 mL of sample has now been diluted 1 part in a total of 10, parts.
The shaking procedure is repeated for the 10 -4 blank and 0. This same procedure is repeated to form a 10 -6 dilution blank from which you will establish 10 -7 0. The plate is swirled to mix the sample with the agar. The agar is made sure does not run over the edges of the plate. The lid is replaced. The agar is allowed to cool and solidity.
After incubation, the colonies are counted on each plate. Both the colonies on the agar surface and the colonies growing within the agar must be counted. The colonies are counted by marking their position on the back of the Petri plates with a marking pen.
This aid in keeping track of those colonies previously counted and avoids recounts. Counts are recorded. If a plate has more than colonies record it as TNTC too numerous to count. From the plate-count data, the concentration of bacteria in the original sample is calculated. For statistical reasons only plates with between 30 and colonies are used in this calculation. Each colony forming unit CFU represents the progeny of a single cell.
Therefore, the number of bacterial cells in the original sample is determined by multiplying the number of colonies on a dilution plate by the corresponding dilution factor. Generally replicates of each dilution are plated, and the mean count recorded. Spread Plate Method Another technique for performing a standard plate count is the spread plate method. As the name implies, serial dilutions of a sample are spread onto the surfaces of agar plates. Three 9 mL dilution water blanks are labeled as 10 -1, , and respectively.
Four nutrient agar plates are labeled through The dilution tube is mixed thoroughly by vortex or vigorous shaking. Using a new pipette, 1 mL from the 10 -1 dilution tube is transferred aseptically to the dilution tube.
The sample is vortexed. Using this same procedure, 1 mL from the 10 -2 dilution tube is transferred to the dilution. Because only 0. Therefore, the greatest dilution plate is Starting with the greatest dilution, the tube is vortexed and 0. Because starting with the greatest dilution, same pipette can be used for each of the tubes. The glass spreader is sterilized by dipping it in alcohol and flaming it in a Bunsen burner flame. Books Video icon An illustration of two cells of a film strip.
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