4-2 Viable plate counts
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Viable plate counts
One of the most common methods of determining cell number is the viable plate count. A sample to be counted is diluted in a solution that will not harm the microbe, yet does not support its growth (so they do not grow during the analysis). In most cases a volume of liquid (or a portion of solid) from the sample is first diluted 10-fold into buffer and mixed thoroughly. In most cases, a 0.1-1.0 ml portion of this first dilution is then diluted a further 10-fold, giving a total dilution of 100-fold. This process is repeated until a concentration that is estimated to be about 1000 cells per ml is reached. In the spread-plate technique some of the highest dilutions (lowest bacterial density) are then taken and spread with a sterile glass rod onto a solid medium that will support the growth of the microbe. It is important that the liquid spread onto the plate soaks into the agar. This prevents left over liquid on the surface from causing colonies to run together and the need for dry plates restricts the volume to 0.1 ml or less. A second method for counting viable bacteria is the pour plate technique, which consists of mixing a portion of the dilution with molten agar and pouring the mixture into a petri plate. In either case, sample dilution is high enough that individual cells are deposited on the agar and these give rise to colonies. By counting each colony, the total number of colony forming units (CFUs) on the plate is determined. By multiplying this count by the total dilution of the solution, it is possible to find the total number of CFUs in the original sample.
Figure 4-1 Dilution plating and viable plate counts
(A) A demonstration of a decimal series of dilutions. The 100 sample is a concentrated solution of methylene blue. A 0.2 ml portion of this was added to 1.8 ml (1:9 ratio) of 0.85% saline to create the 1:10 dilution. After mixing, 0.2 ml of the 10-1 dilution was added to a second tube containing 1.8 ml to create the 10-2 dilution. This was continued to generate the dilution series.
One major disadvantage of the viable plate count is the assumption that each colony arises from one cell. In species where cells grow together in clusters, a gross underestimation of the true population results. One example of this are species of Staphylococcus, which is known to form clumps of microorganisms in solution. Each clump is therefore counted as one colony. This problem is why the term CFUs per ml is used instead ofÃ¢ bacteria per mlÃ¢ for the results of such an analysis. It is a constant reminder that one colony does not equal one cell. Great care must also be taking during dilution and plating to avoid errors. Even one error in dilution can have large effects on the final numbers. The rate at which bacteria give rise to an observable colony can also vary. If too short an incubation time is used, some colonies may be missed. The temperature of incubation and medium conditions must also be optimized to achieve the largest colonies possible so that they are easily counted. Finally, this technique takes time. Depending on the organism, one day to several weeks might be necessary to determine the number of CFUs that were present when the experiment started. Such information may no longer be useful for many experiments.
Despite its shortcomings, the viable plate count is a popular method for determining cell number. The technique is sensitive and has the advantage of only counting living bacteria, which is often the important issue. Any concentration of microorganism can be easily counted, if the appropriate dilution is plated. It is even possible to concentrate a solution before counting, as is often done in water analysis, where bacterial populations are usually at low density. The equipment necessary for performing viable plate counts is readily available in any microbiology lab and is cheap in comparison to other methods. Finally, by using a selective medium it is possible to determine the number of bacteria of a certain class, even in mixed populations. These advantages have made viable plate counts a favorite of food, medical, aquatic and research laboratories for the routine determination of cell number.[Prev] | [Next]