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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.

(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. (B) A series of pour plates demonstrating the appearance of a viable plate count. The 3

plates show a 10-7, 10-8, and 10-9 dilution of a natural sample. Note how the number of colony forming units decreases 10 fold between the plates. 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 of bacteria per ml 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.

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OVERVIEW OF DIRECT PLATE COUNTING METHODOLOGY When one decides to count the number of cells in a sample, the issues of viable numbers, and the total numbers should come to mind. There are many methods used for counting cell numbers, some of which count total cell numbers (live cells + dead cells), and others that count only viable, or live cells. Direct plate counting is a method used to count the number of viable cells in a sample. By the very nature of the procedure, the dead cells are unable to be included in the count. Once the cells to be counted have been isolated, they are to be diluted due to the fact that too many cells will cause the Petri plate to be so densely populated with colonies, that they would be impossible to count. After the cells have been diluted, they are incubated on an agar medium until colonies form. It is at

this time that the cells may be counted. The image on the left shows a soil dilution plate containing bacteria and fungi. This plate is countable, but on the difficult side. 2. OVERVIEW OF DIRECT PLATE COUNTING PROCEDURES There are two main methods of direct plate counting: spread plate method and pour plate method. The spread plate method consists of evenly spreading the diluted sample over an agar plate. When using this method, a volume of 0.1 ml of the diluted sample should not be used since the agar will not be able to absorb the excess. Using this method yields colonies that form on the surface of the agar.

Picture obtained from Biology of Microorganisms by Madigan, Martinko, and Parker When using the pour plate method, a diluted sample is pipetted into a sterile Petri plate, then melted agar is poured in and mixed with the sample. Using this method allows for a larger volume of the diluted sample. Usually in the range of 0.1 - 1.0 ml. This method yields colonies that form colonies throughout the agar, not just on the surface. Caution must be taken with this method to ensure that the organism to be counted can withstand the temperatures associated with the melted agar.

Picture obtained from Biology of Microorganisms by Madigan, Martinko, and Parker

3. PROS AND CONS Direct plate counting is a good method of counting cells. A couple of advantages are that it works well for cells that separate in a short amount of time after they divide, and it counts only viable numbers. However, direct plate counting is not without its flaws. It does not work so well for those cells that stick together after cell division. Another problem that may arise is the very specific nutritional needs of some organisms. This is a special consideration when dealing with mixed samples such as those from a soil, because one cannot be sure the media has sufficient nutritional value for all of the cell types present. Sampling error is another possible problem. Sampling error refers to the uneven distribution of the sample on the agar surface. Direct plate counting has been described as the best available method for determining viable numbers, despite the large chance of the occurrence of an error (175, Claus). 4. ADDITIONAL SOURCES OF INFORMATION Claus, G. W. 1989. Understanding Microbes. W. H. Freeman and Company, New York, NY. pp. 175-185. Heggers, J. P., and Robson, M. C. 1991. Quantitative bacteriology: its role in the armamentarium of the surgeon. CRC Press, Boca Raton, FL. Madigan, M. T., Martinko, J. M., and Parker, J. 1997. Biology of Microorganisms. Prentice Hall, Upper Saddle River, NJ. p. 156.