If you directly plate a bacterial culture, the number of cells that grow on the plate are too large to allow counting. Therefore, it is important to use a serial dilution process to ensure that the number of colonies on the plate is around 300 to 500. In some cases such as samples of highly purified water, there may be too few organisms in the sample. Such samples need to be concentrated before they can be plated for counting.
The plate provides a nutrient medium for growth of microorganisms streaked on it. All organisms therefore receive the same nutrients. While this is fine for cultures that consist of a single organism, it poses a problem in case of mixed cultures. The different organisms present within a mixed culture may differ quantitatively and qualitatively in their nutritional requirements. When such a culture grows, cells that don't receive sufficient nutrition do not grow and multiply. Therefore, although the organisms are present in the culture, their numbers do not reflect in the plate count.
The accuracy of the plate count depends on an even distribution of organisms on the surface of the agar plate. If these cells are distributed unevenly on the surface, it causes errors in the count obtained. For example, in some species, organisms may occur not as single cells, but as clusters, pairs, or packets. Therefore, the assumption of the plate count that each colony has arisen from a single cell is erroneous and the estimated population of organisms is much less than the true levels.
Prior to plating, the culture is rendered sufficiently dilute to allow the isolation of separate cells on the plate. An error in this process of dilution has a major impact on the final viable plate count. The incubation temperature and time are also critical factors because organisms differ in their optimum incubation temperature and period. In the case of mixed cultures, where there is a wide difference in these values across organisms, there can be insufficient growth of a particular species, leading to an error.
After plating the culture, the agar plates are incubated to allow colonies to develop. This incubation period can range from days to weeks depending on the nature of the organisms. In certain instances, the results obtained at the end of this period may be of no practical value. For example, using plate counts for monitoring the environmental in a sterile parenteral manufacturing unit has no real-time benefits although it is of value in monitoring trends in the microbial load of the area.