Determination of cell count
Many studies require the quantitative determination of bacterial populations. The two most widely used methods for determining bacterial numbers are the standard, or viable, plate count method and spectrophotometric (turbidimetric) analysis. Although the two methods are somewhat similar in the results they yield, there are distinct differences. For example, the standard plate count method is an indirect measurement of cell density and reveals information related only to live bacteria. The spectrophotometric analysis is based on turbidity and indirectly measures all bacteria (cell biomass), dead or alive. The standard plate count method consists of diluting a sample with sterile saline or phosphate buffer diluent until the bacteria are dilute enough to count accurately. That is, the final plates in the series should have between 25 and 250 colonies. Fewer than 25 colonies are not acceptable for statistical reasons, and more than 250 colonies on a plate are likely to produce colonies too close to each other to be distinguished as distinct colony-forming units (CFUs). The assumption is that each viable bacterial cell is separate from all others and will develop into a single discrete colony (CFU). Thus, the number of colonies should give the number of live bacteria that can grow under the incubation conditions employed. A wide series of dilutions (e.g., 10–4 to 10–10) is normally plated because the exact number of live bacteria in the sample is usually unknown. Greater precision is achieved by plating duplicates or triplicates of each dilution.