The heterotrophic plate count (HPC), formerly known as the standard plate count, is a procedure for estimating the number of live, culturable heterotrophs in a sample. Heterotrophs are not taxonomically defined, but are a broad heterogeneous group including, bacteria, yeasts, and molds that require organic carbon for growth.1 Because no single culture technique satisfies all the growth requirements of all heterotrophs in a water sample, the microorganisms that are isolated from a water sample vary based on the culture conditions and inoculation technique used.
Although HPC media generally are not selective in nature, each medium and technique described in this section enriches a different subpopulation of heterotrophic bacteria present in a sample. Thus, each method gives different results for the same matrix because of differences in medium composition, incubation temperatures and times, and inoculation technique used.2,3 The type of heterotrophs recovered from a water matrix can vary between locations, seasons, and even consecutive samples taken from the same location.1
The heterotrophic plate count method is a traditional microbiological technique described nearly 150 years ago by Robert Koch and has been included in Standard Methods since its first edition. The HPC method is used as a general assessment of the sanitary quality of a water matrix, and as a measure of the efficacy of water treatment and distribution. If measured routinely, the HPC can be used to identify changes in bacterial density thus indicating changes in the quality of treated waters.1-3
The bacteria that are detected by HPC can also interfere with the detection of indicator organisms, such as total coliforms. They may be present in higher numbers than coliform bacteria and, therefore, may outcompete any coliform bacteria present in a water sample for nutrients. This phenomenon is termed coliform suppression and is suspected to occur in coliform tests where there is obvious growth, but a lack of results that can be clearly interpreted as positive or negative. Coliform suppression generally occurs when HPC bacteria are >500 CFU/mL.4
Most media described in these methods are used to plate samples, which permits a direct colony count and visualization of various colony morphologies. Colonies may arise from pairs, chains, clusters, or single cells—all of which are included in the estimate of colony-forming units. Interactions among the various microorganisms in the sample also influence the final colony count.5,6
Because the HPC count depends on the growth medium and inoculation technique used, choose the inoculation method and medium that best suits the purpose of the resulting information. In general, high nutrient media and high incubation temperatures are believed to be better for enumerating bacteria from animals and humans, while low nutrient media and low incubation temperatures (such as R2A) are better for culturing autochthonous bacteria.3 The spread plate technique generally recovers more bacteria than the pour plate method due to the lack of heat exposure. In general, however, lower incubation temperatures and extended incubation times allow greater recovery.7
Three inoculation techniques and three media are described, along with one MPN method using enzymatic detection. Only compare data generated using the same procedure and medium.