Enumeration of bacteria 293

FIG. 10-7 The spread plate technique for isolating and enumerating microorganisms.

It is assumed that each colony arises from an indi­vidual bacterial cell. By counting the number of colonies that develop, the colony forming units (CFUs), and by taking into account the dilution fac­tors, the concentration of bacteria in the original sam­ple can be determined. Preferably two or three plates are counted to determine numbers of bacteria in a sample. Each plate counted should have 30 to 300

FIG. 10-8 The pour plate technique for isolating and enu­merating microorganisms.

colonies. If bacterial numbers in a sample are low, it is sometimes necessary to filter the suspension to concentrate the bacterial cells by collecting the cells on a membrane filter. The typical membrane filter for collecting bacterial cells is made of nitrocellulose or cellulose acetate and has a pore size of 0.2 to 0.45 |лт, which is small enough to trap most bacterial cells. The membrane filter with the trapped bacteria is then placed onto a suitable medium so that bacterial re­production can occur, and the colonies that develop on the filter are counted. Countable plates are those having between 30 and 300 colonies. Less than 30 colonies is not acceptable for statistical reasons and more than 300 colonies on a plate are likely to pro­duce colonies too close to distinguish as individual CFUs. Such samples are noted as TNTC (too numer­ous to count).

A major limitation of the viable plate count proce­dure is that it is selective. There is no single combina­tion of incubation conditions and medium composi­tion that permits the growth of all bacterial types. The nature of the growth medium and the incubation con­ditions determine which bacteria can grow and thus be counted. Viable counting measures only cells that are capable of growth on the given plating medium under the set of incubation conditions that are used. Sometimes cells are viable but nonculturable unless rigorous steps are taken to acclimate the microorgan­isms to laboratory culture conditions. The viable plate

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