Enumeration of bacteria 295

depth can be introduced between the slide and the cover slip. Consequently, the volume of the sample overlying each square is known.

It is often desirable to stain the cells. This helps in visualizing bacterial cells. Alternatively, a known volume of a sample containing a suspension of bac­teria is poured through a filter, such as a nitrocellu­lose 0.2 |xm pore size filter. The bacteria are stained on this filter, often using a fluorescent stain, and counted under a microscope. Many fluorescent dyes, such as acridine orange, stain all cells, making it im­possible to differentiate living from dead bacteria. The difficulty in establishing the metabolic status of the observed bacteria is a major limitation of this pro­cedure.

Bacterial cells can be enumerated by direct micro­scopic count procedures.

Instruments also are available for direct counting of bacterial cells. Particle counters, such as a Coulter particle counter, permit the discrimination of parti­cles based on size so that particles the size of bacteria are counted automatically. As long as there are no nonliving interfering particles in the same size range of bacteria, this is a rapid counting method.

Most Probable Number (MPN) Procedures

Another approach to bacterial enumeration is the de­termination of the most probable number (MPN). MPN is a statistical method based on probability the­ory. In a most probable number enumeration proce­dure, multiple serial dilutions are performed to reach a point of extinction. The point of extinction is the di­lution level at which not even a single cell is de­posited into one or more multiple tubes (FIG. 10-10). A criterion is established for indicating whether a particular dilution tube contains bacteria. Such a cri­teria can be development of cloudiness or turbidity in a liquid growth medium or gas production (de­tected by its accumulation in an inverted tube). The pattern of positive and negative test results are then used to estimate the concentration of bacteria in the original sample, that is, the most probable number of bacteria, by comparing the observed pattern of re­sults with a table of statistical probabilities for ob­taining those results (Table 10-1). Often a 95% confi­dence limit is given in MPN tables.

The most probable number procedure is a statistical method based on probability theory used to deter­mine bacterial populations by diluting bacterial samples to the point of extinction.

FIG. 10-10 The most probable number (MPN) procedure involves inoculation of multiple tubes with replicate samples of dilutions. The pattern of tubes that show growth (brown) and those that do not (orange) are compared with a statistical table to calculate the MPN of bac­teria in the original sample. In this example all 5 tubes at the 10° show growth, 4 of 5 tubes at the 10~^! dilution show growth, and only 1 of 5 tubes at the 10~² dilution show growth. Therefore the MPN bacteria in the original sample is 170 per 100 ml (see Table 10-1).

296 CHAPTER 10 BACTERIAL REPRODUCTION AND GROWTH OF MICROORGANISMS

^Ш TABLE 10-1 ^^^Я

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