## V V V V

Figure 11.13 MPN method schematic. Positive tubes are shown as dark after incubation.

tubes will end up positive. At the 100-fold dilution, on average only 0.05 cell is added to each tube, and hence all may be expected to be negative.

The pattern of positive tubes that results (in this case, perhaps 5-3-0) is an indication of the cell numbers originally present. Through a mathematical procedure, in fact, the most probable number (MPN) of cells originally present resulting in that pattern can be estimated. These values have been tabulated for several different numbers of replicates (e.g., 3, 5, and 10 tubes per dilution), and also for different dilution factors (e.g., two fold as well as 10-fold serial dilutions). Note that a positive or negative result in each tube is used to arrive at a quantitative estimate of cell numbers. The estimate from a pattern of 5-3-0 is an MPN of 8.0 mL-1 of the original sample. (A response of 5-2-0 gives an MPN of 5.0, the starting value used in the example.)

An obvious disadvantage of this approach is that it produces a statistical estimate rather than an actual count. For example, with the 5-3-0 example, there is a 95% likelihood that the actual number is between 3.0 and 25, an undesirably broad range. On the other hand, the MPN is potentially useful for a wide variety of samples and can be adapted for many types of microorganisms, including some for which other methods have not been successful.

Turbidity and Absorbance A method that can be used in laboratory cultures, especially pure cultures, grown on soluble (and preferably colorless) media is measurement of turbidity (cloudiness) or absorbance. Turbidity is the amount of light dispersed 90° from the path of incident light passing through a material. It is measured with a turbidi-meter using a photocell at right angles to the light path. Absorbance is the reduction in the transmission of light along the light path and may occur due to both dispersion and absorption of light. Absorbance is also called optical density (OD) and is usually measured with a spectrophotometer (also called a spectrometer). Absorbance is used more often than turbidity because spectrometers are more commonly available than turbidi-meters.

If a small amount of microbial suspension is placed in a sample tube, its turbidity, or the amount of light dispersed toward the photocell, is proportional, up to a point, to the number of particles in the suspension. Similarly, for absorbance, the amount of light that passes through the suspension will be inversely proportional to the concentration of organisms, provided that particle size does not change. This is a form of Beer's law (absorbance is proportional to concentration), which is the basis of most quantitative spectrophotome-try (although in this case a substantial amount of the light is refracted rather than actually absorbed). Thus, increases in either turbidity or absorbance can be used as a surrogate measure of growth, or correlated through use of a calibration curve to microbial counts obtained by other methods.

One advantage of using turbidity or absorbance is that they are not destructive of the culture. In fact, special flasks with sidearms are available so that the turbidity of a pure culture can be determined over time without the need to open the flask and risk contamination. However, if particle size increases, through flocculent growth or filament elongation, turbidity and OD will underestimate the cell count or biomass. Also, this approach generally cannot be used with environmental samples.

Counting Viruses and Bacteriovores Viruses are too small to see under any light microscope for direct counting. Also, since they grow only within cells of other organisms, they

cannot be cultured directly like bacteria. Thus, alternative methods are needed to enumerate them.

If a suspension of the bacteria Escherichia coli is spread over the surface of a plate containing an appropriate medium, a uniform bacterial lawn will develop. If at the same time that the culture is added, a sample containing coliphage (viruses that attack E. coli) is included, "holes" in the lawn will develop as the viruses infect and kill cells and then spread to neighboring cells. These clear areas, where the bacteria have been lysed by the phage, are referred to as plaques (Figure 11.14). The number of such plaques is then an estimate of the number of plaque-forming units (PFUs) present in the volume of sample added. Similarly, other phage can be enumerated using appropriate host bacteria for the lawn. Analogous to CFUs, it is often expected that each PFU stems from a single initial phage.

Many animal and plant viruses can be counted in similar ways using appropriate host cell cultures. In these cases, however, individual infected cells may be counted rather than plaques. Alternatively, an MPN type of procedure might be used, or if necessary, the development of infection in exposed whole organisms.

Plaque formation can also be used to count some organisms that feed on bacteria, such as amoeba. Host cell lysis caused by Bdellovibrio, the bacteriovorous proteobacteria (Section 10.5.6), also forms plaques.