Enumerating Microorganisms: Viable Counts

Viable bacteria may be counted by diluting samples, plating the dilutions on solid medium, and counting the colonies that arise. Results are usually expressed as colony-forming units (CFU), since some bacteria may not produce colonies on the medium selected.

Only plates (or replicate plates from the same dilution) with 30-300 colonies are counted. Plates with fewer than 30 colonies give statistically unreliable results, while plates with more than 300 colonies are too crowded to allow all the bacteria to form distinct colonies. Usually, more than one dilution in a series is plated, just to be sure that results in a countable range will be obtained. Ignore dilutions giving results outside of the countable range.

The concentration of bacteria in the original sample is calculated as:

CFU ml-1 (or g-1) = (colonies on plate)/(final plate dilution)

Frequently, volumes other than one ml are used to inoculate the plate. For example, 0.1 ml is often used when surface-plating, as larger volumes may not be absorbed by the agar. Plating 0.1 ml of a given dilution is mathematically identical to plating a 1 ml of a further 1:10 dilution.

For this reason, the size of the inoculum is usually incorporated with the dilution factor to give the "final plate dilution" (d x i). When 1.0 ml of a 10-4 dilution is plated, the final plate dilution is 10-4. When 0.1 ml of the same dilution is plated, the final plate dilution is 10-4 X 10-1 = 10-5.

The formula becomes:

CFU ml-1 (or g-1) = (colonies on plate)/(d x i)

Express answers in scientific notation with two significant figures. Watch for division errors:

1/(4 X 10-6) = 2.5 X 105, NOT 4 X 106, and 1/10-6 = 106, NOT 10-6.

Example:

1 g feces + 99 ml saline = 10-2 dilution (A)

1 ml A + 99 ml saline = 10-2 X 10-2=10-4 dilution (B)

0.1 ml B + 9.9 ml saline=10-4 X 10-2 = 10-6 dilution (C)

1 ml C + 9 ml saline = 10-6 X 10-1 = 10-7 dilution (D)

Triplicate 0.1 ml samples of dilutions C and D are plated. The plates are incubated and the colonies are counted. C gives 320, 300, 298 colonies (average, 306). Ignore dilution C, since it averages more than 300 colonies/plate. D gives 34, 30, 29 colonies (average 31). Use the average number of colonies from dilution D to calculate the CFU/g feces:

CFU/g feces = 31/(10-7 X10-1)=31 X 108 = 3.1 x 109.

The final plate dilution was 10-8 and there were 3.1 x 109 CFU/g fecal material.

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