Quantifying the bacterial level in the sample can be done in several ways. New rapid diagnostic tests can identify particular bacteria in a sample, but the total bacterial count is informative and important in a manufacturing or diagnostic process; there are several ways to do this. Later in the course you will use a statistical technique on water samples called the Most Probably Number (MPN).
- Direct microscopic counts of cells can be done, visually, and by various types of electronic particle counters.
- Viable bacterial counts can be determined by the Standard Plate Count (SPC) method. A sample is diluted several times (serial dilution) and then plated over the agar surface of a petri plate. After incubation the number of colonies on the plate can be counted and are directly related to the number of bacteria in original sample, based on the magnitude of the dilutions. Each colony arises from a single bacterium, or group of bacterium depending on the typical arrangement (tetrad, staph, diplo, etc.) and is referred to as a Colony Forming Unit (CFU).
- Indirect counts of a sample can be performed by using a spectrophotometer and measuring the turbidity (aka absorbance or Optical Density) of the sample. This is called the Turbidimetric method. A standard curve must be created using the known count of a sample, and then counts of various dilutions can be extrapolated.
There are advantages and disadvantages to each method. Direct counts sample small amounts of a larger sample and one can’t distinguish between live cells and dead cells. One could however, see a variety of cell types. Likewise, the Turbidimetric method would also count live and dead cells, though one could not distinguish different types of cells. In addition, the Optical Density (OD) of a culture may not always be linear especially at high cell density (twice the number of cells may not cause twice the turbidity). The Standard Plate Count does represent live cells, and sometimes a variety of colony types indicate the composition of the original sample. The SPC would not account for cells that might not grow well on the type of media being used, or cells that might be inhibited by other bacteria or other environmental factors. This is not a problem in a sample of known bacteria.
Contributors and Attributions
Kelly C. Burke (College of the Canyons)