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In microbiology, colony-forming unit (CFU) is an estimate of viable bacterial or fungal numbers. Unlike direct microscopic counts where all cells, dead and living, are counted, CFU estimates viable cells. The appearance of a visible colony requires significant growth of the initial cells plated - at the time of counting the colonies it is not possible to determine if the colony arose from one cell or 1,000 cells. Therefore, the results are given as CFU/mL (colony-forming units per milliliter) for liquids, and CFU/g (colony-forming units per gram) for solids to reflect this uncertainty (rather than cells/mL or cells/g).
The purpose of plate counting is to estimate the number of cells present based on their ability to give rise to colonies under specific conditions of nutrient medium, temperature and time. Theoretically, one viable cell (viable defined as able to multiply via binary fission under the controlled conditions) can give rise to a colony through multiplication. However, solitary cells are the exception in nature, and most likely the progenitor of the colony was a mass of cells deposited together. In addition, many bacteria grow in chains (e.g. Streptococcus) or clumps (e.g. Staphylococcus). Estimation of microbial numbers by CFU will, in most cases, undercount the number of living cells present in a sample for these reasons.
The plate count is linear for E. coli over the range of 30 - 300 CFU on a standard sized petri dish. Therefore, to ensure that a sample will yield CFU in this range requires dilution of the sample and plating of several dilutions. Typically ten-fold dilutions are used, and the dilution series is plated in replicates of 2 or 3 over the chosen range of dilutions. The CFU/plate is read from a plate in the linear range, and then the CFU/g (or CFU/mL) of the original is deduced mathematically, factoring in the amount plated and its dilution factor.
An advantage to this method is that different microbial species may give rise to colonies that are clearly different from each other, both microscopically and macroscopically. The colony morphology can be great use in the identification of the microorganism present.
A prior understanding of the microscopic anatomy of the organism can give a better understanding of how the observed CFU/mL relates to the number of viable cells per milliliter. Alternatively it is possible to decrease the average number of cells per CFU in some cases by vortexing the sample before conducting the dilution. However many microorganisms are delicate and would suffer a decrease in the proportion of cells that are viable when placed in a vortex.
The plate count method is the standard method used in microbiology to estimate cell numbers. There are a variety of variations on this method which include:
Counting colonies is traditionally performed manually using a pencil and a click-counter. This is generally a straightforward task, but can become very laborious and time consuming when many plates have to be enumerated. Alternatively semi-automatic (software) and automatic (hardware + software) solutions can be used.
Colonies can be enumerated from pictures of plates using software tools. The experimenters would generally take a picture of each plate they need to count and then analyse all the pictures (this can be done with a simple digital camera or even a webcam). Since it takes less than 10 seconds to take a single picture, as opposed to several minutes to count CFU manually, this approach generally saves a lot of time. In addition, it is more objective and allows extraction of other variables such as the size and colour of the colonies.
Completely automated systems are also available from some biotechnology manufacturers. They are generally expensive and not as flexible as standalone software since the hardware and software are designed to work together for a specific set-up. Alternatively, some automatic systems use the spiral plating paradigm.