COLONY FORMING UNIT (CFU) CALCULATION

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A colony forming unit (CFU) is defined as a unit that is commonly used in microbiology to determine or estimate the actual concentration of bacteria or microorganisms that is present in each test sample such as food, water, soil, or clinical sample. It can also be used to estimate the amount or concentration of living (viable) cells in each turbid suspension of bacteria in a tube or broth culture. A CFU can also be defined as a single, viable cell that produces a single colony on an appropriate solid culture media plate. Colony forming unit is a measure of viable (living) bacterial or fungal cells in a sample.  A colony is a population of cells visible to the naked eye.

Since it is not possible to know how many bacteria or microbe that is present at any given time in a test sample, the CFU calculation helps us to determine how many bacteria or microorganisms that is present in the test sample or turbid bacterial suspension per milliliter (mL). CFU helps us to determine the number of viable bacteria present in a sample, and they are seen as visible colonies after plating out the samples in agar culture and placing the plates in the incubator at the optimal temperature required for that particular experiment. After incubation, visible colonies appear on the agar plate. The visible colonies are counted one after the other, and the final count is used to estimate or determine the CFU per mL. The number of visible colonies (CFU) present on an agar plate can be multiplied by the dilution factor to provide a CFU/ml. CFU measures only living or viable cells in a test sample or microbial suspension. It does not measure dead cells.

The formular for determining the CFU is as follows:

CFU/mL = number of colonies counted x dilution factor / volume of bacterial suspension or sample plated

For example,

Suppose your culture plate of the 105 dilution yielded 120 colonies after incubation and counting of the colonies, and you plated a volume of 100 µl (equivalent to 0.1 mL) of cell suspension or sample, the number of bacteria (i.e., the CFU) can be calculated using above formular as follows:

Number of colonies counted = 120

Dilution factor = 105

Volume of cells plated = 0.1 mL

Therefore, CFU = 120 x 105 / 0.1 = 120,000,000 CFU/ml or 1.2 x 108  CFU/ml

After incubation, it is important to observe the plates carefully and count only the countable cells or colonies i.e., plates with countable bacterial colonies (Figure 1). Plates with too many colonies that are not countable (Figure 2) are referred to as too many to count (TMTC). TMTC plates (Figure 2) are not used for estimating the CFU/ml because the colonies are too many and are uncountable (Figure 2); only countable plates can be used to count and estimate the CFU/mL of the sample or turbid bacterial suspension because the colonies in this particular agar culture plate are countable (Figure 1).

Figure 1. Agar culture plate with countable colonies
Figure 2. Agar culture plate with too many to count (TMTC) cells.

To achieve a countable plate (Figure 1) for the estimation of CFU, it is important to serially dilute the sample or microbial (bacterial) suspension several times and maybe up to 103 cells before plating out. Only the last tube or final dilution (e.g., 103 or 102) should be used for plating out. If a higher concentration of cells or sample is used for plating out (e.g., 106 or 108), then you will end up having a plate with colonies that are too many to count (Figure 2).

CFU is important because it helps you to know the actual number of viable or living cells present in the sample or bacterial suspension. While the CFU (obtained by plating out samples on agar plates) gives you the number of living cells, the counting done using the microscope or haemocytometer counts all cells, both living and dead bacterial or microbial cells. Counting with colony-forming units (CFUs) requires culturing the microorganism or bacteria or sample on an agar culture plate; and this technique allows you to count only viable cells. This is in contrast with microscopic examination (where we use haemocytometer) which counts all microbial cells, both living and dead cells. The visual appearance of a colony in a cell (suspension) requires significant growth of that organism; so, when counting colonies, it is uncertain if the colony arose from one cell or a group of cells. The only way we can remove doubt and express such results is by expressing them as colony-forming units (CFUs).

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