The Colony Forming Unit (CFU) estimates the number of viable bacteria or fungal cells within a sample. This unit is based on the premise that a single living cell, or a cluster of cells, will multiply and form one visible colony when grown on a nutrient agar plate under controlled conditions. Quantifying microorganisms is essential for quality control in food and water safety, monitoring contamination levels in pharmaceutical production, and assessing infection magnitude in clinical settings. The CFU count provides a quantitative picture of the living microbial population.
Understanding Serial Dilutions
The concentration of microorganisms in an original sample is frequently too high to count accurately on a single plate. To resolve this, microbiologists use serial dilution, which systematically reduces the number of organisms across a series of tubes. This process involves mixing a small, measured volume of the initial sample into a larger volume of a sterile diluent, such as saline or buffer. A common practice is a tenfold dilution, where one milliliter of sample is added to nine milliliters of diluent, resulting in a 1:10 dilution.
This diluted mixture is used as the starting material for the next dilution step, repeating the process to create a series like 1:100, 1:1,000, and so on. The dilution factor is the inverse of the dilution (e.g., 10 for a 1:10 dilution). The total dilution factor for any tube is determined by multiplying the individual dilution factors of all preceding steps. Calculating this factor is important because it is used to back-calculate the original concentration of the sample.
Identifying Countable Plates
After serial dilution, small volumes from the diluted samples are plated onto agar and incubated. Only plates within a specific, statistically validated range are appropriate for counting. This standard guideline is the 30-300 rule, which dictates that the ideal number of colonies on a petri dish should be between 30 and 300. Plates with fewer than 30 colonies are statistically unreliable, as a single counting error can disproportionately skew the final result.
Plates containing more than 300 colonies are too crowded for accurate counting. Excessive colony growth leads to overlapping or merging colonies, making it difficult to distinguish individual Colony Forming Units. Plates outside this range are designated as Too Few To Count (TFTC) or Too Numerous To Count (TNTC) and are not used in the final calculation. The goal of serial dilution is to produce at least one plate within the 30–300 window to ensure the CFU/mL calculation is representative of the original sample.
Step-by-Step CFU/mL Calculation
The concentration of microorganisms in the original sample, expressed as CFU per milliliter, is calculated using the formula: \(\text{CFU/mL} = (\text{Number of colonies}) / (\text{Volume plated in mL} \times \text{Dilution Factor})\). For example, assume a sample was diluted \(10^{-5}\) (a dilution factor of 100,000). If \(0.1 \text{ mL}\) of this sample was plated, and the resulting colony count was 125, these three values are the inputs.
The calculation first involves multiplying the volume plated by the dilution factor. In this example, the product is \(0.1 \text{ mL} \times 100,000\), which equals 10,000. Next, the number of colonies is divided by this product to find the CFU/mL.
The calculation is \(125 / 10,000\), yielding a raw result of \(0.0125 \text{ CFU/mL}\). This is converted into scientific notation for readability. Therefore, the final result is \(1.25 \times 10^4 \text{ CFU/mL}\), or 12,500 CFU/mL. This systematic approach scales the count back to the concentration of the initial sample.
Interpreting and Reporting Results
Once the CFU/mL value is calculated, the final step involves interpreting and reporting the results. For plates outside the 30–300 countable range, a specific format reflects the data limitations. A plate that is Too Numerous To Count (TNTC), having more than 300 colonies, indicates the concentration exceeds the reliable limit of the method. This result is reported as greater than the calculated upper countable limit, such as \(>3.0 \times 10^5 \text{ CFU/mL}\).
A plate that is Too Few To Count (TFTC), with fewer than 30 colonies, suggests the concentration is near or below the detection limit of that dilution. If no growth is detected, the result is expressed as less than the lowest limit of detection for the plated volume and dilution (e.g., \(\)<10 \text{ CFU/mL}[/latex]). Countable results are reported using scientific notation to handle large numbers. Final reported numbers should be limited to two significant figures to reflect the precision of the plate count method.