Plate counting, or viable count, is a foundational laboratory technique used in microbiology to estimate the number of living bacteria in a sample. This method relies on the principle that a single viable microorganism, or a cluster of them, will multiply and form a visible mass (a colony) on a solid nutrient medium. The result is a quantitative measure of the microbial population, widely applied in food safety, clinical diagnostics, and environmental testing.
The Concept of Colony Forming Units
The measurement of microbial concentration is reported in Colony Forming Units (CFU), which estimates the number of viable cells present. A CFU does not necessarily represent a single bacterial cell, but rather the progenitor unit (or cluster) that gave rise to the visible colony. Since bacteria often exist in clumps or chains, CFU reflects the number of reproductive units capable of growth under specific laboratory conditions.
To ensure the colonies are distinct and countable, the original sample must undergo a process of serial dilution. This stepwise dilution reduces the microbial density until a small, manageable number of organisms are plated onto the agar.
A statistically significant count requires plates to fall within a specific range, typically between 30 and 300 colonies. If a plate has fewer than 30 colonies, the count is considered unreliable due to the small sample size and is designated Too Few To Count (TFTC). Conversely, a plate with more than 300 colonies is too dense for accurate counting, as colonies will overlap and merge, leading to a Too Numerous To Count (TNTC) designation.
Step-by-Step Counting Procedure
The physical act of counting colonies requires a systematic approach to ensure accuracy and avoid double-counting. The first step is to select a plate from the dilution series that falls within the acceptable 30 to 300 colony range. The plate is typically inverted and placed onto a colony counter, an instrument that often provides a magnified view and a gridded surface.
For manual counting, a specialized marking pen is used to tap the bottom of the Petri dish over each visible colony. Many manual colony counters feature an electronic pressure pad that registers this tap, automatically tallying the count on a digital display and providing an audible confirmation. This marking technique prevents double-counting and helps maintain focus during the repetitive task.
To manage high colony numbers efficiently, the plate’s gridded surface can be used to divide the agar into quadrants or smaller sections. The technician can then count the colonies in one or two representative sections and extrapolate the total count, though counting the entire plate is preferable for maximum accuracy.
While manual counting is flexible for distinguishing between different colony shapes, automated colony counters use image analysis software to provide rapid and consistent results, especially when processing many plates.
Determining Bacterial Concentration
Once the raw colony count (N) is obtained from the statistically valid plate, the next step is to convert this number into the original sample’s concentration, typically expressed as CFU/mL. This calculation requires accounting for the volume of the diluted sample plated and the total dilution factor applied. The concentration is calculated by dividing the number of colonies by the volume plated (in mL), and then multiplying by the dilution factor.
The dilution factor is the reciprocal of the total dilution of the sample plated; for instance, a \(10^{-5}\) dilution has a dilution factor of \(10^5\). If a plate from the \(10^{-5}\) dilution yielded 125 colonies and a volume of \(0.1 \text{ mL}\) was plated, the calculation becomes \((125 / 0.1 \text{ mL}) \times 10^5\). This simplifies to \(1.25 \times 10^8 \text{ CFU/mL}\).
Final concentrations are reported in scientific notation with an appropriate number of significant figures, usually two, to maintain precision and manage the large numbers involved. This mathematical step provides the final, quantifiable measure of viable microorganisms in the initial, undiluted sample. If multiple plates from the same dilution are counted, the colony counts should be averaged before applying the formula to increase the reliability of the final reported concentration.
Troubleshooting and Quality Control
Several issues can compromise the accuracy of a plate count, necessitating careful quality control throughout the procedure. Plates outside the 30–300 range, designated as Too Numerous To Count (TNTC) or Too Few To Count (TFTC), must be excluded from the final calculation. A TNTC result indicates that the original sample was not diluted enough, leading to confluent growth where colonies merge into an indistinguishable lawn of bacteria.
Another common source of error is the presence of “spreader colonies,” which are motile bacteria that grow across the agar surface, obscuring or inhibiting the growth of other colonies. If a spreader covers more than 25% of the plate surface, the result is generally considered invalid and must be rejected. Contamination, identifiable by colonies with unusual morphology or color, also invalidates a count, as it suggests the introduction of microorganisms from an external source.
To mitigate random errors and confirm the reproducibility of the assay, standard protocol dictates that each dilution should be plated in duplicate or triplicate. Comparing the counts across these replicate plates helps confirm consistency in the plating and counting technique. If the counts between replicates vary significantly, it suggests a technical error occurred, and the entire dilution series may need to be repeated to achieve a reliable result.