Preparing for the Count
A Colony Forming Unit (CFU) count is the standard method used in microbiology to estimate the number of viable microorganisms, such as bacteria or fungi, within a sample. This measurement counts living cells or clusters of cells capable of multiplying to form a visible colony under specific conditions. The CFU count determines microbial concentration, which is applied in quality control for food safety, water testing, and pharmaceutical production.
To obtain a reliable count, the concentration of microorganisms must be drastically reduced through serial dilution. This involves a stepwise, geometric dilution of the sample, typically in ten-fold increments, using sterile diluents. This procedure ensures that at least one resulting plate contains an appropriate number of isolated colonies, preventing a dense, uncountable lawn of growth.
The goal is to achieve a “statistically significant” plate count, generally accepted to fall between 30 and 300 colonies. If a plate has more than 300 colonies, the count is inaccurate because colonies may merge or overlap. Conversely, plates with fewer than 30 colonies are not statistically representative of the overall population due to large relative error.
Proper plating technique is necessary for an accurate count, using either the spread plate or the pour plate method. In the spread plate method, a small volume of the diluted sample is spread onto the surface of solidified agar. The pour plate method involves mixing the diluted sample with molten agar before it solidifies, allowing colonies to grow both on the surface and embedded within the medium. Plates are then incubated for 24 to 48 hours, allowing each viable CFU to develop into a visible, countable colony.
Standardized Counting Techniques
Once colonies have developed, the physical act of counting requires selecting the plate that falls within the acceptable range of 30 to 300 colonies. Plates exceeding the upper limit are designated as Too Numerous To Count (TNTC). Plates falling below the lower limit are designated as Too Few To Count (TFTC) and are excluded from the final calculation.
The simplest manual technique involves inverting the Petri dish and using a fine-tipped marker to place a dot over each colony as it is counted. This marking ensures each colony is counted only once. For a more organized approach, the plate can be placed on a Quebec colony counter, a specialized device that offers an illuminated surface and often a magnifying lens to improve visibility.
Using the Quebec Counter
The Quebec counter frequently incorporates a Wolffhügel grid etched onto the counting surface, which helps systematically track the count by dividing the plate into sectors. The counter is equipped with a digital display and a pressure-sensitive marking stylus. Pressing the stylus against the plate to mark a colony simultaneously increments the digital counter.
Estimation Techniques
If a plate contains too many colonies to count manually but is not completely TNTC, an estimation technique called quadranting can be used. This method involves dividing the plate into four equal sections. The colonies in one or two sections are counted, and that number is multiplied by the appropriate factor to estimate the total count for the entire plate.
Calculating Colony Forming Units (CFU/mL)
The final colony count obtained from the selected plate must be converted back to the concentration in the original, undiluted sample, which is expressed as Colony Forming Units per milliliter (CFU/mL). This conversion is necessary because the count reflects the number of microbes in the diluted volume that was plated, not the initial sample. The standard formula used to determine this concentration is: CFU/mL = (Number of Colonies) / (Volume Plated in mL $\times$ Dilution Factor).
The dilution factor is the inverse of the dilution that was plated. For example, a sample diluted $10^{-5}$ has an inverse dilution factor of $10^5$. The volume plated must be in milliliters; 100 microliters must be converted to 0.1 mL for the calculation. If a $10^{-5}$ dilution plate yielded 125 colonies after plating 0.1 mL, the calculation is $125 \div (0.1 \text{ mL} \times 10^{-5})$, which simplifies to $125 \div 10^{-6}$.
Performing the division results in $125 \times 10^6$ CFU/mL, which represents the concentration in the original sample. This mathematical step reverses the effect of the serial dilution and the small plating volume, providing the absolute concentration of viable microbes.
Dealing with Anomalies and Reporting
During the counting process, microbiologists must address common growth anomalies that can interfere with an accurate result. A major issue is the presence of spreading colonies, which are characterized by microbial growth that rapidly extends across the agar surface, often obscuring other discrete colonies. If spreading growth covers more than one-quarter of the plate surface, the count is invalidated and reported as a spreader, necessitating a repeat of the entire plating procedure.
In cases where a plate contains multiple types of microorganisms, such as in environmental samples, the presence of mixed cultures must be noted, but the total count includes all colony types.
For reporting the final CFU/mL result, standard scientific notation is required to manage the large numbers generated. The final reported value should be rounded to two significant figures. For example, a calculated result of $125 \times 10^6$ CFU/mL is formally reported as $1.3 \times 10^8$ CFU/mL.