If the plating procedure yields only plates with greater than 200 colonies, obtain an estimated count as follows. Count colonies in representative portions (determined subjectively) of the plate receiving the highest dilution. Using a lighted colony counter with gridlines that are 1‐cm apart (Figure 3.2) assists in choosing a representative area of the plate to count.
If there are fewer than 10 colonies per cm2, then count 13 squares chosen as follows: 7 consecutive horizontally and 6 consecutive vertically; see Figure 3.3 for a visual example of plate counting field. The sum of the colonies in the 13 squares multiplied by 5 equals the estimated count per 65 cm2 plate, which is the area of a typical Petri dish, assuming its inner diameter is ~90 mm.TABLE 3.1 Microbial population count in cooked meat.Dilution FactorNumber of ColoniesPlate 1 Plate 2 10–2 {least dilute}12 16 10–33 1 10–4 {most dilute}0 0 Figure 3.3 Procedure to estimate microbial population count in samples using crowded plates (> 200 colonies/plate).
If the number of colonies per cm2 are from 10 up to 100 colonies, count colonies in 4 representative squares and multiply the average by 65 to give estimated count per plate (Figure 3.3).
If there are greater than 100 colonies per cm2, then record the count as > 6500 CFU/plate (Figure 3.3).
In all cases, equation 3.7 is used to estimate population counts (CFU/g, est.). Never report the final count in the food sample as too numerous to count (TNTC). Plates with different surface areas, such as Petrifilms, are counted using the same principle. Count a minimum of 4 squares and average those counts, then multiply that count by the area of the plate being used.
Plates with spreaders
Count a chain of colonies that are not too distinctly separated as a single colony. If colonies can be distinguished, then it is not considered a spreader for counting purposes. If chains of colonies appear to originate from separate sources, count each chain as one colony. If the spreader is greater than 25% of the plate, report the results as spreaders (Spr.) rather than as a number.
Which counting rule to use
The previous discussion is generally sufficient for determining which rule to follow for counting colonies and population resulting from a particular analysis. Alternatively, the counting rule to follow may be determined using a systematic approach, such as the decision tree described in Figure 3.4. Examples for applying most of these counting rules are shown in Figure 3.5. Note that slightly different rules are applied for counting fungal colonies; this is explained in the chapter dealing with enumeration of this group of microorganisms.
Figure 3.4 Decision tree for applying microbial colony and population count rules.
Figure 3.5 Applying colony and population counting rules.
Population Counting
After the colony count is determined, the analysist should be able to determine the concentration of a microorganism (or a microbial group) in the food. The concentration is often expressed as CFU per gram of that food. As inidicated before, this concentration will be referred to as “population count,” and it is governed by the following general equation:
To calculate the denominator of equation 3.5, one should know the volume of diluted homogenate that has been plated and the concentration of food in that dilution.
The volume dispensed in a Petri plate is commonly 0.1 ml for spread‐plating and 1.0 ml for pour‐plating, but volumes other than these can be used. The concentration of food in this diluted homogenate can be considered to equal the dilution factor. For example, if a food has been homogenized and diluted 1:100 (i.e., 10–2 dilution factor), this results in a concentration of 0.01 g of food per g (or mL) of diluted homogenate. Therefore, equation 3.6 can be rewritten as follows:
As previously stated, the density of food, diluted homogenate and diluent is approximated as 1 g/ml. Note that volume of sample dispensed is quantified in ml. If analysts have dispensed a volume measured in μl, this value will need to be adjusted to yield a correct population count.
Important Considerations
The following reminders should help in avoiding the common pitfalls encountered during enumeration of microorganisms in food:
Notice the difference in usage of “colony count” and “population count.”
When a specific microbial population is counted, the generic word “population” is replaced with a word or a phrase that describes the population. For example, in subsequent chapters, the “population count” is reported as aerobic mesophilic count, spore count,