Analytical Food Microbiology. Ahmed E. Yousef
depends on the microorganisms of interest and their natural habitat, as well as presence of competing microbes.
In food microbiology, several incubation temperatures are typically used. For potentially pathogenic organisms, such as Salmonella enterica, incubation occurs at 32–37°C, a temperature range suitable for mesophiles. A somewhat cooler temperature (e.g., 25°C) is more preferred by spoilage organisms such as yeasts, molds, and psychrotrophic bacteria. “Room temperature” is typically taken to mean 22°C, but this temperature may vary depending on the room used for incubation and even the season and area of the world. Refrigeration at 4°C is typically used to maintain cultures without allowing further growth. A refrigerated incubation can also be used for cold enrichments of psychrotrophic microorganisms such as Listeria monocytogenes.
Plates containing inoculated agar media are typically inverted before incubation. If plates are incubated with lids upward, water condensate falls on the agar surface causing the spreading of colonies. When plates are inverted, condensed water (from moist agar) accumulates on the plate lid. Excessive water condensation on the lid, however, is undesirable and should always be minimized. Pouring hot agar (> 50°C) aggravates this problem. In the case of spread plates, it is preferable to pour the agar in these plates 24–48 hours before use. Some microbiologists choose to “dry” the spread plates soon after preparation for several hours in a warm clean incubator. For the safety of the analyst, plate lids with excessive water condensation should be replaced with dry sterile lids.
Colony Counting
“Counting” in food microbiology refers to the determination of the size of a microbial population within a specific quantity of food (i.e., population concentration). Enumerating the number of colonies on agar plates may also be referred to as “counting,” therefore careful distinction between these two usages is urged. Throughout this manual, the former will be referred to as “population count” and the later as “colony count.”
Some enumeration techniques, such as the direct microscopic counting method, allow determination of the number of cells per unit volume or weight of the sample. The plate count technique, however, determines the number of cells or cell clumps capable of forming colonies on agar plates. Since it is impossible to distinguish colonies arising from individual cells and those from cell clumps, the final population count determined by this method is expressed as colony forming units per unit volume or weight, i.e., CFU/ml or CFU/g.
To begin the counting process, the analyst should lay out the incubated plates in order of dilution to evaluate the executed dilution scheme and technique. The lowest dilution plated should have yielded the plate with the most colonies, and the number of colonies are expected to decrease by approximately a factor of ten as dilutions increase, provided that a decimal dilution scheme is used. If this is not the case, analysts should be cautious when interpreting results. Counting colonies on plates can be done visually, preferably with the help of a colony counter. Colony counters, such as the darkfield Quebec colony counter (Figure 3.2), provide background lighting and magnification so that small colonies are not overlooked. To carry out this process accurately and rapidly, the analyst should mark the counted colonies with a marker pen on the bottom of the plate, to make sure that the same colonies are not counted repeatedly.
Counting rules
In order to obtain counts that can be compared among different laboratories, it is necessary to establish consistent guidelines for counting colonies. In some circumstances, however, different counting or calculation methods may be used in place of, or in conjunction with, the standard counting rules. The following are the rules that will be applied throughout this book for counting bacterial colonies.
Figure 3.2 Darkfield Quebec colony counter with a Petri plate mounted for colony counting.
Plates with colonies in the range of 20–200 (the best possible scenario)
Although other references may use 30 to 300 or 25 to 250 colonies as suitable countable bacterial colonies on a plate, the range 20–200 will be used throughout this book. If plating yields plates with colony counts in the range of 20–200/plate (as judged by a preliminary estimation), discard all remaining plates and count colonies only on the plates in this range. Calculate the CFU/g using equation 3.7. Familiarity with equation 3.7 is important. Examples of dilution factors are 1/10 (i.e., 10–1) and 1/100 (i.e., 10–2), and the volume plated is commonly 1 or 0.1 ml.
Duplicate plates with colony counts in the 20–200 range are ideally obtained from one dilution only. When this condition is not met, the following rules are applied.
One plate in the 20–200 range. If the exercise yields only one plate with a colony count in the range of 20–200, calculate the CFU/g in the original sample using the number of colonies on that plate instead of an average.
Consecutive dilutions producing plates in the 20–200 range. If plates from two consecutive dilutions yield 20–200 colonies, compute the CFU/g resulting for each of the two dilutions. If the two population counts are not appreciably different (e.g., 1.5×104 and 1.2×104 CFU/g), average the numbers and report the average as sample population count. If the numbers are substantially different (e.g., the higher CFU/g is more than twice the lower one), report only the lower computed CFU/g.
Plates with < 20 colonies
If the plating procedure results in only plates with fewer than 20 colonies, record the actual number of colonies on the plates receiving the lowest dilution (i.e., the highest concentration of sample plated) and apply equation 3.7. In addition, report the number as “estimated” or “est.” For example, a sample of cooked meat was analyzed by pour‐plating 1 ml of sample dilutions 10–2 to 10–4. Incubated plates produced less than 20 colonies and thus the count of microbial population in meat is calculated and reported as shown in Table 3.1.
Plates with no colonies
When all plates produce no colonies, the count is estimated to be smaller than the minimum detection limit of the procedure followed. The minimum detection limit is the count that would result from the presence of one colony on the plate receiving the lowest dilution of the sample. Apply equation 3.4 to estimate the count using the lowest dilution plated and substitute the numerator of the equation with < 1. For example, if no colonies appeared on any of the plates receiving the dilutions shown in the previous example (Table 3.1), then
It is not necessary in this case to report the count as “est.” because the presence of the less than (<) sign indicates the uncertainty of the count.
Plates