population using the plate count method often involves homogenizing a sample, preparing dilutions of the homogenized product, plating appropriate dilutions on a suitable medium, incubating the inoculated medium, counting resulting colonies, and calculating the concentration of the targeted population. For determining the concentration of microbial population accurately, it is imperative that the analytical sample is appropriately obtained and prepared. Information about sampling and sample preparation (including homogenization) has been discussed in Chapter 2.
Dilution
Differences in food’s microbial populations span several orders of magnitude, hence dilutions should be made before these populations can be measured with reasonable accuracy. To accomplish this task, an analytical sample is typically weighed, dilutions are made, and the count of microorganisms in the diluted sample is determined. The degree of dilution should be tracked carefully so that concentration of microorganisms in the undiluted food can be calculated. The degree of dilution (i.e., dilution factor) can be represented, generically, by the following equation:
(3.1)
Although weights can be measured with great accuracy, microbiologists prefer volumetric over gravimetric measurements because in the former, the analysis can be completed more quickly and aseptic techniques can be applied more easily. Furthermore, dilution is completed in multiple steps, typically in a decimal dilution series. To simplify the volumetric dilution procedure, the following approximations will be applied:
1 Food density is equal to 1 g/ml (at ambient temperature), therefore, food volume and mass will be considered numerically equal.
2 Final volume of diluted sample equals the sum of the volumes of the sample to be diluted and the diluent to be added.
With these practical considerations in mind, the equation above can be approximated as follows:
For example, a ten‐fold dilution (i.e., decimal dilution) of a food sample is prepared by mixing one part of the food with nine parts of a diluent, which commonly is a physiological saline solution or peptone water. Applying equation 3.2, the “dilution factor” for this diluted sample is 1/10 (i.e., one tenth) or 10–1.
The subsequent dilution, in a decimal dilution series, is made by mixing 1 ml of the first diluted sample with 9 ml diluent. The new mixture will have a total dilution of 1/100 (one hundredth) or 10–2. Note that exponents in a ten‐fold dilution scheme are additive, i.e., a 10–1 dilution followed by a subsequent 10–1 dilution yields a total dilution factor of 10–2. Additional dilutions are prepared as needed and the dilution factor, at any step of the series, can be calculated using equation 3.3.
Decimal dilution series are recommended for ease of calculation, but other ratios of weight (or volume) of a sample and diluent can be used. If a dilution in the series is not decimal (e.g., two‐fold dilution), the dilution factor of the new mixture can also be calculated using equation 3.3.
Dilutions suitable for plating
Portions of the prepared dilutions are used to inoculate agar media in Petri plates and the process is called “plating” (Figure 3.1). The questions that should be considered by the analyst are: How many dilutions should be prepared, and which dilutions should be selected for plating? In other words, what dilution scheme should the analyst prepare and follow? Preparing all possible dilutions and plating these dilutions is a waste of resources and effort. On the contrary, preparing a limited number of dilutions may lead to the failure of the analyst to accurately determine the population of the organism in the food.
To answer the questions just presented, the dilution scheme should be based on the projected concentration of the targeted microorganism in the product. A product expected to contain a high load of the microorganism should be diluted further than that expected to contain a small load. The microbial load is sometimes easy to predict if the analyst has prior experience in analyzing the same product. In most cases, however, the analyst should check published literature for microbial populations expected in the product. In other situations, the analyst may seek information about the product from the producer, manufacturer, processor, or retailer. Considering that determining a dilution scheme is often based on the analyst’s best guess, the scheme should accommodate a reasonable margin for error. For example, if the food is presumed to contain 107 CFU/g of the microorganism subject to analysis, the dilution scheme should allow determination of populations in the range of 106 to 108 CFU/g. This can be achieved by the scheme shown in Figure 3.1, assuming that an ideal countable plate contains 100 colonies. This scheme can be verified using the population count equations described later in this chapter.
Figure 3.1 Example of a dilution scheme, showing the dilutions (prepared from a homogenized food) and the dilutions selected for plating, presuming the targeted population in the food is 1.0×107 CFU/g.
Pipetting
Pipetting is an important activity for completing dilutions successfully. Quantitative transfer of broth culture, diluent, homogenized food, liquid food, or similar materials requires accurate pipetting. This can be accomplished by using a pipette in combination with a pipetting device (pipette dispenser, pipetaid, etc.). The pipetting device is an essential tool in analytical laboratories and may vary from simple rubber bulbs to automated pipetters. Simple mechanical pipetting aids are often used with glass or plastic pipettes. These pipettes, often referred to as serological pipettes, are maintained sterile in canisters or individual wrappers. In this book, it is suggested that individually wrapped sterile serological pipettes, along with a hand‐held pipette pump, are used to make the initial dilution (from the homogenized food)
