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CHAPTER 3 ENUMERATION OF MICROORGANISMS IN FOOD: DILUTION SERIES. COLONY COUNTING. MOST PROBABLE NUMBER.

      INTRODUCTION

      How can one evaluate the microbiological quality of a food? Enumeration of microorganisms in the food is the answer that often comes to mind. The important follow up question then is: Can this enumeration be done accurately so that the results are used reliably to measure the microbiological quality? Reliability of the results, obviously, depends on how the enumeration is executed. This chapter addresses this topic with the goal of familiarizing the analysts with the methods and techniques used in enumeration and helping them apply these to produce repeatable and reliably results.

Foods vary in microbial load depending on how they are produced, processed, transported, stored, and handled. Microbial load, which is sometimes referred to as microbial burden, bioburden, or microbiota, is made of a predominant microorganism, a group of microorganisms having common characteristics, or a number of unrelated microorganisms. Determining the microbial load involves counting or enumerating the population of microorganisms in the food. “Counting” or “enumeration” in food microbiology refers to process of determining the concentration of a population of a microorganism (or microorganisms) of interest in a food. The counting exercise, therefore, determines the number of microbial cells, the number of colony forming units (CFU), or the most probable number of CFU present in a unit volume, weight, or surface area of a given sample. There are several methods used in counting or enumerating microbial populations in food. Counting using the “plate count” and the “most probable number” methods will be discussed, as these are the primary means of enumeration presented in the remainder of this book. Other counting techniques are also used in quality control laboratories (e.g., direct microscopic count and spiral plating); however, these techniques will not be discussed in this chapter.

PLATE COUNT METHOD

      The procedure for determining the count of a microbial 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:

With these practical considerations in mind, the equation above can be approximated as follows:

(3.2)

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.

(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

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