Analytical Food Microbiology. Ahmed E. Yousef

in the original unopened containers. In case of bulk food, or when the original container is too large for submission to the laboratory, a subsample is aseptically transferred to a clean, sterile container.

       Use Sterile Sampling Utensils. In the event of repackaging, suitable sterile plastic or metal containers are preferable over glass containers. These containers must be clean, dry, leak‐proof, and of a size suitable for the sample. Sampling tools such as forceps, spatulas, and scissors should be appropriately wrapped and autoclaved prior to use.

       Label Samples Appropriately and Create a Sample Record. A proper label should be developed to identify sample contents, date of sampling, sample collector’s name, and other pertinent information (e.g., sample temperature or storage conditions, and type of package from which subsample was taken). The simplest form of labeling is using masking or labeling tape, on which information is written with a permanent marker; this is preferred over writing directly on sample container. In addition to the information on the label, a record should be created to document additional pertinent information, such as the times of collection and of arrival at the laboratory, condition of sample at the time of arrival, etc. In some food inspection agencies, the label on the sample package is replaced with a barcode that is linked to a record in an electronic database.

       Deliver Samples Promptly and Control Temperature During Transportation. Samples of refrigerated food should be kept refrigerated and those of frozen food should be handled and transported in the frozen state. However, samples of refrigerated food should not be frozen at any time; freezing can alter sample microbiota. Holding these samples for considerable time before analysis may alter the microbial burden or profile.

      Sample Preparation

      The food inspector or sample collector delivers laboratory samples to the analytical facility. The delivered sample could be a retail package, a consumer‐size container, or a portion of a food bulk. Sample preparation refers to the reduction of the laboratory sample into a test sample (or analytical sample) and preparation of the latter for analysis. Therefore, sample preparation includes: (i) withdrawal and measurement of a representative test sample from the laboratory sample; (ii) homogenization to distribute microorganism uniformly in the test sample; and (iii) dilution of the sample homogenate to decrease food microbiota to a countable or detectable level.

       Withdrawing the test sample

      Microbiological results are often reported quantitatively, therefore, sample mass (or volume) should be carefully measured and reported. The test sample, which is used directly in microbiological analysis, could weigh 10, 25, or 50 g, but a 25 g test sample is commonly used in the detection of pathogens. Larger sample size means more accurate representation of the food lot and greater ability to recover scarce contaminants. Many analysts, however, prefer smaller samples, since these are easier to handle and less costly to analyze.

      If the recommended sample mass cannot be easily obtained (e.g., food difficult to mix before weighing), analysts should be able to modify the analytical procedure to accommodate this deviation. Analysts occasionally opt to combine several test samples into a single “composite sample.” For example, if 15 portions (25 g each) are taken from 15 one‐pound meat packages, and these packages are expected to be similar in microbiological quality, the analyst may combine these into a 375 g composite sample. The composite sample is then diluted (10^‐1) and analyzed. Composite sampling is a cost‐saving practice, but it could conceal an abnormally contaminated sample.

      The physical characteristics of food dictate the technique suitable for withdrawing the test sample:

      1 Pourable liquid, powder, and some shredded foods are easy to mix in original packages. Withdrawing a test sample form these types of food involves thoroughly mixing the contents of the package, aseptically measuring a predetermined portion, and transferring this portion to sterile container.

      2 For pasty and thick products (e.g., packaged ground meat or multilayered cake), the package contents may be transferred to a bigger sterile container, and the contents are mixed using an appropriate sterile implement.

      3 Solid foods that cannot be mixed manually include blocks or wheels of hard cheese and similar products. Pieces or wedges of the cheese may be cut with a sterile knife and aseptically shredded, using a sterile shredder. The shredded cheese should be mixed thoroughly before a test sample is withdrawn. Alternatively, a sterile cheese trier may be used to extract several core samples from the cheese wheel or block. The cheese pieces extracted by the trier can be easily cut into small pieces with a knife or a spatula, and the test sample can be taken from them.

      4 Frozen bulky food (e.g., frozen meat) may be thawed in a refrigerator (2–4°C) overnight (~18 hrs) before a test sample is withdrawn. However, it is sometimes preferable if a test sample can be aseptically withdrawn directly from the frozen food package. In the latter case, sampling equipment may include a drill with sanitized pits, or a band saw with sanitized stage and blade.

      5 Bulky food, with contaminants residing predominantly on the surface, is tricky to sample. Such foods include whole chicken carcasses and cantaloupes. Grinding a piece of chicken should distribute contaminants evenly and makes it easy to withdraw a 25‐g test sample; however, results of analysis expressed as CFU/g could be misleading, since the microbiota were not evenly distributed in the initial sample. It may be preferable to take a surface sample from this type of food, by swabbing a defined area and expressing the results as CFU/cm2. Limited surface area, however, may not produce a test sample sufficiently large enough to detect low levels of pathogen on these products. In this case, the whole food unit (e.g., the whole chicken carcass) may be thoroughly mixed with a diluent in a large sterile bag, and the pathogens washed off the surface are detected in a portion of the diluent. Note that in this case, results are reported per unit, not per area or mass.

      6 Bulky but easy‐to‐cut foods such as vegetables and fruits require careful handling during sample preparation. These could be aseptically cut in a biological cabinet using a sterile cutting board and knife, and the cut pieces mixed in a sterile container or bag before a test sample is withdrawn.

      The mass of the transferred portion is measured on an analytical or a top‐loading balance. If the test sample is measured in volume, a sterile pipette or graduated cylinder is used. It should cautioned that fast but careful sample withdrawal and measurement is critical for minimizing contamination by the analyst or laboratory environment. For example, weighing the test sample to the nearest 0.1 gram takes less time and offers less chance for contamination than a process that produces a test sample weight with two‐decimal digit accuracy.

       Homogenizing the test sample

Most test samples require homogenization before analysis. The goal of homogenization is to release microorganisms from food into suspension. Blenders, stomachers, sonicators, shakers, and hand massaging of bagged food are different approaches for homogenizing a food sample. Liquid food samples are manually mixed before analysis, whereas solid foods commonly require mechanical stirring (homogenization) in a suitable diluent to break food clumps and release microorganisms from the food matrix. Methods of homogenization may vary in ability to recover entrapped microorganisms. Blenders and stomachers (Figure 2.2) are commonly used homogenizers to prepare the food sample for analysis. The revolving blades of the blender divide the food into small particles and mix them with the diluent. The same goal may be achieved using a stomacher, depending on the characteristics of the food. The stomacher is a mechanical device that agitates a food sample placed in sterile plastic bag. The back and forth mechanical action of the stomacher paddles mimics the stomach action (hence the name) and helps incorporate the sample particles into the diluent.

Figure