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 The shredded cheese should be mixed thoroughly before a test sample is withdrawn.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bulky food (e.g., frozen meat) may be thawed in a refrigerator (2–4°C) overnight (~18 hrs) before a test sample is withdrawn. However, it is sometimes preferable if a test sample can be aseptically withdrawn directly from the frozen food package.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using a sterile cutting board and knife, and the cut pieces mixed in a sterile container or bag before a test sample is withdrawn.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 2.2 Food blender (left) and stomacher (right).
Dilution of food homogenate
When pathogens are present in food, they are often found in very small numbers. Foods analyzed for detection of such pathogens are homogenized in an enrichment broth and the homogenate is not diluted further. The mixture is incubated to enrich the pathogen population so that it becomes easy to detect.
Other types of food are analyzed to determine the size of a certain microbial population (e.g., coliform count). In this case, the food is homogenized in a suitable diluent (e.g., sterile saline solution) and the homogenized food is appropriately diluted, usually decimally. A diluent is a liquid used to release the microorganisms from the food matrix, resulting in a suspension that represents the food. The suspension can be further analyzed by enumeration techniques. Ideally, the diluent should be compatible with the food system (i.e., allows for ease of homogenization) and should allow for maximum recovery of the microorganisms by not inducing biological stress (i.e., pH should be near neutral and osmolarity should be close to the microbial physiological level). Diluents include peptone water, citrate buffer, saline solution, and neutralizing buffer. Peptone water is the most commonly used diluent for food analysis; however, it may not be appropriate for some foods. Foods that are naturally acidic or contain antimicrobial compounds should be diluted with neutralizing buffer to prevent the inactivation of microorganisms during the recovery step. A buffer, such as warm (40ºC) citrate solution, is the preferred diluent for microbial analysis of hard cheeses, which will not mix evenly in cold aqueous solutions.
A subset of dilutions is selected for plating on a microbiological medium suitable for sustaining the targeted population. The extent of dilution and selection of the dilutions to be plated depends greatly on the analyst’s expectations of the size of microbial population in the food. The larger the population, the greater the degree of dilution required. An example of a dilution scheme is shown in Figure 2.3.
Figure 2.3 Decimal dilution of food homogenate.
ENVIRONMENTAL SAMPLING
Food microbiota include pre‐harvest contaminants as well as microorganisms introduced from the processing environment. Assessing the microbial load and variety of microorganisms in the factory environment provides insight into the quality and safety of the finished product produced in the facility. Consequently, the processing environment is often sampled, and results of analysis are carefully considered by the facility management. Samples are often taken from floors, drains, and equipment surfaces, particularly those that are food contact surfaces. Refrigerators and other storage sites also should be sampled frequently. The sampling procedure depends on the nature of the site, degree of contamination, and the microbiological information sought. There are two broad categories of samples that are commonly taken from food processing environment: surface and air samples.
Surface Sampling
Surfaces in processing facilities are commonly designated by their likelihood to cause food contamination. Food contact surfaces (Zone 1) are important sites that are often considered during environmental sampling. A food contact surface could be part of a piece of equipment, packing material, storage tank, ripening room, conveyer belt, or any other item that is expected to touch the food item. Zone 2 is an area where surfaces do not directly contact food but are usually located in the same room as those of zone 1. If zone 2 is contaminated with pathogens, it is likely that zone 1 also becomes contaminated. Contaminant transfer is often caused by human or machine action. Surfaces in zone 2 include walls or floors located near processing equipment and overhanging pipes or equipment. Zone 3 is an area that
