Analytical Food Microbiology. Ahmed E. Yousef
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 may cause contamination of zone 2. Zone 3 includes warehouses, employee locker rooms, and loading docks. Listeria monocytogenes is an example of pathogens that are commonly transmitted to food from the processing environment.
Sampled surfaces could be rough or smooth, flat or with curves and corners, continuous or with cracks and crevices, and accessible or difficult to reach. Therefore, the choice of a method to sample a surface depends not only on the surface zone but also on its characteristics.
Swab method
A sterile cotton swab may be used for limited surfaces or on hard‐to‐reach crevices. The swab is typically made of a wound cotton head (~0.5 cm diameter and 2 cm long) and a 12–15 cm long wooden stick. The swab may be prepared in the laboratory, sterilized, and kept in a sterile container until the time of use. Alternatively, cotton swabs may be purchased as individually wrapped sterile units. In addition to the swab, a sterile rinse solution in a test tube is needed for surface sampling. Many commercially available products include a sterile swab packaged with appropriate diluent in a shatterproof (plastic) tube. A predetermined area (e.g., 100 cm2) of the surface to be sampled is swabbed with the moistened cotton swab, which is returned to the rinse solution tube. The rinse solution may be diluted serially, and selected dilutions are spread on the surface of a suitable agar growth medium. The inoculated plates are incubated, and populations of the targeted microorganisms are counted. In this scenario, when quantitative results are desired, the rinse solution is considered the undiluted analytical sample.
Sponge method
If the surface to be swabbed is large, or if a microorganism of a low incidence rate in processing environment is sought, a sponge may be used instead of a cotton swab. A natural or synthetic sponge with ~5 × 5 cm contact surface that is free from antimicrobial agents is suitable for this purpose. The sponge can be packed in a heat‐resistant bag or wrapped in aluminum foil and sterilized by the analyst, or purchased prepacked and presterilized from a commercial source. During sampling, the sponge is held aseptically, moistened with 10 ml rinse solution, rubbed against the surface to be sampled and returned to a sterile plastic bag. The sample should be transferred to the laboratory under refrigeration and analyzed without delay. If the purpose of sampling is to detect pathogens, the sponge is transferred to a suitable enrichment broth and the mixture is incubated. When sampling is carried out to quantify environment microbiota, the sponge is mixed with 50‐ or 100‐ml diluent and further dilutions are made. Selected dilutions are then spread on the surface of a suitable agar growth medium, plates are incubated, and the population of the targeted microorganism is counted.
Replicate organism direct agar contact (RODAC) method
The replicate organism direct agar contact (RODAC) method may be used on easily accessible flat surfaces. In this method, Petri plates are filled with an agar medium suitable for the microbiota to be analyzed. These plates may be prepared in the laboratory or purchased from a commercial source. The RODAC plates should contain enough agar medium so that the surface of the medium is convex and rises above the rim of the plate. At the sampling site, the agar medium in the RODAC plate is exposed to the surface being sampled. This exposure is accomplished by pressing the plate against the sampled surface and rolling the plate while applying some pressure. The cover is replaced, and the plate is incubated at a temperature and for a time appropriate to the targeted microorganism or microbiota. After incubation, the colonies on each plate are counted and colony subculturing may follow. Since no sample dilution takes place, the RODAC method is suitable for sampling pre‐cleaned or sanitized surfaces. If the surface is contaminated heavily, incubated RODAC plates will be crowded with colonies and results will be difficult to interpret.
Air Sampling
Microorganisms may become airborne due to activities