(β). The chance of failing to reject the null hypothesis when the alternative hypothesis is true (Type‐II error). The commonly used values of β are 0.1 and 0.2 (i.e., 10% and 20%). The smaller the value of β, the larger the sample size needed to produce statistically meaningful experiment.Power of the experiment (1‐β): Once β is selected, the power is determined. Power is the probability that the analysis can detect a difference between the two groups, when this difference is truly present. Although it is always desirable to have a high power, 1‐β is often set at 0.8 (80%) to make the sample size manageable.Effect size: A meaningful effect size should be considered. In the example above, one may assume that if the percentage of Salmonella‐positive eggs from the less‐infected hen group is 10% (p 1 ), the more infected group should produce Salmonella‐positive eggs at the rate of 20% (p 2 ) or more, for the difference to be meaningful. Additionally, if 1‐β is chosen to be 0.8, it means that we have 80% chance of detecting the stated difference between these two groups. Note that the smaller the p 1 and p 2 values, the larger the size of sample needed to produce statistically meaningful results.Population standard deviation: This is needed for continuous data, such as changes in pathogen populations. However, in the example described above, the variation between the two groups is not continuous, it is dichotomous (i.e., results reported as Salmonella‐positive or Salmonella‐negative) and expressed as incidence rate or proportion. Therefore, determination of standard deviation is not relevant to the example presented here, but it will be needed for other types of analyses.Calculate sample sizeThe information collected so far is sufficient to determine sample size per group, i.e., number of eggs to be tested for S. enterica for each of the two hen groups. Based on this information, α = 0.05, 1‐β = 0.8, p 1 = 0.1, and p 2 = 0.2. These values can be entered in an appropriate statistical equation (e.g., Dell et al., 2002) for calculating sample size. Alternatively, commercial statistical programs (e.g., Minitab or SAS) contain modules that automate the sample size calculation once the above parameters are determined. In the example at hand, 218 eggs for each of the two groups need to be analyzed to determine if incidence of this pathogen is different in the two groups of hens. The sample size in this example is quite large because of the small rate of contamination of S. enterica on eggs. The rate of incidence of the pathogen inside the egg (i.e., in the yolk) is even smaller and thus requires much larger sample size to detect any differences between the two groups.
It should be obvious from the previous discussion that sample planning is not limited to size determination. The plan should be tailored to address food characteristics, reason for sampling, lesson to be learned or action to be taken, and other factors. Food inspection, mandated by regulatory agencies, should be based on sound sampling plans considering the health and economic impact of the outcome of the analysis. The frequency of inspection and sampling may depend on the quality or safety history of the food produced or processed by a given establishment. Inspection and sample collection are minimized for establishments with a recent history of producing good quality food or food that posed no or low safety risks. Therefore, a vendor’s recent history should be considered when developing a sampling plan.
SAMPLING TECHNIQUES AND SAMPLE PREPARATION
Sampling Tips
After developing the sampling plan, actual sampling should be carefully executed. The following are some tips to be observed during withdrawing, handling, and transporting of samples.
Collect Laboratory Sample in Original Container and Repackage Only if Necessary. Samples are ideally submitted to the laboratory 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
