Some Advantages and Disadvantages of Dip, Spot, and Spray Inoculation Methods for Determining the Efficacy of Sanitizer Washes on Produce
Mimics contamination from highly contaminated irrigation, run-off, or flume water
Delivered volume of inoculum known. Population in inoculum can be accurately calculated. Efficacy of sanitizer can be compared on different tissues within the same produce item. Most consistent inoculum applied of three inoculation methods
Mimics contamination from aerosols
Volume of inoculum and number of cells delivered to each produce item are unknown. Some inoculum may be internalized, complicating interpretation of data. Large volumes of high inoculum containing populations of pathogens are difficult to manage safely, even in highly experienced laboratories May not reflect contamination that would occur from contaminated irrigation, run-off, or flume water
Accurate delivery of inoculum is difficult, especially with smaller produce items. Aerosols generated are difficult to manage safely, even in highly experienced laboratories various types of produce and between different types of tissues on the same produce. Infiltration of the inoculum into cavities, wounded tissues, or porous areas on the produce surface, e.g., cut tissue, stem scar tissue, stomata, or lenticils [58,59], can result in conditions that may inhibit or enhance growth. Cells lodged in these areas may be protected against contact with sanitizers, particularly those with little or no surfactant activity. Analysis of produce inoculated by dip or spray methods requires a large number of units for each treatment, as random error values can be unpredictably large. Thus, efficiency of recovery or log10 changes in viable populations during subsequent storage or as a result of treatment with a sanitizer cannot be accurately calculated. Alternatively, fruits and vegetables can be inoculated by applying a known volume of cell suspension, e.g., up to 100 of known population to the surface. Spot inoculation represents contamination from a point source such as contact with soil, workers' hands, or surfaces of equipment and has been recommended for testing the efficacy of sanitizers in killing foodborne pathogens on tomatoes , lettuce, and parsley .
Temperature differentials between produce and inocula can affect the number of cells that infiltrate tissues. A negative differential, i.e., when the temperature of the produce is higher than the temperature of the inoculum, can result in enhanced infiltration of microbial cells [27,54,60]. A standard temperature at which both the produce and the inoculum are adjusted before inoculation should be selected for sanitizer efficacy studies. Otherwise, exposure of test cells to sanitizers and the efficiency of retrieval of cells may be affected.
In studies to determine the efficiency of retrieval of cells or efficacy of sanitizers, the inoculum applied to produce should be dried for a set period of time at a controlled temperature and relative humidity before treatment is applied and samples are analyzed. Fluctuations in temperature and relative humidity should be minimized between the time of drying and treatment or analysis. Three or more replicate experiments, each including four or more samples for each set of test parameters in each replicate, should be done. More samples may be needed, depending on specific objectives. Negative controls should always be included.
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