When a manufacturing facility conducts microbial testing internally, the lab is expected to have hygiene and pathogen control programs in place and verified for effectiveness. These internal labs are to operate under their own quality systems, which may or may not be operated under the scope of an ISO 17025 platform.
Either way, the lab follows its own standard operating procedures and cross-contamination mitigation techniques with goals in keeping with the food safety systems employed by the facility that the lab is serving. A documented and carefully implemented laboratory environmental monitoring program (EMP) will provide the scientific evidence that sanitation activities are being conducted and pathogens are being controlled within the lab.
Hygiene monitoring and pathogen monitoring programs fall under the environmental monitoring umbrella and contain the same general elements of a manufacturing facility EMP including:
- Site selection and site lists;
- Assigning zones to each site;
- Determination of test organisms and test assays;
- Specification setting;
- Site selection randomization;
- Designated sampling times;
- Result tracking and trending;
- Corrective/preventive actions; and
- Verification activities.
It may seem as if you are seeing double—“hygiene monitoring” and “pathogen control programs” are the same thing, right? In a word, no. The main difference relates to the organisms that are tested. In a hygiene monitoring program indicator organisms are tested, while in a pathogen monitoring program all pathogen assays that are routinely run in the lab are tested. Similarities between the programs are that they both verify that cleaning and sanitation are effective as written and applied.
Assays and organisms tested. A hygiene monitoring program will use assays that yield quantitative as opposed to qualitative results. Quantitative results will give a numerical value and are reported in colony forming units (CFU/swab, CFU/sponge or unit of time for air plates). The assays include aerobic plate count (synonyms: total plate count, total viable count), Enterobacteriaceae, coliforms, E. coli, Enterococcus faecalis, yeast, and mold.
Sampling times and result interpretation. There are three sampling times, each of which will have a different objective:
- To assess cleaning/sanitation efficacy: Samples are taken after cleaning and before the application of a sanitizer, and then again after application of the sanitizer;
- To assess potential soil accumulation points: Sites include scales, water baths, and under and within pieces of equipment; and
- To assess the efficacy of Good Laboratory Procedures during lab operations: Sample times include after sampling powders, during and at end of operations.
Organisms. An aerobic plate count (APC) measures the number of bacteria that grow aerobically in media at the ideal growth temperatures of most bacteria. Results are obtained in 48 hours. An APC assay can assess the efficacy of cleaning and sanitization when sponges or swabs are taken immediately after the cleaning or sanitizing event. When the results are out-of-specification (OOS), management should view how cleaning/sanitation procedures are performed along with reviewing chemicals and application frequency. When areas such as door handles, carts, or incubator racks are swabbed during lab operations, the results can be used to provide an indication not only of their current microbial load, but also how often the sites are cleaned/sanitized and disassembled.
Enterobacteriaceae, coliform, E. coli, and E. faecalis test results are used for the same purposes as APC in ensuring hygienic conditions. They are particularly useful as indicators of environmental control (i.e., the removal of soil accumulation), whether the lab routinely runs tests for these organisms or not.
Yeast and molds are an indicator of air quality. The ventilation system in a lab may not be exclusive to the lab and monitoring can be used to assess air vent buildup that may impact the lab or the external environment. This is expressly indicated if the lab does not have HEPA filtration units. Mold is a common indication of water leaks and can be found in areas that have been exposed to water, especially when pooled.
There are a number of methods to monitor air quality. The use of an air sampler that draws air and impales it onto a petri dish is a valuable tool because it draws in large quantities of air. Alternatively, passive air monitoring (settling plates) is a common, inexpensive method to collect samples wherein media is exposed to operational air for a predetermined amount of time (commonly 15-45 minutes) and the plates are then incubated. Normal operations should be taking place during air monitoring.
Pathogen Control Programs
Assays and organisms tested. A pathogen control program will use assays that yield qualitative as opposed to quantitative results. Qualitative results will yield a negative or positive and are reported per gram weight of the sample tested or per sponge/swab. The assays usually include an enrichment step that will repair injured cells and allow cells, if present, to multiply to a concentration that can be detected by the assay. Detection limits, the lowest concentration required for a positive result, varies according to the assay. Some assays are more sensitive than others. Cross-reactivity (false positives) may occur with some organisms that have genetic similarities to the target. Test kit manufacturers have information on detection limits and false positive rates available. With all assays, confirmation that the assay is suitable to the matrix (sample tested) is always an important component prior to testing.
Organisms. Organisms tested within a pathogen control program are the same as those used for the samples tested in the lab. However, it is not necessary to run all testing platforms if the lab uses multiple methods when testing samples. Select one platform and assay sponges/swabs taken at various lab locations for all the pathogens that are tested in the lab. Select sites where the pathogens are sampled, incubated, handled, and discarded within the lab. Do not exclude technician lab coats, gloves, or common areas such as sinks, cart paths, and locations where hand contact may occur.
Laboratory EMP Components
Compiling site list. Each lab area where the samples are logged-in/staged, media prepared, dishes washed, assays conducted, plates read, and transfer points must have representation within the site list. Within each area, all equipment used, environmental areas (walls, floors, cabinets), and utensils are to be accounted for on the site list. The site list should be reviewed on a periodic basis to account for equipment added or removed, construction or site modifications, and any facility issues such as roof leaks. Plotting all sites on a lab diagram is a step to help determine that each area and piece of equipment has been represented.
Assignment of zones. Similar to the manufacturing plant, site zones are designated as Zones 1 through 4:
- Zone 1: Any surface where a sample has contact (e.g., pipette tip, sampling scoops, mixing vessel);
- Zone 2: Adjacent to sample contact (e.g., lab coat, gloves, stomacher, centrifuge, handles, laminar flow hoods and bio-safety cabinets, scale, control panels);
- Zone 3: Equipment and infrastructure where samples are exposed (e.g., cabinets, carts, hand wash sinks, floor/floor mat); and
- Zone 4: Hallways leading into or out of the lab, air ducts exterior to the lab, pathways to remove waste, offices.
Sample selection and frequency. Sample selection from the site list should be randomized. The use of a random number generator is recommended so that all sites will have an equal chance of selection. The goal is for each sample site to be tested at a desired frequency. For example, if the lab has 200 sites on the site list and the lab would like each sample to be tested within a quarter and samples taken on a weekly basis, then approximately 13 samples are to be tested per week (See Table 1). This volume will increase if the same site is sampled immediately after cleaning, after sanitation, during lab operations, and at the end of the operational day.
Additionally, swabs/sponges should be taken whenever a pathogen is recovered from a sample or when proficiency check samples are run in the lab. Segregated rooms (areas) where pathogen isolates are handled will require sponges/swabs to be taken whenever the room is in use—often on a daily basis, and especially after cleaning and sanitation.
Setting specifications. Zone 1 post-sanitation specifications should be set at <10 cfu/sponge for hygiene assays and negative for pathogens. Zone 2 and 3 specifications may be set initially and then change depending on the baseline data. Six to 12 months of baseline data will be needed to create specifications that are achievable and provide insight throughout high and low sample volumes, seasonal changes, and varying technician scheduling.
Data collection and review. Each month, the site list and results are to be reviewed by laboratory management, ensuring that sampling is conducted as per the SOP, that OOS results have been addressed, and that corrective/preventive actions were effective by verification sampling. This review also allows identification of developing trends and assurance that the program is working as intended. All data should also be reviewed in a historical context (e.g., comparison of present data to the last 30 days, last several months, previous year’s results).
If any pathogen result is positive, Salmonella serological testing or identifications to the species level is to be performed. The information can be compared to the positive strains used in the lab for quality control (QC) to determine if there was a cross-contamination. If the strain is not from the QC program, this may point to a routine lab sample that was run in the near past that tested positive or to a harborage point within the laboratory that will need to be isolated and removed. Without species-level identifications, corrective/preventive actions are difficult to conduct.
- Increasing counts over several weeks;
- Increasing OOS results;
- Multiple OOS results on similar sites, within a zone, or across several zones;
- Identifications that point to QC or sample cross-contamination; and
- Multiple identifications of the same organism that may point to a harborage site within the lab.
Corrective actions. When presumptive and/or OOS results are returned, corrective actions are conducted immediately to minimize/contain the risk. This usually involves intensive cleaning/sanitizing that may also require equipment disassembly if appropriate. It is important to note that prior to initiation of any corrective actions, a review of the laboratory area must be conducted noting hygienic state, activity and equipment in the area. Investigative swabbing must be conducted prior to any cleaning/sanitizing activity to preserve “evidence.”
Investigative process. Investigative samples are taken with the objective of identifying the cause of the OOS result. Investigations are conducted after a corrective action and will lead to appropriate preventive actions. Samples are taken in a vectoring manner (in 360-degree radius, if possible, surrounding the site). Vector samples should include locations adjacent, under, and above the initial site. In addition to vector sampling, the investigative team should observe the sample site and lab operations nearby. The team is looking at the process, lab environment, equipment, and utensils and identifying circumstances that led to the results.
Preventive actions. As opposed to corrective actions, preventive actions are performed only after the cause of the OOS has been identified. Actions are preventive in nature, such as making repairs, sealing against water incursions, or increasing equipment disassembly.
An effective laboratory EMP verifies that good laboratory practices are in place and that pathogen cross-contamination is prevented within the laboratory. Both a quantitative hygiene monitoring and a qualitative pathogen monitoring program are essential to providing the evidence that the laboratory environment is in control. Having a documented system and trained staff in place is an essential component of effective and efficient laboratory operations.
Dr. Deibel, a Food Quality & Safety Editorial Advisory Panel member, is the chief scientific officer at Deibel Laboratories where she is responsible for leading the technical staff in research, food safety, and regulatory issues. Reach her at VirginiaDeibel@DeibelLabs.com. Dr. Post, director, food safety and regulatory affairs at Deibel Laboratories, is an expert in pathogen control programs for low moisture foods and processes.