Environmental Monitoring in Microbiological Labs

It does not seem right that food production plants work so hard taking multiple samples of various types of surfaces, at all times of the day, looking to verify the efficacy of good manufacturing practices and sanitation protocols. The laboratory appears to have the easy part, just testing the samples, seemingly unaware of the how slow the hours pass waiting for results, the relief when they are in-specification, or the panic when they are not. It must be nice to be a laboratory! Well, not so fast. Lab personnel work through the same processes for the same reasons because similar protocols are followed. Laboratories will often

carry out environmental testing to ask: Are the good laboratory practices (GLPs) followed by the lab staff and management effective? Are the cleaning and sanitation practices effective? Has there been cross-contamination within the laboratory?

The laboratory environmental monitoring programs (EMP), as in a plant, will vary according to the predominant assays performed, lab areas, traffic patterns, air flow, equipment conditions, and even the season. Careful consideration is made in the development of the program so that it meets the needs of the individual laboratory, while covering the key components that every lab should follow. As those in a food production environment will see, an EMP followed by an in-house or a third-party contract lab adhere to similar steps to ensure that the data generated are accurate, reproducible, and dependable.

Key Components

Environmental monitoring zones and site list selection. In any EMP, it is critical to define what constitutes highest risk areas, such as sample contact areas (zone 1), to lower risk areas, such as floors and walls (zone 3). The importance of developing a thorough site list and establishing zones cannot be underestimated. Each lab area where samples are logged-in/staged, media prepared, dishes washed, samples incubated, assays conducted, plates read, and materials autoclaved must have locations represented within the environmental monitoring site list.

If the trending data suggests that some zone 3 sites have been identified as “hot spots,” then site lists should represent this zone in proportions related to the data. The site list should be reviewed on a periodic basis to account for added or removed equipment, new test methods, substantive construction or site modifications, and any facility issue.

Since each lab may have different processing areas, equipment, and utensils, site lists may be unique for each lab even if they are under the same ownership. However, the principle of including sites within each critical lab area, representing all zones, remains across all labs. Table 1 provides a general overview of environmental zones in a lab.

Assays selected. The assays selected for each sample are those that are conducted on samples. For example, if incoming lab samples are tested for Listeria spp., Salmonella, coliforms, E.coli, and coagulase-positive Staphylococcus then those are the assays to be included in an EMP. Whatever organisms are tested, the same methods that the lab is asked to run are to be used for the EMP. When yeast and mold are tested, see Air Monitoring section below.

Sampling modalities include swab, sponge, amplicon, and air sampling. Amplicon sampling withstanding, samples are either categorized as indicator or specific organism.

Indicator organisms are used to assess the overall microbial load of a sample site. Aerobic plate count (APC) measures the number of bacteria that grow aerobically. Results are obtained in 48 hours. An APC assay can assess the efficacy of cleaning and sanitization when sponges are taken immediately after the event. When the

results are out of specification, cleaning/sanitation procedures should be reviewed and revised. When areas such as door handles, carts, pipettor handles, or control panels are swabbed between cleaning and sanitation, the results can be used to provide an indication of not only their current microbial load, but how often the sites should be cleaned/sanitized. In this regard, close monitoring, or tracking, of data is a key function of environmental monitoring programs. APC, coliforms, and Enterobacteriaceae are examples of indicator organisms.

One note about indicator samples is they may be tested to assess cleaning/sanitation efficacy on a more frequent basis or at specific times.

Air monitoring. Just as in a production environment, laboratory air quality can be monitored when yeast and mold is tested. Mold spores, in particular, can be spread via air

currents. Many times, yeast and mold counts have seasonal variations. Summer months may show higher counts than winter, especially in states that experience winter freezes. Increased counts may also indicate water leaks, clogged air filters or doors left open. There are a number of methods for monitoring air quality. The use of an air sampler that draws air and impacts it onto a petri dish is a valuable tool. Alternatively, passive air monitoring wherein settling plates are used is a common, inexpensive method of air collection—media is exposed to operational air for a pre-determined amount of time. During air monitoring, normal operations should be taking place.

Sampling frequency. The frequency depends on the number of sites and how often the lab wants each site sampled. For most labs, at least weekly sampling is a good place to start. Sampling should take place on different days of the week to account for differing sample loads and activities. Table 2 outlines an example of a sampling frequency program. When samples are selected, they should be randomized by using a random number generator such that all sites have an equal chance of selection. Keep in mind that with randomized sampling, some samples may be tested two or more times and some may not be tested at all in a given timeframe.

Setting specifications. For any program, setting specifications is not an exact science. Zone 1 post-sanitation results should be less than 10 colony-forming unit/sponge or negative depending on the type of assay. Zone 2 and 3 specifications may be set initially and then change depending on baseline data that is collected over multiple months. Tracking and trending consecutive data from six to 12 months is a good rule-of-thumb to set specifications. After this period of time, the lab management reassesses and possibly revises specifications. Table 3 provides examples of specifications for zones 1 to 3.

Data collection and review. Each month, the site list and results should be reviewed by lab management to ensure sampling is conducted as per the SOP, that out-of-specification results have been addressed, and that corrective actions were effective and verified. This review also allows identification of developing trends. All data should be reviewed in a historical context and relevant corrective/preventive actions should be included in the monthly review.

Corrective actions. If presumptive and/or out-of-specification results are observed, involvement of varied management levels provides a diversity of perspectives and ideas for corrective activities.

Corrective actions are activities that are conducted as soon as possible after result notification. For example, the sample site and surrounding area will be cleaned and sanitized before the close of business on the day the results are observed. After cleaning and sanitizing, the sample site(s) are to be sampled to verify the efficacy of the cleaning/sanitizing event. Sampling of the site will continue each day until three consecutive in-specification results are obtained. When this qualification is met, the sample is returned to the site list for routine monitoring. These samples are to be taken after cleaning and sanitation of the site has been performed. If any of these samples are out-of-specification an investigation should be launched.

The investigative process can be initiated for an out-of-specification result or when a client result is positive to verify there was no cross-contamination event. A team will determine sites of additional samples and review past records. The investigative samples, or vector samples, should be collected in a 360-degree radius, if possible, from the initial out-of-specification. Vector samples may include locations adjacent, under, and above the initial site. In addition to vector sampling, the investigative team observes the site and lab operations. The team is looking at the process, lab environment, equipment, and utensils. Any abnormalities or adjustments made should be documented.

Summary

An effective laboratory EMP will help verify cleaning and sanitation effectiveness, employee practices, and air quality in the laboratory. The program also incorporates direction when there is an out-of-specification result and the appropriate investigative process. These practices ensure accountability and quality results that clientele can depend on.

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Dr. Deibel is director of microbiology at Covance. Reach her at [email protected]. Dr. Sridhar is the principal scientist at CavinKare Pvt. Ltd. in Chennai, India. Reach her at [email protected].

References Furnished Upon Request