FREQUENTLY ASKED QUESTIONS – Updated 07/16/2014

Environmental Monitoring & Sampling Questions, Answered by Dr. Ann Draughon

Q1. If you were to select one environmental microbial test medium for evaluating surfaces (swab or sponge) in a food processing plant which organism would you target? And why?

A1. If I only had the resources to do one test to evaluate surfaces in a food processing plant, it would have to be an ATP swab assay system and not a microbiological growth test. With the ATP assay, we will at least know whether our contact surfaces are clean and sanitary and suitable for food processing.

Q2. What is the best “indicator” and what is the best “pathogen” to test for in a food processing plant?

A2. To choose the best “indicator” or “pathogen” assay for your processing plant, you must know what it is you are looking for and what types of microbial problems you anticipate based on historical precedence and regulatory actions.

For fully cooked refrigerated ready-to-eat foods, the key concern is usually Listeria monocotogenes. The indicator bacterium is Listeria species.

For raw meat and poultry, raw vegetables (raw fruit), dried powders, dry cereals, nut butters and any foods historically associated with Salmonella or verotoxic E. coli illness, the best indicator would be Enterobacteriaceae which includes both Salmonella and E. coli or the use of the more traditional coliform/E. coli indicator assay.

Q3. Do you believe that allergen testing will someday be regulated? At what permissible exposure limit or threshold?

A3. There are still unanswered questions concerning reported allergen reactions to foods, the diagnostic procedures and quantities of allergen required to elicit a response. There is also great biological diversity in the human population in our response to allergens or food sensitivities. Only two of the eight recognized allergens in the U.S. currently have AOAC performance tested methods (ELISA). A publication by the University of Nebraska Food Allergen Center titled “Effective Allergen Control Plan: A Framework for Food Processors” states that FDA does not intend to set acceptance specifications or methods for validating cleaning processes.

 www.3m.com/Foodsafety Page 1 of 6 Trying to regulate levels of a chemical when the methodology of analysis is still in development and when so many types of equipment and food processes are involved would be impractical for a regulatory agency.

The exposure limits or threshold that might be set for regulatory purposes would have to be carefully evaluated in a variety of food matrixes and research studies before government agencies could even consider setting those levels.

I believe that companies should use a quantitative ELISA method to validate the absence of peanuts or a particular allergen in their food product. The protein swabs that can be used in ATP assay systems also offer the processor a useful tool for validating that equipment is free of protein residues (true allergens are usually protein-based). These validation studies will provide both the company and the regulatory agency with scientific support for a “visually clean standard”.

Q4. Will allergen testing and action levels be regulated at some point in food in the future?

A4. Probably, but we do not have the science to do so effectively right now and it is up to the food processor in the meantime to do validation studies of the cleaning effectiveness of their facility if they handle allergens.

Q5. What should I look for in a good ATP system? Since there are so many options, how can I select one for my plant?

A5. Look for a system that is user-friendly, easy to read and that has good customer support to answer my questions or offers training. The disposables should have a reasonable shell-life at room temperature so no need to discard expired swabs. The neutralizer for the sanitizer should be a part of the swab system. Many of the systems are quite similar but one key difference is the software availability that comes with the ATP system. The software can help you establish baselines for your facility and immediately let you know if one or more of your swabs is out of specification and can also track RLU and trends over time and by location so that potential problems can be quickly identified as they develop.

Q6. Do food plants routinely test the environment for viruses in the U.S.? Are specific food processing segments more apt to test for viruses?

A6. Since diagnostic methods are not readily available to test viruses in food environments, it would be very unlikely that anyone would try to do so right now. Since most common foodborne disease viruses are carried in feces, an indicator such as Enterobacteriaceae or E. coli might be used. Some have suggested using a bacteriophage to E. coli as an indicator. However, it should be pointed out that there is no direct experimental evidence at this time that links occurrence of certain bacteria to that of foodborne virus. As virus testing evolves, I think the raw shellfish and seafood industry may lead the way. Water is generally a simpler matrix to test compared to foods.

Q6. What resources are available to help me select a reputable contract laboratory?

A6. The most widely accepted validation authority globally is ILAC. Over 50 countries from all continents have signed the International Laboratory Accreditation Cooperative (ILAC) arrangement. For each country, a specific program has been formally approved within the country to provide laboratory accreditation. You should look for a laboratory within the country of interest that has been accredited by these bodies or agencies. The agency for each country

 www.3m.com/Foodsafety Page 2 of 6 (some have more than one) is listed on the ILAC website in a pdf document: https://www.ilac.org/documents/Signatories_to_the_ILAC_Arrangement.pdf

Reputable contract labs in order to be accredited by these agencies will have to be ISO 17025 certified.

Q7. Is there a difference between EB and Total EB testing? A7. They are the same.

Q8. Are there any really robust Listeria environmental sampling plans to ensure the plant is being adequately swabbed?

A8. A really robust sampling plan for Listeria certainly exists but is unique for each facility. There is no “out of the box” plan. It is a plan that you and your team develop using facility mapping, developing baselines, selecting appropriate assays and sampling sites as discussed in the webinar, using appropriate data management to follow data, etc. These issues are addressed in 3M Food Safety’ Environmental Monitoring Webinars: Logical and Systematic Use of EMS Program Data to Reduce Risk and Developing an Effective Environmental Monitoring, Sampling and Testing Program

Q9. All of our finished products require further processing in the form of cooking, which is also a kill step. Should I have to worry about environmental swabbing for listeria, or if it is present on food contact equipment?

A9. This would depend on the type of product you are working with in your operation and whether your product is processed inside an airtight container without subsequent exposure to the environment. If the product is water cooled after processing, is there ever a possibility of occasional leakage due to improper seals? Completing a Risk Assessment would be a good step in identifying if an environmental listeria swabbing program (or another type of sampling program) is warranted. Reviewing your plant blueprints and product flow through the plant would be a wise step to see is you can limit cross contamination between zones. Many plants with a kill step in the process employ an environmental listeria swabbing program in order to manage and mitigate the risk of cross contamination occurring.

Q10. What is your testing method of choice for dry areas? Wet areas?

A10. Swabs can be used either wet or dry. Sponges are normally pre-moistened and can be used in either wet or dry plants. Some dry plants severely limit the introduction of any moisture in the process environment. Some processors in dry plants collect samples with air sampling devices, vacuums and/or dustpans and then transport the "samples" to the lab for preparation and plating. We sometimes use scrapers in dry plants and wet areas to sample crevices, welds and areas where there is build-up of solid material. Sponges are normally used for sampling larger areas and swabs work best for small spaces, corners and inside tubing. If using a moist sponge in a dry plant, it is imperative that no moisture remain in sampling area. When doing any type of sampling, it is important to leave no residue behind of any kind.

Q11. How to avoid cross contamination in environment and incubator when microorganisms experiments such as mold and bacteria experiment.

A11. Employ solid GMP's and SOP's when handling all collected samples whether they are product or environmental samples. Incubators should be cleaned on a regular basis and sanitized. Any spills should be cleaned immediately and sanitized. Quats are normally used for all laboratory surfaces for disinfection and should be used before and after experiments are conducted in the laboratory. Chlorine based sanitizers are also effective and cheap but can

 www.3m.com/Foodsafety Page 3 of 6 damage equipment. In laboratories working with both mold and bacteria, personnel must be taught the proper way to view a fungal plate to prevent spore dispersal and the lids of petri dishes containing filamentous fungi (mold) should be secured when sitting on a bench. Tossing plates of mold without securing lids into a biohazard container will quickly disperse mold spores throughout the air. Something as simple as scotch tape over the stack of mold plates to hold them together for disposal is very helpful. The ideal situation is to purchase a laminar flow hood for working with bacterial experiments. Air spray sanitizers can sometimes help reduce bacteria and spore loads in laboratory air and ideally it is best to use them at the end of the day when everyone has left the lab.

Q12. We work in the Microbiology Lab in a Soft Drink Company (Carbonated, no carbonated, Bottled water). Do you recommend sanitation process every 48-72 hours?

A12. Extended run times are more in vogue in CSD plants than ever before. Daily cleaning and sanitizing has evolved to every other day or during flavor changeovers for some processors. The FDA guidelines are minimal beyond good GMP and a validated sampling program.

Since three types of products are being processed: carbonated, non-carbonated and bottled water, it is essential that each type of product be considered individually in regard to a risk assessment and cleaning schedule. A sweetened non-carbonated beverage may be higher risk than a carbonated one or a beverage with no sugar added such as bottled water. Additionally, it may only be specific parts of the processing line that may need more intensive cleaning schedules than others ie. Bottle or can warmer/washer area, filler, syrup tank lines during changeover, caps or closures, etc.

A good baseline study that tells you how much build-up (if any) of microorganisms is occurring between sanitation periods (for example, sampling at startup and every eight hours until you stop processing at accessible locations) will give you the data to help validate your cleaning schedule. Sampling at less accessible locations could be done at the beginning of the run and at the end of the run several times (24, 48, 72h). This way you know how your facility and how your product performs over time microbiologically.

Q13. What, if any, is the typical pathogen load needed to find Listeria monocytogenes on a stainless steel surface with a standard sponge (100 sq cm)?

A13. This is difficult to answer as there are so many variables to consider. You can look long and hard on the internet without finding the answer. Theoretically, you would need only one live cell in a 100 sq cm area if you use an FDA or USDA approved enrichment method for recovery. In reality and in my experience with relatively clean stainless steel surfaces such as blades, we can regularly recover Listeria monoctogenes with 10 to 100 cells per 100 sq cm IF other Listeria are not present. We also found that the presence of Listeria innocua can mask that of L. monocytogenes and we needed 10 times more L. monocytogenes than L. innocua to detect L. monocytogenes. This is one reason we often test for Listeria species and follow up with L. monocytogenes if the generic Listeria are present. There are issues that may affect your detection of L. monocytogenes such as the method you are using, sampling procedure, type of food manufactured, grade, condition and cleanliness of stainless steel (scratches, dents, etc.), age of processing plant and types of cleansers/sanitizer used.

Q14. Should condenser units in coolers be evaluated and included in an EMS program?

A14. Condenser units in coolers and elsewhere in the facility are great harborage points for Listeria and other bacteria that like cold. They are difficult to clean and can easily contribute to contamination of food and equipment.I would strongly recommend monitoring condenser units used in coolers and in spaces where they drip.

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Q15. If including Enterbacteriacae in your Plan, what would the acceptable limit be? What are the limits for bacteria counts such as TPC, coliforms, and Yeast & Mold in food and direct food contact areas? Are there any guidelines available to set our limits?

A15. A criterion that is often used for Enterobacteriaceae (EB) in environmental sampling plans is as follows for a 3-class sampling plan with FIVE SAMPLES taken:

. Below 10 EB per 100 cm2 is considered NEGATIVE and ACCEPTABLE . Between 10 and 100 EB per 100 cm2 is considered POSITIVE . If more than 2 of the 5 samples are POSITIVE – then that site fails inspection . If any sample is above 100 EB/cm2 – the site fails inspection

Yeasts and Molds, APC or Staph:

. Pass – sample < 100 CFU/cm2 . Fail - any sample > 1000 CFU/100 cm2 . Fail – More than two samples are > 100 CFU/100 cm2 but < 1000 CFU/100 cm2

This criterion is not a law, just a guideline. Please refer to ICMSF (International Commission on Microbiological Specifications for Foods) for more charts and larger sampling sizes if you are interested. They provide a wealth of guidance not just on EB but on other common indicator bacteria and pathogens (YM, coliform, etc). You may find that you want looser or tighter control on your facility. You can also start with a higher acceptable criterion and work toward lowering it over time by using your sampling data and more rigorous sanitation as needed.

Food manufactured (low risk vs high risk such as powdered baby food), age of plant, surface sampled – contact surfaces, drains vs. conveyor belts, type of sanitizer used, robustness of your sanitation program, environmental controls, bacteria kill step in the process, etc. You would first have to map you plant and conduct testing for a period of time in order to understand the baseline level of enterobacteriaceae present in the plant. Once established you can develop control limits and implement continuous process or sanitation improvement steps to reduce levels.

3M Food Safety Environmental Monitoring Product Questions, Answered by John Wadie

Q1. If I use 3M™ Petrifilm™ Plates as a direct contact method should I sanitize the surface after sampling?

A1. Yes, this is an industry best practice. When sampling with 3M Petrifilm Plates growth media can be left behind and surface should be re-cleaned.

Q2. I would like to know our long I should expose my 3M™ Petrifilm™ Plates to compressed air.

A2. We do not have a standard time recommendation for compressed air. An in-house test should be completed to determine appropriate time which will be affected by the pressure of the system and volume of air delivered

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Q3. If a plant is using an air exposure plate for Total Plate count and Yeast and Molds with an exposure time of 15 minutes, is that enough?

A3. 15 minutes is our validated time for air sampling with Petrifilm Plates. Since you do not know the cubic feet of air that actually impacts your air exposure plate (Petrifilm or standard agar plate), when it is sitting out for 15 minutes, it is impossible to come up with a specific criterion. Taking one sample now and then is really useless since you do not know what the number means. However, this is a useful Qualitative (not Quantitative) method for monitoring air. You do this by putting you plates in specific locations that you want to monitor at designated times and then recording the data over time. Create graphs as you collect the data. You will be able to tell if the area sampled is higher or lower than normal. If there is a steep increase in counts you can take action to determine the problem. There are air samplers that provide quantitative measurement of specific volumes of airs if needed.

Q4. Where can I get the additional information on the specific 3M Petrifilm Plates and use limitations?

A4. Information regarding 3M Petrifilm Plates and all other 3M Food Safety products can be found at www.3M.com/foodsafety. You can also contact your local sales representative for additional information and support.

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