SOP: Environmental Sampling for the Detection of Microorganisms

GIFSQ — Global Institute for Food Safety & Quality

Principle

This procedure applies to sampling the environment for microorganisms in food establishments in support of compliance activities relative to Section 7 of the Food and Drugs Act, and Health Canada policies such as the Policy on Listeria monocytogenes in ready-to-eat (RTE) food and Canadian Food Inspection Agency (CFIA) directives and guidelines. It applies to both food contact surfaces (FCS) and non-food contact surfaces (NFCS) in a food manufacturing plant, and describes the procedures for the collection and handling of environmental samples for microbiological assessment. This revised procedure replaces MFLP-41A and MFLP-41B, dated September 1992 and July 2006, respectively.

With a well designed sampling plan, analysis of environmental samples will aid in demonstrating whether there is adequate control over good manufacturing practices and whether the sanitization process is adequate. Environmental sampling is an important tool to verify whether additional pathogen control measures are needed, provides information over time for trend analysis, and provides some measure of confidence that the products being manufactured are made under sanitary conditions. If a quantitative method is used, it is essential that microbiological analysis be initiated as soon as possible after sampling in order to avoid any loss due to die-off of viable microorganisms.

Apparatus

• Sponges (approximately 4 cm by 8 cm), sterile
• Sterile container, for holding moistened sponges
• Screw-capped plastic vials, sterile, for swab tubes
• Sterile transport-media tubes, screw-capped
• Swabs (approximately 2 cm head firmly attached to an applicator stick 12 to 15 cm long), sterile dry or pre-sterilized
• Sterile gloves
• Sterile forceps, in an envelope, for manipulating sponges
• Sponge material with an attached handle (e.g., sponge on a stick), as an alternative to forceps
• Sealable plastic bags
• Markers, for labelling
• Ice packs
• Cooler, clean and sanitized
• Thermometer, to check the internal cooler temperature on arrival at the laboratory
• Spoons and forceps, sterile, for dried-material sampling
• Sterile containers such as cups, Whirl-Pak, or jars, for dried-material samples
• Head cover, facial hair cover, disposable overalls and overshoes, for sampling inside large equipment
• Paper packing material, for the cooler
• Sterile material in the kit must be assembled using aseptic technique.

Reagents

• Neutralizing Buffer, or other buffered rinse solution as appropriate — use 10 to 15 mL to completely moisten each sponge; also used for swab vials. Commercially available, or prepared by laboratory personnel.
• Sterile dry swab preparation: prepare screw-capped plastic vials containing 20 mL of sterile Neutralizing Buffer or any other buffered rinse solution containing neutralizers, autoclaved at 121 °C for 15 min. Place the sterile swab in the neutralizing buffer before sampling.
• Sterile transport media: prepare screw-capped tubes of sterile transport media, autoclaved at 121 °C for 15 min. After swabbing the environmental surface, place the swab in this medium for transport.
• Calcium alginate fibre swabs: swabs made of calcium alginate fibres are soluble in aqueous solutions containing 1% sodium hexametaphosphate (or, alternatively, sodium glycerophosphate, sodium citrate, or 1% of any mixture of these), allowing the release of the captured organisms. Pre-sterilized swabs in various transport media are commercially available.
• Sponge preparation: put sterile sponge(s) in a sterile container containing 10 to 15 mL of Neutralizing Buffer or other buffered rinse solution as appropriate to completely moisten each sponge; autoclave at 121 °C for 15 min. Individually packaged sterile sponges, pre-moistened with neutralizing buffer, are also commercially available, and sponges and neutralizing buffer can be purchased separately and used together at the time of sampling.
• Note: do not use sponges or neutralizing buffer past the expiry date. Keep bacterial carriers refrigerated (2–8 °C) until needed.
• Note: it is recommended that each lot of bacterial carriers (sponges, swatches, swabs) be tested for inhibitory properties against the targeted bacteria by using the method of Libras and Rose (References) or another acceptable method. If kits are purchased from commercial suppliers, information from the company should be obtained.

Neutralizing media

Where sampling is carried out on surfaces previously subjected to chemical germicide treatment, appropriate neutralizers must be incorporated into the medium; neutralizing media are commercially available. Industry must consult with the appropriate regulatory program (e.g., CFIA or other government agencies) to determine whether commodity-specific requirements impact the choice of neutralizer. The method refers to its Appendix 2 for the full list; the table is carried below.

Note: other manufacturers may offer neutralizing buffer or media; it is the responsibility of the laboratory to verify the composition of the medium and to ascertain that the medium is appropriated for the intended use.

Procedure

Before sampling

The person responsible should take the following steps before commencing environmental sampling:
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Draw a process flow chart and plant traffic pattern
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Determine target organism(s)
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Identify the critical control points
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Determine sampling objectives (e.g., FCS or NFCS)
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Arrange for sterile equipment and material as required
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Identify the sampling material and record according to sampling sites (e.g., label each sample that is taken with the date, site sampled, FCS/NFCS)
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Identify other hazards associated with the sampling area
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Note: aerobic colony counts and other indicator microorganisms (e.g., faecal coliforms) can be used to assess overall sanitation. If information is needed regarding cleaning and sanitation, it is recommended that samples be taken immediately before production, just after cleaning and sanitation have been completed. If information is needed regarding the microbiological environmental conditions during production, it is recommended that samples be taken during production. Consult the appropriate regulatory program guidelines from the CFIA, provinces, etc. for specific instructions that may apply.

Personnel hygiene and clothing

(The method’s Appendix 1 personnel protocol is carried here as procedure content.)

• If more than one plant is to be visited during the same day, an adequate number of coveralls must be brought along so that a clean pair is worn in each plant; all inspection gear to be used in a subsequent plant visit must be sanitized before visiting the next plant.
• Personnel doing environmental sampling are expected to follow principles of good personal hygiene. This includes keeping hands, fingernails, hair, beard and clothing (including shoes and boots) scrupulously clean. All jewellery, such as watches, rings, earrings, bracelets, tie clips, etc., must be removed prior to entering the processing area of a plant.
• Personnel doing environmental sampling must not carry out sampling when suffering from a cold, flu, etc., or if afflicted with skin infections or gastro-intestinal illness.
• Inspection clothes, footwear and other equipment are potential vectors of contamination. Personnel must place special emphasis on the adequacy and cleanliness of their inspection gear before bringing it into the plant.
• Disposable coveralls are preferred; if not available, laundered, individually wrapped coveralls (preferably white) are acceptable. Pockets should be below the waistline only and deep enough to hold flashlights, spatulas, pens, etc. Only snap buttons, zippers or velcro bands should be used for “buttoning”.
• Hair nets, beard and moustache snoods should be disposable, preferably white and individually wrapped.
• Personnel doing environmental sampling should wear steel-toed rubber safety boots with a slip resistant sole (preferably white). Disposable footwear is acceptable as long as it does not present a safety hazard; re-usable stretchy rubber overshoes are also acceptable.
• Hard hats in addition to hair nets should always be worn.
• During sampling: use foot baths and other precautionary measures the plant may have in place; wash hands after breaks, after use of washrooms, after lunch, at the end of sampling, and any time during sampling when a potential source of cross-contamination (i.e., drains, dismantled equipment, etc.) has been touched; only proper tools should be used during sampling (e.g., do not use pens, pencils, etc., to examine drains or dismantle equipment, since this could be a potential vector for cross-contamination).
• After sampling: place coveralls, shoes, hats, etc., back into bags or containers to avoid contamination of briefcases, vehicle, etc.; discard disposable equipment at the plant in proper garbage receptacles.

Sampling sites

Sampling sites depend on the purpose of the sampling. Examples include, but are not limited to:

• Food contact surfaces (FCS — any surface or object that comes into contact with the RTE product): chill brines; containers; racks for transportation; conveyor belts; slicers, dicers, shredders, blenders, etc.; table and equipment used to assemble/package product; packaging equipment; hand tools, gloves, aprons, etc.; metal surfaces with gaps (bad welding, etc.); food residue sites and other hard-to-clean areas.
• Non-food contact surfaces: drains and aerosols; standing water; cracks in floors and walls; smokehouses; floors in heavily-trafficked areas; tires on fork-lift trucks; food and wheel baths that are not in “good shape”; high-pressure hoses; cleaning tools (mops, squeegees, brushes, etc.); trash cans; under-side of conveyor belts; hollow rollers; roller guards, bearings, etc.; chill tanks; refrigerators, cold rooms; ice makers; overhead pipes; drip pans; wet insulation; maintenance tools, dust from construction, air filtration.

Qualitative (presence/absence) sampling

Larger swabs (e.g., sponges) are preferred for walls and floors larger than 1 m². Use swabs or cotton-tips for hard-to-sample crevices, and sponges for equipment and small food contact surfaces (up to 30 cm by 30 cm, or 900 cm²).
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While the swab method is preferred, contact plates and air sampling may also be used to detect both indicator microorganisms and pathogens. The type of swab used (e.g., swatches, sponges or cotton swabs, etc.) depends on the nature of the surface to be sampled and the material available.
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It is very important that personnel doing the environmental sampling have a good knowledge of hygienic practices and aseptic techniques. Conduct the environmental sampling in a counter-flow manner: unless specified otherwise, start in the finished product areas of a plant, then proceed to the potentially contaminated processing areas, and the raw product storage and receiving areas.
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For sampling horizontal surfaces (floors, tables, etc.) and vertical surfaces (walls, etc.), sterile gloves may be used to hold the swab/sponge. It is recommended to use sterile forceps to manipulate the sponge, or to use sponge material with an attached handle (e.g., sponge on a stick) which is available commercially. Wearing sterile gloves, aseptically open the envelope containing the sterile forceps, and ensure that the tip of the instrument does not become contaminated when it is removed from the envelope.
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Inside large equipment: sampling would very seldom have to be done from inside large equipment. However, if sampling has to be done from inside large pieces of equipment (e.g., milk drier), dress entirely in clean clothing: head cover, facial hair cover (if needed), disposable overalls and overshoes. NOTE: never bring non-sterile material inside clean equipment.
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Residual diluents should not be present in the container(s) containing the swabs. Aseptically remove a swab, using appropriate precautions in order to avoid contaminating either the container or other swabs. Squeeze excess liquid from the sponge/swab prior to collecting the environmental sample.
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Wipe or swab the area to be sampled with firm and even pressure. Wipe or swab the surface area up and down until the whole target surface is covered, then turn over the wipe or swab and swab or wipe the surface area from left to right until the whole target surface is covered. For surfaces which are difficult to wipe or swab because of their contour, it may not be possible to wipe or swab a defined area using this procedure.
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For an irregular surface such as a conveyor belt, wipe or swab the area to be sampled with firm and even pressure. Wipe or swab the surface area up and down until the whole target surface is covered, then turn over the swab or wipe the surface area from left to write until the whole target surface is covered. Turn over the swab or sponge once again and wipe or swab the surface area in a diagonal pattern. This allows the sampling of every plane of the surface that will likely come into contact with the food.
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Place the swab back into its sterile container and pack the container in a sealable bag such as a Whirl-Pak bag. Disinfect the sampling area and any areas that were touched during sampling using a procedure in accordance with the establishment’s procedures. Dispose of gloves used in the sampling procedure before the next sample site, to insure contaminants are not carried over.
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Once all of the swabs are collected, pack the individual bags into a larger sealable bag. Place an ice-pack at the bottom of the cooler. Cover the ice-pack with paper packing material so that the swabs are not touching the ice-pack, and then place the samples on top. Place more paper packing material on top of the sample and place one more ice-pack on top of the paper. The sample can now be shipped to the laboratory.
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Environmental samples must be refrigerated (2–8 °C) when received by the laboratory and analysed as soon as possible.
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Note: if using a method from the Compendium of Analytical Methods, it is recommended that the “Application Section” be reviewed before use to determine the method’s applicability to the food or environmental sample in question. Applicability of methods to new matrices requires validation as outlined in the Compendium of Analytical Methods.

Quantitative (enumeration) sampling

• Contact plates used to quantify microorganisms may be utilized as appropriate. Refer to the Compendium of Analytical Methods for suitable contact plate methodology. Alternate methods to quantify microorganisms may be considered if validation data is reviewed by the Microbiological Methods Committee (MMC) and approved for use in regulatory testing.

Sampling of dried material (e.g., scrapings, sweepings, etc.)

Put on sterile gloves
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Take a sample at each site selected using appropriate sterile equipment (spoons, forceps, etc.) and insert into sterile containers, such as cups, Whirl-Pak, jars, etc.
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If sufficient sample is available, the quantity collected should be close to the amount used in the analytical method that detects the target microorganism in the finished product.

Transportation of samples

Samples should be kept at refrigeration temperatures and brought to the laboratory as soon as possible
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This is usually accomplished using clean and sanitized coolers with clean and sanitized ice packs. An adequate number of ice packs should be used to ensure proper refrigeration temperature is maintained (e.g., 0 to 10 °C). A check should be made, using a thermometer, to verify the internal cooler temperature when the cooler arrives at the laboratory. Do not freeze.

Analysis

• Analyse upon receipt following the appropriate methodology for the organism(s) of concern. Refer to the Compendium of Analytical Methods for a list of methods. The appropriate regulatory agency (e.g., CFIA, provincial authority, etc.) should also be consulted for specific method requirements.

Precautions

• Non-sterile equipment used for sampling should be sanitized before sample collection (e.g., cooler, sampling boxes, ice packs).
• Sterile gloves must not come into contact with sanitizing agents before use. It is recommended to wash or sanitize hands prior to opening the sampling kit. Sterile gloves should be replaced as often as necessary.
• If sampling is to be carried out on surfaces previously subjected to chemical germicide treatment, appropriate neutralizers must be incorporated into the medium.
• Do not use sponges or neutralizing buffer past the expiry date.
• Keep bacterial carriers refrigerated (2–8 °C) until needed.
• Never bring non-sterile material inside clean equipment.
• Do not freeze samples during transportation.
• It is recommended that all sampling activities, including sampling site and corresponding results of analysis, be recorded for trend analysis.

References

• Health Canada, Health Products and Food Branch. Environmental Sampling for the Detection of Microorganisms. Compendium of Analytical Methods, Volume 3, Laboratory Procedure MFLP-41, July 2010.
• American Public Health Association. 2001. Compendium of Methods for the Microbiological Examination of Foods; Fourth Edition. F.P. Downes and K. Ito (eds.). American Public Health Association Inc.
• Daley, E.F., Pagotto, F. and J.M Farber. 1995. The inhibitory properties of various sponges on Listeria spp. Lett. Appl. Microbiol. 20, 195–198.
• Difco Laboratories. 1998. Difco Manual, Dehydrated Culture Media and Reagents for Microbiology; Eleventh Edition. Difco Laboratories Inc.
• Health Canada Compendium of Analytical Methods, Volumes 1, 2, and 3, including Appendix G and Appendix L.
• Libras, C.M. and Rose, B.E. 1989. Antibacterial Properties of Retail Sponges. J. Food Protect. 52(1):49–50.