From: Food Quality & Safety magazine, April/May 2013

Hygiene Monitoring Strategies that Hit the Mark

by Charles Giambrone, MS

Hygiene Monitoring ­Strategies that Hit the Mark

The emphasis on validation had begun in 2002 with the advent of the Microbial Sampling of Ready-To-Eat Products for the FSIS Verification Testing program, the subsequent revisions in June 2003 with 9 CFR Part 430. Then on March 15, 2006 with the alternative control measures coupled with the EIAOs and a new type of risk based sampling programs for Lm for food contact and environmental surfaces.

Also, due to the requirements by the newly established Food Safety Modernization Act (FSMA) enacted into law by Congress in early 2012, a number of market sectors have already have mandated enforcement including validations using hygiene monitoring technologies.

The rise in implementation of the Global Food Safety Initiative (GFSI) programs, especially in the latest GFSI-approved versions of SQF, BRC, and now FSSC 22000 here in North America, has made validation mandatory via the various hygiene monitoring modalities.

Particularly, the new SQF edition 7 has an increased emphasis on Validation-Verification of hygiene monitoring/sanitation in module 11 and in module 12 as well as “mandatory elements.” This includes documented and frequency for validation of chemical concentrations and procedures (SSOPs) as well as the food contact and environmental surface hygiene monitoring validation methods and procedures.

Similarly, with the new BRC Issue 6, there is this enhanced enforcement on Validation-Verification in Clause 4.11.4 in Housekeeping & Hygiene. The Housekeeping & Hygiene clause is also considered a Fundamental Clause. Also, 4.9.1 emphasizes Chemical & Physical Contamination Control.

Zone Sampling Considerations

In both food processing plants as well as food service operations, many food safety experts and knowledgeable sanitarians view pre-requisite program controls as a multi-barrier system akin to a dartboard. We all know the Zone 1 or “bull’s eye” is the actual food contact zone. Obviously, all hygiene control programs must prevent pathogens and spoilage microbes from compromising this critical zone.

However, the sound proactive approach is to establish and maintain microbial control outward from Zone 2, which are the indirect food contact areas, to Zone 3, the immediate environmental zones around the food processing area, finally to the Zone 4 area that is the general environmental environs of the food plant. The objective of any sound hygiene monitoring program is to not merely focus on the bull’s eye, but be as assiduous in your program as you can to control Zones 4 and 3, thereby minimizing the risk upon Zones 2 and 1.

Therefore your hygiene monitoring validation programs must include both the Food Contact (Zones 1 and 2) and Environmental (Zones 3 and 4) for soil removal, as well as indicator and pathogen microbe validations. The frequencies will of course vary based upon the risk assessment of the plant’s HACCP program for each and every product manufactured at a specific plant site.

For example, on a post kill/cooked-RTE processing piece of direct food contact equipment, the frequency that equipment unit will be assayed on a weekly basis will be far greater than a raw meat blender or mixer under the hygiene sampling plan for that facility.

The selection of sites either during a pre-operational sampling or during a scheduled shift cleanup or even operational assessments must be selected based upon a program’s risk assessment of each site, but must be selected by the sampling team in a manner not to tip off the sanitation staff. The sampling matrix in its entirety must cover all critical food contact and environmental sites within the program’s mandated frequency. This is vital in order to generate a validation history that accurately reflects the realities of the facility’s design and operation.


There are a myriad of systems and devices to assess hygiene levels on both food contact and environmental surfaces. Some employ sophisticated microbial detection methods utilizing polymerase chain reaction (PCR) with sophisticated instrumentation. However, I will focus on some of these companies that have both soil detection, indicator microbial detection, and pathogen detection systems that are designed for hygiene monitoring programs in concert with plant sanitation.

There are three primary tools to sample a surface: the classic swab, the sponge, and the wipe. Many studies have been done through the years to ascertain which extracts the most soil or microbes for sampling. But the critical factor is that one must understand the limits of the sampling system.

For example, a swab is an excellent tool to assay a gasket or a tight area, but has a lower recovery of viable microbes than a sponge or wipe. Meanwhile a sponge swab or RODAC plate is effective for a flat surface, while a curved tank wall may prohibit the use of RODACs but is conducive to sampling by a sponge system.

A comprehensive national study published in Applied and Environmental Microbiology utilized both sponge sticks (3M), and wipes to recover Bacillus anthracis spores showed that the gauze wipes had 35 percent rate of spore recovery while the sponge sticks had a recovery range of 26 percent to 36 percent.

Using both methods, the sponge, as is typically done, is placed into a sterile sampling bag or stomacher bag and is transported with either a peptone water or other recovery broth. A recovery or resuscitation broth is critical for stressed/sublethally injured vegetative microbes, but is not essential for spores.

The utilization of the sponge stick or solar-cult devices enables one to use one sponge face for horizontal direction of a surface, the other for vertical, and the edges for the diagonal of the same sampling surface. This is done omitting the variable of handling the sponge directly with gloved hands.

Best recovery of low numbers for enumeration is achieved via membrane filtration and plating. For pathogen presence/absence sequential incubations in AOAC BAM approved broths is done coupled with confirmation by selective platings. Sponge systems are also utilized for carcass detection of EHEC microbes in numerous USDA plants.

Soil detection systems. There are many different ATP units with varying features. All have evolved into more compact units, with self-containing ATP swab pens coupled with excellent software to download and interpret data for your hygiene validation verification programs.

QAC sanitizers have been known in some instances to create false positive readings with ATP, while oxidizing ­sanitizers can create false negatives when a freshly sanitized surface is ­sampled for ATP.

There are many units on the market, but there has been consolidation of some companies the past 10 years. Examples of hygiene monitoring with ATP units include: AccuClean (Neogen), Charm, CleanTrace (3M), Ensure (Hygiena), HyLite (EM Millipore), and SystemSure Plus (Hygiena). Most utilize a swab that is pre-moistened. The AccuClean has a small sponge-like sampling area instead of a swab. Some utilize a lyophilized pellet of the enzymes that gets reconstituted; others, like Hygiena’s Ensure and SystemSure Plus use a liquid system.

Once the surface is assayed, the ATP swab pens are activated by twisting, cracking the chamber containing the enzymes needed to activate the firefly reaction, then placed into the ATP luminometer chamber where the photodiode measures the Relative Light Units (RLUs) released. The higher the RLU reading, the greater the soil. Unfortunately, RLU scales vary widely between ATP luminometer. This historically was done by manufacturers to tie the user into one unit since all the surface ATP swab history is based upon a luminometer type’s specific scale.

Years ago, faulty attempts were made to correlate ATP with microbial levels. This is highly problematic because ATP assays measure organic soil load that is lowest in bacteria. Yeasts and molds have much higher levels of ATP per microbe than do bacteria, so if a soil matrix has active spoilage fungi, the RLU reading will be higher than with vegetative bacteria.

Establishing a baseline for pass/fail for your food contact and environmental sites is critical. All these ATP luminometers enable you to set multiple pass/fail RLU readings for specific sites, plant areas, or equipment depending again upon the nature, surfaces, and dynamics of the processing operation with that equipment or environmental site. Baseline ­studies need to be done very carefully when an ATP hygiene monitoring program is set up in a specific facility so the pass/fail limits are meaningful, reliable, and reproducible.

There are specifically designed ATP pens to sample water and clean-in-place (CIP) rinsate systems. Hygiena’s AquaSnap is one example to sample the final rinse of a CIP cycle (prior to sanitization) and assess the level of ATP soil in the final rinse. This is a useful tool for hygiene monitoring of CIP system cleaning validations.

The other cautionary note both for ATP systems and for any other soil enzyme detection system is the point in the sanitation process it is best utilized. Many sanitizers can and will interfere with the enzyme reactions. Quaternary ­ammonium compound (QAC) sanitizers have been known in some instances to create false positive readings with ATP, while oxidizing sanitizers can create false negatives when a freshly sanitized surface is sampled for ATP.

I recommend that since ATP is a barometer of soil level, it’s best utilized after the cleaners have been completely rinsed but before the sanitizer is applied. That way if there is a fail reading, the surface can be re-cleaned prior to sanitization. This omits the chance of reactions between ATP pen enzymes and the sanitizer. It is also applicable for protein detection enzyme systems. Some will give a dramatic false positive when a clean surface sanitized with peracetic acid is sampled.

Speaking of non-ATP soil detection systems, these are qualitative, colorimetric swab pens that can detect, using a color scale, the presence of protein residues. Neogen has its AccuClean for protein while Hygiena has its ProClean unit. While these do not provide a quantitative validation of the hygiene monitoring, they are user-friendly tools that permit sanitation supervisors or managers to ascertain if a critical piece of equipment must be re-cleaned. However, these protein swabs are not to be used for allergen validation since they measure general protein, and do not target the specific allergen you are seeking to remove via the SSOP for that equipment or area. Rather specific allergen detection systems like Neogen’s Reveal Rapid 3D kits using lateral flow technology are required for allergen hygiene monitoring.

Hygiena also has an enzyme detection swab for lactose or sucrose soil residue: SpotCheck Plus, which turns green in the presence of either sugar residue. This is useful for dairy plants, confectionary candy, and beverage plants.

Indicator Microbe Testing. The typical methodology for sampling either food contact or environmental surfaces for indicator microbes involves a standard swab, in some cases with a neutralizing buffer or a Letheen neutralizing broth that is needed to neutralize any residual sanitizer. In a hygiene monitoring program, one should be sampling for either indicator or pathogenic microbes, after the sanitizer(s) have been applied.

Indicator microbes will depend upon the nature of the operation. In addition to Total/Aerobic Plate Count, indicator microbial tests include coliform-E.coli, Enterobacteriaceae, yeast and mold, and Staphyloccus spp. For example, meat, poultry, or dairy plants will test either for coliform-EC or Enterobacteriaceae, while bakery operations will typically assay for yeast and mold.

Contact slides have been around for some time but some user-friendly versions include the Envirocheck line from EM Millipore or the Hylab dip slide paddles marketed by Neogen. In addition, as discussed, RODAC plates can be manufactured with a variety of agar media for the targeted indicators.

Hygiena, using its multipurpose hygiene reader Ensure, has the capability with its Microsnap swab system of providing a facility with a result for E.coli or Enterobacteriacea in less than seven hours. It relies on a “bioluminogenic” reaction involving specific enzymes generated by the specific indicator group to measure and detect the indicator

Like ATP, pass/fail levels for indicator microbes depend upon the food contact or environmental site being sampled. However, a guideline I prefer is one advocated in the British Columbia Centre for Disease Control’s document on environmental hygiene monitoring that has an interpretation of a “clean” surface being <45 cfu (colony forming units) , “contaminated” surface being 140-260 cfu, with “very contaminated” being > 260. This document is an excellent primer on how to approach hygienic monitoring, critical environmental sites to assay in generic environmental terms, and sampling using both swab and sponge assay methods.

Air monitoring is also a vital component of your hygiene monitoring program. While many plants utilize the classic air sedimentation method employing petri dishes left exposed to a specific area for 15 minutes, this method relies on the serendipitous deposition of microbial particles landing onto an agar surface.

Air sampling units that either utilize an agar plate, or an agar strip that actively pulls a defined volume of air within the area/room to be sampled are preferred. Particularly air sampling units like the RCS units (Reuters Centrifugal Samplers) like the type manufactured by EM Biotest enable you to actively sample and count an airborne microbial density for total count, coliform, or yeast and mold. They provide a cfu count/cubic meter or cubic foot of sampled air. Seasonal air sampling enables a plant’s program to validate sanitation of air handling units, intake vents, and cooling tunnels/spiral units. Therefore, air sampling has become a critical component of a facility’s hygiene monitoring program.

Pathogenic Detection Systems. The sponge swab methods mentioned previously for indicator microbes are also typically used for environmental and food contact hygiene monitoring for Salmonella and environmental Listerial species.

Neogen, using the same lateral flow technology utilized for allergen validation, has a series of Reveal systems to detect Listerial spp., Salmonella, and E. coli 0157:H7. These are rapid detection systems and provide the end user with a result after 15 minutes for environmental Salmonella, 20 minutes for environmental Listeria, and eight hours for E.coli 0157:H7. Because of its lateral flow design, the Reveal systems are strictly presence or absence, but are very rapid in their result for presumptive positives.

Hygiena has a presence-absence test that employs a self-contained swab medium with a large swab head, InSite. The InSite swab gets incubated at 37 degrees Celsius for 30 to 48 hours and provides a color scale to qualitatively assess the severity of environmental Listerial species for presumptively positive Listeria. The quicker the growth medium turns a brownish color, the higher the level of environmental Listeria sampled. This method is cost effective and is user friendly, and can successively grow stressed/injured Listerial species even amongst a high microflora.

3M has a modification of their Petri­film line (3M Petrifilm Environmental Listeria plates) that quantifies the level of environmental Listerial species and is AOAC approved. This is significant because in many instances for environmental or even food contact surfaces, a presence or absence test does not provide sufficient information. By quantifying, you can correlate the severity of the contamination levels with the zone sampled in the plant. The results are achieved in a 27 to 31 hour time frame, so corrective actions can be implemented into the plant hygiene program.

Now What?

All the methods discussed have accuracy, reproducibility, and a sense of timeliness. How we interpret the data and track the trends in a hygiene monitoring program will either improve a program, let it remain stagnant, or, at worse, cause a major food safety crisis in a plant. We can use the hygiene monitoring program for both food contact and environmental zones in a reactive or a proactive manner. The software programs to organize and track the data for many of these systems or devices are already available.

If we are truly promoting the continual improvement of a facility’s food safety program, the data must be utilized in a proactive manner to teach staff on every level how to continually improve the facility’s hygiene programs. To be sure, the GFSI programs and the regulatory bodies now do demand that your data serve as roadmap for continual improvement of your PRPs and HACCP programs.

Giambrone is the global technology and regulatory manager for Rochester Midland Corp.’s food safety division. He can be reached at

References Furnished Upon Request



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