Industrial Utility Efficiency

Microbiological Testing Considerations for Compressed and High Risk Ambient Air Systems in Food Plants

Introduction - The Microbial Air Biosphere [1]

Air borne microbes are transported via a combination of three methods: spores [bacterial/fungal], water droplets, & biofilms [via sloughing].

Micro-aerosolized droplets are how many members of the microbial world become cross-contaminants via the air mode of transmission.  Food borne viral pathogen Hepatitis A and the ubiquitous Norwalk are very often transported via micro- aerosols. It is well known that many viral or bacterial pathogens or spoilers are transmitted via respiratory bursts [coughs/ sneezes] from people or air handling system, condensate, and splash back from floors. Strict cGMPs  can limit  and control transmission in terms of personal & environmental  hygiene.

Spores or sporocysts are a significant mode of microbial airborne transport., Airborne transport is an important mode spores/ or sporocysts rely on air currents as a transport and survival mechanism for the microbial species in question. Protozoal sporocysts from genera like Cyclospora / Cryptosporidium spp.  are very hardy, biocide resistant but are not associated via airborne trans- mission routes.

Airborne contamination is most easily examined via microbial spores... which evolved to develop a sporulation cycle, just like the lower and higher plant kingdom: for propagation and species survival.  Bacterial spore formers like the pathogenic Bacillus cereus or the more infamous Clostridium botulinum are often associated as either part of a biofilm community [see below] or direct contamination of a food product or residual organic matter. Bacterial spores are designed to be hardy to withstand chemicals [microbial soil toxins or biocides, and to dry environments like other spore-like entities.

The fungal world’s yeasts and molds also produce spores to survive in quite hostile environs. The fungal world’s diversity created a diverse variety of spore casing and lifecycles that the more primitive bacteria simply lack. There is an impressive portfolio in the beauty and varieties of the fungal fruiting bodies/ sporangia that many fungi possess. These fruiting bodies are the unique airborne transport mechanism for the fungal world.

Thankfully, out of the more than 50,000 fungal species that produce fungal spores, only roughly 50 are known pathogens.

Yeasts which are our unicellular also have direct impact on the functionality of many food products. For example, spoilage yeasts like Zygosaccharomyces bailii  or the multi-species villains called ‘Wild Yeasts’ attack in a variety of food products in a stealth mode, The products range widely from salad dressings, baked products  to non-fermented beverages, to the fermented beer and wine,  All of these food industry markets each have vested interests in controlling bioaerosols in their processing facilities.


Regulatory Standards/Considerations

ISO Standards [2]: There are several key regulatory and certification bodies that discuss and impact both ambient and compressed air testing. The International Standards Organization speaks directly about compressed air testing, and the methods utilized to assay compressed [and ambient air] handling systems.

ISO 8573.1:2010 standard for compressed air was developed in 1991 and updated in 2010. It has multiple components. Part 1 describes 3 primary contaminant types with 9 classes with 9 most liberal to 0 the most exacting. Also, compressed air purity is classified as A, B, or C. Class A describes solid particles, B, liquid water/humidity; C oil particles. It also delineates the origin of the three class contaminants on the components of a compressed air handling system. 

Since we are focusing here on microbiology, the Class A solid particles are our discussion. Over 75-80% of the air particles are less than 10 microns [micrometers] in size.  For example, a Class 2 filtering system regarding solid particles, requires filters to remove particles [including microbes] that are 1 micron is size. 

There is also the ISO 12500 standard which focuses directly on filtering systems with 1: Oil particles, 2: Oil vapors, 3: Particulates. All deal with test methods to measure respective filtering efficiencies. For example, regarding particulates, fine sized filters are challenged with particles in the 0.5 to 5 micron ranges.

Meanwhile ISO 8573-7 focuses on Microbial testing of compressed air.  While ISO 8573-7 does not require a specific sampling method, it does require Colony Forming Unit [CFU] enumeration.  A specific type of Impactor sieve sampler, the SAS Pinocchio Super II which is utilized by Trace Analytics it can provide a quantitative sampling procedure for Compressed air. This is a Surface to Air Sample (SAS) that can utilize RODAC or full Petri dishes.

A third option for qualitative analyses of compressed air is the CAMTU unit [Parker-Balston] using a specialized donut shaped petri dish that easily integrates into an existing air-line. More on these methods detailed below.  In any case one should employ a sampling device & method that best suits the respective program’s requirements.

However, any of these samplers should employ proper Aseptic technique throughout the sampling protocol and utilize “blind” controls before and after actual sampling coupled with a negative control.  Also, any of these samplers can grow the colonies on an agar surface not only for CFU enumeration but biochemical identifications. Any reliable sampling unit has to be fully validated before actual air sampling

Under ISO 8573-7 proper disinfection of the actual unit, and the table or cart it sits upon should be mandated along with air feed lines, and valves preferably sampling 1000 liters of air in a 10-12.5 minute sampling time period.


GFSI Standards [3]:

The two primary GFSI Standards in North America are BRC and SQFI, and they both address compressed air testing.

In BRC Version 8, with regards to BRC clause 4.5.3 under Utilities [Water, Ice, Air & other Gases] states:  “Air and other gases used as an ingredient or that are in direct contact with products shall be monitored to ensure this does not represent a contamination risk. Compressed air that is in direct contact with the product shall be filtered at point of use.”

However, BRC does not delineate methodology or actual compressed air microbial specifications.  Only that the program must document compressed air used in direct contact with food products must be tested.

Concerning SQF Edition 9 several things have been clarified and expanded concerning air testing, as with Edition 8, Module : Compressed Air and other gases shall be clean and present no risk to food safety.  

Also states “Compressed air systems…… that come into contact with food or food contact surfaces shall be maintained and regularly monitored for quality and applicable food safety hazards. The frequency of analysis shall be risk based and at a minimum annually. “

Furthermore, with regards to Ventilation 11.1.6 there now is a requirement to provide adequate ventilation in food processing areas preferably positive air pressure should be installed.  The other addition is in High-Risk Processes [] mandates that “ambient” air in high-risk areas shall be tested at least annually to confirm that it does not post a risk to food safety.”

As with BRC, SQF does not stipulate which air sampling methods to employ nor provide actual microbial parameters either as guidelines or regulations. [4]


FSMA / SCFA Standards

With Preventative Controls, and the need to conduct a proper Hazard Analysis identifying the Critical Control Points, will prioritize where Compressed Air is utilized in a medium to high-risk process impacting zones 1 & 2 and product itself.  Consequently, indirect compressed air contact has risk, with the highest risk being direct contact with compressed air.

Process Control is the only Preventive Control that FSMA mandates Validation, and since Process Controls include all compressed air applications like drying, sorting, freezing, moving, carbonating, culturing, inert packaging, & MAPs it’s a critical feature of the program. So, yearly to biannual Validation of the Compressed Air quality for Microbes & other Particulates, along with moisture and oil is strongly recommended.

These compressed air system contaminants can be derived anywhere in the system ranging from the compressor itself, all joints, valves, and unions. The ambient air intakes and filters can be a source of microbial contamination if the Preventative Maintenance [PM] programs are not assiduously maintained. Condensate traps and receiver tanks can accumulate water vapor making the compressed air too humid and conducive to microbial growth.  

In terms of material design for the compressed air system FDA prefers Nylon /Conductive Polymer or Stainless Steel [preferred] with Welded seals being the top pick followed by Stainless compression seals. Valving should be Stainless steel Shut-off, or Particle Free Stainless steel. [5]



Other Key Standards

3 A Standard [604-05-3A] requires a filtration level of 99.9999% for sterile air, and non sterile air requires a 99% filtration level.  The British Compressed Air Society [BCAS] Section 6 has dew point, oil removal and a particulate removal specification range @ 0.1 to 0.5 microns [which includes microbial spores].

Air Sampling Technologies [1, 6]

As described above in the ISO standards discussion, there are a variety of microbial air samplers designed to test ambient air.  Some of these have been adapted to test compressed air systems. All began under regulatory and international standards for pharmaceutical and clinical/microbiology clean rooms under ISO 14698-1/2.

Impingers utilize a liquid capture media tube to collect the microbes to approximate a human’s respiratory capture system. The air being sampled is sucked through a slit tube the into a flask or tube with liquid media. The inlet tube’s diameter controls sample flow rates.  The sampling media is then volumetrically plated and a quantified count can be calculated using sample time vs. flow rate.   

With Impingers some drawbacks include the damaging of the microbial cells being hurtled into the liquid media collection device. Many collection devices are glass which are verboten in food and beverage plants. A positive is that the liquid medium can be microbiologically assayed in a variety of ways including PCR .

Some examples of Impingers include SAS-PCR from VWR utilized primarily for clean rooms and special labs. It is employed mostly for pathogen detection.  Another is the Bertin Technogies Coriolis sampler that employs a cyclonic sampling of the air using centrifugal force on the vessel’s cone like walls to get the microbes into liquid media.

Impactors in contrast to Impingers utilize an adhesive or solid agar media to collect the microbial particulates. A quantified sample of air per set velocity via a two types: Slit sampler or Sieve Sampler [a perforated plate] Both Slit or Sieve types will generate a laminar air flow onto the agar surface[s]. The slit’s width or the sieve’s hole diameters dictate the quantifiable air velocity. There is a directional change in both units’ design that will compel the microbes to “impact” the agar interface. 

With an Impactor air sampler once the proper/desired air volume is achieved the units will stop sampling and either the agar plate or strip can be removed for incubation and counting. All provide a quantified Colony Forming Unit [CFU] count per set volume of air [cubic meters or cubic feet].

A positive of the Impactors is that sterile media is readily prepared or obtained from a supplier.  Another benefit is that the Impactors can handle a variety of flow rates from moderate to high flow and high sample air volumes.  Impactors do rely on classical microbiological media and 1 to 4 days incubation for colony enumeration.

There are numerous varieties of Impactors. They include the following:

  • Casella Slit sampler: Has an agar plate turntable positioned below a slit
  • Andersen sampler: a cascade sieve that has layers of plates from larger to smaller holes

Here are the Impactor types primarily utilized for Compressed Air Microbial Analyses: 

  • Surface Air System [SAS] Sieve Samplers: SAS  180, Super Pinocchio II or the CAMTU II unit {Parker Bolston] all can use a large agar plate while the Super Pinocchio can use 90 mm petri dishes, but has an adapter to use a RODAC type plate [55 mm diameter] . All these are utilized for compressed air systems.
    • The Super Pinocchio II is the most precise and provides accurate, quantifiable counts. However, it is very cumbersome, heavy and somewhat difficult to utilize in a food & beverage plant environment.
      • Media is relegated primarily to Total Count but can create Differential media plates for Fungal or Coliform enumerations.
      • Sampling volume is usually 1000 liters
  • CAMTU unit, is lightweight and portable and utilizes an ingenious donut shaped agar plate to sample compressed air.  Parker Balston created this CAMTU unit, and states it’s good for a “qualitative” analysis of compressed air systems.
    • User friendly to set up and operate, I believe that although its data is qualitative, it is still very helpful for a lot of food & beverage plants to employ. [Since there are no quantified requirements for microbial counts of compressed air by any regulatory standard!]
  • Reuters Centrifugal Sampler [RCS] is a Slit type Impactor using a impeller creating a vortex via centrifugal force, and a specialized agar strip containing 34 autonomous agar wells. Versatile, the High Flow Touch model can be used for ambient air and compressed air testing in Food & Beverage plants.
    • Developed by Merck Biotest, and now owned by Millipore Sigma this is a sampler I have employed for nearly 30 years for both ambient, and using a recent innovation, an adapter, quantified sampling for compressed air systems.
    • Are reasonably lightweight and semi-portable and are easier to use in a food & beverage plant environment. 
    • Agar strips are easy to store refrigerated, and easy to ship for analyses to a microbiology lab for incubation and counting.
    • Agar strips are of three types Total Count, Yeast Mold, and TSM [for coliforms] and colonies can be cultured and identified off these strips just like an agar petri dish.
    • Sampling can be hand held or use a tripod with remote for hard to sample production areas.
    • Programmable for a variety of air volumes.  For Compressed air typically a 1000 liters @ 15 psi [air pressure adjusted] is used, but I have successively utilized 500 liters for many compressed air system samples.


Reuters Centrifugal Sample

Reuters Centrifugal Sample

The Reuters Centrifugal Sampler [RCS] is a Slit type Impactor.

Membrane Filtration:  alternate approach that sampled compressed air or ambient air flows through a quantified velocity through a microbial membrane filter which is then cultured [Akin to sampling water systems for microbes]. This method is employed in Pharmaceutical industry applications.


Remarks on Data Analysis

In order for the data to be reliable and scientifically valid, the respective air sampler must be periodically validated and calibrated [Annually].

The efficiency to collect microbial particles and the ability of the technology to maintain a very high level of viability to grow the microbes into colonies on media.  This includes the flow rate under large air volume sample sizes. The more the flow rate at large air volumes makes the unit’s sampling more accurate.

Validation protocols are developed by the units’ manufacturers. Meanwhile existing units must be periodically recalibrated either by the end user or the manufacturer. For example, Millipore Sigma with the RCS High Flow mandates yearly re-calibrations of field units.

As stated above to date of this article there are no set standards in the U.S. or mandated ranges for either High, Medium or Low Risk microbial density per cubic unit of air. This is true both for ambient air measurement as well as for compressed air measurement in food & beverage programs. Exception to this are the British Compressed Air Society [BCAS] Section 6 and 3 A as discussed above. This is why ISO and 3A are trying to define microbiological specifications for compressed air and needed also for ambient air in high to low risk food processing modes.

So, due to a lack of regulatory guidelines or standards for food & beverage air samples, I have employed “guidelines” with varying levels of Unsatisfactory, to Substandard to Satisfactory. The results obtained from microbial air sampling both for ambient and compressed air systems are reviewed on the basis of the risk assessment for the sampling area or site. Any result that finds less than 100 CFU per cubic meter air is considered the goal and is deemed Satisfactory. Of course, this is not applicable to sterile / aseptic processes. 

The testing of air handling systems [ambient & compressed air] will continue to mature and evolve with increasing automation and accuracy of air sampling systems.  The food & beverage processing markets in the past 20 years have increasingly considered air system quality a major component of their HACCP plans.  Stay tuned!



[1] Giambrone, C.  Airborne Microbial Control Program’s Role in Food Plant Hygiene.  Food Safety Magazine, Signature Series, June-July 2005.

[2] International Standards Organization: ISO 8573.1, ISO 12500: Air Quality Standards, ISO 8573-7: Microbial Testing and Compressed Air Standards.

[3] Scott, Lee,  Parker Gas Separation and Filtration Division: Compressed Air GMPs for GFSI Food Safety Compliance.

[4] British Retail Consortium [BRC] Version 8 Clause 4.5.3 and Safe Quality Food Institute [SQFi]  Edition 9, Module 11.5.5

[5] International Food Safety & Quality Network:  FSMA, HACCP, and Your Compressed Air System  May 17, 2018

[6] Rapid Microbiology: Air Samplers for Microbiological Monitoring of Air Quality


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