Something in the air – techniques for monitoring airborne microorganisms
Controlling levels of microbial contamination in the air around sensitive processing and packing operations has become an increasingly important part of hygienic food manufacturing in recent years. The use of high-efficiency particulate air (HEPA) filtered ventilation systems is common and even self contained clean rooms now feature in some particularly vulnerable production processes. Despite this, the methods routinely used for monitoring the microbiological quality of the air in food factories have lagged behind those available for other applications, with rudimentary qualitative methods still being widely used. But sophisticated monitoring systems designed for the medical and pharmaceutical industries are available and can easily be adapted for use by food manufacturers.
There are a number of important sources of microbiological contamination in any food manufacturing operation, but most microbiologists would probably rank people, raw materials and processing water as their top three. It is difficult to argue with that view, but it does overlook another important factor. The atmosphere in food factories may not be the first concern as a source of microbial contamination, but in most cases it is certain to contain airborne microoganisms, some of which may cause product spoilage, or be foodborne pathogens. Some types of bacteria and fungal spores survive remarkably well in the atmosphere – bacterial spores can be isolated from the jet stream several miles above the Earth’s surface. Even so, routine monitoring of numbers of microorganisms in the air around processing and packing lines is often a low priority – an attitude sometimes reflected in the continued use of outdated and inadequate methods.
Microbiological air quality is critical in the medical and pharmaceutical sectors, where maintaining sterility is the aim, but in most food processing environments it is less of an issue. Nevertheless, the last 20 years have seen much more attention given to airborne microbial contamination in the food industry. The ongoing quest for longer and longer shelf life has seen the introduction of sophisticated ventilation systems supplying HEPA filtered air under positive pressure designed to exclude contaminated outside air and limit the microbial population in sensitive areas. Some manufacturers have even adopted clean room installations of the type common in the medical sector in an attempt to reduce contamination levels during processing and packing and to increase shelf life. The types of foods that can benefit from this approach include bakery goods, cheese and other dairy products, fresh pasta, ready meals and chilled ready-to-eat foods. These developments highlight the need to update methods for monitoring airborne microbes. Using the latest technology could provide opportunities to gain lot more useful information about airborne contamination.
The microbiology of the air
Fortunately, the atmosphere is not a very welcoming environment for many microorganisms. The joint effects of desiccation and sunlight cause many microbial cells to die rapidly when suspended in air. This is especially true of Gram-negative bacteria, including food borne pathogens like E. coli and Salmonella. Nevertheless, some Gram-positive bacteria and fungal spores can survive for long periods in the atmosphere and can be widely dispersed by air currents. The typical microflora of the air is usually made up of pigmented Gram-positive bacteria and bacterial and fungal spores, which are resistant to the drying effects of the air and to radiation. Unfortunately, it can include some pathogenic bacteria, such as Staphylococcus aureus and Bacillus cereus, and common food spoilage fungi, notably species of Penicillium and Aspergillus.
Many of these microorganisms are present in the air on minute dust particles, which can remain suspended for long periods, but they may also be dispersed in the very small water droplets of aerosols. Aerosols can be produced from people, through coughing and sneezing, and by many other mechanisms. But the most important source of aerosols in most food factories is wet cleaning, especially where high-pressure water jets are used. The droplets formed during cleaning operations can disperse even relatively fragile bacterial cells, such as Listeria, for short periods before they begin to die. Aerosols from cleaning may be a contributor to the spread of Listeria around factories where it has become endemic and difficult to remove. In other words, cleaning operations may sometimes actually help to recontaminate the production environment.
Monitoring air quality
The survival of micro-organisms in the air is dependent on a number of interacting factors, and is not easy to predict. This is one of the main reasons why routine monitoring of airborne bacteria and fungi can be important, especially where vulnerable products are manufactured. Microbiological monitoring of the air in facilities where pharmaceuticals and medical devices are produced is well established. In most countries it is a regulatory requirement, and international standards have been published for biocontamination control in clean rooms and other controlled environments (ISO 14698-1/2). While there are published recommendations for microbiological air quality in food processing plants, such as those produced in the USA by the American Public Health Association (APHA), regulatory standards are largely lacking.
In the absence of mandatory standards, it makes good sense for individual food businesses to examine the need for control and monitoring of airborne microbes using a HACCP approach, taking into account the vulnerability of product and process, the surrounding environment and other factors. Once an acceptable contamination level has been established, monitoring airborne contamination regularly is a useful means of identifying problems before they affect product quality and safety. In many cases, simply monitoring the overall population of airborne microbes may provide sufficient information, but in more sensitive operations it may be helpful to look for specific pathogens, or for spoilage organisms. The results of monitoring allow trend analysis and provide early warning if levels of contamination are seen to be rising. Where HEPA-filtered positive pressure ventilation systems and clean rooms are in use, they are likely to have strict specifications for acceptable levels of airborne micro-organisms. Here monitoring is a vital part of verifying that the system is operating effectively.
There are both passive and active methods for monitoring the microbial population of the air. Active sampling methods have become an essential environmental monitoring tool in the pharmaceutical and medical device sectors, but much of the food industry still relies on passive monitoring.
Passive monitoring typically employs ‘settle plates’ – petri dishes containing appropriate culture media, which are opened and exposed for a given time and then incubated to allow visible bacterial and fungal colonies to develop and be counted. Settle plates are only capable of monitoring those viable biological particles that sediment out of the air and settle onto a surface over the time of exposure. They will not detect smaller particles or droplets that remain suspended in the air and they cannot sample specific volumes of air, so the results can only be considered semi-quantitative at best. They are also vulnerable to interference and contamination from other sources and may easily become overgrown in heavily contaminated conditions.
On the other hand, settle plates are inexpensive and easy to use, requiring no special equipment. By using different culture media, they can also be used to estimate the numbers of specific groups of micro-organisms, such as spoilage fungi, in the air. They are moderately useful for qualitative analysis of airborne micro-organisms and the data they produce may provide some indication of underlying trends in airborne contamination and an early warning of problems. They are also useful for directly monitoring airborne contamination of specific surfaces. In a low risk food factory, where airborne contamination is of limited significance, settle plates may provide an adequate means of monitoring microbiological air quality, but more sensitive operations need a more sophisticated approach.
Active monitoring differs from passive monitoring in that it requires the use of a microbiological air sampler to physically draw a known volume of air over, or through, a particle collection device. There are two main types.
Impingers use a liquid medium for particle collection. Typically, sampled air is drawn by a suction pump through a narrow inlet tube into a small flask containing the collection medium. This accelerates the air towards the surface of the collection medium with the flow rate being determined by the diameter of the inlet tube. When the air hits the surface of the liquid, it changes direction abruptly and any suspended particles are impinged into the collection liquid. Once the sampling is complete the collection liquid can be cultured to count any viable micro-organisms in the sample. Since the sample volume can be calculated using the flow rate and sampling time, the result is quantitative.
Impingers have some important disadvantages for routine microbiological monitoring of the air. Traditional designs are usually made of glass, which would not be permitted for use in most food manufacturing environments. Impingement into liquids may also damage some microbial cells and overlong sampling times may even allow some cells to multiply in the collection medium. However, collection in a liquid medium means that the sample can be analysed using a variety of methods, including molecular techniques such as PCR, so that results can be obtained more rapidly.
Recently, instruments have been developed using variations on the basic impinger principle, such as the Coriolis®µ sampler made by Bertin Technologies in France, and the SAS-PCR sampler from pbi International. These samplers are not constructed from glass and so can be used to sample the air in controlled environments. The Coriolis sampler uses a cyclone effect to accelerate the sampled air into the collection liquid. Any suspended particles in the air are thrown out by centrifugal force, collect on the walls of the conical collection vessel and concentrate in the collection liquid. The SAS-PCR device is designed specifically to collect pathogens for subsequent detection by molecular methods and circulates the collection liquid to prolong contact time with sampled air. Although these samplers have been designed with the pharmaceutical and medical sectors in mind, they may have applications in the food industry, especially for high risk manufacturing where there is a need to monitor specific pathogens or spoilage organisms.
Impactor samplers use a solid or adhesive medium, such as agar gel, rather than a liquid for particle collection. Typically air is drawn into a sampling head by a pump or fan and accelerated, usually through a perforated plate (sieve samplers), or through a narrow slit (slit samplers). This produces a laminar air flow onto the collection surface, often a standard agar plate filled with a suitable medium. The velocity of the air is determined by the diameter of the holes in sieve samplers and the width of the slit in slit samplers. When the air hits the collection surface it makes a tangential change of direction and any suspended particles are thrown out by inertia, impacting onto the collection surface. When the correct volume of air has been passed through the sampling head, the agar plate can be removed and incubated directly without further treatment. After incubation, counting the number of visible colonies gives a direct quantitative estimate of the number of colony forming units in the volume of air sampled.
Impaction samplers offer benefits in terms of convenience and they are also able to handle the higher flow rates and large sample volumes necessary to monitor air quality in controlled environments where the number of microbes present is likely to be very low. However, microbial cells may be damaged by mechanical stress during the sampling process and lose viability. Furthermore, impaction samplers do not allow the use of rapid methods to enumerate and characterise microorganisms, but rely on conventional culture methods taking several days to obtain a result. This problem can be overcome to some extent by the use of a water-soluble polymer gel instead of agar. This does allow the sample to be analysed by rapid techniques such as PCR or cytometry.
Hand-held SAS air sampler (with kind permission of Cherwell laboratories Ltd.)
A wide variety of commercial instruments have been developed using the impaction principle. One of the best known is the Andersen sampler, a multi-stage ‘cascade’ sieve sampler that uses perforated plates with progressively smaller holes at each stage, allowing particles to be separated according to size. Another is the Casella slit sampler, in which the slit is positioned above a turntable on which is placed an agar plate. As air is drawn through the slit, the agar plate rotates, so that particles are deposited evenly over its surface. Both of these instruments have been used for many years, but more recently a number of highly portable and convenient impaction samplers have been developed specifically for monitoring the air in sensitive areas. Most of these are sieve samplers, such as the Surface Air System (SAS) samplers made by pbi International in Italy, and use agar contact plates or full-sized culture plates as the collection surface. These portable samplers can be hand-held, or mounted on a tripod during sampling, and can be programmed to sample a specific volume of air, or sequential samples at pre-set times.
Automated air sampling
Several manufacturers of microbiological air samplers have developed semi-automated systems, usually based on sieve type impaction samplers, for monitoring clean rooms and controlled production areas. An example of this type is the AirCapt® MP8 Multipoint Biological Monitoring System from US-based Particle Measuring Systems. These devices typically use a number of sampler heads linked to a central control unit, which can be programmed to follow a pre-set sampling programme. In some medical applications the sampler heads can be fitted permanently in place so that they undergo the same sterilisation regime as the rest of the clean room. It is also possible to set up a wireless network of portable air samplers controlled by a central PC, with no need for any electrical or vacuum line connections. Semi-automated systems offer the possibility of integration with environmental monitoring and QC software packages, providing the basis of a paperless system for recording microbiological data. There is no technical reason why similarly sophisticated systems cannot be used to monitor microbiological air quality in food processing areas where the risk from airborne contamination is considered sufficiently high.
There is little doubt that monitoring airborne micro-organisms is generally a more critical issue in the medical and pharmaceutical sectors than it has traditionally been in the food industry. This has driven the development of a range of advanced techniques and instruments designed specifically for the purpose and aimed mainly at medical and pharmaceutical manufacturers. The food industry has had no real incentive to adopt the same techniques up to now, but the move towards food manufacture in increasingly clean environments to minimise contamination and extend shelf life has narrowed the gap between the food and the medical sectors. The microbiology of the air is becoming a much more important consideration for food manufacturers and that is creating a demand for better data collection methods. Fortunately, many of the instruments and test protocols designed for other industries can be easily adapted for food production. Businesses that adopt these techniques will have opportunities to collect a lot of valuable data about the airborne microbes in their production environments, which old-fashioned settle plates could never provide.