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Air Sampling 101
Consider the benefits and problems of each method to determine which method and sampler are best.
by Julie Buddemeyer
Proactive companies from all sectors; pharmaceutical, food and beverage, biotechnology, hospitals and environmental protection, are realizing the importance of an active air sampling program. There are several options in sampling methods, some more efficient than others.
Sedimentation or settle plates are the most primitive method for sampling airborne microorganisms. A Petri dish containing a suitable agar is exposed to the atmosphere and the agar medium will collect bacteria-laden particles that eventually settle by gravity. It is a passive, non-volumetric method and imprecise by over-representing larger particles due to their rapid settling rate.
This method is inefficient for collecting small particles because air turbulence around a plate can affect the results and small particles may never settle. In order to counteract this air turbulence effect, the plate has to be left out longer, which can cause desiccation. Agar drying out leads to poor bacterial growth and reduces the viable count of stress-sensitive microorganisms. Settle plates are also impossible to validate because there is no way to measure the volume of air sampled.
The impactor is essentially a jet that draws air into the sampler, which then directs the air stream at the collection agar. The different types of impactors are listed and discussed below.
In a slit-to-agar impactor, a known volume of air is drawn by vacuum through a slit opening and then accelerated and directed toward the surface of a Petri dish containing agar media. The plate rotates on a turntable at a selected rate of speed and the impacted microorganisms are separated spatially by the plate's rotation, providing an analysis based on time. The microorganisms, because of their higher mass, become impacted on the agar surface, while the rest of the air mass flows around the plate and exits the air sampler.
While this type of impactor offers the unique benefit of time analysis, it also presents several efficiency flaws. A specific, large plate is necessary for the rotating turntable, which could conflict with evaluation methods designed for standard (90mm) Petri dishes, such as colony counters. Also, between the vacuum action and the rotating plate, the media often dehydrates; which as mentioned above, can lead to poor bacterial growth and reduce the viable count of stress sensitive microorganisms. Because air enters into the sampler device and is then directed toward the collection medium, not all particles will impact on the intended, rotating plate. Particle deposits onto surfaces other than the impaction medium can account for up to 10 percent of total particles, skewing the selected sample from a true representation of the air quality. Perhaps the greatest drawback, however, is the slit-to-agar sampler's inefficiency of collecting smaller particles. While larger particles with sufficient inertia will deviate from the streamlines and impact on to the agar surface, smaller particles, less than .5 µm will follow the gas streamlines, missing the agar surface altogether and being exhausted back into the atmosphere.
There are several types of sieve impactors. One-stage impactors have only one perforated plate set in front of on agar plate, while stacked sieve samplers can have up to six stages of perforated plates and agar plates. In stacked sieve, or cascade samplers, each perforated plate is held above an agar plate with successive plates having smaller holes. At a constant flow, larger particles impact on the first stage, whereas smaller particles impact on the last impaction stage. The major advantage of a stacked sieve impactor is that it can provide data on particle size.
Sieve impactors are not without their own set of specific problems. Unless sampling times are short or the relative humidity high, the areas of nutrient agar directly under each hole of a stage can rapidly dry out, adversely affecting the growth of fastidious microorganisms. Using plastic dishes may cause electrostatic effect, deflecting particles away from the dish to deposit on other internal parts of the sampler. While these problems are significant, much like the slit-to-agar sampler, the major problem with sieve impactors is their inefficiency at collecting smaller particles. In order to impact smaller particles, the holes need to be small and the air flow rate accelerated, however, if the flow rate is too fast, the smaller microorganisms will be killed due to the shear force of impact.
Centrifugal samplers create a vortex in which particles with sufficient inertia leave the airstreams to be impacted upon a collection medium. Air is drawn into the sampler by an impeller housed inside an open shallow drum. The air is then accelerated by centrifugal force toward the inner wall of the drum. Lining the inner wall is a plastic strip supporting a thin layer of agar medium, onto which airborne particles are impacted. The major advantage of centrifugal samplers used to be that they were battery operated and small enough to be hand-held. However, other samplers are now comparative in portability to the centrifugal samplers.
Centrifugal samplers are generally known to be efficient in collecting particles 15 µm and larger, but have trouble with smaller particles. Less than 10 percent of particles under 2 µm are deposited. The use of a strip instead of a standard Petri dish presents evaluation problems and colony counters may have difficulty counting the strip, requiring counts to be done manually. Because the sampler exhausts the air stream from the same opening used to create the vortex, the surrounding atmosphere is disrupted, affecting the accuracy of the collected sample. This single opening system also creates another problem relating to validation. With the air entering and leaving the same opening, flow rate quantification is only theoretical. The vortex itself can also affect sampling accuracy. As the particles enter the vortex through the rotating impeller the CFUs (colony forming units) will break apart showing higher counts and giving a false higher efficiency rating that is not representative of the actual atmosphere.
With an all glass impinger, air is drawn through a curved suction tube (curved to simulate the nasal passage) and accelerated through a jet at the bottom. The bottom part of the impinger is filled with a suitable liquid that captures the particles. The liquid is filtered after sampling and then processed as usual. Impingement is an efficient method for collecting various sizes of microorganisms and is therefore suitable for determining the initial air quality of an area.
Although effective, impingement is not considered a convenient air sampling method. The glass containers are not disposable and must be prepared every time and depending on the area to be sampled, this can be very time consuming. Impingement can not be reliably validated because flow rate is very low and can't be measured. While impingers are efficient for collecting microbes, the collection time must be carefully monitored to decrease the effects of evaporation of the collecting fluid as well as cooling of the sample. Evaporation and cooling can both negatively affect the retention rate of the microorganisms. Also, microbes grow while sampling and can be disrupted or separated during sampling, which can create higher counts similar to that of the centrifugal impactor.
Gelatin Membrane Filtration
The most innovative air sampling technology is the gelatin membrane filtration method. Air is sampled at a programmable flow rate and passes through the gelatin membrane filter which captures the microbes. The filter is 300 µm thick, making it capable of capturing even the smallest microorganism. It is very complex and porous, so microbes will collect not only on the surface, but will become entangled within the filter. This is the only sampling method with an absolute retention rate and the only method that also reliably captures airborne viruses. The filter is composed of 50 percent water which protects the captured microbes and completely eliminates the problems associated with desiccation. Another important feature is that all of the air entering the sampler must first pass through the filter; thereby ensuring microbes are not reintroduced into the atmosphere. The exhaust air is not located close to the filter sampling the air, so air flow is not affected.
Processing the filter is also convenient and easily read with most automated colony counters. After sampling, the filter is placed directly on a standard (90mm) agar plate and dissolves, leaving the microorganisms in direct contact with the agar. The filter can also be dissolved into a sterile solution for special evaluations, such as when inhibitors (i.e. disinfectants or antibiotics) are present in the air being sampled, very high colony counts are expected or the microorganisms collected are to be incubated on several different media types at the same time.
When selecting an air sampling method, it is important to consider the benefits and problems connected with each method and to determine which sampler is best suited for specific needs and requirements. Actively monitoring air quality is becoming increasingly important for production facilities and wit h HACCP in many instances it is already mandatory.
Julie Buddemeyer is the marketing and sales assistant for Microbiology International. She can be reached at 800-396-4276 or info@800EZmicro.com.