Sampling for Airborne Biological Contaminants:
A RATIONAL APPROACH
Nothing we do raises as many questions as microbial air sampling.
While the science of microbial air sampling is fairly straightforward, somewhere along the line our approach to it has become much like that of a backward soothsayer who divines the question after first being given the answer. For example, we often first buy the sampler and then try to fit it into our operation
Sampling strategies differ when applied to quality control, validation, or research. Each requires a differing degree of sampling sophistication and a different approach to data analysis. All microbial aerosol samplers must be judged in terms of their capability to collect microbes under different operating conditions while minimizing the environmental stress on the organisms collected. There is, therefore, no single sampling method that is suitable to all occasions.
Another point to remember: rarely is microbial air sampling conducted as a quality control point of a product. More often than not, air sampling is done to determine the cleanliness, or absence thereof, of the environment in which a product is manufactured. Or in this instance, to answer the familiar question: how clean is clean?
Microbial air samplers are characterized by:
* Mode of capture,
* Flow rate and flow characteristics, and
* Collection efficiency as a function of particle size and shape.
As a rule, aerosol collection devices that exhibit the lowest shear forces collect samples where microorganisms have the highest viability. Conversely, these samplers usually have the lowest physical efficiencies in terms of numbers of airborne particles collected. The microbiological collection efficiency, therefore, depends largely upon the sampling method used.
The primary objective of any sampling program is to produce a set of samples that are representative of the source under investigation, and, that these samples are suitable for subsequent analysis. Because the air we sample in any environment is not homogeneous even in a clean room that exhibits good laminar airflow, there can be no duplicate samples. We therefore need to consider sampling conditions, sampling time, and sample size as limitations in our data collection scheme.
Collecting a representative sample of airborne microbes is probably the most difficult to achieve. Apart from the inherent absence of microbial uniformity in air, is the problem of ensuring that particles of all sizes have an equal probability of entering the sampler. This can be partially remedied when the sampling rate is chosen so that the velocity of air entering the sampler inlet equals the velocity of the air being sampled.
Achieving or approaching isokinetic sampling conditions is particularly important in an area in which air sampling is done under dynamic conditions. If the velocity of the air entering the sampler is greater than that of the room’s air movement, small particles will predominate because they move more easily across the streamlines. Conversely, if the velocity is slower, larger particles predominate because, unlike smaller particles, they do not follow the curvature of the streamlines around the sampler inlet.
Anisokinetic sampling may result in sampling errors that range from 20% to 300%, depending upon particle size and varying environmental conditions. Ideally, to overcome the effect of dynamic air movement on the capture of microbial aerosols, stagnation point sampling can be used. In the absence of any air movement, smaller particles are efficiently captured and a particle size profile estimated.
To further complicate this issue, consider the following: air is not a natural environment for most microbes. Survival of microorganisms in air is affected by a large number of environmental factors, the most important of which are temperature and humidity. Under natural conditions these numerous factors operate simultaneously. Consider also that force is required to generate an aerosol, and likewise, to capture particles within that aerosol. These forces can damage or even fracture fragile structures such as microbes in their vegetative state.
The fragile nature of airborne microorganisms is largely species dependent, and determined by its physiological condition. Once airborne, microbes become stressed through desiccation or hydration depending upon the condition of their natural growth site. Radiation, oxygen, ozone and various other gaseous and particulate pollutants, if not lethal, may further stress these organisms. Some stressed and injured microorganisms may, however, fully recover when given a suitable environment. This property of reversible injury or repair in microorganisms is widespread and the implications of it are important in developing the testing protocol.
The agar medium selected for use in all microbiological sampling devices should therefore be fresh and pre-screened for sterility by placing it in an incubator at 36°C for 24 hours. In conducting the initial microbial assays, malt extract agar is recommended for the general detection of fungi while agar containing casein peptone, soy peptone, and sodium chloride is used for bacterial sampling. Trypticase soy agar has probably the most universal applicability for the collection of aerobic bacteria and fungal species, whereas, for the detection of anaerobic species, a thioglycollate medium is recommended. Once initial bioburden estimates are established and a greater specificity is required to target certain organisms, specialized agars containing antibiotics and/or other inhibitory and growth regulating compounds are available from commercial sources. However, when these selective media are used in an air sampling devices, the collection efficiency may be severely hindered. These media are generally inhibitory to small inocula; even of the organisms for which they are “selective” by retarding recovery of those that are injured or stressed.
Because the organisms found in air come from different environments, the temperatures used to enhance their growth on an artificial medium should approach that of their normal habitat. Most of the organisms found in the air fall within two distinct temperature preferences. The psychrophilic organisms, or those that prefer cold, thrive at low temperatures between 0° and 30°C, while the mesophyllic organisms prefer moderate temperatures between 15° and 43°C. Therefore, to recover the maximum number of organisms in any air sample, consider incubating the medium at 22°C for 24 hours (or 48 and 72 hours as necessary) to recover psychrophilic organisms, immediately followed by incubation at 36°C to recover the mesophyllic organisms.
The outcome of a well-planned sampling strategy depends on good science, logic and to a lesser degree, a measure of good luck. Taking the time to estimate the types of organisms that may be present; describing the static, dynamic and physical characteristics of the clean room and its air, and, hopefully conducting a viable/nonviable particulate profile of the space to be sampled, will yield data that becomes the basis for the entire microbiological sampling scheme.
Since there is no single agar medium on which all microorganisms will grow, no single incubation temperature that will encourage growth, and no single assay procedure that can completely characterize the microbial contamination in all environments. Likewise, there is no universal sampling device.
Because of the different modes of collection used by the various microbiological air sampling devices, it is both difficult to compare and contrast their sampling efficiencies. Sieve samplers, for example, provide particle size distribution, while slit-to-agar samplers are used to determine the airborne bioburden as a function of time. Settling plates are suitable to locate a point source where larger particles are generated; centrifugal samplers provide an easy, rapid means of taking numerous samples of the gross airborne bioburden where size and temporal considerations are unimportant; low shear-force liquid impingers are ideal for the recovery of stressed organisms and therefore provide the best recovery of the widest range of airborne microorganisms.
The following is a brief review of the types of microbial air samplers and their use.
* Gravitational Samplers. Settling plates are the simplest forms of collection of airborne biological particles. A Petri dish containing agar medium will collect particles that settle by gravity. It is passive, non-volumetric, and imprecise by over-representing larger particles due to their rapid settling rate. Collection in turbulent air is seriously affected by shadowing or turbulent deposition. Settling plates, however, can be used as an adjunct to other sampling methods or as a pre-screening tool in areas suspected of containing a significant bioburden where deposition is most likely. For example, placing settling plates around water purification equipment during servicing may indicate the need for improved aseptic technique if viable droplets are deposited in decreasing concentric concentrations from the aerosol source. Settling plates can be useful to initially evaluate the airborne microbial release of cooling towers and condenser units.
* Impingers (AGI-30). Most impingers are designed to operate by drawing aerosols through an inlet tube that is curved to simulate the nasal passage. The air is subsequently passed through a jet into a liquid medium. The jet, which is positioned 30 mm above the bottom of the glass impinger, consists of a short piece of capillary tube. When the pressure-drop across this capillary tube attains a minimum of half an atmosphere, the flow through it becomes sonic and may therefore be rate-limiting. The most efficient sampling rate for the AGI-30 to capture particles in the 0.8µm to 15.0 µm range is 12.5 l/min. Larger particles are collected on the curved inlet and are recovered by pipetting a known volume of collecting fluid into the impinger inlet; which then flows slowly through the jet into the impinger.
The usual volume of collecting fluid is 20 ml, but depending on application may be reduced to as little as 2 ml to increase the concentration of collected microorganisms. The collecting fluid can be plated, filtered or diluted as necessary. Sampling time should be adjusted to prevent evaporation of the collecting fluid and cooling of the sample. The collection medium used with an impinging type sampler should prevent osmotic shock of vegetative organisms. Because deionized water may promote lyophilization, a dilute poly peptone-peptone broth or a physiological saline transport medium may demonstrate greater recovery efficiency.
The AGI-30 was designated as the reference standard sampler and the confidence in this device is largely historical. For this reason, the all-glass impinger is the best unit to determine the initial bioburden of an area.
* Impactors. Inertial forces are responsible for impaction action. The impactor is basically a jet, under which is an agar impaction surface. Particle-laden air sucked through the jet is directed at the collection agar surface so that particles impact onto it. In a cascade impactor, particles are captured by inertial force. Particles impact in cascading order: larger particles are captured first while those smaller remain airborne and follow the streamlines; each successive stage having increased air velocity through the corresponding jet. This type of sampler is the most commonly used in cleanroom air sampling. Several types are designed to serve various applications.
* Stacked Sieve Samplers consist of a series of six stages each composed of a plate having 200 holes. Each plate is held above a Petri dish containing nutrient agar with successive plates having smaller holes. At constant flow (28.3 l/min), largest particles impact on the first stage whereas smallest ones impact on the last impaction stage. 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.
Fastidious microorganisms consequently may fail to grow on the upper stages due to the desiccation of the agar, but grow on the lower stages because the relative humidity of air increases as it passes through the sampler. Covering the agar with a water-evaporation retardant, or increasing the water content of the agar can reduce this problem. The precision with which airborne microbes are captured depends on the collection surface. The agar containing Petri dishes need to be level and filled precisely with the recommended amount of agar to give the correct plate to agar surface distance. Using plastic Petri dishes may give rise to an electrostatic effect, but it can be argued that the small problems of wall losses can be offset by the practicalities of use. The major advantage of multiple stage cascade impactor, is that it can provide particle size data.
There is a two-stage version of the impact sampler that separates viable particles into “respirable” and “non-respirable” fractions. The upper stage corresponds roughly to deposition on stages 1 and 2 of the six-stage version and the lower stage, to deposition on stages 3 to 6. However, side-by-side comparisons with the six-stage version revealed an approximately 40% lower recovery rate of bacteria with the two-stage sampling device, except when sampling dilute concentrations of particles larger than 1 µm. Using the correction factors provided by the manufacturer when completing the count can minimize this difference in efficiency.
There are several variants of the sieve sampler, including one-stage devices. While these units will not provide particle size distribution, they are efficient collectors across the entire size spectrum. These units have the advantage of being rapid and consistent but should be used only as a validation tool, once the scope of the bioburden is known.
* Slit-to-Agar Samplers. In slit-to-agar samplers, air is drawn through a narrow slit, then accelerated and directed toward the surface of a Petri dish containing agar media. The collection surface is often placed on a rotating turntable. Airborne microorganisms impact onto the agar surface and are separated spatially by the plate’s rotation; thereby providing a stochastic analysis. The sampler is operated at a fixed sampling rate of 50 l/min; rotational speeds can be varied and the collection efficiency is for particles 0.5 µm and higher. This sampler is best used to monitor the effect of materiel movement or operational variations of support and production equipment.
* Centrifugal Samplers. The principle of collection of these sampling devices is centrifugation, which involves the creation of a vortex in which particles with sufficient inertia leave the airstreams to be impacted upon a collection surface like a semi-solid medium. The most frequent example of this type is the Reuter centrifugal air sampler. 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 or agar medium, onto which airborne particles are impacted. These strips are subsequently removed and incubated. The motor for the impeller is battery operated and the whole sampler unit is small enough to be hand-held.
There has been some concern expressed about its effective flow rate and collection efficiency. Air enters and leaves the sampler through a single opening; therefore flow rate quantification is theoretical. The sampling rate is calculated from the impeller revolution of 4092 rpm; yielding an effective sampling rate of 40 l/min. Studies have shown, however, that the effective sampling rate may be nearer 100 l/min under certain conditions. While these devices collect larger particles (15 µm) quite efficiently, less than 10% of particles under 2 µm in diameter are deposited. Therefore comparisons to the stacked sieve, slit-to-agar, and liquid impinger samplers cannot be made. A newly redesigned Reuter sampler allows separate entry and exit airflow that may improve its performance, but provides an actual flow rate and can more closely approximate isokinetic conditions.
If different groups of organisms are to be studied, which necessitates the inoculation of different media, repeat samples must be taken at each sample point. This increases the sampling time and may result in errors if rapid and large variations occur in the air. This sampler however has the distinct advantage of ease of use. Additionally, if the sampling protocol is properly defined, it can be used as its own control and the need for volumetric sampling becomes moot. Rapid comparisons of qualitative microorganism profiles in the intra- and extramural air can be made to determine the proper sampling strategy via quantitative instruments. This makes the Reuter centrifugal sampler the most versatile instrument as a pre-screening tool.