Bioaerosol Evaluation in Indoor Environments

Assessment of bioaerosol exposure offers challenges distinct from those for inorganic aerosols and chemical agents.

INDOOR air quality (IAQ) typically refers to the quality of air inside buildings where people work or live. Indoor air quality issues and specific building-related health concerns may result from a complex combination of physical, chemical, biological, ergonomic, and behavioral risk factors. Air quality can be compromised when there is inadequate ventilation, when chemicals are used in the building, when gas furnaces malfunction, when outdoor pollutants enter fresh air intakes, or when mold or other microorganisms grow inside the building or in the heating and ventilation (HVAC) system. The term "indoor air quality" is usually used in reference to non-industrial workplaces, such as office buildings, governmental institutions, hospitals, libraries, schools, and in residential structures.

Federal and state laws generally do not regulate indoor air quality, although certain regulations do protect employees from exposures to specific substances with potentially harmful effects. These regulations specify concentrations of certain substances that must not be exceeded and are called Permissible Exposure Limits. However, PELs are usually higher than levels found in most office buildings or other non-industrial environments, are established for standardized eight-hour time-weighted average exposures (TWAs), and are not appropriate for use in evaluating non-occupational exposures. Readers should familiarize themselves with the state and local regulations specific to their locale.

Experience in air monitoring has shown that levels of chemicals in the air rarely exceed current PELs for routine activities. Exceptions may include situations where the types of chemicals in use or of concern, along with current work activities, indicate a more aggressive evaluation protocol. Knowledge of facilities, equipment, and materials in the affected environment, along with familiarity with operations, is crucial to an effective indoor air quality evaluation.

Visual inspections are expedient for initial screening and for identifying appropriate opportunities for sampling and analysis. Monitoring for molds or other microorganisms in indoor air should not be performed initially because of technical difficulties associated with this type of sampling and the lack of established standards for levels of mold or mold spores in the air. Instead, it is recommended that ongoing sources of water (roof leaks, leaking pipes) be repaired, mold-contaminated material be removed or cleaned by qualified personnel, and/or the pathway between the mold source and building occupants be identified and removed or blocked.

Evaluating Bioaerosols in the Indoor Environment
Airborne and/or settled particulate material of microbial, plant, or animal origin is often referred to collectively as bioaerosols or organic dust. Bioaerosols can consist of pathogenic and non-pathogenic microorganisms, toxins, and allergens.

The effects of exposure to bioaerosols can include infectious diseases, acute toxic effects, allergies, stress, and other disorders. Infections result from the multiplication and growth of microbes in the human body, while allergies result from exposures to antigens. Antigens are capable of stimulating the production of antibodies that induce allergic reactions. Simply put, an allergic reaction is the result of the body's immune system responding to an antigen.

Assessment of bioaerosol exposure offers challenges distinct from those for inorganic aerosols and chemical agents. Measurement of microorganisms relies upon collection of a sample into or onto solid, liquid, or agar media followed by microscopic, microbiologic, biochemical, immunochemical or molecular biological analysis.

Bioaerosols may exist in both viable (living) and nonviable states. Generally, there are two approaches for evaluation of microbial exposure: culture methods and non-culture methods. Most methods attempt to sample for only viable particles because these can be cultured so that they multiply, making identification easier. Counting culturable microorganisms is a very sensitive quantification method that also permits identification of species. However, viable sampling is limited to short sampling times (to reduce loss of viability) and may introduce significant measurement error. Although dead or non-culturable microorganisms and specific microbial components are not detected by culture methods, bioaerosols in the nonviable state may have potential toxic or allergic properties. Spores, which can be formed by fungi and by certain bacteria, can be both viable and nonviable and are capable of causing disease in both forms.

Non-culture methods attempt to enumerate organisms without regard to viability, using microscopy for counting spores or cells. They allow full work shift measurements but have specific problems, such as a limited potential for quantitative identification and low counting accuracy.

Advanced methods such as PCR-based technologies, FISH, and immunoassays have opened new avenues for detection and speciation regardless of whether organisms are culturable. Also, specific bioaerosol associated agents can be measured using specific immunoassays, other bioassays, or GC/MS (gas chromatography and mass spectrophotometry.) These agents may either be directly pathogenic (such as allergens, bacterial endotoxin, and fungal mycotoxins) or may be general markers of exposure (such as ergosterol, fungal extracellular polysaccharides). Unfortunately, there are no currently available methods for many bioaerosols.

Storage and extraction procedures are critical in bioaerosol measurements, particularly for agents such as viruses, proteins, endotoxins, etc. Variability in bioaerosol exposure (both in space and time) is often great, and thus requires quite large measurement series to detect dose-response relationships and/or differences between contaminated and background areas. Additionally, bioaerosol exposures are ubiquitous, and all are exposed throughout their lives, which greatly complicates risk assessment. Therefore, few if any legal exposure limits exist for bioaerosol exposures.

Considerations of Bioaerosol Sampling Methods
Bioaerosol sampling is conducted in an attempt to discern whether the agents of concern are being generated within a building or from a naturally occurring, external source. The characteristics of viable bioaerosol agents require specialized sampling instruments in order to preserve the organisms for laboratory culture and evaluation. Their temperature, moisture, nutrient needs, and their relative fragility are primary considerations when selecting sampling instrumentation. Users' knowledge of the strengths and imitations of each sampling method is crucial to achieving valid results and to implementing safe, healthful, and effective control strategies.

Methods for collecting bioaerosol samples in the indoor environment are classified as passive or active. Passive air sampling methods are straightforward and can be done by simply setting out plates containing appropriate culture media. Such settling plates may be compromised by exogenous air currents and by contamination from external and human origins. Passive methods, moreover, are not as effective as more active sampling methods. Active sampling methods use traditional sampling trains consisting of a personal air sampling pump connected by tubing to appropriate media, along with a flow calibration device. Specialized instruments may also be available.

Active air sampling instrumentation falls into two main categories, inertial impactors and a group consisting of impingers, rotating impactors, and filters. Most active air sampling methods rely on impaction. The slit sampler and the cascade impactor are examples of inertial impactors and are typically used for screening evaluations because they have short sampling times and accommodate a wide variety of organisms to be collected. Collection can be performed over a short sampling time, because the media can be examined directly under a microscope or a variety of strips or plates containing different culture media can be used sequentially. Impingers collect material from the air in a liquid medium that can be transported to the laboratory for appropriate analysis. Filter instruments collect samples directly onto filter paper media.

Sampling of Bioaerosols on Inertial Impactors
Inertial impactors--such as the multi-stage cascade impactor--can separate viable particles into respirable and non-respirable particles. They are available from several reputable manufacturers.

The primary use of these impactors is in environments where airborne concentrations are expected to be low, and thus they are particularly suited to indoor air evaluations. The typical impactors designed for collecting microbial samples are six-stage, two-stage, and single-stage impactors. Others, such as the eight-stage impactor, may be available for specialized purposes and collection of nonviable particles.

Cascade Impactors are made up of classification stages consisting of a series of nozzles and an impaction surface. At each stage an aerosol stream passes through the nozzles and impinges upon the surface. Particles in the aerosol stream with a large enough inertia will impact upon the plate. Smaller particles pass on to the next stage. By designing stages with higher aerosol velocities in the nozzles, smaller diameter particles are collected at each stage. The particle size range collected at each of the stages depends on the orifice velocity of the specific stage, the distance between the orifices and the collection surface, and the collection characteristics of the preceding stage.

The combination of a constant flow rate and successively smaller diameter orifices increase the velocity of sample air as it cascades through the sampler, resulting in the impaction of progressively smaller particles in the succeeding stages. At 1 ACFM, the particle fractionation ranges from 10.0 to 0.4 micrometers in diameter. Particles too small to be impacted on the last collection plate are collected in a backup filter.

As air is drawn through an inertial impactor, it passes through orifices and strikes the surface of an agar collection medium. As air passes through the orifices at a calibrated rate of 1 cfm, the velocity increases at a rate inversely proportional to the area of the orifice: the smaller the orifice, the higher the velocity. When the velocity of a particle in the air stream is sufficiently large, its inertia will overcome its aerodynamic drag and the particle will leave the air stream to be impacted on the surface of the agar media. Particles not reaching a sufficient velocity for impaction will remain in the air stream and proceed to the next lower stage of the impactor.

In a multi-stage impactor. the orifice size decreases in each subsequent stage. Each stage of a standard six-stage inertial impactor contains a metal plate with 400 orifices of the same size. The impactor separates particles into six aerodynamic ranges, approximating the settling ranges in the human respiratory system. Thus, probable point of respiratory deposition, particle behavior, type of control equipment needed for particle collection, and compliance with OSHA, EPA regulations, and Threshold Limit Values (TLV) can be determined. A standard petri dish containing agar (a nutrient media) is located below each stage of the impactor. The stages are held together by a series of clamps or pins.

The two-stage impactor has only 200 orifices per stage; the collected particles on the first stage are considered to be in the non-respirable range and particles on the second stage are considered to be in the respirable range. When compared to the six-stage impactor, the two-stage impactor is considered to have decreased counts. Impactors with fewer stages are expedient for conducting screening evaluations of large areas, due to the shorter sampling times and reduced media requirements. The single stage impactor is an efficient bioaerosol sampler, collecting airborne bacteria and fungi from 0.65 microns to 22 microns.

Small impactor devices are available for personal microbial sampling. The personal impactor is designed to be worn by an individual, giving aerodynamic particle size distributions from 0.4 to 21 microns. It is adaptable to commercially available sampling pumps. Flow enters the inlet cowl and accelerates through radial slots in the first impactor stage. The cowl eliminates ash and debris from the sampler. Particles larger than the cut-point of the first stage impact on the precut collection substrate. Then, the air stream flows through the narrower slots in the second impactor stage, smaller particles impact on the second collection substrate, and so on. The width of the radial slots is constant for each stage but smaller for each succeeding stage. Thus, the jet velocity is higher for each succeeding stage, and smaller particles eventually acquire sufficient momentum to impact on one of the collection substrates. A final filter collects remaining aerosol analyte.

Prior to sampling, collection substrates and back-up filters are weighed and placed in the impactor. The sampling flow rate of the personal sampling pump is set at 2 lpm. The impactor's personal mounting bracket is attached to the individual's lapel or pocket. After sampling, the substrates and filter are weighed. Weight increases on each substrate are the mass of particles in the size range of that impactor stage. The total weight of particles on all stages and filter is added and the percent particle mass in each size range is calculated. Respirable particle mass fraction is determined from the particle size distribution.

Devices such as the slit impactor and the rotating impactor are typically used for screening evaluations becuase their short sampling times allow for a wide variety of microorganisms to be collected over a short period of time. The slit impactor is a type of inertial impactor for collecting samples of bioaerosols. The slit sampler is a battery-powered, field-portable device for collection of samples directly onto microscope slides. Airflow is typically calibrated at 10 lpm. The slides may be used without an adhesive if the sample is not to be preserved for future reference. The slides are examined directly under a microscope. A variety of sampling strips of the rotating impactor may be used sequentially.

Successful bioaerosol sampling in a problem building depends on what the investigator determines to be relevant issues, the availability of sampling instrumentation and associated services, and the needs and/or preferences of building management. As in all other investigations, the likelihood of obtaining meaningful results is dependent on clearly defined objectives.

The selection of the most appropriate exposure assessment method(s) for bioaerosols is highly dependent on the specific goals of the evaluation and the specifics of the affected environment. The interpretation of bioaerosol measurement results is difficult without detailed information about the sampling and analytical procedures used. In addition, the development of legal exposure limits is restricted by uncertainty in exposure assessment methods and the complexity of risk assessment.

For further information, readers are referred to "Bioaerosols: Assessment and Control," a comprehensive publication on biologically derived airborne contaminants available from the American Conference of Governmental Industrial Hygienists (

This article originally appeared in the May 2003 issue of Occupational Health & Safety.

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