Bioaerosols and Disease Donald E Gardner PhD

7 Sampling and Identifying Airborne Microbial Contaminants

Before commencing extensive sampling for the presence of airborne microorganisms, medical evidence should suggest the occurrence of infectious or allergic disease among the workers that appears to be related to the workplace. Significant exposure to aerosolized infectious agents in the workplace can be suspected when there are (1) several important sources or reservoirs, (2) amplifiers or conditions favoring microbial survival, (3) highly susceptible individuals or known carriers, (4) complaints or epidemics of disease, and (5) a microbiological laboratory report of positive cultures. Of special concern are those environments with high occupant density possibly resulting in increased risk of the airborne transmission of infectious agents between individuals. An on-site inspection may aid in uncovering the potential source of biological contamination, which can be confirmed using standard microbiological techniques. The ventilating system should be examined for appropriate design, operation, and maintenance. Evidence of microbial growth in cooling coils and on wet surfaces can indicate a source of biological contamination and a possible microbial reservoir. In some cases, bioaerosol sampling may not be necessary if there is substantial evidence of visible microbial growth. With evidence of microbial growth on floors, walls, or ceilings, or in the ventilation systems, sampling for bioaerosols and appropriate remediation should be considered. Sampling for microorganisms should be undertaken, especially when medical evidence suggests the occurrence of disease. A careful on-site inspection can be of significant value in designing appropriate air sampling methods. Once the purpose or the goal of bioaerosol sampling is determined, appropriate sampling method(s) should be chosen. Sampling for bioaerosols in the workplace can provide useful information necessary to characterize the exposure conditions, determine whether the contamination represents a potential or real hazard, and establish the need for control measures.

Bioaerosol monitoring is a rapidly emerging area of industrial hygiene. Airborne sampling for bioaerosols is complex, expensive, and may include measuring bacteria, viruses, fungal spores, endotoxins, allergenic and toxic substances of plant and animal origin, and protein aerosols (3). The airborne concentration may be expressed in different ways, depending on the type of particle. Bacteria and fungal spores, for example, may be expressed in terms of the number of bacteria entities of a given type per unit volume of air (i.e., number per m of air). On the other hand, viable particles may be expressed in terms of their ability to reproduce—that is, the number of "colony-forming units" per unit of volume of air (i.e., cfu per m of air). For endotoxin and allergenic materials, it is appropriate to express concentration in terms of the mass of the active component per unit volume of air (i.e., mg per m of air) Such diversity presents many difficulties in sampling and measurement methodology for exposure assessment.

Although there are no standard methods for sampling and analysis of microbiological agents in air, the same principles that apply to measuring and collecting any particulate aerosol also govern air sampling for microorganisms (101). The sampling methods most commonly used to collect airborne microbes are described in detail elsewhere (101-103). Methods commonly used include glass impingers, cascade impactors, sedimentation, and real-time samplers. A recent comprehensive review compares the various experimental, theoretical, and physical characteristics of the commonly used bioaerosol samplers (104).

The selection of the sampling methods and subsequent laboratory analyses are determined by the medium to be sampled (air, water, surface) and the type of agent to be detected (6). Samplers used to isolate viable microbes in the air must be capable of collecting the aerosol with high efficiency, must minimize injury to the organism during the collection process, and must maintain the culturability of the collected microorganisms. In monitoring viable microorganisms, only culturable microorganisms are enumerated and identified, thus possibly leading to an underestimation of bioaerosol concentrations.

Aeroallergens (pollen and fungi) may be sampled without culturing the microorganisms. This can be done using passive aerobiological monitors that rely on gravity collection on an appropriate medium or on some collection surface. Then the collected microorganisms can be enumerated and identified using microscopy, classical microbiology, molecular biology, or immunochemical techniques. With this method, large particles are collected more effectively than small particles. These passive monitors can be used either as area or personal monitors, but they provide only semiquantitative information because only particles of certain dimensions will settle onto the surface in a given time and there is no way of knowing the volume of air that is being sampled.

In sampling culturable bacteria and fungi, the bioaerosol is generally collected by impaction onto the surface of a solid medium (agar), filtration through a membrane filter, or impingement into an isotonic liquid medium. Such an air sampler consists of a pump that draws a known quantity of air over or through a collection surface that contains appropriate growth or collection medium for the organism suspected of being present.

After impaction onto a medium surface and incubation, the organisms may be transferred onto selective or differential media and incubated at different temperatures for identification and enumeration of the microbes (105). When using collection fluids, the sample can be placed directly on agar or serially diluted and plated, or the entire volume of fluid can be filtered through a membrane filter. The membrane filter is then placed on an appropriate growth medium. Microbes may be identified by using microscopy, classical microbiology, or molecular biology techniques such as restriction fragment length polymorphic (RFLP) analysis. Classical microbiology techniques include observation of growth characteristics, cellular or spore morphology, simple and differential staining, and biochemical, physiological, and nutritional tests for bacteria. DNA analytical techniques which may be applied to both nonviable and viable microorganisms include polymerase chain reaction and enzyme-linked immunosorbent assay. Such methods may be used to identify specific microorganisms and to locate areas of contamination. Concentrations of endotoxin determined by using the Limulus amebocyte lysate assay method have been correlated with patient symptoms in some studies (105).

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