indicates a need to identify and eliminate the source of contamination. Concentrations ranging from 4,500-10,000 CFU/m3 have been suggested as the upper limit for ubiquitous bacterial aerosols [Nevalainen 1989; ACGIH 1989, Bacteria]. These exposure limits, however, do not apply to pathogenic microorganisms. Actinomycetes (mesophilic and thermophilic) are commonly found in agricultural areas. Their presence in indoor environments is an indicator of contamination [ACGIH 1989, Bacteria; Banaszak et al. 1970; Lacey and Crook 1988]. Thermophilic Actinomycetes at concentrations above 70 CFU/m3 in an affected person's work area have been regarded as the threshold for triggering remedial action [Otten et al. 1986].
5.
ADDITIONAL IDENTIFICATION AND ENUMERATION PROCEDURES FOR NONVIABLE OR VIABLE BIOAEROSOLS Classifying nonviable and nonculturable microorganisms cannot be performed using the methods described in the previous section. Identification of nonviable or nonculturable microorganisms or components of microorganisms can be performed using microscopy and molecular biology techniques. In addition, microscopy techniques may be used for enumeration of suspensions of viable and nonviable microorganisms [McCrone 1973]. a.
Microscopy (1)
Bright-Field or Light In bright-field or light microscopy, an ordinary microscope is used for simple observation or sizing. Visible light from an incandescent source is used for illumination and the specimen appears against a bright backfield. Objects smaller than 0.2 µm cannot be resolved. The image contrast (visibility) decreases as the refractive index of the substance/microorganism under observation and the mounting medium become similar. To maximize the contrast, the mounting medium should have the same refractive index as glass or the immersion oil. Membrane filters are often "cleared" by using the appropriate immersion oil. This method is commonly used to observe various stained (killed) specimens and to count microorganisms. In addition, pollen grains and fungi spores are often identified and enumerated in this manner [Eduard et al. 1990].
(2)
Phase Contrast Phase-contrast microscopy is used when the microorganism under observation (e.g., Escherichia coli) is nearly invisible and an alternative mounting medium is not possible or permissible. A phase-contrast microscope uses a special condenser and diffraction plate to diffract light rays so that they are out of phase with one another. The specimen appears as different degrees of brightness and contrast. One cannot see an object exactly matching the refractive index of the mounting liquid; however, very slight differences produce visible images. This type of microscope is commonly used to provide detailed examination of the internal structures of living specimens; no staining is required.
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