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Fluorescence Fluorescence microscopy uses ultraviolet or near-ultraviolet source of illumination that causes fluorescent compounds in a specimen to emit light. Fluorescence microscopy for the direct count of microorganisms has been described in a number of studies. Direct-count methods to enumerate microorganisms found in soil, aquatic, and food samples have been developed using acridine orange [Palmgren et al. 1986a, Palmgren et al. 1986b, Karlsson and Malmberg 1989]. More recently, this method was applied to airborne microorganisms and it was concluded that it is of the utmost importance to combine viable counts with total count enumeration in the study of microorganisms in work-related situations [Palmgren et al. 1986a].

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Electron Electron microscopy uses a beam of electrons instead of light. Because of the shorter wavelength of electrons, structures smaller than 0.2 µm can be resolved. Scanning electron microscopy (SEM) is used to study the surface features of cells and viruses (usually magnified 1,000-10,000X); and the image produced appears three-dimensional. Also, SEM permits visualization of microorganisms and their structure (e.g., single spores or cells, clumps, chains, size, shape, or other morphological criteria). Viable microorganisms cannot be distinguished from nonviable microorganisms [Donham et al. 1986; Karlsson and Malmberg 1989]. Transmission electron microscopy is used to examine viruses or the internal ultrastructure in thin sections of cells (usually magnified 10,000-100,000X); and the image produced is not three dimensional.

b.

Endotoxin Assay A virulence factor possessed by all Enterobacteriaceae (as well as other Gram-negative bacteria) is the lipopolysaccharide, endotoxin, found in the outer membrane of the cell wall. Individuals may experience disseminated intravascular coagulopathy, respiratory tract problems, cellular and tissue injury, fever, and other debilitating problems. Amebocytes are carried “within the blood-like circulating fluid” of the Limulus polyphemus (horseshoe crab). After exposure to the lysed amebocyte cells, a liquid suspension containing trace levels of endotoxin (lipopolysaccharides) will gel. This test is called the Limulus amebocyte lysate (LAL) assay. Clinical microbiology laboratories use this assay to test for contamination by Gram-negative bacteria [Baron and Finegold 1990]. Airborne endotoxin has been found in high concentrations in agricultural, industrial, and office environments [Milton et al. 1990; Rylander and Vesterlund 1982]. Endotoxin aerosol measurement techniques lack comparability between results obtained in different laboratories because of differing sampling, extraction, and analytical methods (generically called the Limulus method) [Rylander and Vesterlund 1982; Olenchock et al. 1983; Jacobs 1989; Milton et al. 1990]. Concentrations of endotoxin, a lipopolysaccharide found in the cell wall of Gram-negative bacteria, determined using the LAL assay method, have been correlated with patient symptoms in very few studies [Rylander and Vesterlund 1982; Milton et al. 1990].

c.

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NIOSH Manual of Analytical Methods