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 air sampled into the adjusted number of colonies observed on the plate (see section 2.b.). (3)

Limitations Bioaerosol collection methods are “grab sample” techniques and, thus, represent only approximations of transient microbial concentrations in problematic atmospheres. Timely ascertainment of bioaerosol involvement is not possible, because of the timedependent nature of the cultivation of samples and the subsequent enumeration of colonies. The methods thus far pertain to culturable microorganisms. Microorganisms that are stressed or injured either by environmental conditions or bioaerosol sampling procedures may be viable, but not culturable [McFeters et al. 1982]. Certain species may be too fastidious to grow in laboratory culture. For instance, some bioaerosols (e.g., Legionella pneumophila, Histoplasma capsulatum or Pneumocystis carinii) are very difficult, if not impossible, to collect and culture [Ibach et al. 1954; Dennis 1990].

c.

Identification of Culturable Bioaerosols The science of classification, especially the classification of living forms, is called taxonomy. The objective of taxonomy is to classify living organisms to establish the relationship between one group of organisms and another, and to differentiate between them. Several criteria and methods for the classification of culturable microorganisms and the routine identification of some are discussed in the subsections that follow. Besides using these methods, the nonviable and nonculturable methods of identification discussed in Section 5 also may be used with culturable microorganisms. (1)

Classical or General Microbiology Classical microbiology includes general methods for classifying or identifying microorganisms. The least specific of these is the observation of growth characteristics. Growth characteristics include the appearance of the microorganisms in liquid medium, colonial morphology on solid medium, and pigmentation. On the cellular level of bacteria, cell shape, cell size, arrangement of cells, and presence (absence) of flagella, capsule, or endospores are characteristic of general classes of microorganisms. Simple and differential staining may be performed on bacteria. Simple staining is a method of staining microorganisms with a single basic dye that highlights cellular size, cellular shape, cellular arrangement, and presence (absence) of flagella, capsule, or endospore using a microscope. Stains such as methylene blue, carbolfuchsin, crystal violet, or safranin may be used for bacteria. A stain that distinguishes among structures or microorganisms based on reactions to the staining procedure is called a differential stain. Two examples of differential stains are the Gram stain and the acid-fast stain. The mechanism of the Gram stain may be explained on the basis of physical differences in the cell walls of these two general groups of bacteria (Gram-positive and Gram-negative). The Gram-positive bacteria possess a cell wall composed of a relatively

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NIOSH Manual of Analytical Methods