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 thick peptidoglycan layer and teichoic acids. Gram-negative bacteria possess a cell wall composed of a thin peptidoglycan layer and an outer membrane which consists of lipoproteins, lipopolysaccharides, and phospholipids [Tortora et al. 1989]. A few of the commercially available identification kits require a Gram-stain prescreening to assure that the correct reagents are used. Some species of bacteria, particularly those of the genus Mycobacterium, do not stain readily. In the acid-fast staining process, the application of heat facilitates the staining of the microorganism. In general, fungi are classified by spore morphology or colonial morphology. Stains such as lactophenol cotton blue, periodic acid-Schiff stain, or potassium hydroxide (10% KOH) may be used. Biochemical, physiological, and nutritional tests for bacteria evaluate cell wall constituents, pigment biochemicals, storage inclusions, antigens, temperature range and optimum, oxygen relationships, pH tolerance, osmotic tolerance, salt requirement and tolerance, antibiotic sensitivity, energy sources, carbon sources, nitrogen sources, fermentation products, and modes of metabolism (autotrophic, heterotrophic, fermentative, respiratory). As a rule, batteries of such tests, rather than any one individual test, are used to identify or classify microorganisms. A few commercially available test batteries are discussed in the following subsection. Fungi are very difficult to classify [Smith 1990]. (2)

Clinical and Environmental Microbiology All identification systems should permit the efficient and reliable differentiation between microorganisms. Several modifications of classical biochemical procedures have been used in recent years to facilitate inoculation of media, to decrease the incubation time, to automate the procedure, and to systematize the determination of species based on reaction patterns. Historically, clinical microbiological techniques are used for analysis of environmental samples. However, clinical strains and environmental isolates may differ, requiring modification of clinically-based techniques. (I)

Biochemical Analyses Several commercial multitest systems have been developed for identification of members of the family Enterobacteriaceae and other pathogenic microorganisms because of the high frequency of isolation of Gram-negative rods in clinical settings. These microorganisms are indistinguishable except for characteristics determined by detailed biochemical testing. These systems require that a pure culture be examined and characterized. Following is a listing of commercially available identification kits: API® 20E (Analytab Products, Plainview, NY); Enterotube II and R/B Enteric (Roche Diagnostics Systems, Nutley, NJ; Hoffmann-La Roche & Co., AG, Basel, Switzerland); Micro-ID (Organon Teknika-Cappel, Durham, NC); Minitek and Sceptor (BBL Microbiology Systems, Cockeysville, MD); and MicroScan (American Microscan, Inc., Sacramento, CA). Automated identification systems include Quantum II (Abbott Laboratories, North Chicago, IL), Autobac IDX (General

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NIOSH Manual of Analytical Methods