Interpret as shown in Figure 45-4.
along with suspensions of the unknown mycobacteria being tested.
Growth Rate. The rate of growth is an important criterion for determining the initial category of an isolate. Rapid-growers usually produce colonies within 3 to 4 days after subculture. Even a rapid-grower, however, may take longer than 7 days to initially produce colonies because of inhibition by a harsh decontaminating procedure. Therefore, the growth rate (and pigment production) must be determined by subculture; the method is described in Procedure 45-7. The dilution of the organism used to assess growth rate is critical. Even slow-growing mycobacteria appear to produce colonies in less than 7 days if the inoculum is too heavy. One organism particularly likely to exhibit false-positive rapid growth is M. flamcens. This species therefore serves as an excellent quality control organism for this procedure.
Pigment Production. As previously discussed, mycobacteria may be categorized into three groups based on pigment production. Procedure 45-7 describes how to determine pigment production. To achieve optimum photochromogenicity, colonies should be young, actively metabolizing, isolated, and well-aerated.34 Although some species, such as M. kansasii, turn yellow after a few hours of light exposure, others, such as Ai. ùmiae, may take prolonged exposure to light. Scoto-chromogens produce pigmented colonies even in the absence of light, and colonies often become darker with Prolonged exposure to fight (Figure 45-4). One member °f this group, M. szulgai, is peculiar in that it is a scoto-chromogen at 35° C and nonpigmented when grown at
25° to 30° C. For this reason, all pigmented colonies should be subcultured to test for photoactivated pigment at both 35° C and 25° to 30° C. Nonchromogens are not affected by light.
Biochemical Testing. Once placed into a preliminary subgroup based on its growth characteristics, an organism must be definitively, identified to species or complex level. Although conventional biochemical tests can be used for this purpose, new methods (discussed later in this section) have replaced biochemical tests for the identification of mycobacterial species because of the previously discussed limitations of phenotypic testing. Although key biochemical tests are still discussed in this edition, the reader must be aware that this approach to identification will be ultimately replaced with molecular methods. Table 45-10 summarizes distinctive properties of the more commonly cultivable mycobacteria isolated from clinical specimens; key biochemical tests for each of the major mycobacterial groupings, including M. tuberculosis complex, are listed in Table 45-11. Key biochemical tests are discussed in the following text; detailed procedures are given in other texts.1,5
Niacin. Niacin (nicotinic acid) plays an important role in the oxidation-reduction reactions that occur during mycobacterial metabolism. Although all species produce nicotinic acid, M. tuberculosis accumulates the largest amount. (M. simiae and some strains of M. chelo-nae also produce niacin.) Niacin therefore accumulates in the medium in which these oiganisms are growing. A positive niacin test is preliminary evidence that an
Figure 45-4 Imtial grouping of mycobacteria based on pigment production before and after exposure to light. In one test ystem, subcultures of each isolate are grown on two agar slants. One tube is wrapped in aluminL foil to Severn exposte,
5" JH k "tUb!iS a,1°Wed Hght eXp0SUre- ^ient growth is present, the JSJS Xis
M?^i Th^ examined'°eether- Photochromogens are unpigmented when grown in the dar^ube A> id S2LP r 8 .TT (tUbe B)" S«>iochromogens are pigmented in the dark (tube C); the color does not ntensHy after exposure to light (tube D). Nonphotochromogens are nonpigmented when grown in the dark (tube E) 1 remam so even after light exposure (tube F). '
organism that exhibits a buff-colored, slow-growing, rough colony may be M. tuberculosis (Figure 45-5). The method is delineated in Procedure 45-8. This test is not sufficient, however, for confirmation of the identification. If sufficient growth is present on an initial L-J slant (the egg-base medium enhances accumulation of free niacin), a niacin test can be performed immediately. If growth on the initial culture is scanty, the subculture used for growth rate determination can be used. If this culture yields only rare colonies, the colonies should be spread around with a sterile cotton swab (after the growth rate has been determined) to distribute the inoculum over the entire slant. The slant is then rein-cubated until light growth over the surface of the medium is visible. For reliable results, the niacin test should be performed only from cultures on L-J that are at least 3 weeks old and show at least 50 colonies; other wise, enough niacin might not have been produced to be detected.
Nitrate Reduction. This test is valuable for the identification of M. tuberculosis, M. kansosii, M. szulgafy and M.fortuitum. The ability of acid-fast bacilli to reduc| nitrate is influenced by age of the colonies, temperature; pH, and enzyme inhibitors. Although rapid-growers can be tested within 2 weeks, slow-growers should be tested after 3 to 4 weeks of luxuriant growth. Commercial^ available nitrate strips yield acceptable results only witl§ strongly nitrate-positive organisms, such as M. tubefc culosis. This test may be tried first because of its ease og performance. The M. tuberculosis-positive control muîÊ be strongly positive in the strip test or the test results vw$ be unreliable. If the paper strip test is negative or if thÉl control test result is not strongly positive, the chemiGÉ
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