Cultivation of Listeria, Corynebacterium, and Similar Organisms
المؤلف:
Patricia M. Tille, PhD, MLS(ASCP)
المصدر:
Bailey & Scotts Diagnostic Microbiology
الجزء والصفحة:
13th Edition , p277-280
2025-06-03
642
Media of Choice
Corynebacterium spp. usually grow on 5% sheep blood and chocolate agars. Some coryneform bacteria do not grow on chocolate agar, and the lipophilic (lipid loving) species (e.g., C. jeikeium, C. urealyticum, C. afermentans subsp. lipophilum, C. accolens, and C. macginleyi) produce much larger colonies when cultured on 5% sheep blood agar supplemented with 1% Tween 80 (Figure 1).
Fig1. Corynebacterium urealyticum on blood agar with Tween 80 (A) and blood agar (B) at 48 hours. This organism is lipophilic and grows much better on the lipid-containing medium.
Selective and differential media for C. diphtheriae should be used if diphtheria is suspected. The two media commonly used for this purpose are cystine-tellurite blood agar and modified Tinsdale agar (TIN). Tellurite blood agar maybe used with or without cystine. Cystine enhances the growth of fastidious organisms, including C. diphtheriae. Both media contain a high concentration of potassium tellurite that is inhibitory to normal flora. Organisms capable of growing on Tinsdale agar are differentiated based on the conversion of the tellurite to tellurium. This conversion results in color variations of grey to black colonies on the two media. C. diphtheriae also produces a halo on both media. C. diphtheriae can be presumptively identified by observing brown-black colonies with a gray-brown halo on Tinsdale agar (Figure2). The brown halo is produced when the organism uses tellurite to produce hydrogen sulfide. The halo produced on cystine-tellurite blood agar appears brown as a result of the organism breaking down the cystine. In addition, Loeffler medium, which contains serum and egg, stimulates the growth of C. diphtheriae and the production of metachromatic granules in the cells. C. diphtheriae grows rapidly on the highly enriched agar and produces gray to white, translucent colonies within 12 to 18 hours. Primary inoculation of throat swabs to Loeffler serum slants is no longer recommended because of the inevitable overgrowth of normal oral flora.
Fig2. Colony of Corynebacterium diphtheriae on Tinsdale agar. Note black colonies with brown halo.
Corynebacterium spp. are unable grow on MacConkey agar. They all are capable of growth in routine blood culture broth and nutrient broths, such as thioglycollate or brain-heart infusion. Lipophilic coryneform bacteria demonstrate better growth in broths supplemented with rabbit serum.
Incubation Conditions and Duration
Detectable growth of corynebacterium on 5% sheep blood and chocolate agars, incubated at 35°C in either ambient air or in 5% to 10% carbon dioxide, should occur within 48 to 72 hours after inoculation. The lipophilic organisms grow more slowly; it takes 3 days or longer to identify visible growth on routine media. For growth of C. diphtheriae, cystine-tellurite blood agar and modified Tinsdale agar should be incubated for at least 48 hours in ambient air. Five percent to 10% carbon dioxide (CO2) retards the formation of halos on TIN agar.
Colonial Appearance
Table 1 describes the colonial appearance and other distinguishing characteristics (e.g., hemolysis and odor) of each clinically relevant genus or species of corynebacteria on blood agar. Colonies of C. diphtheriae on cystine tellurite blood agar appear black or gray, whereas those on modified Tinsdale agar are black with dark brown halos (see Figure 2). C. diphtheriae colonies may be recognized by one of four varieties of colony morphologies. These colony types are referred to as gravis, intermedius, belfanti, and mitis, based on the phenotypic characteristics of size, texture, color, hemolysis, and the presence of metachromatic granules.
Table1. Gram Stain Morphology, Colonial Appearance, and Other Distinguishing Characteristics
Table1. Gram Stain Morphology, Colonial Appearance, and Other Distinguishing Characteristics—cont’d
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