Cultivation of Enterobacteriaceae
المؤلف:
Patricia M. Tille, PhD, MLS(ASCP)
المصدر:
Bailey & Scotts Diagnostic Microbiology
الجزء والصفحة:
13th Edition , p314-317
2025-07-08
525
Media of Choice
Most Enterobacteriaceae grow well on routine laboratory media, such as 5% sheep blood, chocolate, and MacConkey agars. In addition to these media, selective agars, such as Hektoen enteric (HE) agar, xylose-lysine-deoxy cholate (XLD) agar, and Salmonella-Shigella (SS) agar, are commonly used to cultivate enteric pathogens from gastrointestinal specimens. The broths used in blood culture systems, as well as thioglycollate and brain heart infusion broths, all support the growth of Enterobacteriaceae.
Cefsulodin-irgasan-novobiocin (CIN) agar is a selective medium specifically used for the isolation of Y. enterocolitica from gastrointestinal specimens. Similarly, MacConkey-sorbitol agar (MAC-SOR) is used to differentiate sorbitol-negative E. coli O157:H7 from other strains of E. coli that are capable of fermenting this sugar alcohol.
Klebsiella granulomatis will not grow on routine agar media. Recently, the organism was cultured in human monocytes from biopsy specimens of genital ulcers of patients with donovanosis. Historically, the organism has also been cultivated on a special medium described by Dienst that contains growth factors found in egg yolk. In clinical practice, however, the diagnosis of granuloma inguinale is made solely on the basis of direct examination.
Table 1 presents a complete description of the laboratory media used to isolate Enterobacteriaceae.
Table1. Biochemical Media used in the Differentiation and Isolation of Enterobacteriaceae
Table1. Biochemical Media used in the Differentiation and Isolation of Enterobacteriaceae—cont’d
Table1. Biochemical Media used in the Differentiation and Isolation of Enterobacteriaceae—cont’d
Incubation Conditions and Duration
Under normal circumstances, most Enterobacteriaceae produce detectable growth in commonly used broth and agar media within 24 hours of inoculation. For isolation, 5% sheep blood and chocolate agars may be incubated at 35°C in carbon dioxide or ambient air. However, MacConkey agar and other selective agars (e.g., SS, HE, XLD) should be incubated only in ambient air. Unlike most other Enterobacteriaceae, Y. pestis grows best at 25° to 30°C. Colonies of Y. pestis are pinpoint at 24 hours but resemble those of other Enterobacteriaceae after 48 hours. CIN agar, used for the isolation of Y. enterocolitica, should be incubated 48 hours at room temperature to allow for the development of typical “bull’s-eye” colonies (Figure 1).
Fig1. Bull’s-eye colony of Yersinia enterocolitica (arrow) on cefsulodin-irgasan-novobiocin (CIN) agar.
Colonial Appearance
Table 2 presents the colonial appearance and other distinguishing characteristics (pigment and odor) of the most commonly isolated Enterobacteriaceae on MacConkey, HE, and XLD agars . All Enterobacteriaceae produce similar growth on blood and chocolate agars; colonies are large, gray, and smooth. Colonies of Klebsiella or Enterobacter may be mucoid because of their polysaccharide capsule. E. coli is often beta-hemolytic on blood agar, but most other genera are nonhemolytic. As a result of motility, Proteus mirabilis, P. penneri, and P. vulgaris “swarm” on blood and chocolate agars. Swarming results in the production of a thin film of growth on the agar surface (Figure 2) as the motile organisms spread from the original site of inoculation.
Table2. Colonial Appearance and Characteristics of the Most Commonly Isolated Enterobacteriaceae*
Fig2. Proteus mirabilis swarming on blood agar (arrow points to swarming edge).
Colonies of Y. pestis on 5% sheep blood agar are pin point at 24 hours but exhibit a rough, cauliflower appearance at 48 hours. Broth cultures of Y. pestis exhibit a characteristic “stalactite pattern” in which clumps of cells adhere to one side of the tube.
Y. enterocolitica produces bull’s-eye colonies (dark red or burgundy centers surrounded by a translucent border; see Figure 1) on CIN agar at 48 hours. However, because most Aeromonas spp. produce similar colonies on CIN agar, it is important to perform an oxidase test to verify that the organisms are Yersinia spp. (oxidase negative). The oxidase test should be performed on suspect colonies that have been subcultured to sheep blood agar (Table 1). Pigments present in the CIN agar will inter fere with correct interpretation of the oxidase test results.
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