Direct Detection Methods for Erysipelothrix, Lactobacillus, and Similar Organisms
المؤلف:
Patricia M. Tille, PhD, MLS(ASCP)
المصدر:
Bailey & Scotts Diagnostic Microbiology
الجزء والصفحة:
13th Edition , p290-291
2025-06-24
751
Gram staining of Arcanobacterium spp. demonstrates delicate, curved, gram-positive rods with pointed ends and occasional rudimentary branching. This branching is more pronounced after these organisms have been cultured anaerobically. Arcanobacterium spp. stain unevenly after 48 hours of growth on solid media and also exhibit coccal forms.
Lactobacillus is highly pleomorphic, occurring in long chaining rods and in coccobacilli and spiral forms (Figure 1).
Fig1. Gram stain of Lactobacillus spp. Note spiral forms (arrow).
E.rhusiopathiae stains as both short rods and long filaments. These morphologies correspond to two colonial types: (1) rough colonies that contain slender, filamentous, gram-positive rods with a tendency to overdecolorize and appear gram negative and (2) smooth colonies that contain small, slender rods. This variability in staining and colonial morphology may be mistaken for a poly microbial infection both on direct examination and culture.
Gardnerella organisms are small, pleomorphic gram variable or gram-negative coccobacilli and short rods. Wet mount and Gram staining of vaginal secretions are key tests for diagnosing bacterial vaginosis caused by G. vaginalis. A wet mount prepared in saline reveals the characteristic “clue cells,” which are large, squamous epithelial cells with numerous attached small rods. A Gram-stained smear of the discharge shows the attached organisms to be gram-variable coccobacilli. In bacterial vaginosis, clue cells are typically present, and large numbers of other gram-positive rods (i.e., lactobacilli), representing normal vaginal flora, are absent or few in number. In addition, the BDaffirm vaginal DNA probe (VDP) may be used for direct detection from genital specimens. Special vials containing transport reagent are used to stabilize the organ ism’s nucleic acids prior to testing (Becton, Dickinson and Company Franklin Lakes, NJ).
Cultivation
Media of Choice. All the genera described in this chapter grow on 5% sheep blood and chocolate agars. They do not grow on MacConkey agar but do grow on Columbia colistin-nalidixic acid (CNA) agar. CNA agar is a nutritional base that may include 5% sheep blood to enhance the growth of fastidious organisms. The antibiotics colistin and nalidixic acid prevent the overgrowth of gram-negative organisms. All genera except Gardnerella sp. grow in commercially available blood culture broths. Gardnerella organisms are inhibited by sodium polyanetholsulfonate (SPS), which currently is used as an anticoagulant in most commercial blood culture media. An SPS-free medium or a medium with SPS that is supplemented with gelatin should be used when G. vaginalis sepsis is suspected.
Isolation of G. vaginalis from female genital tract specimens is best accomplished using the selective medium human blood bilayer Tween agar (HBT). HBT is CNA agar with amphotericin B added to prevent the growth of yeasts and filamentous fungi. Human blood is layered over the top to enhance the beta-hemolytic pattern of G. vaginalis.
Incubation Conditions and Duration
Detectable growth of these organisms should occur on 5% sheep blood and chocolate agars, CNA, and HBT incubated at 35°C in 5% to 10% carbon dioxide (CO2) within 48 hours of inoculation.
Colonial Appearance
Table 2 describes the colonial appearance and other distinguishing characteristics (e.g., hemolysis) of each genus on sheep blood agar. G. vaginalis produces small, gray, opaque colonies surrounded by a diffuse zone of beta-hemolysis on HBT agar (Figure 2).
Table2. Colonial Appearance on 5% Sheep Blood Agar and Other Characteristics
Fig2. Gardnerella vaginalis on human blood bilayer Tween (HBT) agar. Note small colonies with diffuse zone of beta-hemolysis (arrow).
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