Diagnosis of Y ruckeri may be achieved by isolation of the pathogen such as on

Diagnosis of y ruckeri may be achieved by isolation

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Diagnosis of Y. ruckeri may be achieved by isolation of the pathogen, such as on the selective media of Waltman and Shotts (1984) or Rodgers (1992), and thence identification. According to Waltman and Shotts (1984), 53/60 isolates hydrolysed Tween 80, but none fermented sucrose. Therefore, typically on the selective medium, Y. ruckeri colonies were green with a zone of hydrolysis (indicated by the presence of insoluble calcium salts) around them. Unfortunately, in our experience with this medium U.K. isolates rarely hydrolysed Tween 80. Therefore, interpretations should be made carefully. Wakabayashi and Egusa (1972) proposed an identification scheme for Ps. anguilliseptica based on a small number of phenotypic traits, principally motility, growth at 37°C, presence of soluble pigment, production of H2S, indole and oxidase, nitrate reduction, gelatin degradation, susceptibility to the vibriostatic agent (O/ 129), and the ability to attack glucose. According to these workers, the tests were sufficient to differentiate Ps. anguilliseptica from Ps.fiuorescens, Ps. alcaligenes, V. anguillarum, Aer. liquefaciens (= Aer. hydrophila), Ph. damselae subsp. piscicida and H. piscium.
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Diagnosis 231 A simplified diagnostic test for V. anguillarum, involving "glucose motility deeps" (GMD) has been reported (Walters and Plumb, 1978). Essentially, GMD is a much modified version of the oxidation-fermentation test medium, comprising: Phenol red broth base (Difco) 1.6% (w/v) Glucose 1.0% (w/v) Yeast extract 0.3% (w/v) Agar 0.3% (w/v) Stab-inoculated media are incubated at 25°C for 24-48 h, when acid production and motility (indicated as a carrot-like diffuse growth around the stab mark) are recorded. It remains for further work to confirm the specificity of the reaction for V. anguillarum. Colony morphology and pigmentation This should be recorded from "young" colonies, i.e. shortly after growth is initially detected. The presence of aerial hyphae may be assessed with a stereo-microscope. The presence of pigment should be assessed from basal medium supplemented with 5-10% (w/v) skimmed milked powder (Oxoid). The Gram-staining reaction With smears from young cultures, this reaction serves also to determine the presence of rods, cocci, mycelia, microcysts and endospores. For convenience, we recommend the use of commercially available staining and decolorising solutions, such as those marketed by Difco. Heat-fixed smears should be stained for 1 min with crystal violet, washed in tap water, covered with Gram's iodine for 1 min, re-washed, decolorised by a few seconds in acetone-alcohol, and counterstained for 30 sec in safranin. The smears are washed thoroughly, and gently blotted dry, prior to microscopic examination preferably at a magnification of x 1,000. The acid-fast staining reaction This reaction highlights the presence of Mycobacterium, Nocardia and possibly Rhodococcus. Heat-fixed smears may be flooded with carbol fuchsin, and heated until the steam rises by means of wafting a source of heat (from a Bunsen burner or cotton wool plug soaked with alcohol) underneath the sHde. After 5 min, the stain
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  • Spring '20
  • Bacteria, representative, gram-negative bacteria

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