A solution was proposed by Evelyn 1977 who advocated the use of a drop plating

A solution was proposed by evelyn 1977 who advocated

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organism. A solution was proposed by Evelyn (1977), who advocated the use of a drop-plating technique (essentially, this is analo- gous to dilution plates, which dilute out potential interference by fast-growing heterotrophs). In a later report, Evelyn (1978) recommended the use of peptone (0.1% w/v)-saline (0.85% w/v) as a diluent to remove any inhibiting factors against the pathogen, which may be present in kidney tissue (Evelyn et al., 1981; Austin, 1986). Some inconsistencies in the performance of KDM2 were attributed to varia- tion in the composition of the commercial peptone (Evelyn and Prosperi-Porta, 1989). To overcome this inconsistency when single lots of peptone were not available, two possible modifications were suggested. First, a "nurse" culture technique was reported. This technique accelerated the growth of the BKD organism, and increased the sensitivity at which the pathogen could be detected. The technique, which was based on satellitism or cross-feeding, involved inoculating the nutritionally fastidious pathogen next to a non-fastidious feeder—the nurse organism. Evelyn et al. (1989) placed drops of a dense suspension of a stock culture of the BKD organism (= the nurse organism) onto the centre of KDM2 plates. Samples, suspected of containing the pathogen, were placed as 25 |il drops around the periphery of the nurse culture. With incubation, the nurse culture grew rapidly, presumably modified the conditions in the KDM2, and thus enhanced the growth of the pathogen in the periphery. For example, colonies of the BKD organism were observable after incubation for 19 days, compared with 25 days for the conventional approach. The second modification involved supplementing KDM2 agar with a small amount of spent (KDM2) broth that was previously used for growing the pathogen. In both cases, it seems that an unknown metabolite serves as a growth stimulant (Evelyn et al, 1989, 1990). Until the advent of selective isolation techniques, initial isolation of the pathogen from fish tissues was an uncertain affair, prone to contamination by fast-growing aerobic heterotrophs. With this in mind, a selective isolation medium, SKDM (Appendix 5.1; Austin et al., 1983a) was devised, which proved to be effective for isolation of the pathogen from dilute samples. SKDM permitted the recovery of the pathogen from seeded river water, and from the kidney and faeces of experimentally infected fish (Embley, 1983; Austin and Rayment, 1985). In contrast, the pathogen was not recovered on corresponding KDM2 plates, which were completely over- grown by other bacteria. Clearly, selective media, such as SKDM, should prove
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158 Bacterial Fish Pathogens useful in further ecological studies on the causal agent of BKD. In a comparison of KDM2, SKDM and the charcoal-containing derivative, it was determined that the selective medium (SKDM) was most effective for the primary isolation of the patho- gen from Atlantic salmon (Gudmundsdottir et al, 1991). In this comparison of positive samples, 91%, 60% and 35% were positive on SKDM, the charcoal contain- ing derivative and KDM2, respectively. Clearly, the selective medium enhanced significantly the ability to recover the pathogen. Moreover, serum was more advanta-
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