This begs the question about the reasons for culturabiHty when the pathogen is

This begs the question about the reasons for

This preview shows page 186 - 189 out of 594 pages.

This begs the question about the reasons for culturabiHty, when the pathogen is recovered from only a proportion of obviously infected animals. Microscopy will often reveal a greater number of bacterial cells than might be deduced from the results
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164 Bacterial Fish Pathogens of plating experiments. Perhaps, as has been argued with L-forms, a threshold number of bacterial cells need to be present to enable some to be capable of pro- ducing growth in broth or on soHd medium. Also, the definition of growth needs to be carefully considered, insofar as the basic criterion reflects observations with the naked eye, i.e. turbidity in broth or clearly visible colonies. The limited growth of micro-colonies may well be missed by classical bacteriological methods. Aeromonas sobria Pure culture growth was obtained from kidney, liver and spleen of moribund animals following inoculation of TSA with incubation at 22°C for possibly one or two days (Toranzo et al, 1989). Aeromonas veronii biovar sobria Scrapings from the ulcer were inoculated on to Aeromonas-SQlQctivQ medium contain- ing 5 |ig/ml of ampicillin with unspecified incubation conditions (Rahman et ai, 2002a). Alteromonadaceae representatives Pseudoalteromonas piscicida Individual diseased eggs were placed on marine agar with incubation at 28°C for 2 days (Nelson and Ghiorse, 1999). Shewanella putrefaciens Bacteria were isolated from the kidney, liver and spleen following inoculation onto BHIA supplemented with 3% (w/v) sodium chloride, with incubation at an un- specified temperature (presumed to be >37°C) for an undetermined period (Saeed et aL, 1987). Campylobacteriaceae representative Arcobacter cryaerophilus Growth occurred on Campylobacter-sdQctiYQ agar and enriched TSA with incubation at 25°C for 1-7 days (Aydin et al, 2002). Enterobacteriaceae representatives Citrobacter freundii Pure culture growth was recovered from kidney on rabbit blood agar (Sato et al, 1982). Subsequently, similar organisms have been obtained from kidney homogen-
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Isolation/Detection 165 ates spread over the surface of BHIA and TSA with incubation at 25 or 37°C for 24-48 h. Edwardsiella ictaluri Isolation has been readily achieved from kidney, liver, spleen, intestine, brain and skin or muscle lesions by inoculation of material into BHIA or blood agar. Following incubation at 26° C for 48 h, smooth circular (2 mm diameter), slightly convex, entire, non-pigmented colonies develop (Hawke, 1979). A selective medium has been described by Shotts and Waltman (1990) [Appendix 5.1]. Edwardsiella tarda Isolation of Edw. tarda from diseased fish is a straightforward procedure involving the use of commonly available media, such as TSA (Meyer and Bullock, 1973; Alcaide et al, 2006) or BHIA (Amandi et al, 1982). On such media, small, round (0.5 mm in diameter), raised, transparent colonies develop in 48 h at 24-26°C (Meyer and Bullock, 1973). The use of thioglycollate broth followed by subculturing on BHIA has also been used successfully (Appendix 5.1; Amandi et ai, 1982). Indeed, this two-step enrichment procedure has proved to be more sensitive than BHIA used alone. In one experiment, this two-step procedure enabled the recovery
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