Subsequently Rose et al 1990a re examined the possibility that Aer salmon icida

Subsequently rose et al 1990a re examined the

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Subsequently, Rose et al. (1990a) re-examined the possibility that Aer. salmon- icida may enter a dormant state in water, using methods modified from the work of Allen-Austin et al. (1984), as described above. However, in their experiments the addition of 0.1% (w/v) TSB to ahquots withdrawn from microcosms after viable counts of Aer. salmonicida had reached zero, did not result in renewed growth of the organism. Thus, Rose et al. (1990a) concluded that the most probable explanation for the results obtained in the previous study (when there appeared to be resuscitation of dormant cells by added nutrients) was the presence of small numbers of viable, culturable cells, which were too few in quantity to be detected by the sampling protocol employed. This conclusion was based on the observation that the addition of 0.1% (w/v) TSB to microcosms after the viable count had reached zero resulted in the re-appearance of viable, culturable cells within 48 h of incubation at 22° C. However, in both studies bacteria enumerated by microscopic techniques remained at levels of approximately 10^/ml in water samples retrieved from the experimental microcosms containing Aer. salmonicida, even after viable counts had apparently reached zero. It is curious that Rose et al. (1990a) proffered no explanation to account for the level of bacteria that were observed microscopically (were the cells alive or could they have been dead?). In a later study, which again addressed the issue of dormancy/NCBV for Aer. salmonicida, Morgan et al. (1991) assessed the survival of the pathogen in lake water, employing an extensive range of techniques, including epifluorescence microscopy, respiration, cell culture, cell revival, flow cytometry, plasmid maintenance and membrane fatty acid analysis. These workers found that Aer. salmonicida became unculturable in sterile lake water, but microscopic and flow- cytometric methods revealed the continued presence of cells. However, attempts to revive these cells by the addition of TBS were unsuccessful. Despite this, it was found that both genomic and plasmid DNA, and also RNA, were maintained in the cells, even though they could not be cultured on conventional media. Morgan et al. (1991) concluded that morphologically the cells remained intact, although their viability could not unfortunately be definitively demonstrated. In addition, they commented (and we strongly agree) that non-culturability of some bacteria from environmental samples maybe as much a function of the ignorance of the parameters necessary for their recovery, as the occurence of a truly non-culturable, specialised survival state. Subsequently, by means of flow cytometry with rhodamine-123, they established a
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Epizootiology: Gram-negative bacteria 259 NCBV state in sterile lake water (Morgan et ai, 1993). However, flow cytometry indicated that cellular properties related with viability were lost shortly after cultur- ability disappeared in distilled water, but not so in lake water (Deere et al, 1996b).
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