252
individual cells, with net negative charges reported on virulent, agglutinating cells,
and net positive charges on avirulent non-agglutinating strains. He suggested that
the negatively charged virulent form of Aer. salmonicida is able to persist, albeit
under starvation conditions, retaining viability in river sediments. It was also proposed that the decline in negatively charged virulent cells in sediments over prolonged periods, also noted by other investigators (e.g. Michel and Dubois-Darnaudpeys
1980 ), may be caused by the spontaneous occurrence of positively charged avirulent
free-living cells of Aer. salmonicida. These cells originate from the virulent ones,
attached to sediment particle surfaces, and subsequently detach from the sediment/
sand particles. This free-living form could be considered to enter a dormant phase,
according to Sakai ( 1986b ), because the viability of these bacteria declines due to a
lack of nutrients. It was further proposed that the free-living cells represent a transitional life stage of the pathogen, which would ultimately lose viability (Sakai
1986b ).
Subsequently, Rose et al. ( 1990a ) re-examined the possibility that Aer. salmonicida may enter a dormant state in water, using methods modifi ed 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addition of 0.1 % (w/v) TSB to aliquots withdrawn from microcosms after viable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
4 /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 which 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 epifl uorescence microscopy, respiration, cell culture, cell
revival, fl ow cytometry, plasmid maintenance and membrane fatty acid analysis.
These workers found that Aer. salmonicida became unculturable in sterile lake
water, but microscopic and fl ow cytometric methods revealed the continued presence of cells. However, attempts to revive these cells by the addition of TSB 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 defi nitively
demonstrated. In addition, they commented (and we strongly agree) that nonculturability of some bacteria from environmental samples maybe as much a func5 Aeromonadaceae Representative (Aeromonas salmonicida)
individual cells, with net negative charges reported on virulent, agglutinating cells,
and net positive charges on avirulent non-agglutinating strains. He suggested that
the negatively charged virulent form of Aer. salmonicida is able to persist, albeit
under starvation conditions, retaining viability in river sediments. It was also proposed that the decline in negatively charged virulent cells in sediments over prolonged periods, also noted by other investigators (e.g. Michel and Dubois-Darnaudpeys
1980 ), may be caused by the spontaneous occurrence of positively charged avirulent
free-living cells of Aer. salmonicida. These cells originate from the virulent ones,
attached to sediment particle surfaces, and subsequently detach from the sediment/
sand particles. This free-living form could be considered to enter a dormant phase,
according to Sakai ( 1986b ), because the viability of these bacteria declines due to a
lack of nutrients. It was further proposed that the free-living cells represent a transitional life stage of the pathogen, which would ultimately lose viability (Sakai
1986b ).
Subsequently, Rose et al. ( 1990a ) re-examined the possibility that Aer. salmonicida may enter a dormant state in water, using methods modifi ed 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 aliquots 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
4 /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 which 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 epifl uorescence microscopy, respiration, cell culture, cell
revival, fl ow cytometry, plasmid maintenance and membrane fatty acid analysis.
These workers found that Aer. salmonicida became unculturable in sterile lake
water, but microscopic and fl ow cytometric methods revealed the continued presence of cells. However, attempts to revive these cells by the addition of TSB 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 defi nitively
demonstrated. In addition, they commented (and we strongly agree) that nonculturability of some bacteria from environmental samples maybe as much a func5 Aeromonadaceae Representative (Aeromonas salmonicida)