251
Survival of Aeromonas salmonicida in the Absence of Culturing The observations for V. cholerae were of especial interest because we found that cells of Aer.
salmonicida could be enumerated by microscopic direct counts when no colonyforming units were recovered on agar plates, raising the possibility that problems
concerning the recovery of Aer. salmonicida may bear similarities to V. cholerae.
Our experiments had shown that if low numbers of Aer. salmonicida were inoculated into sterilised reservoir water the organism could not be recovered using plating methods, after 72 h. However, when microscopic direct counts were done the
number of bacteria underwent an initial increase 6 h post-inoculation, and maintained these numbers until the conclusion of the experiment. Unfortunately, it was
not possible to know from these results whether the cells observed by microscopy
were, indeed, viable. Thus, in a follow up study, the hypothesis that Aer. salmonicida may enter a non-recoverable but viable state was tested. However, these data
bore some similarity to survival studies with other pathogens, namely that there is
often is an initial increase in the number of cells as visualised by microscopic methods despite culturing indicating a progressive decline in numbers.
The Non Culturable But Viable (NCBV) State It was confi rmed by Allen-Austin
et al. ( 1984 ) that small inocula of cells rapidly declined in fi lter-sterilised river
water, when enumerated using total viable count procedures on agar plates, such
that the system appeared to be totally devoid of viable cells by day 17. However, the
microscopic procedures showed that, after an initial decrease, the number of Aer.
salmonicida cells remained constant at approximately 8.0 × 10
2 /ml. TSB to 0.01 %
(v/v) was added to the experimental system 7 days after the plate counts reached and
remained at zero, and the sample was split into three equal volumes, incubated at 22
and 18 °C in addition to 15 °C. At 22 °C, 150 colony-forming units of Aer. salmonicida/ ml of sample were recovered on TSA 6 h after supplementation with the nutrient. There was no apparent increase in the direct microscopic count at 6 h, but by
24 h the microscopic fi elds contained too many cells to count. This result
demonstrated that 6 h after nutrient addition, a proportion of the cells had regained
the ability to produce colonies on TSA, despite the fact that for the previous 7 days
none had been capable of colony formation. At 18 °C, the response to the added
nutrient was much slower, insofar as colonies were not detected until after 4 days
had elapsed. There was, however, a pronounced increase in the direct counts at 24 h
after nutrient was added. This continued up to 4 days, when the fi rst colonies were
cultured on solid medium. Similarly, there was a lag of 5 days before colonies were
recoverable at 15 °C. This coincided with an increase in the direct count. In comparison, it is emphasized that plate counts remained at zero in unsupplemented river
water at 15, 18 and 22 °C. The factors involved in triggering the return of Aer. salmonicida to a culturable state need careful evaluation. As demonstrated in the
experiment reported here, temperature and nutritional changes appear to be responsible for reactivating cells of Aer. salmonicida (Allen-Austin et al. 1984 ).
Sakai ( 1986b ) also proposed a mechanism for the long-term survival of Aer. salmonicida in the aquatic environment based on electrostatic charge differences on
Aeromonas salmonicida
Survival of Aeromonas salmonicida in the Absence of Culturing The observations for V. cholerae were of especial interest because we found that cells of Aer.
salmonicida could be enumerated by microscopic direct counts when no colonyforming units were recovered on agar plates, raising the possibility that problems
concerning the recovery of Aer. salmonicida may bear similarities to V. cholerae.
Our experiments had shown that if low numbers of Aer. salmonicida were inoculated into sterilised reservoir water the organism could not be recovered using plating methods, after 72 h. However, when microscopic direct counts were done the
number of bacteria underwent an initial increase 6 h post-inoculation, and maintained these numbers until the conclusion of the experiment. Unfortunately, it was
not possible to know from these results whether the cells observed by microscopy
were, indeed, viable. Thus, in a follow up study, the hypothesis that Aer. salmonicida may enter a non-recoverable but viable state was tested. However, these data
bore some similarity to survival studies with other pathogens, namely that there is
often is an initial increase in the number of cells as visualised by microscopic methods despite culturing indicating a progressive decline in numbers.
The Non Culturable But Viable (NCBV) State It was confi rmed by Allen-Austin
et al. ( 1984 ) that small inocula of cells rapidly declined in fi lter-sterilised river
water, when enumerated using total viable count procedures on agar plates, such
that the system appeared to be totally devoid of viable cells by day 17. However, the
microscopic procedures showed that, after an initial decrease, the number of Aer.
salmonicida cells remained constant at approximately 8.0 × 10
2 /ml. TSB to 0.01 %
(v/v) was added to the experimental system 7 days after the plate counts reached and
remained at zero, and the sample was split into three equal volumes, incubated at 22
and 18 °C in addition to 15 °C. At 22 °C, 150 colony-forming units of Aer. salmonicida/ ml of sample were recovered on TSA 6 h after supplementation with the nutrient. There was no apparent increase in the direct microscopic count at 6 h, but by
24 h the microscopic fi elds contained too many cells to count. This result
demonstrated that 6 h after nutrient addition, a proportion of the cells had regained
the ability to produce colonies on TSA, despite the fact that for the previous 7 days
none had been capable of colony formation. At 18 °C, the response to the added
nutrient was much slower, insofar as colonies were not detected until after 4 days
had elapsed. There was, however, a pronounced increase in the direct counts at 24 h
after nutrient was added. This continued up to 4 days, when the fi rst colonies were
cultured on solid medium. Similarly, there was a lag of 5 days before colonies were
recoverable at 15 °C. This coincided with an increase in the direct count. In comparison, it is emphasized that plate counts remained at zero in unsupplemented river
water at 15, 18 and 22 °C. The factors involved in triggering the return of Aer. salmonicida to a culturable state need careful evaluation. As demonstrated in the
experiment reported here, temperature and nutritional changes appear to be responsible for reactivating cells of Aer. salmonicida (Allen-Austin et al. 1984 ).
Sakai ( 1986b ) also proposed a mechanism for the long-term survival of Aer. salmonicida in the aquatic environment based on electrostatic charge differences on
Aeromonas salmonicida