cells remained constant at approxi mately 80 X 10ml TSB to 001 vv was added to

Cells remained constant at approxi mately 80 x 10ml

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cells remained constant at approxi- mately 8.0 X 10^/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 CPU of Aer. salmonicida/m\ of sample were recovered on TSA 6h after supple- mentation with the nutrient. There was no apparent increase in the direct microscopic count at 6 h, but by 24 h the microscopic fields 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 first 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 respons- ible 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 individual cells, with net negative charges reported on virulent, agglutinating cells.
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258 Bacterial Fish Pathogens 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 viabiHty 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 viabiHty of these bacteria decHnes due to a lack of nutrients. It was further proposed that the free-living cells represent a transitional Hfe stage of the pathogen which would ultimately lose viability (Sakai, 1986b).
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