In situ enumeration methods utilizing fluorescent probes were used to estimate the number of physiologically active bacteria in river water. Two fluorogenic compounds, 5-cyano-2, 3-ditolyl tetrazolium chloride (CTC) and 6-carboxy fluorescein diacetate (6CFDA), were chosen for direct epifluorescent microscopic detection of active bacteria. CTC is a soluble redox indicator which is reduced by respiring bacteria to fluorescent CTC-formazan crystals. 6CFDA is hydrolyzed by nonspecific esterases to produce the fluorescent compound, 6-carboxy fluorescein. Estimates of the number of active bacteria identified by these fluorescent probes were compared with those obtained with the plate count method. Bacteria with respiratory activity, as determined by CTC reduction, accounted for approximately 10% of the total bacteria at oligotrophic sites and 15 to 20% at eutrophic sites. These values exceeded those obtained with the plate count method. Estimates of enzymatically active bacteria, as determined by 6CFDA hydrolysis, were also higher than those obtained with the plate count method, constituting 40 to 50% of the total bacteria at both oligotrophic and eutrophic sites. These results indicate that some non-culturable bacteria retain physiologic activity and may play an important role in the ecosystem.

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We examined the correlation between the extracellular polysaccharide (EPS) production and salt tolerance of rhizobia. Twelve rhizobial isolates from six different legume hosts were used in this study. Six isolates were found to be salt-tolerant to various degrees and other isolates were salt-sensitive in the presence of 0.5% or higher concentrations of NaCl in Mannitol-salt-yeast extract (MSY) culture broth. Salt-sensitive isolates developed dry and larger colonies on MSY agar plates, except TAL619 and CWP2 which developed larger and gummy colonies similar to the NaCl-tolerant isolates. Salt-sensitive isolates excreted lesser amounts of EPS even when they grew under stress-free conditions, except TAL619 and CWP2 which showed significant level of EPS production. On the other hand, salt-tolerant strains produced notable amounts of EPS. Among the six salt-tolerant isolates, RCSt1 and CPL3 showed sharp and significant rates of increase of EPS production with increasing salt concentration. Three other strains, RJL3, RMB3 and CWP2S showed less of an increase, while for TAL309L, the EPS production remained unaltered with increasing salt concentration. From the results, it appears that extracellular polysaccharide may have some influence on salt tolerance of certain rhizobial strains.

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