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There are reports in the literature that the long-term storage (e.g. >30 d) of fixative- preserved seawater samples causes significant decreases in bacterial (BA) and viral abundances (VA). However, the effects of storage on the frequency of visibly infected bacteria (FVIB), and conse- quently on bacterial mortality due to viral lysis (BMVL), remain to be evaluated. First, to determine the variables that facilitate the prediction of the FVIB value at the time of storage (i.e. FVIBi), we con- sidered a bacterial community composed of 2 groups (i.e. visibly infected bacteria and the others) and assumed an exponential decay relationship for the bacteria in each group during the storage of pre- served samples. In the hypothetical model, the FVIBi could be well estimated in terms of BA at the time of storage, a decay rate of BA, and FVIBf and BAf (i.e. FVIB and BA measured at the end of storage, respectively). Further, we tested this idea by applying it to 7 seawater samples that were pre- served with 2% glutaraldehyde (final conc.) and stored at different temperatures for ca. 30 d. For the 3 preserved coastal samples, considerably better estimates of BMVL were obtained by applying the theoretical consideration to estimate the FVIBi (the BMVL was estimated to be 89.9 to 118.7% of the BMVL at the time of storage (BMVLi), which was the value calculated with FVIBi) than if FVIBf was used (the BMVL was estimated to be 45.5 to 89.9% of the BMVLi value). Interestingly, estimates of the BMVL obtained for the 4 preserved offshore samples were comparable to the FVIB values mea- sured at 3 to 5 d after the start of the experiments, as in the case of preserved coastal samples in the present study, suggesting that it may be possible to estimate FVIBi values for preserved offshore sam- ples. It is recommended that the above-mentioned variables be measured in order to reliably esti- mate the FVIB value at the time of preservation for fixative-amended and frozen seawater samples that may be stored for a long time, such as oceanic cruises.

Effects of storage on the estimates of virus-mediated bacterial mortality based on observation of preserved seawater samples with transmission TEM