Influence of storage mode and duration on the microscopic enumeration of Synechococcus from a cold coastal ocean environment

Abstract

Photosynthetic picoplankton of the genus Synechococcus can represent a substantial proportion of planktonic community biomass and production in many oceanic provinces. These cells are typically enumerated by visualizing the autofluorescence of phycoerythrin using epifluorescence microscopy. Detailed studies of bacterioplankton and preliminary studies with photosynthetic picoand nanoplankton suggest that the number of cells which can be visualized changes with mode and duration of sample storage. Inaccurate estimates of Synechococcus abundance may bias the interpretation of the distribution and turnover of microbial stocks. We carried out a comprehensive, long-term (-0.9 yr) time-course study to determine if storage mode and duration influence microscopic estimates of Synechococcus abundance. Seawater samples preserved with gluteraldehyde were either stored at 4°C untd counting (i.e. RS-refrigerated in suspension) or slides were prepared and stored at -20°C until counting (i.e. FF-filtered and frozen). Over time, both methods converged on an apparent cell loss (ACL; i.e. loss of epifluorescence-detectable cells) of ca 45%. Significant (p 2 0.05) ACL occurred within 1 mo for the RS method, whereas cell numbers were unchanged for the first 2 mo for the FF method. Apparent cell loss was hyperbolic for both storage modes and the rate constants for decay were similar. Our results are consistent with the suggestion in the literature that ACL may have been due to persistence of intracellular autolytic enzymes in the preserved cells. We also examined the influence of the excitation wavebands on the estimation of Synechococcus abundances. About twice as many Synechococcus were observed using green (490 to 545 nm) compared to blue (450 to 490 nm) excitation epifluorescence microscopy and this increase was significant (p < 0.001). Based on our results, we recommend that samples for the enumeration of Synechococcus be immediately preserved, filtered, frozen, and counted using green excitation within 2 mo.