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

Photosynthetic picoplankton of the genus Synechococcus can represent a subs tantial proportion of planktonic community biomass and production in many o ceanic provinces. These cells are typically enumerated by visualizing the a utofluorescence of phycoerythrin using epifluorescence microscopy. Detailed studies of bacterioplankton and preliminary studies with photosynthetic pi co- and nanoplankton suggest that the number of cells which can be visualiz ed changes with mode and duration of sample storage. Inaccurate estimates o f Synechococcus abundance may bias the interpretation of the distribution a nd turnover of microbial stocks. We carried out a comprehensive, long-term (similar to 0.9 yr) time-course study to determine if storage mode and dura tion influence microscopic estimates of Synechococcus abundance. Seawater s amples preserved with gluteraldehyde were either stored at 4 degrees C unti l counting (i.e. RS-refrigerated in suspension) or slides were prepared and stored at -20 degrees C until counting (i.e. FF-filtered and frozen). Over time, both methods converged on an apparent cell loss (ACL; i.e, loss of e pifluorescence-detectable cells) of ca 45%. Significant (p less than or equ al to 0.05) ACL occurred within 1 mo for the RS method, whereas cell number s were unchanged for the first 2 mo for the FF method. Apparent cell loss w as hyperbolic for both storage modes and the rate constants for decay were similar. Our results are consistent with the suggestion in the Literature t hat ACL may have been due to persistence of intracellular autolytic enzymes in the preserved cells. We also examined the influence of the excitation w avebands 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 fo r the enumeration of Synechococcus be immediately preserved, filtered, froz en, and counted using green excitation within 2 mo.