Long-term preservation of Tetraselmis indica (Chlorodendrophyceae, Chlorophyta) for flow cytometric analysis: Influence of fixative and storage temperature.

Immediate enumeration of phytoplankton is seldom possible. Therefore, fixation and subsequent storage are required for delayed analysis. This study investigated the influence of glutaraldehyde (GA) concentrations (0.25%, 0.5%, and 1%) and storage temperatures (-80°CLN2, -80°C, -20°C, and 5°C) on Tetraselmis indica for flow cytometric analysis. Cell recovery, granularity, and membrane permeability were independent of GA concentration whereas cell size and chlorophyll autofluorescence were concentration dependent. After an initial cell loss (16-19%), no cell loss was observed when samples were stored at 5°C. Cell recovery was not influenced by storage temperature until 4months but later samples preserved at -80°CLN2, -80°C, and -20°C resulted in ~41% cell loss. Although maximum cell recovery with minimal effect on cell integrity was obtained at 5°C, autofluorescence was retained better at -80°CLN2 and -80°C. This suggests that in addition to fixative, the choice of storage temperature is equally important. Thus for long-term preservation, especially to retain autofluorescence, the use of lower concentration (0.25%) of GA when stored at a lower temperature (-80°CLN2 and -80°C) while a higher concentration (1%) of GA when stored at a higher temperature (5°C) is recommended.