Hawaii Ocean Time-series (HOT)
in the School of Ocean and Earth Science and Technology at the University of Hawai'i
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BACTERIA AND CYANOBACTERIA
SUMMARY: Picoplankton populations are subsampled from seawater collected in Niskin bottles. The cells are fixed in the field by the addition of paraformaldehyde and, in the laboratory, stained with Hoechst 33342 (a DNA specific dye) and enumerated by dual-beam flow cytometry.
Flow cytometric enumeration is based on the method of Monger and Landry (1993). This method increases substantially the precision of bacterial counts, relative to epifluorescence microscopy. Hoechst 33342 is used because binding to DNA substantially alters its fluorescence spectrum, which facilitates separation of cell fluorescence signals from the background fluorescence of the unbound dye. A 225 mW UV laser is aligned colinearly with a 1 W visible (488 nm) laser to permit enumeration of both heterotrophic and autotrophic picoplankton. Methods for enumeration of autotrophs are given by Campbell and Vaulot (1993) and are not described in detail here.
Because this is a procedure for enumerating preserved cells, sterilization is not a guarantee against contamination. However new, sterile plastic containers tend to be among the cleanest containers available. All reagents (preservative and stain) must be prefiltered through 0.2 µm filters and the sample should not come in contact with fingertips or other potentially contaminating surfaces. No drawing tubes are used.
Because of the small sample volume (1 ml), frozen samples thaw quickly. Therefore they should be kept in liquid nitrogen during transport from the ship to the shore-based laboratory.
Add 10 g paraformaldehyde (be careful not to breathe dust) to 90 ml boiling distilled water and stir (use magnetic stir bar). Add 1 M NaOH, dropwise, with constant mixing, until the solution clears. Cool in ice to room temp, add 10 ml filtered sea water (can be cooling in ice while on stirrer). Adjust pH to 7.5 (careful, pH changes very rapidly below about 8.8; i.e., only one or two more drops needed). Use a Sterifil apparatus with 47 mm GF/F filters to filter this solution. Filter in two equal aliquots,changing the filter between aliquots to avoid clogging. The final preservative strength is about 10%. This solution should be prepared fresh for each cruise and pH checked before use.
6. Equipment and Supplies