(Section Protistology and Aquatic Ecology)
Filters for HPLC pigment analysisA known volume of water collected between at 3 m depth using a Niskin sampler is filtered over 47 mm diameter GF/F filters. Prolonged exposure of the water to direct sunlight or high artificial light levels is avoided as this may alter accessory pigment composition of the phytoplankton. A low vacuum filtration pressure is used and filtering water stops when flow rate through the filter begins to decrease.
When all water has been filtered, the vacuum is maintained for a couple of seconds to remove as much water as possible from the filter. Filters are plied in the middle and dry patted between blotting paper. The more water has been removed from the filter, the better the preservation of the pigments.
Filters are wrapped in alu-foil and marked containing the station code, the date and the volume of water filtered.
Filters are collected in a plastic bag containing the date of the cruise and stored immediately in a freezer at -20°C.
Samples for enumeration and identification of phytoplankton250 ml of water collected at 3 m depth using a Niskin sampler is transferred to 250 ml tissue culture flaks.
Lugol is added until the sample has a ‘cognac-like’ color (less than 1 ml).
The samples are stored at room temperature in the dark.
Samples for isolation of diatoms30 liters of seawater is collected by means of bucket hauls and poured through a 10 µm plankton net. If the net is blocked at less than 20 liter (which may be the case during a phytoplankton bloom), less water is filtered. During winter more than 50 liters is collected.
The sample is transferred to a 250 ml tissue culture flask that is stored in a cooler box in the dark filled with seawater to maintain the phytoplankton at ambient temperature. Samples are brought to the lab in Gent within the next three days for isolation of the dominant diatom species.
Pigment analysis using High Performance Liquid ChromatographyAfter filed sampling the GF/F filter used for seawater filtration during sampling is transferred to -80°C. Prior to analysis, pigments are extracted in 2 ml of a 90 % acetone solution and sonicated during 30 s. Extraction of the pigments is carried out in a dark room to avoid photo degradation of pigments. Before injection of the extract into the HPLC system, the extract is filtered over a 0.2 mm filter to remove small particles. The set up of the HPLC system used is that described by Wright et al. (1991). The analysis yields a chromatogram including individual peaks for all phytoplankton pigments present in the sample. To identify the pigments peaks, their retention times are compared to those of pure pigment standards and by inspection of the absorption spectrum by means of a Diode Array Detector. The concentration of each pigment is estimated from the area of the peak using a calibration based on pure pigment standards. Chlorophyll a is used as an indicator of total phytoplankton biomass while concentrations of accessory pigments allow evaluating the composition of the phytoplankton community present. For example, the accessory pigment fucoxanthin is specific for diatoms (Bacillariophyta) while lutein is a marker pigment for green algae (Chlorophyta).
Wright S.W., Jeffrey S.W., Mantoura R.F.C. , Llewellyn C.A., Bjørland T., Repta D., and N. Welschmeyer. 1991. Improved HPLC method for the analysis of chlorophylls and carotenoids from marine phytoplankton. Mar. Ecol. Prog. Ser. 77: 183-196.
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