Method adapted from
Lesley Clementson CSIRO (April,
2002)
Water samples collected for chlorophyll analysis from the field and from cultures are filtered through 47 mm and 25mm GF/F filters respectively. For field samples, if filtering directly from the Niskin bottle, the filtrate must be collected so the volume of water filtered can be measured. The pressure used during filtering should be low (@ 5 mm Hg) to prevent cell breakage. Filtering and then handling of filters should be performed under dimmed lighting.
|
|
Volume to filter |
Filter size |
|
Field Sample |
no less than 2 litres and preferably 4 or more litres |
47mm GF/F |
|
Culture Sample |
Typical coloured culture may only need 25 mL |
25mm GF/F |
After
filtering, remove the filter from its holder (with the vacuum still applied, if
possible, to remove as much moisture as possible from the filter) and place in a
cryo-tube. Label each tube with cruise number, station number, depth and pigment
number. (When filtering directly from the Niskin bottle under pressure rather
than vacuum, the excess moisture in the filters must be removed before freezing.
This can be done by using a second pump that can apply a vacuum to the filter
holder or by folding the filter in half and blotting dry with white paper towel.
All
glassware is cleaned in dilute Extran solution, rinsed three times with MQ water
and once with acetone (AR). Scissor blades are wiped clean on tissues between
samples.
Frozen
filters are cut into halves and placed in a clean 10 ml centrifuge tube. 3 ml of
100% acetone (see note on solvents
below) is added to the tube. Cover tube with parafilm and vortex for 30
seconds before placing the tube in an ice-water bath whilst the filter and
acetone are sonicated for 15 minutes. The filter and acetone are then stored for
at least 18 hours at 4°C. After this time, 0.2 ml MQ water is added to each
tube (solvent » 90:10 acetone : water) and the filter and solvent sonicated for
another 15 minutes. Solvent and filter are then transferred quantitatively to a
Biorad column (see figure x) containing a small GF/F filter acting as a plug.
The sample tubes are rinsed with 2 x 0.5 ml of acetone/water (90:10)
which is quantitatively added to the Biorad column.
Each Biorad column is fitted into a centriguge tube and centrifuged for 5
minutes at 5000 rpm. The filtrate is
stored in the cool and dark (small foam esky) just prior to analysis.
The absorbance of the filtrate is measured using a U.V./visible
spectrophotometer with 10 mm path length optical glass cells (40 mm cells can be
used if the colour of the extracts is very pale).
Absorbance is read at wavelengths of 750, 664, 647 and 630 nm.
The absorbance at 750 nm is subtracted from the absorbance at each of the
other three wavelengths and substituted into the following equations:
[chl. a]extract = 11.85A664/l - 1.54A647/l
- 0.08A630/l
[chl. b]extract = 21.03A647/l - 5.43A664/l
- 2.66A630/l
[chl. c]extract = 24.52A630/l - 1.67A664/l
- 7.60A647/l
A =
corrected absorbance.
l = path
length in cm.
The
concentration of each chorophyll in the sample in µg/L is obtained by the
following equation:
[chl.x]sample = [chl.x]extract * (v/V)
v = volume
of extract in ml.
V = volume
of seawater filtered in litres.
The total
concentration of chorophyll in the sample in µg/L is obtained by the following
equation:
[chl.]total = [chl.a]sample + [chl.b]sample
+ [chl.c]sample
Solvents
used for phytoplankton pigment extraction are many and varied.
Dimethyl formamide has the highest extraction efficiency but is
particularly toxic and therefore difficult to handle.
Methanol is another highly efficient extractor, particularly for hard to
extract species such as some cyanobacteria. Unfortunately accurate equations to
determine chlorophyll a, b and c are
not available for methanol pigments. Acetone
(either 90 or 100%) is therefore regarded as the most suitable solvent for a
broad range of marine phytoplankton species and is widely used for both field
samples and cultures. If
in doubt, protocols in Jeffrey et al, 1997 detail the suitability
of various solvents for a range of different classes and species of microalgae
(full ref below).
“Phytoplankton
pigments in oceanography: Guidelines to modern methods. 1997. Ed. S. W. Jeffrey,
R. F. C. Mantoura, S. W. Wright. Unesco Publishing”