Biological field and laboratory methods for measuring the quality of ...

BIOLOGICAL METHODS
5.1.1 Dry weight
Place the aliquot of concentrated sample in a
tared porcelain crucible, and dry to a constant
weight at 105°C (24 hours is usually sufficient).
Subtract the weight of crucible to obtain the dry
weight.
5.1.2 Ash-free weight
After the dry weight is determined, place the
crucible in a muffle furnace at 500°C for 1 hour.
Cool, rewet the ash with distilled water, and
bring to constant weight at 10S°e. The ash is
wetted to reintroduce the water of hydration of
the clay and other minerals that, though not
driven off at 105°C, is lost at SOO°C. This water
loss often amounts to 10 percent of the weight
lost during ignition and, if not corrected for, will
be interpreted as organic matter. Subtract the
weight of crucible and ash from the dry weight
to obtain ash-free weight.
5.2 Chlorophyll
All algae contain chlorophyll a, and measuring
this pigment can yield some insight into the
relative amount of algal standing crop. Certain
algae also contain chlorophyll band c. Since the
chlorophyll concentration varies with species and
with environmental and nutritional factors that
do not necessarily affect the standing crop,
biomass estimates based on chlorophyll measurements
are relatively imprecise. Chlorophyll can
be measured in vivo fluorometrically or in acetone
extracts (in vitro) by fluorometry or
spectrophotometry.
5.2.1 In vitro measurement
The algae differ considerably in the ease of
pigment extraction. The diatoms extract easily,
whereas the coccoid greens extract with difficulty.
Complete extraction of pigments from all
taxonomic groups, therefore, requires disruption
of the cells with a tissue grinder or blender, or
by freezing or drying. Generally, pigment is
more difficult to extract from old cells than
from young cells.
Concentrate the algae with a laboratory centrifuge,
or collect on a membrane filter (O.4S-M
porosity) or a glass fiber filter (0.45-M effective
pore size). If the analysis will be delayed, dry
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the concentrate and store frozen in a desiccator.
Keep the stored samples in the dark to avoid
photochemical breakdown of the chlorophyll.
Place the sample in a tissue grinder, cover with
2 to 3 milliliters of 90 percent aqueous acetone
(use reagent grade acetone), add a small amount
(0.2 ml) of saturated aqueous solution of magnesium
carbonate and macerate.
Transfer the sample to a screw-capped centrifuge
tube, add sufficient 90 percent aqueous
acetone to bring the volume to 5 ml, and steep
at 4°C for 24 hours in the dark. Use the solvent
sparingly, avoiding unnecessary pigment dilution.
Agitate midway during the extraction
period and again before clarifying.
To clarify the extract, centrifuge 20 minutes
at 500 g. Decant the supernatant into a clean,
calibrated vessel (lS-ml, screw-capped, calibrated
centrifuge tube) and determine the volume.
Minimize evaporation by keeping the tube
capped.
Three procedures for analysis and concentration
calculations are described.
Trichromatic Method
Determine the optical density (00) of the
extract at 750, 663, 645, and 630 nanometers
(nm) using a 90 percent aqueous acetone blank.
Dilute the extract or shorten the light path if
necessary, to bring the 00663 between 0.20 and
0.50. The 750 nm reading is used to correct for
turbidity. Spectrophotometers having a resolution
of 1 nm or less are preferred. Stopper the
cuvettes to minimize evaporation during the
time the readings are being made.
The chlorophyll concentrations in the extract
are determined by inserting the corrected I-em
OD's in the following equations. (UNESCO
1966).
C a = 11.64D66 3 - 2.16D645 + 0.10D63o
cb = -3.94D66 3 + 20.97D645 - 3.66D630
Cc =-5.53D663 - 14.810645 + 54.22D630
where Ca, Cb, Cc are the concentrations, in
milligrams per liter, of chlorophyll a, b, and c,
respectively, in the extract; and 0663,0645,
and 0 630 are the I-em OD's at the respective
wavelengths, after subtracting the 750-nm blank.