156
Exercise 10
away, the filters should be folded carefully in half with their inner surfaces coming
against each other, placed into labeled, folded absorbent pads (e.g., Millipore), stored
in a small darkened desiccator, and immediately frozen. Pigment samples may be
stored for a few days when frozen at - 20 to - 60°C. Storage of samples in cold,
darkened acetone is not recommended because significant degradation of pigments
occurs.
Extraction. Although chlorophyllous and carotenoid pigments may be extracted
readily in organic solvents, for many algae extraction is not complete unless the cells
are ruptured. Therefore filters should be placed with forceps into the base of a
glass/glass or preferably Teflon/glass tissue grinder; add approximately 3 to 4 ml of
90% alkaline acetone solution (see "Apparatus and Supplies," p. 163). Blue-green algae
are difficult to disrupt; the addition of a small amount of glass powder assists in the
extraction. Millipore filters will dissolve immediately. Grind the sample vigorously
but carefully in a hood for 45 sec; remove the pestle slowly while it is still rotating at
the end of the period. Decant into a stoppered graduated centrifuge tube or cylinder.
Add about 3 ml more of the acetone solution to the grinding tube and grind vigorously
for another 15 sec. Add this rinse solution to the same centrifuge tube and record
the total volume to at least the nearest tenth of a m!. Immediately stopper the
centrifuge tube tightly; hold in darkness until an even number of filters has becn
extracted. Centrifuge at maximum speed in a clinical centrifuge (3000 to 4000 rpm;
ca. 1000 g) for 5 min. Use a refrigerated centrifuge at 5 to lOoC, if available. When
glass fiber filters are used, fibers may cling to the walls of the tube. Remove the tube
in this case and gently splash the walls by flicking the tube, thus disloddging any fibers
adhering to the sides. Centrifuge for another 2 min.
Carefully remove the clear supernatant so as not to disturb the sedimented residues.
Use a rubber bulb and curve-tipped pipet. Be certain to expel the air from the bulb
before placing the pipet into the centrifuge tube, otherwise air bubbles will cause
turbulence. Place the liquid into clean (must be nonacidic) cuvettes of the spectrophotometer and measure the absorption -emission characteristics, as discussed below,
without delay. Small-volume cuvettes holding 10 ml or less with light path lengths of
5 or 10 cm, are recommended strongly to avoid dilution of the pigments.
Spectrophotometric Determination of Chlorophyll a, b, and c,
and Plant Carotenoids
The plant pigments of algae consist of the chlorophylls and carotenoids (carotenes
and xanthophylls). The three major chlorophylls, a, b, and c, absorb light maximally
at specific wavelengths when dissolved in organic solvents. From these absorption
characteristics an estimate can be made of the concentrations of the pigments.
Chlorophyll a is by far the most dominant chlorophyllous pigment and occurs in
greatest abundance. Thus often chlorophyll a alone is used to estimate algal biomass.
The spectrophotometric estimate of concentrations of the chlorophyll c moieties
in the trichromatic method treated below is only approximate. More elaborate and
accurate analytical methods are available for chlorophyll determinations. In particular, analyses by high performance liquid chromatography (H PLC) will likely
supercede spectrophotometric methods in the future. Chlorophyll concentrations are
expressed in .ug/l (or mg/m 3 ). Plant carotenoid pigments are estimated collectively in
somewhat arbitrary pigment units that approximate mg/m 3 .
Exercise 10
away, the filters should be folded carefully in half with their inner surfaces coming
against each other, placed into labeled, folded absorbent pads (e.g., Millipore), stored
in a small darkened desiccator, and immediately frozen. Pigment samples may be
stored for a few days when frozen at - 20 to - 60°C. Storage of samples in cold,
darkened acetone is not recommended because significant degradation of pigments
occurs.
Extraction. Although chlorophyllous and carotenoid pigments may be extracted
readily in organic solvents, for many algae extraction is not complete unless the cells
are ruptured. Therefore filters should be placed with forceps into the base of a
glass/glass or preferably Teflon/glass tissue grinder; add approximately 3 to 4 ml of
90% alkaline acetone solution (see "Apparatus and Supplies," p. 163). Blue-green algae
are difficult to disrupt; the addition of a small amount of glass powder assists in the
extraction. Millipore filters will dissolve immediately. Grind the sample vigorously
but carefully in a hood for 45 sec; remove the pestle slowly while it is still rotating at
the end of the period. Decant into a stoppered graduated centrifuge tube or cylinder.
Add about 3 ml more of the acetone solution to the grinding tube and grind vigorously
for another 15 sec. Add this rinse solution to the same centrifuge tube and record
the total volume to at least the nearest tenth of a m!. Immediately stopper the
centrifuge tube tightly; hold in darkness until an even number of filters has becn
extracted. Centrifuge at maximum speed in a clinical centrifuge (3000 to 4000 rpm;
ca. 1000 g) for 5 min. Use a refrigerated centrifuge at 5 to lOoC, if available. When
glass fiber filters are used, fibers may cling to the walls of the tube. Remove the tube
in this case and gently splash the walls by flicking the tube, thus disloddging any fibers
adhering to the sides. Centrifuge for another 2 min.
Carefully remove the clear supernatant so as not to disturb the sedimented residues.
Use a rubber bulb and curve-tipped pipet. Be certain to expel the air from the bulb
before placing the pipet into the centrifuge tube, otherwise air bubbles will cause
turbulence. Place the liquid into clean (must be nonacidic) cuvettes of the spectrophotometer and measure the absorption -emission characteristics, as discussed below,
without delay. Small-volume cuvettes holding 10 ml or less with light path lengths of
5 or 10 cm, are recommended strongly to avoid dilution of the pigments.
Spectrophotometric Determination of Chlorophyll a, b, and c,
and Plant Carotenoids
The plant pigments of algae consist of the chlorophylls and carotenoids (carotenes
and xanthophylls). The three major chlorophylls, a, b, and c, absorb light maximally
at specific wavelengths when dissolved in organic solvents. From these absorption
characteristics an estimate can be made of the concentrations of the pigments.
Chlorophyll a is by far the most dominant chlorophyllous pigment and occurs in
greatest abundance. Thus often chlorophyll a alone is used to estimate algal biomass.
The spectrophotometric estimate of concentrations of the chlorophyll c moieties
in the trichromatic method treated below is only approximate. More elaborate and
accurate analytical methods are available for chlorophyll determinations. In particular, analyses by high performance liquid chromatography (H PLC) will likely
supercede spectrophotometric methods in the future. Chlorophyll concentrations are
expressed in .ug/l (or mg/m 3 ). Plant carotenoid pigments are estimated collectively in
somewhat arbitrary pigment units that approximate mg/m 3 .