340
Exercise 27
3. Remove a 10-ml portion of the extract for the measurement of chlorophyll
derivatives. Measure absorbance with a spectrophotometer at the red maximum, at
or near 667 nm, for the plant chlorophyll derivatives. A correction for background
absorbance may be substracted from the chlorophyll peak by drawing a baseline
between 520 and 800 nm.
4. For the isolation of carotenoids, add a 25-ml sample of the acetone extract to an
equal volume of 20% (w/v) ofmethanolic potassium hydroxide. Agitate the samples
on a shaker for 2 h.
5. Separate the carotenoids into epiphasic pigments (without hydroxyl groups) and
hypophsic carotenoids (with two or more hydroxyl groups) by partition in a
separatory funnel with petroleum ether (30 to 60°C) and with aqueous 90%
methanol, respectively. Measure the absorbance with a spectrophotometer at 450
nm of each of the carotenoid derivatives in their respective solvents.
6. Pigment concentrations are expressed as units p~r gram of organic matter, one unit
being equivalent to an absorbance of 1.0 in a to-cm spectrophotometer cell when
dissolved in 100 ml of the appropriate solvent (Sanger and Gorham, 1972).
Diatom Analyses
1. To a sediment sample of known wet weight (ca. 250 mg), carefully add 50 ml of
concentrated nitric acid and about 109 of potassium dichromate. Add several
boiling chips and boil the mixture gently in a hoodfor 20 min (Hohn and Hellerman,
1963). Note: Use extreme caution and wear protective shielding. Allow the material
to cool and settle.
2. Decant and discard the supernatant, add distilled water to the residue, mix, and
allow the materials to resettle. Repeat until the pH of the mixture tests neutral with
litmus paper.
3. Place microscopic coverslips (ca. 18 x 18 mm) on an aluminum block or similar hot
plate that will maintain a uniform, very low heat (e.g., a slide warmer).
4. Thoroughly mix the diatom-distilled water mixture of known volume in the beaker
or flask. Immediately remove an aliquot of 200 to 500,u1 with an automatic
micropipet. Use a fresh pipet for each sample. Place on the coverslip and allow
mixture to dry slowly.
5. After all of the samples are distributed on the coverslips and have dried, increase the
temperature of the hot plate for 20 min.
6. Place a drop of the mounting medium Hyrax (refractive index 1.65) onto a
microscope slide. Invert the coverslip and place onto the drop of Hyrax. The mount
can be made permanent by evaporating most of the Hyrax solvent with moderate
heating. Label the slide.
7. Microscopically examine the diatom slides from the several sediment layers.
Attempt to identify major genera and species. Determine the proportions (percent)
of major groups of diatoms for a given amount of sediment, e.g., centrales;
araphidine pennate diatoms.
Pollen Analyses
Pollen grains of higher plants and spores of lower plants typically develop a resistant
layer outside the cell wall. This layer, the exine, is waxy or resinous and very resistant
chemically. The surface of the exine is variously sculptured and assists, along with
general shape and size and the shape and arrangement of wall apertures, in identifying
Exercise 27
3. Remove a 10-ml portion of the extract for the measurement of chlorophyll
derivatives. Measure absorbance with a spectrophotometer at the red maximum, at
or near 667 nm, for the plant chlorophyll derivatives. A correction for background
absorbance may be substracted from the chlorophyll peak by drawing a baseline
between 520 and 800 nm.
4. For the isolation of carotenoids, add a 25-ml sample of the acetone extract to an
equal volume of 20% (w/v) ofmethanolic potassium hydroxide. Agitate the samples
on a shaker for 2 h.
5. Separate the carotenoids into epiphasic pigments (without hydroxyl groups) and
hypophsic carotenoids (with two or more hydroxyl groups) by partition in a
separatory funnel with petroleum ether (30 to 60°C) and with aqueous 90%
methanol, respectively. Measure the absorbance with a spectrophotometer at 450
nm of each of the carotenoid derivatives in their respective solvents.
6. Pigment concentrations are expressed as units p~r gram of organic matter, one unit
being equivalent to an absorbance of 1.0 in a to-cm spectrophotometer cell when
dissolved in 100 ml of the appropriate solvent (Sanger and Gorham, 1972).
Diatom Analyses
1. To a sediment sample of known wet weight (ca. 250 mg), carefully add 50 ml of
concentrated nitric acid and about 109 of potassium dichromate. Add several
boiling chips and boil the mixture gently in a hoodfor 20 min (Hohn and Hellerman,
1963). Note: Use extreme caution and wear protective shielding. Allow the material
to cool and settle.
2. Decant and discard the supernatant, add distilled water to the residue, mix, and
allow the materials to resettle. Repeat until the pH of the mixture tests neutral with
litmus paper.
3. Place microscopic coverslips (ca. 18 x 18 mm) on an aluminum block or similar hot
plate that will maintain a uniform, very low heat (e.g., a slide warmer).
4. Thoroughly mix the diatom-distilled water mixture of known volume in the beaker
or flask. Immediately remove an aliquot of 200 to 500,u1 with an automatic
micropipet. Use a fresh pipet for each sample. Place on the coverslip and allow
mixture to dry slowly.
5. After all of the samples are distributed on the coverslips and have dried, increase the
temperature of the hot plate for 20 min.
6. Place a drop of the mounting medium Hyrax (refractive index 1.65) onto a
microscope slide. Invert the coverslip and place onto the drop of Hyrax. The mount
can be made permanent by evaporating most of the Hyrax solvent with moderate
heating. Label the slide.
7. Microscopically examine the diatom slides from the several sediment layers.
Attempt to identify major genera and species. Determine the proportions (percent)
of major groups of diatoms for a given amount of sediment, e.g., centrales;
araphidine pennate diatoms.
Pollen Analyses
Pollen grains of higher plants and spores of lower plants typically develop a resistant
layer outside the cell wall. This layer, the exine, is waxy or resinous and very resistant
chemically. The surface of the exine is variously sculptured and assists, along with
general shape and size and the shape and arrangement of wall apertures, in identifying