Historical Records of Changes in the Productivity of Lakes
341
the plant species from which the pollen originated [see, e.g., Faegri and Iversen (1964)
and Kapp (1969)].
Chemical treatment of a sediment sample greatly facilitates the analysis of pollen by
removing the extraneous sedimentary materials and by concentrating the pollen. With
care, such methods are quantitative for a given amount of sediment. Many special
techniques have been devised [see Brown (1960), Kummel and Raup (1965), and Faegri
and Iversen (1964)]. The brief procedures discussed below are used commonly for
sediment samples.
1. The samples are treated with acid to remove salts such as calcium carbonate. Expose
the sample to dilute (3N) hydrochloric acid at room temperature for a brief period;
then centrifuge at a high speed and decant the acid.
2. Organic colloids are removed by boiling the sample with 10% potassium hydroxide
for about 10 min. Then shake the sample vigorously (a few drops of alcohol will
dissipate the froth) and strain through a sieve of a mesh opening of ca. 0.2 mm. Wash
the residue with a jet of distilled water. Then concentrate the fine suspension that has
passed the sieve by centrifugation.
3. The final treatment of the sample is acetolysis, to remove cellulose materials, the
cellular contents, and the cellulose wall (intine) of the pollen. Wear gloves and use
caution during this procedure.
a. Dehydrate the sample by adding glacial acetic acid and mix with a stirring rod.
b. Centrifuge and decant the acetic acid.
c. Slowly add about 10 ml of a mixture of 9 parts acetic anhydride: 1 part of
concentrated sulfuric acid. Stir and leave the stirring rod in the tube.
d. Heat gently to the boiling point by immersion for one minute in a boiling water
bath, stirring occasionally.
e. Centrifuge and decant the acetolysis mixture.
f. Wash with glacial acetic acid.
g. Wash twice with distilled water by successive stirring, centrifuging, and
decanting.
4. The pollen now may be examined directly with a microscope, but it is better to
mount on permanent slides.
a. Wash the sample (step 3g above) with a few drops of water and 95% alcohol,
centrifuge, decant, and discard the supernatant.
b. Wash the residue with absolute alcohol, centrifuge; decant.
c. Wash the residue with benzene, centrifuge, and decant.
d. Add about 1 ml of benzene to the residue, transfer the mixture to a small vial, add
high viscosity silicon oil, and allow to evaporate for 24 h.
e. Transfer a small amount of the silicon oil to a microscope slide and cover with a
coverslip. Slides may be sealed with nail polish along the edges.
f. One wash with tertiary butyl alcohol can be substituted for the alcohol and
benzene washes (steps a, b, and c) (Kapp, 1969).
5. Proceed with identification of major groups of pollen at high magnification (400
x or greater), using reference sources such as Kapp (1969). Attempt to discern the
changes in relative abundance of known indicator species. For example, increases in
pollen of the ragweed (Ambrosia) commonly are associated with landscape
disturbance such as deforestation and initiation of agriculture.
341
the plant species from which the pollen originated [see, e.g., Faegri and Iversen (1964)
and Kapp (1969)].
Chemical treatment of a sediment sample greatly facilitates the analysis of pollen by
removing the extraneous sedimentary materials and by concentrating the pollen. With
care, such methods are quantitative for a given amount of sediment. Many special
techniques have been devised [see Brown (1960), Kummel and Raup (1965), and Faegri
and Iversen (1964)]. The brief procedures discussed below are used commonly for
sediment samples.
1. The samples are treated with acid to remove salts such as calcium carbonate. Expose
the sample to dilute (3N) hydrochloric acid at room temperature for a brief period;
then centrifuge at a high speed and decant the acid.
2. Organic colloids are removed by boiling the sample with 10% potassium hydroxide
for about 10 min. Then shake the sample vigorously (a few drops of alcohol will
dissipate the froth) and strain through a sieve of a mesh opening of ca. 0.2 mm. Wash
the residue with a jet of distilled water. Then concentrate the fine suspension that has
passed the sieve by centrifugation.
3. The final treatment of the sample is acetolysis, to remove cellulose materials, the
cellular contents, and the cellulose wall (intine) of the pollen. Wear gloves and use
caution during this procedure.
a. Dehydrate the sample by adding glacial acetic acid and mix with a stirring rod.
b. Centrifuge and decant the acetic acid.
c. Slowly add about 10 ml of a mixture of 9 parts acetic anhydride: 1 part of
concentrated sulfuric acid. Stir and leave the stirring rod in the tube.
d. Heat gently to the boiling point by immersion for one minute in a boiling water
bath, stirring occasionally.
e. Centrifuge and decant the acetolysis mixture.
f. Wash with glacial acetic acid.
g. Wash twice with distilled water by successive stirring, centrifuging, and
decanting.
4. The pollen now may be examined directly with a microscope, but it is better to
mount on permanent slides.
a. Wash the sample (step 3g above) with a few drops of water and 95% alcohol,
centrifuge, decant, and discard the supernatant.
b. Wash the residue with absolute alcohol, centrifuge; decant.
c. Wash the residue with benzene, centrifuge, and decant.
d. Add about 1 ml of benzene to the residue, transfer the mixture to a small vial, add
high viscosity silicon oil, and allow to evaporate for 24 h.
e. Transfer a small amount of the silicon oil to a microscope slide and cover with a
coverslip. Slides may be sealed with nail polish along the edges.
f. One wash with tertiary butyl alcohol can be substituted for the alcohol and
benzene washes (steps a, b, and c) (Kapp, 1969).
5. Proceed with identification of major groups of pollen at high magnification (400
x or greater), using reference sources such as Kapp (1969). Attempt to discern the
changes in relative abundance of known indicator species. For example, increases in
pollen of the ragweed (Ambrosia) commonly are associated with landscape
disturbance such as deforestation and initiation of agriculture.