POLLEN ANALYSIS
Pollen grains are the microscopic male reproductive particles released from flowering
plants. As these grains are vital for the survival of flowering species, evolution has
ensured that individual species produce different types of pollen grains. Pollen grains
are also made of a resistant organic material (sporopollenin) that means they are often
remarkably well-preserved in sediments many thousands of years old.
This combination of factors means that over time as sediments accumulate, pollen
grains from plants growing at the site, nearby, and occasionally at considerable
distance, will be incorporated and preserved. Individual species and communities of
flowering plants grow within limits of tolerance to environmental conditions, such as
moisture and temperature. Changes that occur in the environment therefore induce
stress in plants and plant communities. If rates of change are slow and no physical
barriers exist then plants will migrate, following the preferred growing conditions. As a
result, changes in plant communities (and the spectrum of pollen grains being released)
reflect changing environmental conditions.
The pollen content of sediments deposited over time therefore has the potential of
recording vegetation and vice versa environmental change.
Pollen grains from Romney Marsh peat samples. From L to R - Betula (birch), Ulmus (elm), Alnus (alder), Pinus
(Pine), Poaceae (grass) and Tilia (lime)
For pollen analysis, samples of sediment are washed and treated with strong acid and
alkaline washes to remove less resistant organic and mineral matter. The small amount
of material left is placed on a slide and examined by a microscope. Pollen grains are
identified by their shape and surface texture. Counting of the different number of pollen
types continues until a statistically reliable number has been achieved. The totals of
each type are usually represented as a percentage of the total number of grains found
in the sample. By counting pollen from consecutive layers of sediment it is possible
therefore to see how vegetation has changed over time.
Interactive Pollen diagram from Pannel Bridge
There are problems with pollen analysis, for example some plant species naturally
produce more pollen, some grains are transported more effectively and other grains are
more delicate and are poorly preserved. These taphonomic considerations have to be
considered in any interpretation of a pollen sequence.
Wetlands and the sediments that form in them provide ideal conditions for pollen
analysis. Water saturated soils have a reduced oxygen content which aids organic
preservation. Low-energy depositional conditions also often dominate in wetlands which
means they act as a natural trap for pollen grains transported by rivers, the sea and the
atmosphere.
http://www.liv.ac.uk/geography/RomneyMarsh/RM%20Tech&Analysis/Analysis/Pollen.htm
Procedure for the preparation of
sediment samples for pollen analysis
Notes on sample preparation -
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Samples should be stored wet at <40C.
If sampling from an unsliced core, all the samples should be taken at
one time to prevent having to re-open the bags and drying out the core
material.
Beware of contamination - clean all spatulas etc. Thoroughly between
samples.
If the core is long but covers a short time period, a large sample volume
(1cm3) may be necessary. However if the core is short, covering a long
time period use 0.5cm3.
If using absolute pollen techniques (quantitative), add weighed tablets
of exotic taxon to a known volume of sediment in a labelled 50 ml
boiling tube.
Removal of Calcium.
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Slowly add about 10 ml of 10% HCl to the sample. When effervescence
stops, place the tube in a boiling water bath and stir well until
effervescence again stops. If the tube threatens to froth over, reduce
the foam with a squirt of acetone. Centrifuge and decant.
All centrifugation should be at 3000 r.p.m. for 4 minutes and about 2 ml
of methanol should be added to reduce the specific gravity and reduce
the losses in decanting.
If sediment is highly calcareous, it will be necessary to centrifuge,
decant and add a further 10 ml of HCl. It is essential to remove all
calcium carbonate at this stage, as the later addition of HF will result in
the formation of insoluble calcium fluoride.
After supernatant has been decanted, thoroughly mix sediment at each
stage.
Wash with distilled water, centrifuge and decant.
If the samples are extremely calcareous, this process should be
repeated 2 or 3 times.
Removal of Humic Acid
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Add 10 ml of 10 % KOH. Place in a boiling water bath for no longer than
5 minutes. Stir occasionally.
Record the darkness of the supernatant as a measure of the degree of
humification of the sample.
Removal of organics.
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Strain and wash the sample through a fine mesh screen (170-180um)
into a 50 ml polypropylene tube. Wash the residue on the screen
thoroughly with a jet of distilled water
Centrifuge and decant.
Place the coarse residue trapped on the screen into a labelled petri dish
and examine under a low power microscope for seeds, fruits, moss
remains, large pieces of charcoal, etc.
Wash and centrifuge at least 5 times with distilled water until no trace
of brown colour remains in the supernatant, remembering to mix
thoroughly each time after decanting. This removes many small organic
and inorganic particles.
Removal of silicates.
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Add 10% HCl, stir, centrifuge and decant.
If sediment contains mineral matter, treat with 40% HF. In a suitable
fume cupboard add about 10 ml HF and place tube in a boiling water
bath for 20 minutes.
Stir occasionally with a polythene rod.
Remove tube from the water bath, add methanol to reduce the specific
gravity and centrifuge.
Decant carefully into a sodium carbonate neutralising solution.
Half fill tube with 10% HCl and place in water bath for 20 mins. Stirring
occasionally, centrifuge and decant.
If supernatant is yellow (or green) repeat this procedure - it is better to
add fresh HF rather than leaving the tubes in the water bath for longer!
Add about 10 ml water, stir , centrifuge and decant.
Removal of water.
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It is essential to remove all water before the acetolysis step as the
acetolysis mixture reacts very violently with water.
Add 10 ml Glacial Acetic Acid, stir, centrifuge and decant.
Repeat this step to ensure all water is gone.
Removal of cellulose (acetolysis)
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Add 9ml acetic anhydride and 1ml concentrated sulphuric acid
(Erdtman's acetolysis solution) and place in water bath for a maximum
of 3 minutes, stirring after 1.5 min.
Remove tube from bath and fill with Acetic acid, stir, centrifuge and
decant.
Wash with a further 10 ml Acetic Acid. (Stir, Centrifuge,Decant!)
This removes the soluble cellulose products of the acetolysis.
Staining and mounting.
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Wash the sample into 15ml glass centrifuge tubes using water.
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Centrifuge and decant.
Add 9 ml water and 1 ml KOH to obtain correct pH for staining (about
pH7). Stir, centrifuge and decant.
Add 1 or 2 drops of 1% aqueous safranin and 10 ml water. Stir,
centrifuge and decant. Do Not Overstain!
Add about 10 ml Tertiary butyl alcohol. Stir, centrifuge and decant Stir,
centrifuge and decant
Wash sediment into a labelled vial with the minimum amount of TBA,
centrifuge and decant
Add silicon oil (200cs viscosity equal in amount to the remianing
residue, stir well and leave unstoppered in a fume cupboard (lightly
covered) to allow residual TBA to evaporate.
Slides are prepared by pipetting a drop of the suspension onto a slide
and covering with a square coverslip. The coverslip is then held in place
using clear nail polish.
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http://www.geog.ucl.ac.uk/~jhope/lab/poll1.htm