Preparation of organic sediments for pollen analysis
By: K. Gajewski
This procedure is based on that in use in the Palynology Laboratory of
the Center for Climatic Research, University of Wisconsin (A Swain, R.
Steventon) and by K. Hadden, University of Toronto as modified by us.
You should be shown this at least once before trying it on your own.
(see the library page complete citations)
- Faegri and Iversen
- Cwynar et al.
NOTE: Read carefully the Materials Safety Datasheets on all the chemicals
that you use. These are available in the lab and up to date versions are
MUST wear gloves, goggles and a lab coat at all times. The entire procedure
must be done under the fume hood. Note the location of the first aid kit,
the shower, the eye-wash station and the burn blanket. There is a telephone
in the lab should you need to call security. You must have proper training
before using the laboratory (see Konrad Gajewski or the lab technician
for specific requirements).
Be especially careful during the HF and acetolysis steps. HF is particularly
dangerous if you inhale or touch it. DO not let any touch your skin. You
cannot smell it, only use it in the hood, with the front down as low as
possible. The acetolysis solution can be explosive when it comes in contact
- Centrifuge tubes (50 ml) with caps
- 7 micron Nitex screens with screen holders
- Hot plate with water bath. Use 500 ml Beakers with a few cm of water.
There should be enough water so it won't boil dry while you are working
(you can add a few ml if you see the water level is too low), but not
so much that it bubbles over into your tubes.
- 10 and 100 ml graduated cylinder: use the two in the acid cabinet
that are used only for acetolysis solution.
- Wood sticks for stirring, or the vortex stirrer.
- Hydrochloric acid (10%HCl is made by putting 90ml of distilled water
in a graduated cylinder, and adding 10 ml of concentrated HCl. Pour
the water in the large bottle labeled 10%HCl, and add the acid. Always
add acid to water, and not the inverse. Use a graduated cylinder that
is used only for clean chemicals and/or distilled water. That is, cylinders
should be used for sample water or chemicals, but never both.)
- Potassium hydroxide (10%KOH is made by mixing 10 g of KOH pellets
with 90 ml distilled water)
- Sulphuric acid H 2 SO 4 (concentrated)
- Acetic Anhydride (Acetolysis solution is made by mixing 10 ml of sulphuric
acid with 90 ml of acetic anhydride. Use the bottles provided. SLOWLY
add the sulphuric acid to the acetic anhydride. Be very careful; this
reaction produces a lot of heat. This solution should never be mixed
with water. The bottle must be kept dry. Never clean it with water.)
- Glacial acetic acid
- Hydrofluoric acid ( Be very careful when using HF. It is extremely
dangerous and you should never get any on your skin, nor breath any
of the vapors.)
- 95% Ethanol
- Saffranin stain
- tert-Butanol (You shouldn't inhale this.) N.B. TBA freezes at room
- Silicone fluid (2000 cs)
The procedure consists of successive addition of chemicals to eliminate
the non-polliniferous material from your sediments.
- sample, spike tablet
- 10%HCl (eliminates the carbonates)
- wash (distilled water)
- 10%KOH (eliminates some organic matter)
- HF, repeated if necessary (eliminates silica)
- glacial acetic acid
- acetolysis (eliminates some organic matter, prepares the pollen)
- glacial acetic acid
- 95% alcohol
- 95% alcohol
- Silicone oil
- coarse sieve after 1 if needed; fine sieve after 6 if necessary.
- Normally, you would subsample your sediment from the core and place
the samples in vials. Transfer the sediment from the vial to the centrifuge
tubes (50 ml). In sediments from boreal or temperate regions, 1 cc should
be sufficient. In arctic or low organic samples, however, 4 cc are typically
used. Note in your lab notebook the correspondence between your lake
name, sample depth and centrifuge tube number. The steps you follow
should be documented in your lab notebook as you go along; see the template.
- When you begin the procedure, start the water baths going. Some steps
require heating the centrifuge tubes. Put 2-3 500 ml beakers with some
water on the heater, and regulate the temperature to keep the water
- The number of samples that you do depends on your experience. You
can run 8 or 12 or up to 24 samples. Some steps have more critical timing,
so you need to be able to work efficiently before you try to run too
many samples at the same time. Normally, we divide the samples in two
batches; one can be in the chemical treatment while the other is in
- Place tablets of Lycopodium spike (we are currently using Batch 307862:
mean (sd) 13,500 (690)) in the centrifuge tubes. Normally, we use 2
tablets, which gives a reasonable ratio of pollen grains to spike. You
may need to adjust this to your sediments. Put the batch number and
number of tablets in the notebook.
- Add some 10%HCl. Add a little at first, since the tablets contain
carbonate, and it will fizz. If the sediment contains carbonates as
well, it will fizz even more, so be careful that it does not bubble
over the top of the centrifuge tube. 95% Ethanol can be used to stop
effervescence, if needed. Fill the tube about 2/3 full, and when the
fizzing has stopped...
- Take the tubes in pairs, equalize the amounts in both and place opposite
each other in centrifuge. Centrifuge 5 minutes at least 4000 rpm. Decant
- Add 2/3 tube of distilled water. Stir the sediment, centrifuge and
- At this point, if there are large sand particles or large organic
particles, you may want to coarse sieve the sample. Use the 150 micron
sieve and the sieve holder and run the sample through, returning the
material that passes through the sieve to the centrifuge tube. This
is awkward, and you will need to use a beaker. Use only the clean beakers
used only for this procedure, and not the beakers from the teaching
lab. You may need to centrifuge a couple of times to get all the water+sediment
that passes through he sieve (that is, if you used more than 50 ml of
water to sieve).
- Add 2/3 tube full of 10%KOH. Stir the sediment. Place in boiling water
bath for 6-8 minutes.
- Repeat 5 and 6.
- Add 1/2 tube of HF. Place in water bath for 10-15 minutes.
- Repeat 5 and 6.
- At this point, if your samples are very silty (as usually occurs in
arctic samples) you may need to sieve them through the 7 micron screen.
Add a few ml of warm 7% Sodium pyrophosphate. It should be warm but
not boiling. You can place the flask with the solution on the hot plate,
and regulate carefully the temperature. Cut a piece of screen the appropriate
size and place in the holder. Put the sample + sodium pyrophosphate
in the container and sieve until the filtrate passing through looks
clear of silt. You can rub the bottom of the sieve or use the engraver
touched to the side (thanks to Jock McAndrews for this advice) to speed
the process. After the sample has been sufficiently sieved, return the
sample to the centrifuge tube.
- Repeat 5 and 6.
- Now you need to get rid of the water in preparation for acetolysis.
Add a 1/2 tube of glacial acetic acid, stir the sediment, centrifuge
- Next step is acetolysis. Mix up in the bottle of acetolysis solution.
Add the sulphuric acid to the acetic anhydride slowly.
- Add the solution to the tubes; 1/2 full should be sufficient. Carefully
place the tubes in the water bath for three minutes . When the time
is up, carefully remove them, equalize the amount in the tubes with
glacial acetic acid, not water and centrifuge. Carefully time your second
batch so as not to exceed the three minutes. at the end of acetolysis,
you can turn off the bath, but see below.
- Add a half tube of glacial acetic acid, stir the sediment, centrifuge.
- Add a 1/2 tube of distilled water, stir the sediment, centrifuge.
- Now you need to do a 95% ethanol rinse. Add 1/2 tube of ethanol, stir
the sediment, centrifuge, decant. You can add a drop of saffranin stain
to the tube if you wish to stain the pollen grains.
- Repeat 20. Don't put in any stain the second rinse, however.
- Now put in a 1/2 tube of tert-butanol, stir the sediment, centrifuge
and decant. See below for comments on butanol.
- Repeat 22. Decant as much as possible to make the next step easier.
- You now need to transfer the sediment to the vials where they will
be permanently. If you rinsed the original vials, and they are dry,
you can use them. Make sure they are carefully labeled, and watch during
this procedure, as the butanol can dissolve some inks. Add the sediment/butanol
mixture and with the butanol wash bottle rinse the tube to get the sediment
into the vial. You may need to repeat this step a couple or more time,
although with practice it can be done in one step. Place the vial in
the centrifuge and spin. Decant and repeat if necessary to get all the
sediment into the vial.
- When the sediment is all in the vial and you have decanted the butanol,
add a couple of drops of Silicone oil and stir. You can leave the applicator
stick in the vial. Place the vials in the Clean hood and allow any remaining
TBA to evaporate. When you can no longer smell the butanol (a couple
of days if you decanted efficiently), the samples are ready to be used.
You can cap them and store them (ensuring they are properly labeled
with the lake name, the depth, and if you want, date and your name)
until you are ready to identify the grains on the slide.
- ALL WASTE MUST BE PLACED IN THE PROPER PAILS. BE VERY CAREFUL NOT
TO MIX WASTES IMPROPERLY.
- For pollen reference samples, use the same procedure with several
flowers or anthers of the plant. You can normally use a 15 ml centrifuge
tube. You can skip the HCl and HF steps.
- You can stop the procedure (overnight) at any stage where the sediment
is in water.
- If the samples are REALLY silty, you may want to use HF twice. You
can leave the sediment overnight in cold HF, then centrifuge and wash,
then hot HF as above. The 7 micron sieving can be done between or after
the two HF steps.
- Acetolysis step: when placing the tubes in the boiling water bath,
be careful not to drip any water into the tubes. You should always be
careful about this to avoid contamination, but especially here to avoid
To make a slide, take a drop of the sample and place on the center of
the slide, usually, you need to mix some more Silicone oil on the slide
to get a good density of material on the slide; this comes with practice.
Make a cross on the slide, and put on a cover slip. It is easier to show
this than explain it. Use two drops on fingernail polish on two opposite
corners to keep the coverslip in place.