Processing Arctic sediments including heavy-liquid (Sodium Polytungstate (SPT)) concentration of pollen grains
By: Susan Zabenskie & K Gajewski.
The protocol elsewhere on these pages should suffice for normal sediments. With inorganic sediments from the Arctic, the 7 micron sieving method may help to concentrate the pollen with a minimum of extraneous sediment. In High Arctic sediments, however, this is rarely sufficient. An alternate to the sieve method is here described, based on heavy liquid separation. The entire protocol is listed here, however, you should review the other page (http://www.lpc.uottawa.ca/resources/pollen.html), especially the section on laboratory safety as well.
- Subsample sediment from core and place in vials (or if possible, directly into 50 mL centrifuge tubes. With Arctic sediments, a maximum of 2 cc can be used at one time, otherwise 1 cc should be sufficient. You may need to modify this based on experience with your core. Lake, depth in sediment and tube number should all be noted in lab notebook. Use the standard notebook template to record the details of your processing.
- In each centrifuge tube, place 2 tablets of Lycopodium spike. Note the batch number and number of grains added in the notebook.
- Add 10% HCl. Use a small amount at first as HCl reacts with the carbonate filler in the Lycopodium spike. If needed, empty and clean out the centrifuge vials into the tubes using the HCl. Stir the sediment and HCl together, trying to rid the sample of large clumps of sediment. (Start the water baths now. Watch these periodically and adjust to keep the water boiling)
- Take the tubes in pairs and balance them on the small plastic balance. Place them in the centrifuge across from one another for 5 minutes at 4000 rpm. Note the proper slots on the diagram on the wall. Make sure that (a) the caps on the 4 buckets are closed and (b) the plastic lid is properly seated. Do not run the centrifuge without the caps and lid, even if the tubes only contain water. After they have spun down, decant the supernatant.
- Add 2/3 tube of deionized water, stir, centrifuge and decant (called washing).
- Add 2/3 tube full of 10% KOH. Stir the sediment and place in a boiling water bath for 6-8 minutes. (Generally 8 minutes)
- Repeat steps 4 and 5.
- Perform another 2 washes (step 5 another 2 times) to rid excess particulates floating in the tubes.
- Repeat steps 3 through 5. Making the solution acidic may aid in removing non-pollen material from suspension forcing it to accumulate with the sediment at the bottom of the tubes. (Takeshi 1998) When decanting the water ensure that the maximum possible amount of water has left the tubes. If not, excess water might decrease the density of the SPT.
- Prepare the Sodium Polytungstate (SPT) solution. This is done by other adding powder to a previously made solution (if necessary), or, if none is available, dissolving the powder in water to form a solution with a density of approximately 1.95. Use a hygrometer to verify your specific gravity. Note that the density of sporopollenin is 1.4. Add 5ml of the SPT solution to each tube and mix on the vortex for five minutes. You can set up a retort holder to hold the tubes in position over the vortex mixer (see picture). Make sure the tubes are capped.
- When samples are vortexed, they can be centrifuged at 1800 rpm for 10 minutes (8 at a time). The speed is decreased from that typically used in pollen preparation so that pollen is not forced down to the bottom. To balance the tubes, use extra tubes filled with deionized water or other vials. DO NOT add more SPT to your samples.
- After centrifuging, decant the pollen - the material that floats into another LABELED centrifuge tube. These are now your samples. Make sure to note the second tube number in the lab notebook. It is sometimes necessary to add a small squirt of water into the tubes to ensure all the pollen was decanted. Take the tubes that have the sediment left in the bottom and rinse them out. These are discarded, although you may wish to occasionally make smear slides of this to ensure you are not losing pollen . Be sure to rinse these tubes again with deionized water.
- Take your pollen (in their new tubes) and the original tubes that you rinsed and split the 5ml of SPT plus pollen mixture between the two. It is now important to fill the tubes up as high as possible with water and to top them off with a squirt of ethanol to decrease the density of the water/SPT mixture, ensuring the pollen sinks to the bottom. Note that the squirt of ethanol is done at several steps (any time the tubes are primarily water) to break the surface tension and prevent pollen from floating on the water.
- Centrifuge at 4000 rpm for 5 minutes.
- When decanting, take the supernatant SPT/water mixture that you decanted and place into the plastic container labeled “SPT-dirty”. This material will be reused. This liquid should be filtered using a Buchner funnel and large coffee filter (see picture). It should be filtered twice, using a second coffee filter. The liquid is then filtered a second time with a Buchner funnel under a vacuum using a 7µm mesh (see picture). The liquid is then placed into a cleaned beaker and placed into the oven at 105ºC to evaporate the water and decrease the density of the liquid. You should evaporate a sufficient quantity of water to ensure the density is greater than 1.9, but it should not be allowed to dry. This cleaned liquid should be placed in the plastic container labeled “Clean SPT”, ready to use.
- The two tubes contain pollen from the same sample, they are combined using water (remember the squirt of ethanol), centrifuged and decanted.
- At this point, it is up to the analyst to decide if HF is needed. If the sample is quite silty, then this is recommended. Recall the HF is extremely dangerous, and all protective equipment must be used. In the centrifuge all three caps (centrifuge, bucket, centrifuge) must be used. See the WHIMIS information in the lab.
- HF is added to each tube, approximately half full, centrifuged and decanted. You can heat it for 12-15 minutes if there is a lot of silt remaining. You can also leave it overnight, cold. If you leave it overnight make sure it is properly labeled.
- Step 5 is repeated.
- N ow 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 and decant. If you need to balance the tubes, use glacial acetic acid.
- Next step is acetolysis. Mix the acetolysis solution in the appropriate bottle. Note that this bottle should have no water. Add the sulphuric acid (10 mL) to the acetic anhydride (90 mL) slowly. See the warnings on the standard pollen protocol page.
- 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, nor should you add the ethanol ) and centrifuge. Carefully time your second batch so as not to exceed the three minutes in the water bath. At the end of acetolysis, you can turn off the water bath, but see below.
- Add a half tube of glacial acetic acid, stir the sediment, centrifuge. Do not add water at this step .
- 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 step 25. 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. The TBA must be warmed (but not hot) to prevent it from freezing at room temperature. This is especially important in step 29. To heat the butanol, fill a beaker with hot tap water and place the bottle in the hot solution. Change the water periodically if needed. Do not heat the butanol, nor place it in the water bath on the hot plate.
- Step 27 is not usually repeated here (as per normal protocol) to decrease the chances of decanting pollen.
- You now need to transfer the sediment to the vials where they will be stored permanently. If you rinsed the original vials, and they are dry, you can use them. Make sure they are carefully labeled (and completely dry), and watch during this procedure, as the butanol can dissolve some inks. You can cover the label information with scotch tape, for example. 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 of times, 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 with a toothpick. You can leave the applicator stick in the vial. Place the vials in the Clean Hood (the green one in the core prep room). The clean-hood room is sometimes quite cold and the TBA may freeze before evaporating, in which case you can let it sublimate or place in an alternate, clean location) 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.
- 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 of fingernail polish on two opposite corners to keep the coverslip in place.
- All waste must be placed in the proper pail. Be very careful not to mix wastes improperly.
- You can stop the procedure (overnight) at any stage where the sediment is in water (or ethanol).
- With any acid step, and especially HF, you should be careful not to breathe the vapours, nor let any acid touch your skin. Use goggles, a plastic face mask, lab coats, and extreme caution.
- 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 the reaction.
- Ethanol may be used to break the surface tension at any washing step.
- Thanks to A López-Higuera for help with the SPT method.
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