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UCL Home  /  Geography  /  Resources  /  Laboratory  /  Laboratory Methods  /  Lake Sediment Analysis  /  Pollen Preparation Protocol for Marine Fossil Pollen

Pollen Preparation Protocol for Marine Fossil Pollen

 

Samples are typically between 6 and 9 grams (if volume is needed use water displacement in a 5 cm3 measuring cylinder). For the Marine Isotope Stage (MIS) 5e project use 7 grams split into 3 equal parts (~2.33 grams each) and place into 50 ml plastic centrifuge tubes. In the prep sheet provided note the exact weight of each sample, the tube number and an estimation of how much sample is left (if any). Note: Marine samples are usually dry. In order to facilitate their disintegration, break down the sediment using softly a pestle without removing the plastic bag.

Add one tablet of lycopodium per sample (i.e. only in one of the 3 tubes) and write down the exotic concentration and batch number (this is used to quantify the concentration of pollen within the samples).

Removal of calcium

· Add 50 ml of 10% HCl. Marine samples are extremely calcium carbonate rich so add HCl very slowly. Try to get the entire sample in contact with the HCl, by using the whirl mixer and sometimes the caps (be careful when opening the caps, since the reaction produces gases).  When the effervescence stops, fill the tube up to the 50 ml mark and place the tubes in the water bath (~70°C) for 1 hour.  If the tube threatens to froth over, reduce the foam with a squirt of acetone or methylated spirits, which break surface tension. This acidifies the samples to pH1. Centrifuge at 3000rpm for 5 mins and decant.

· Wash with H20: decant off the "yellow" or "brown" clear liquid supernatant in one fluid movement from tubes in the fume cupboard, and thoroughly mix the solid residue left in the bottom of the tubes. Add slowly distilled water up to the 50 ml mark. Note: Make sure that all the sediment is mobilised and broken down (use the caps and if needed a glass rod). Centrifuge; whilst this is spinning prepare sieves i.e. place an 180μm sieve into a 1 litre beaker. Decant off the cleaned sample.

Removal of humic acid

· Add 35 ml of 10% KOH and place in the water bath for 4 mins.  This takes the pH up to alkaline (pH14) taking humic acids into solution. Do not whirl mix or decant.

Removal of organics

· Pour each sample through the sieves, wash with distilled water (using a spraying jet and a nozzle restrictor on the washing bottle). Sieving should be carried out wearing safety glasses as even a small amount of KOH can seriously damage your eyes. Transfer all the produced liquid into the tubes, centrifuge and decant (if there is any remaining liquid in the beaker, this step will have to be repeated; try to split the remaining amount into equal parts so that all your samples have less than 2.5 grams of sediment since larger samples might not get completely digested by the chemicals). If there is anything interesting on the sieve keep it in a small vial.

· Wash with H20 until the liquid that is being decanted off is clear. This removes humic acids (brown) and clays (usually grey) and might take up to 15 washes. Only in case the samples are very “hazy” exceed 13 washes. When all the KOH has been removed (either after the 1st or the 2nd wash) add 2 drops of 10% sodium hexametaphosphate (calgon); this helps break the electrostatic bonds.

Removal of silicates

· Wash with 50 ml of 10% HCl. This is to acidify the samples before HF, assist the action of HF on samples and to make sure there is no residual carbonate. Prepare a large beaker containing a saturated solution of sodium carbonate (which neutralises HF) in the fume cupboard.

· Add 30 ml of 40% HF and place in the water bath for 1 hour. Stir twice every 20 mins using a plastic pipette. After this time put caps on tubes, centrifuge and decant. HF removes silicates from the samples.

· Wash with 35 ml of 10% HCl and place in the water bath for at least 40 mins. This helps to remove silicate residues and fluorosilicates (heating increases the solubility). Centrifuge and decant.

· Add another 30 ml of 40% HF and place in the water bath for 1 hour. Stir twice every 20 mins using a plastic pipette. After this time put caps on tubes, centrifuge and decant.

· Wash with 40 ml of 10% HCl and place in the water bath for at least 1 hour. Centrifuge and decant.

· After decanting the supernatant liquid and using the pipette get a small amount of sample and prepare a slide by adding distilled water. Check the slides at a light microscope under x40 magnification for the amount of remaining silicates. If large pieces and more than 3 can be seen in each field of view repeat the HF and HCl steps but with smaller amounts.  If samples are silicate free, put the sample back in the tubes using distilled water.

· Transfer samples into 1 tube, clean black dry debris from the walls of the tubes and add a small amount of 10% HNO3 to remove sulphides and pyrites. I aimed for 3ml of concentrated HNO3 and 30 ml of H2O, but larger amounts can be used as long as you keep the proportion 1/10. Place in the water bath for 4 minutes. Centrifuge and decant. Note: some of the sample stays on the tube walls, so before centrifuging use methylated spirits to incorporate it with the rest of the sample.

· Wash with H2O, centrifuge and decant. Meanwhile put the wash bottle of TBA into the onto the water bath to melt (remember to open the lid since TBA expands with heat).

Staining and mounting

· Add 22 ml of 1% KOH (2 ml of 10% KOH and 20 ml of H2O). Centrifuge and decant. The purposes of this stage it to take the pH from slightly acid to slightly alkaline. (Safranin turns blue in acidic solutions!!).

· Wash with H2O. If supernatant liquid is brown repeat this step until it becomes clear (maximum 3 washes).

· Add 3 drops of Safranin and 50 ml of H2O. Centrifuge and decant.

· Add 10 ml of TBA, centrifuge and decant (TBA removes water from samples so silicone fluid/oil can be added).

· Transfer into vials: label vials (site once and depth three times since TBA erases marker labels). Put a small amount of TBA into TUBE 1 (not vial 1) and Whirl mix as normal. Then pour into VIAL 1, repeat until all the residue is in vial 1. Once completed for all the samples place the vials into the centrifuge ‘tube holders’ (add tissue paper at the base of the holders to make them “softer”) using tweezers and spin at 3000 rpm for 3 mins. If you cannot transfer the entire sample at once, centrifuge, decant and then add the remaining sample.

· Decant TBA from the vials and add silicon oil (roughly equal to the quantity of the residue) with a disposable pipette. Mix well with a cocktail stick leaving one in each vial, place safely in a beaker lined with tissue and cover to prevent contamination. The TBA will evaporate off over a couple of days, the samples need to be checked and stirred to make sure that they have not dried out and stirred during this period.

· When all the TBA is evaporated put labelled caps on.

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