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UCL Home  /  Geography  /  Resources  /  Laboratory  /  Laboratory Methods  /  Lake Sediment Analysis  /  Pollen Analysis of Terrestrial Sediment

Pollen Analysis of Terrestrial Sediment

Notes on Sample Preparation

  • Samples should be stored wet at <4oC.
  • 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

  • Slowly add about 10ml 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 rpm. for 4 minutes and about 2ml 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 10ml 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

  • Add 10ml 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

  • Strain and wash the sample through a fine mesh screen (170-180um) into a 50ml 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

  • Add 10% HCl, stir, centrifuge and decant.
  • If sediment contains mineral matter, treat with 40% HF. In a suitable fume cupboard add about 10ml 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 10ml water, stir , centrifuge and decant.

Removal of water

  • It is essential to remove all water before the acetolysis step as the acetolysis mixture reacts very violently with water.
  • Add 10ml Glacial Acetic Acid, stir, centrifuge and decant.
  • Repeat this step to ensure all water is gone.

Removal of cellulose (acetolysis)

  • 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 10ml Acetic Acid. (Stir, Centrifuge,Decant!)
  • This removes the soluble cellulose products of the acetolysis.

Staining and mounting

  • Wash the sample into 15ml glass centrifuge tubes using water.
  • Centrifuge and decant.
  • Add 9ml water and 1ml 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 10ml 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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