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Pollen Preparation Protocol for Fossil Pollen

Pollen Preparation Protocol for Fossil Pollen

Fossil Pollen Preparation Protocol (V. 1-1)

Wear a lab coat, latex or vinyl gloves and have a pair of safety goggles accessible.

Samples are typically, between 0.5 and 2 cm3 and can be sampled using a calibrated volumetric sampler or using water displacement in a 5 cm3 measuring cylinder. These should be placed into 50ml plastic centrifuge tubes (small tubes can be used if samples are particularly small).

To quantify the concentration of pollen within the samples an exotic spike can be added, 1-3 lycopodium tablets depending on the nature of the sediment ie. 3 if lots of pollen likely. A ratio of 1:1, or 1:2 is desirable for statistical accuracy, although 1:5 is acceptable. Remember to note the batch number of the lycopodium tablets which relates to the concentrations of spores contained within the tablets which is used in the later calculations.

1. Hot 7% HCl, heat up water bath in fume cupboard to ~90oC (takes 20-30 mins). Fill tubes 2/3 full with HCl and place in the water bath for 20-30 mins so HCl dissolves carbonate, there may be a violent reaction a drop of methylated spirits can be used to break surface tension and stop samples foaming. Stir occasionally with glass rods (one for each tube). This acidifies the samples to pH1.

- if samples are very calcareous it may be necessary to remove carbonate using a small amount of concentrated HCl.

Balance and centrifuge, large tubes 3000rpm/5 mins, small tubes 3000rpm/3 mins.

2. Wash H20, decant off the "yellow" or "brown" clear liquid supernatant in one fluid movement from tubes into a large waste beaker (the contents of the waste beaker can be emptied down the sink and washed away with water), solid residue should be left in the bottom of the tubes. Whirlimix and top up with deionised water, ensure that all the sample is mobilised, use a glass rod if necessary.

Balance and Spin.

- whilst this is spinning prepare sieves ie. Flame with gas burner (using tongs) and quench in water, also prepare funnels and second batch of tubes with labels.

3. Hot 10% NaOH, Decant and Whirlimix. Fill tubes 1/3 to 1/2 full with NaOH. This takes the pH up to alkaline (pH14) taking humic acids into solution. Place in water bath for 2-4 mins. Do not Whirlimix or decant.

4. Sieve, put second batch of tubes into a rack with funnels and sieves. Sieving should be carried out wearing safety glasses as even a small amount of NaOH can seriously damage your eyes.

Pour each sample through the sieves, wash through using deionised water (a nozzle restrictor may be useful to increase the pressure of water and restrict the amount used). Wash through sieve thoroughly using the "corner method" if you want ie. working across the sieve from top to bottom and then left to right. Place used tubes, sieves and funnels into sink after use. Difficult samples may be worked through the using a clean gloved finger or glass rod (care must be taken not to damage the sieve mesh). Alternatively a few water washes prior to sieving will remove humic material, dilute the NaOH so making sieving safer.

5. Wash H2O, same procedure as 2, repeat until the liquid that is being decanted off is clear (this usually takes 5-6 washes, but if youre unlucky upto ~16!!). This removes humic acids (brown) and clays (usually grey). Sticky samples can be difficult to whirlimix and should be stirred with a glass rod, very clay rich samples may benefit from the use of 4% sodium pyrophosphate to help break the electrostatic bonds (placing in the water bath for ~10 minuets will further aid this process), a couple of water washes may be required to fully remove particles.

- The metal Sieves should be washed thoroughly in hot water with detergent, these are not be washed in Decon, which is corrosive. Funnels should be washed in detergent and then placed in a bowl containing Decon (see washing up protocol).

6. Wash 7% HCl, same as 2, except using a smaller amount of HCl instead of H2O. Only do this once. This is to acidify the samples before HF, assist the action of HF on samples and to make sure there is no residual carbonate.

7. HF cold/hot, VERY DANGEROUS! Use the buddy system. Wear long brown rubber apron, latex or vinyl gloves, black rubber gloves and full face mask. Prepare a large beaker containing a saturated solution of sodium carbonate (which neutralises HF) in the fume cupboard. Make sure that you know the location of all the related safety equipment and the
procedure in case of a spill;

Calcium gluconate tablets to be taken if you come into contact with HF in fridge.

Calcium gluconate gel to take to hospital for application to and injection under HF burns - in fridge.

Slaked lime to neutralise large spills of HF - in large canister.

The water hose in case you become contaminated, in which case remove all contaminated clothing and hose yourself down for 15 mins with cold running water.

All work MUST be carried out in the fume cupboard. Open sash slowly as a brief reversal of air flow can be created which draw fumes out into the lab if opened quickly.

Hot - Carefully pour HF into each tube 1/2 - 2/3 full and leave for ~2 hours in the 90oC bath in the fume cupboard. Wash everything well in sodium carbonate. Particularly silicate rich samples may require multiple HFs or even leaving overnight with the water heater off, the fan on and good labelling.

Cold - Alternatively samples can be left well labelled and with tube caps on at the back of the fume cupboard for a longer period of time, it is best to leave the fan on during this period.

After this time put caps on tubes before removing them from the water bath. Balance (add water into tube containers to balance) centrifuge with caps on. Remove tube caps and decant carefully down the FUME CUPBOARD sink. Dip the whole tube into the saturated Sodium Carbonate solution to remove drips of HF from the tube. Do not allow any sodium carbonate into the tube.

Wipe down the floor of the fume cupboard with a cloth soaked in Sodium Carbonate solution, in case of any unnoticed splashes.

HF removes silicates from the samples. This can also be achieved using density separation which floats off organic material leaving the silicate minerals behind.

8. Hot 7% HCl, place into water bath for 20-30 mins to remove silicate residues and fluorosilicates. Heating increases the solubility.

10% HNO3, dilute Nitric Acid may be required at this stage to remove sulphides. Follow procedure as 2.

9. Wash H2O, same procedure as 2.

10. Wash CH3COOH, Concentrated (glacial) Acetic acid (smells of vinegar). Same procedure as 2 except use CH3COOH instead of water. However when decanting pour waste down the sink in the fume cupboard and try a keep the samples in the fume cupboard at all times, and between you and the fume cupboard when centrifuging, use tube caps if necessary. This is used to remove water so that acetolysis can be carried out.

11. Acetolysis, use green nitile gloves. This mixture reacts exothermically (produces heat) and can be both violent and dangerous, especially when it comes into contact with water.

To make up the acetolysis mixture combine 9 parts of Acetic Anhydride (ie. 45 ml) with 1 part of Conc. sulphuric acid (ie. 5 ml) (pour into a smaller beaker first as the bottle is unwieldy) in a measuring cylinder. Place 5 ml of the mixture into each tube, working in the fume cupboard, and incubate in waterbath for 3 mins.

This procedure removes cellulose and polysaccarides from samples by oxidation.

12. Wash CH3COOH, remove from waterbath and top up with Acetic acid to cool and stop the acetolysis reaction and balance. Spin and decant down the sink in the fume cupboard.

13. Wash H2O, same procedure as 2. Meanwhile put the wash bottle of TBA into the onto the water bath to melt. Loosen top to avoid explosion due to a pressure build up!.

14. Wash ~1% NaOH, after decanting Whirlimix and then add a squirt of 10% NaOH, Whirlimix again and top up with H2O. Balance, spin and decant down sink or into a beaker with water in it. Whirlimix.

The purpose of this stage it to take the pH from slightly acid to slightly alkaline. (Safranin turns blue in acidic solutions!!).

15. Wash H2O, same procedure as 2.

16. Wash H2O + 0.2% aqueous Safranin, for wash same procedure as 2, but add 1-4 drops of dye (depending on sample), Whirlimix, top up with H2O, balance, spin and decant.

17. Wash TBA, remove warmed TBA from the waterbath and 1/2 fill tubes best done in the fume cupboard. Be careful not to get the TBA on you as it is toxic or breath the fumes. Balance (top up with TBA), spin and decant into a beaker with water in it. Whirlimix. TBA removes water from samples so silicone fluid/oil can be added, decant down fume cupboard sink.

18. Vial of remainder, label vials (No. three times top and bottom, depth, site and sample no.). Put a small amount of TBA into TUBE 1 (not vial 1) and Whirlimix 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 using tweezers and spin as normal. Place tubes into the sink for washing.

Decant vials into the waste beaker with water in it (to control TBA fumes) and add silicon oil (1-6 drops roughly equal to the quantity of residue) to the tubes with a disposable pipette or cocktail stick. 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. Then you will be able to make slides and begin enjoying counting pollen!!!

Compiled by William Gosling

with the assistance of Ian Lawson, Katy Roucoux and Steve Boreham

WASHING UP PROTOCOL


All equipment that has come into contact with pollen, except the sieves should be washed in this way.

1) Wash and scrub vigorously in normal detergent and warm water, then placed under running water.

2) Transfer to a bowl containing deionised water and 50-100ml of Decon 90 (Alkaline and will burn skin). Soak for 12-48 hours.

3) Rinse in deionised water and fully immerse in a 1% HCl solution (cover bottom of bowl with 7% HCl and half fill with deionised water.

4) Stack in drying basket and place in ~50oC in glassware drying oven. Put your tubes away!!!!

HYDROFLUORIC ACID AND YOU!


Hydrofluoric acid is corrosive. As a gas it is highly irritating to the eyes and the respiratory tract. In solution it can cause sever burns. If you get it on your skin you may not feel the pain at once.

PROTECTION

  • ALWAYS use the protection provided.
  • ALWAYS wash your gloves and other impervious clothing before you remove them.
  • TEST gloves for pinholes by filling them with water, before drying and putting them away ready to use again.
  • ALWAYS wash your hands before you leave the work area.
SPILLAGES
  • Spillages on tools, bench - neutralise with slaked lime
  • The contaminated area must be hosed with plenty of water.
  • Spillages on clothing - neutralise with sodium bicarbonate solution.
FIRST AID

1) Gassing

  • Remove the casualty to fresh air.
  • If necessary, resuscitate the causality.
  • Send to Accident and Emergency Unit.
2) Skin
  • Remove contaminated clothing (NB protect your hands with gloves).
  • Flood skin with clean cool water to remove acid. Send to Accident and Emergency Unit without delay, continuing the following during transit. Apply calcium gluconate gel on and around the affected area and massage it continuously until pain is relieved. This will take at least 15 minutes. Cover area with dressing soaked in gel. Bandage lightly.
3) Eyes
  • Irrigate with clean cool water for at least 20 minutes. This can be continued during transit to hospital.
  • Send to Accident and Emergency Unit or local eye hospital.
  • In all cases inform hospital of the cause of the injury.
  • Report splashes on skin and eyes to supervisor/employer.
FIRST-AID TRAINING

It is the responsibility of your employer to ensure that there is an adequate number of employees on site trained in appropriate first-aid procedures.

Training should be given by organisations approved by HSE. If you have an occupational health department, the doctor or nurse will be able to advise you on how to carry out the different procedures in your workplace.

LEGAL REQUIREMENTS

The Control of Substances Hazardous to Health (COSHH) Regulations 1988 apply to hydrofluoric acid - see Approved Code of Practice COP29, Control of substances hazardous to health, available from HMSO (ISBN 0 11 885468 2).

The Health and Safety (First Aid) Regulations 1981 apply to all aspects of first aid at work - see Approved Code of Practice COP42 First aid at work, available from HMSO (ISBN 0 11 885536 0).