POLLEN EXTRACTION PROCEDURES
FOR QUATERNARY SEDIMENTS

Inorganic Sediment (Archeological, Arroyo)
          M. Trout's detailed instructions
Reference Specimens
Packrat Middens
Lake and Bog Sediments
Acetolysis
Density Separation
References

GENERAL CONSIDERATIONS

The various pollen-extraction procedures are based on the small size and other unique physical properties of pollen. Palynomorphs are extremely resistant to strong acids, but sensitive to oxidizing agents. Extraction procedures rely on these properties to remove the silicate and carbonate sediment matrix investing the pollen. Weak organic compounds can be removed with mild bases, but strong bases destroy the palynomorphs. The density (specific gravity) of pollen is less than that of clastic materials, so liquids with a specific gravity of 2.0 floats pollen, while the clastics sink. Screens can be used to remove particles larger than or smaller than pollen.

SAFETY

The chemicals and procedures used in pollen extraction are hazardous, and many fatalities have resulted from accidents in pollen extraction laboratories. HF, Hydrofluoric acid is particularly dangerous, and exposure to the skin, or inhaling the fumes can be fatal. Pollen extraction should be done only with a high-quality fume hood, and Calcium Gluconate should always be available to treat HF burns. Full face masks, rubber gloves, plastic aprons, and long sleeves should be worn when working with HF.
Precautions
How does HF kill?
How does HF kill?
Example
Example

Calcium Gluconate Gel
Pharmascience Laboratories Inc.
175 Rano Street
Buffalo NY 14207
1 - 800 - 207 - 4477
www.pharmascience.com

Nitric Acid Treatment vs. Acetolysis

Nitric Acid (HNO₃) is routinely used in the extraction of arroyo and archeological samples. This procedure cannot be combined with acetolysis because some of the pollen will be destroyed. Nitric acid (without acetolysis) may be used to remove ferro-magnesian crystals that are resistant to HF and HCL. These crystals are common in playa sediment where sulfur-reducing bacteria are common below the sediment surface.

Screens and Cloth

Large-mesh (160 µm) metal screens are used to remove coarse mineral and plant debris from the sample. Larger mesh adds sand-sized particles to the sample which must be be dissolved by HF, smaller sizes remove large pollen grains.

Fine-mesh (10 µm) nylon cloth (NYTEX) is used to remove extraction-resistant organic particles (e.g., charcoal) or inorganic (clay) materials from the sample. Before attempting NYTEX screening, the samples should be thoroughly disaggregated with sodium pyrophosphate (Na₄P₂O₇) or a similar detergent.
1. suspend sample in 10 ml of 5% solution
2. 7 micrometer NITEX screen, centrifuge, decant, wash twice
3. gently swirl, vibrate with a metal scriber, or rub the underside of the screen gently

CLEANING

Pollen is readily oxidized. To thoroughly clean test tubes, hydrogen peroxide or other oxidizing agents can be used. In fume hood add a few drops of HCl to hydrogen peroxide. This evolves chlorine gas which will destroy any pollen in this liquid. Also a sodium permangenate - sulfuric acid solution can be used to clean test tubes and other glassware.

TRACERS

Thomas Persson
Lund University
Department of Quaternary Geology
Tornavagen 13
S-223 63 LUND, Sweden
Fax: +46 46 2224830
Thomas.Persson@geol.lu.se

GENERAL PREPARATION FOR SEDIMENT SAMPLES

Taking Sediment Samples:

Materials:
Lab notebook, color chart, meter stick, scales, volumetric spoons, spatulas or knives, plastic vials, centrifuge (optional)

Wet Sediment. The core may have been wrapped in plastic wrap and aluminum foil in field, it may be in an aluminum tube (e.g., vibracore) that must be sawed open, or the sediments may paleomagnetic samples in plastic boxes.

Clean all materials and counter top thoroughly. Work in a clean room with filtered air. Scrape the sediment surface clean, or split the core in half to expose a fresh surface. Describe color, texture, and other features of the sediemts. Sample volumetrically and record the location of each sample taken from the core (e.g., depth). Samples can be stored in clean plastic vials, or placed directly in clean test tubes. Sample mass can be determined by weighing the vials before and after adding sediment. Volumetric samples (1 cm³) can be taken with spoon or with a volumetric plunger. Label vials or test tubes carefully.

Dry sediment. Dried cores, arroyo sediments, dried well cuttings, dry surface samples, or dry archeological samples. Use larger volumes (5 - 20 cm³) for archeological, arroyo, or surface (pinch, moss) samples. Indurated samples can be placed in a weak detergent solution in test tubes. This should be should mixed thoroughly, centrifuged and decanted several times. Clay rich samples can be centrifuged and mixed many times to remove clays until the supernatant is no longer milky.

Preparing samples for extraction:

Materials:
Lab notebook, 50 ml plastic tubes, 250 ml beakers, Lycopodium tracer tablets, 10% HCl, centrifuge.

Enter the date, site, and other information in the lab notebook, and record each sample. Prepare 50 ml plastic tubes and beakers by cleaning thoroughly and labeling each beaker and test tube according to the sample it will contain. Ordinarily, 12 samples per set.

1. add the volumetric sediment sample to ca. 50 ml 10% HCl in 250 ml beakers, mix thoroughly
2. add Lycopodium tracers tablets to acidic solution, allow to disolve and mix thoroughly
3. swirl the solution and screen into second beaker
4. transfer screened solution to 50 ml nalgene test tubes
   repeat "d", centrifuging, until transfer complete

TYPICAL EXTRACTION OF INORGANIC SEDIMENT

Materials:
Samples in 50 ml plastic test tubes, clean Pyrex 15 ml test tubes, conc. HCl, 40% HF, 10% HNO3, 10% KOH, safranin stain, glycerin, 1 dram shell vials

1. 10 ml conc. HCl, mix, add 30 ml H₂O, mix
   rinse with hot water
2. HF overnight or 1 hr in boiling water bath
   rinse with hot water
3. transfer to 15 ml pyrex test tubes
4. 10% HNO3 2 min. boiling water bath
   rinse with hot water
5. 10% KOH 2 min. boiling water bath
   rinse with hot water until clear
6. stain with safranin "O"
7. transfer to 1 dram shell vials
8. add a few drops of glycerin
9. store in vacuum jar over desiccant, 1 day - 1 week

REFERENCE SPECIMENS

(centrifuge and decant between steps)

1. wet sample in weak detergent solution, or sodium pyrophosphate (or EtOH)
2. swirl solution and screen into second beaker
3. transfer screened solution to 10 ml glass test tubes repeat "b" centrifuging until complete
4. ACETOLYSIS*
5. 10% KOH 2 min. boiling water bath rinse with hot water until clear
6. stain with weak safranin "O"
7. two TBA (tertiary-butyl alcohol) rinses
8. transfer to 1 dram shell vials
9. add a few drops of silicone oil 50 cs
10. store in vacuum jar over desiccant, 1 day - 1 week

TYPICAL EXTRACTION OF PACKRAT MIDDEN

(centrifuge and decant between steps)

1. 10% KOH 2 min. boiling water bath
   rinse with hot water until clear
2. 10 ml conc. HCl, mix, add 30 ml H₂O, add tracers (5 tablets), mix
   rinse with hot water
3. HF overnight or 1 hr in boiling water bath
   rinse with hot water
4. transfer to 15 ml pyrex test tubes
5. ACETOLYSIS*
6. 10% KOH 2 min. boiling water bath
   rinse with hot water until clear
7. stain with weak safranin
8. two TBA (Tertiary-Butyl Alcohol) rinses if silicone oil
9. transfer to 1 dram shell vials
10. add a few drops of silicone oil (or glycerin)
11. store in vacuum jar over desiccant, 1 day - 1 week

TYPICAL EXTRACTION OF LAKE AND BOG SEDIMENTS

1. 10 ml conc. HCl, mix, add 30 ml H₂O, mix
   rinse with hot water
2. HF overnight or 1 hr in boiling water bath
   rinse with hot water
3. transfer to 15 ml pyrex test tubes
4. 3 ml conc. HCl, mix, add 10 ml H₂O, mix
   rinse with hot water
5. ACETOLYSIS*
6. 10% KOH 2 min. boiling water bath
   rinse with hot water until clear
7. stain with weak safranin
8. if silicone oil: two TBA (tertiary-butyl alcohol) rinses
9. transfer to 1 dram shell vials
10. add a few drops of silicone oil (or glycerin if no TBA)
11. store in vacuum jar over desiccant, 1 day - 1 week

*ACETOLYSIS

1. 5 ml glacial acetic acid
   centrifuge and decant
2. stir sample, add 5 ml acetic anhydride (volumetric dispenser)
3. add 0.55 ml H₂SO₄ to acetic anhydride solution (volumetric pipet),
   mix centrifuge, decant into glacial acetic acid
4. 5 ml glacial acetic acid
   centrifuge and decant

DENSITY SEPARATION

1. add volumetric (100 cm³) sample of sediment to ca. 250 ml water in 1000 ml beaker.
   Add tracers to solution (ca. 2 tablets)
   Add conc. HCl (5 ml increments) until sample stops fizzing.
   Swirl and wait 45-60 sec, pour liquid through tea strainer into 300 ml centrifuge bottle.
2. Centrifuge and decant.
3. Add ZnBr₂ (1.5 times volume of sediment, density = 2.0), to sample; shake vigorously.
4. Centrifuge and decant ZnBr2 (with pollen) into fresh 300 ml centrifuge bottle.
   Discard inorganic sediment in bottom of centrifuge bottle.
5. Dilute ZnBr₂ 3-4 X volume with water.
   Centrifuge and decant ZnBr2 into beaker to be filtered and boiled down.
   Pollen sample is on bottom of centrifuge bottle.
6. Transfer pollen sample to 50 ml nalgene tubes
7. HF 1 hr
   rinse with hot water
8. 10 ml conc. HCl, mix, add 30 ml H₂O, mix
   rinse with hot water
9. Transfer to 1 dram shell vials
10. Add 100% EtOH (or stain, add a few drops of glycerin)
11. store in vacuum jar over desiccant, 1 day - 1 week

REFERENCES

Anonymous 1963. Processing samples for palynological study. Geochronology laboratories, University of Arizona.

Dohrer, L.I. 1980. Palynomorph preparation procedures currently used in the paleontology and stratigraphy laboratories, U.S.Geological Survey. Geological Survey Circular 830.

Cwynar, L.C., Burden, E. and McAndrews, J.H. 1979. An inexpensive sieving method for concentrating pollen and spores from fine-grained sediments. Can. J. Earth Sci. 16:1115-1120.

Gray, J. 1965. Extraction techniques. pp. 530-587 IN: Kummel, B. and Raup, D. HANDBOOK OF PALEONTOLOGICAL TECHNIQUES. W.H. Freeman Co., San Francisco.

Stockmarr. J. 1971. Tablets with spores used in absolute pollen analysis. Pollen et Spores 13:615-621.