Phytoliths: Background Information

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Extraction from soils  

• Soil samples are separated into fractions with gravity sedimentation, if necessary.
• Phytoliths are to be found mostly in the silt fraction (some in the fine sand and the coarse clay). 
• Phytoliths can be extracted from the silt fraction by using flotation in heavy liquid (zinc bromide, zinc iodide or soidum polytungstate are commonly used).

Phytolith Extraction Process From Modern Soil Sediments at SCSU Phytolith Facility

 1.                  Measure approximately 5 g of soil sample into a crucible.
2.                  Dry overnight at 65°C and weigh in the morning (we use a scale that measures down to 0.0001 g precision)
3.                  Record exact weight by sample# down to the last digit in the log book
4.                  Remove sand by dry sieving through a standard #10 seive
5.                  UNDER HOOD: Remove carbonates On the hot plate heat sample in a 400 ml beaker (near 100°C) in 10-15 ml HCl (approximately 10% solution) for 15-20 minutes, or until bubbling ceases
6..                  UNDER HOOD: Remove organics
a.      Add 30 ml 70% nitric acid (and a couple of times during the hour about a pinch of KClO₃), heat for 1 hour covered with hourglass to avoid splashing (hourglass also prevents loss of liquid)
b.      Dilute with distilled water 1:1, cool for 15 min, pour into 2 test tubes
7.                  Centrifuge at 2000 RPM for 3 min in 50 ml plastic tube, 2 tubes may be required depending on how much sample you have
8.                  Decant the liquid carefully, add distilled water to the top of the test tubes, spin again (x2), decant the liquid again
9.                  Add 5% sodium pyrophosphate solution into the test tube almost to the top, tighten lid, shake vigorously
10.      Centrifuge for 3 min at 2000 RPM, decant as much as possible without losing any sediment!
11.                Prepare ZnI₂ to a specific gravity of 2.3 g/cm³.
a.      For six samples: measure 281 g of ZnI₂ + 80 ml of water  + 20 ml conc. HCl on warm plate, take care to add ZnI₂ powder slowly while mixing until all dissolves (see Pearsall’s handout)
b.      Check specific gravity using densitometer (ask Julius or use our own floating one). To increase specific gravity, as zinc bromide granules. To decrease, add some distilled water
12.              Phytolith flotation process:
a.      Add 10 ml of heavy liquid to each sample (assuming there is little to no liquid left in the residue) into a 50 ml test tube
b.      Centrifuge for 5 minutes at a moderate speed (@3000 RPM).  Materials which are >2.3 g/cm³ will sink to the bottom.  Phytoliths should rise to the top   CAREFULLY siphon off the floating phytoliths with the Pasteur pipette into a dry 10 ml test tube. 
13. Transfer samples into pre-weighted glass vials, oven dry overnight. vials
13.2.              [Recycle used heavy liquid if necessary using 1 micron micropore film filters or store in a used dark bottle in a dark place.]
14.              Determine dry weight of phytoliths by subtract the empty weight from the sample weight to determine the phytolith weight
15.              Mounting phytoliths for light microscope analysis
a.      Extract a very small amount of dry phytoliths (less than the size of a pinhead) onto a microscope slide
b.      For temporary use, add one drop of immersion oil.  For permanent use, add one drop of Permount or Naphrax
c.      Place slide cover on slide, being careful to avoid air bubbles
16. Tally at least 300 morphotypes followign standard microscopy procedures as you would with pollen.