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Sample preparation for diatom analysis

After: Bouchard, G. 2004. Freshwater Diatom Biogeography of the Canadian Arctic Archipelago. M. Sc. Thesis, Ottawa-Carleton Geoscience Centre and University of Ottawa. 170 pp.

Note that the process takes up to 10 days, if you are using gravity settling.

Diatom Concentration

  1. Measure out 0.5 or 1 cc of sediment using a volumetric sampler.
  2. Weigh it and place it in a labeled 20 ml glass vial with leak proof lid.
  3. Add a drop of 10% HCl to determine if carbonates are present. If fizzing reaction occurs (carbonates are present), then add 15 ml HCl to the vial. Add slowly if you are getting a reaction. For samples that did not react, add 15 ml deionized (DI) water.
  4. Wait 24 hours.
  5. Aspirate the water or acid waste, leaving the bottom 5 ml of sample intact.
  6. Wash samples four times. The procedure for washing is as follows: Add DI water to sample, stir with a glass rod (a clean one for each sample), allow sufficient time for diatoms to settle if you are using gravity settling (24 hours), aspirate.
  7. When aspirating, take care not to remove any portion of the diatom slurry.
  8. Add 15 ml of strong acid: a 50:50 mixture of concentrated sulphuric (H2SO4) and nitric (HNO3) acids. (Conc. H2SO4 is 98% and conc. HNO3 is 63%). To make the 50:50 molar ratio, add 470 ml H2SO4 to 530 ml HNO3.
  9. Leave covered in the fume hood for 24-48 hours.
  10. Place samples in a 85 - 92 C hot water bath for approximately 2 hours, more if your samples are high in organic matter.
  11. Let the samples sit at room temperature for 24 hours.
  12. Wash samples 7 to 9 times using the washing procedure detailed above, leaving a final slurry of 5 ml upon completion. Make sure the solution is neutral by using a piece of litmus paper.

Making serial dilutions

  1. Pipette 1 ml of the original slurry into a known volume of DI water (e.g. 20 ml).
  2. Place 1 ml of the new slurry into a centrifuge tube of known volume.
  3. Pipette out 1 ml from this slurry into a known volume of water and proceed this way until four or five dilutions are made for each sample.
  4. The cloudiness of the slurry and the density of diatom valves on the slides will determine which dilution will be used for counting. For very clastic sediments, you may have to dilute even further.

Making slides

  1. Shake vials and then place them in a vortex mixer for 5 seconds (see note below re shaking).
  2. Pipette 1 ml of the diatom slurry from each sample onto 18x18 mm cover slips.
  3. Allow to air dry in the fume hood (usually at least 24 hours).
  4. Heat hot plate.
  5. Place slide on hot plate to warm it up.
  6. Use a glass rod to apply 1 drop of Naphrax (the mounting medium) to the slide (keep the slide on the hot plate, as this makes the Naphrax less viscous).
  7. Place cover slip over the Naphrax.
  8. Allow the Naphrax to boil, apply pressure to remove air bubbles (about 15 seconds), with the slide on the hot plate.
  9. Allow to dry before looking at the slide under the microscope as this can dirty the lens (ideally 24 hours).
  10. Scrape off excess mounting medium from around the coverslip to protect the microscope.

Note about organic-rich sediment

  • The process described here is used for inorganic sediment, such as from many Arctic lakes. If sediments are high in organic matter, H2O2 can be just as effective as the H2SO4:HNO3 mixture, and safer to use. If using the H2O2 : begin by adding only 5 ml. Strong reaction with organic matter can cause the tubes to overflow if you add more than this. Add 2 ml every 24 hours until you do not see further reaction. Otherwise proceed the same way as outlined above for inorganic sediment.

Other Notes

  • It can be acceptable to use the centrifuge instead of a 24 hour settling period. If you choose to centrifuge, run the centrifuge at 2000 rpm for at least 5 minutes.
  • Shaking the vials or putting them in a vortex mixer results in many diatom frustules splitting apart into their separate valves. Some researchers prefer this for identification purposes as debris can become lodged inside the frustule, obscuring key features. Others prefer to see the whole frustule, as each valve of the same diatom can be compared which is useful for identification (e.g. Achnanthes species have a raphe on one side and not the other). This is a matter of personal preference.
  • When processing sediments for diatom analysis, wear lab coat, safety goggles and use the fumehood. Read Materials Safety Datasheets for all chemicals you will be using.

Reference

Battarbee, R.W., Jones, V.J., Flower, R.J., Cameron, N.G., Bennion, H., Carvalho, L., Juggins, S., 2001. Diatoms. In: Smol, J.P., Birks, H.J.B., Last, W.M. (Eds.), Tracking Environmental Change Using Lake Sediments. Volume 3: Terrestrial, Algal, and Siliceous Indicators. Kluwer, Dordrecht, pp. 155-202.

See also the Paleolimnology section of our library page.

 

 

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