Diatoms of North AmericaThere are many methods that can be used to remove organic matter from diatoms. The best method depends on the type of sample and the amount of organic material present. In general, use the least aggressive method possible. Here, methods are arranged in order from most gentle to most aggressive.
Be safe. The first method can be done using a hot plate, but all of the other methods require a laboratory and safety equipment.
Be sure to use distilled water, not deionized water. We have seen many poor preparations, with a characteristic film of organic material that comes from the exchange resins of deionizing columns.
1. Incineration method, also termed "burn mount"
Many diatoms have fine silica structures that are joined by organic parts of the cell. These diatoms are best prepared by using heat to remove the organic matter, which does not destroy the fine structures. The incineration method is quick, requires no oxidizing chemicals, and has the advantage of leaving frustules intact. It is appropriate for fresh material (phytoplankton) or samples with diatoms with lightly silicified valves. This method is also useful for species that form colonies, such as Asterionella, Aulacoseira, and Tabellaria, because the cells remain joined in their colonial forms.
To make a burn mount, place a drop of raw sample on a clean coverslip and allow the diatoms to settle and the water to dry. If the sample is from seawater or waters high in salts, pick up the dry cover slip with a forceps, rinse gently in a beaker of distilled water to dissolve salts - but not remove too many diatoms - and then dry the coverslip a second time. Finally, heat the coverslip on a hot plate at high heat for at least one hour.
Be safe. The following methods should only be attempted in a laboratory and with appropriate eye protection, lab coats, closed toe shoes, and laboratory gloves. Processing using acids must be carried out in a fume hood. Laboratory waste must be properly treated and disposed of as hazardous material.
2. Hydrogen peroxide method
Hydrogen peroxide can be used to oxidize organic matter in samples with a low amount of organic matter.
Place ~0.1 cm3 wet sample into a small test tube, then place the test tube in a test tube rack. If carbonates are present, add a little 10% HCl to dissolve. Add 1.0 mL 30% hydrogen peroxide (H2O2) and allow the sample to oxidize for 4-6 hours.
Later (or the next day), add another 1.0 mL 30% H2O2 to the test tube and place the test tube rack in a hot water bath (85°C). Heat for 1-3 hours. Allow the samples to cool and rinse with distilled water. Allow the samples to settle 4-6 hours, then aspirate the supernatant, being sure to leave the diatoms undisturbed on the bottom of the test tubes. Add distilled water, and repeat settling and rinsing 5 times.
Renberg, I. 1990. A procedure for preparing large sets of diatom slides from sediment cores. Journal of Paleolimnology 4: 87-90.
3. Nitric acid cleaning
Samples with a moderate amount of organic matter can be oxidized using concentrated nitric acid (HNO3). Add 20-25 mL of HNO3 to a beaker containing the sample. Boil for an hour, or until the organic material is oxidized. Fill the beaker with distilled water and let the sample settle 4-6 hours. Decant the supernatant (leave 2 cm of liquid in bottom of beaker) and refill the beaker with distilled water. Repeat the settling and distilled water rinsing and settling until all by-products are removed (5-6 times).
Patrick, R. and C. W. Reimer. 1966. The Diatoms of the United States, Exclusive of Alaska and Hawaii. Volume I. Monographs of the Academy of Natural Sciences of Philadelphia, Philadelphia. 688 pp.
4. Lake sediments high in silt and organic material
Add 25 mL of 30% hydrogen peroxide (H2O2) to a beaker containing the sample. Boil for 1 hour on a hot plate. Add 30 mL concentrated nitric acid (HNO3).
Reaction occurs quickly; submerge beaker in cold water to halt the reaction. Fill the beaker with distilled water and let settle for 4-6 hours. Decant the supernatant (leave 2 cm of liquid in bottom of beaker) and refill the beaker with distilled water. Repeat the settling and distilled water rinsing and settling until all by-products are removed (5-6 times).
Stoermer, E. F., M. B. Edlund, and C. H. Pilskaln. 1995. Siliceous microfossil distribution in the surficial sediments of Lake Baikal. Journal of Paleolimnology 14: 69-82.
