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UCL Home  /  Geography  /  Resources  /  Laboratory  /  Laboratory Methods  /  Lake Sediment Analysis  /  Diatom Preparation

Diatom Preparation

It is necessary to remove any organic matter from diatom samples in order to make microscopic identification easier. The cleaning process also allows unwanted mineral material to be removed and the concentration of diatoms to be adjusted [a full discussion of diatom analysis is available in Battarbee (1986)].

Safety

Hydrogen peroxide is a very powerful oxidising agent. Contact with the skin should be avoided and rubber gloves and eye protection should be worn when handling it. If hydrogen peroxide comes into contact with eyes, skin or clothing, wash the spillage under running water. Spills on bench tops, floors etc. should also be diluted with water before mopping up with paper towel. Mopping up concentrated hydrogen peroxide with paper towel can cause fires.

Method for the preparation of samples using a waterbath

This method is particularly suitable for large numbers of sediment samples (Renberg, 1990). It can also be safely left without any risk of explosion and there is less risk of samples boiling dry. It may not be suitable for samples which tend to react vigorously with hydrogen peroxide, such as large epilithon and epiphyton samples.

In this procedure centrifuging the washed sample may be replaced by settling out in a cool place overnight. This is convenient when handling a large number of samples in glass test-tubes and reduces breakage of fine, filamentous diatoms. However, if the samples are prepared in plastic centrifuge tubes they may be centrifuged if preferred.

Equipment

Plastic centrifuge tubes in a rack, water bath filled with distilled water, hydrogen peroxide (H2O2) 30% (100 volume), distilled water, hotplate, 50% hydrochloric acid (HCl).

Digestion procedure

  1. Place approximately 0.01 grams of dried sediment (or 0.1 gram of wet sediment) in each tube, weighing to four decimal places if diatom concentrations are to be calculated. Moistening dried sediment with a few drops of H2O2 will help its dispersal when the rest of the peroxide is added.
  2. Add 5 mls 30% H2O2 to each tube and place in a rack in the water bath (in a fume cupboard) at room temperature. If the sediment does not react violently with the sediment, the temperature of the water bath can be increased to 80oC . 'Blank' tubes containing only H2O2 can be placed at intervals in the rack and analysed to check there is no cross contamination between the tubes during digestion. Evaporation from the water bath is reduced using floating plastic spheres.
  3. Heat samples , checking the level of H2O2 from time to time, until all organic material has been removed. This process may take several days, depending upon the organic content of the samples.  Do not allow the samples to dry. Also keep the water level in the bath topped up with distilled water. The heater cuts out and a red alarm light comes on if the water level drops too low.
  4. Remove the tubes from the bath and add just 1-2 drops of 50% HCl to each tube, which will eliminate any remaining H2O2 and any carbonates. The fizzing which occurs also helps to unstick any diatoms which may have become attached to the side of the test-tube.
  5. Top up test-tubes with distilled water and centrifuge for 4 minutes at 1200rpm. Alternatively leave to settle out overnight at 4oC. The resulting supernatant liquid is then decanted off and the diatoms resuspended in more distilled water.
  6. Repeat this washing process four more times, either allowing the diatoms to settle out overnight at 4oC between each wash or by centrifugation. 1-2 drops of weak ammonia (NH3) solution added to each sample with the final wash will help keep any clays in suspension and will also prevent the diatoms clumping together when making up slides.

 

Method for the preparation of samples on a hotplate

This method is particularly suitable for preparing bulky samples such as epiphyton. It tends to be quicker than the waterbath method (see below) but needs closer supervision. If beakers are allowed to boil dry, diatoms can be difficult to dislodge from the glass. There is also a risk of explosion when very concentrated, hot hydrogen peroxide is rapidly oxidising samples with a high organic content. For this reason it is essential that the fume cupboard window is fully lowered if the heating samples are left unattended, and that the window is lowered to the safe working height and eye protection worn whilst working at the fume cupboard.

Equipment

100ml or 250ml beaker for each sample, hydrogen peroxide (H2O2) 30% (100 volume), distilled water, hotplate, 50% hydrochloric acid (HCl), centrifuge tubes, centrifuge.

Digestion procedure

  1. Place about 0.1g (dry weight, 1 g wet weight) sediment into a beaker. If diatom concentration is to be assessed, the sediment should be weighed to three decimal places.
  2. Add about 20 mls H2O2.
  3. Heat on a hotplate set at 90oC in a fume cupboard until all organic material has been oxidised (1-3 hours). Coarse plant material in macrophyte samples may be removed after half an hour.
  4. Remove the beakers from the heat. Add a few drops of HCl (50%) to remove remaining H2O2 plus any carbonates and wash down sides of beaker with distilled water.
  5. Allow to cool in the fume cupboard (chlorine is generated from the HCl) and pour into centrifuge tubes, leaving any coarse sand in the beaker. Top up with distilled water.
  6. Centrifuge at 1200 rpm for 4 minutes.
  7. Decant off supernatant and resuspend pellet by tapping the base of the tube. Top up with distilled water and centrifuge as before.
  8. Repeat washing process at least three times. Clay may be removed during the last wash by adding a few drops of very weak ammonia solution (1%) to the sample. The clay is then decanted off with the supernatant. The sample is now ready to make into slides.