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UCL Home  /  Geography  /  Resources  /  Laboratory  /  Laboratory Methods  /  Lake Sediment Analysis  /  Use of Microspheres in the Determination of Diatom Concentration

Use of Microspheres in the Determination of Diatom Concentration

It is useful to add a known number of microscopic markers (in the same size range as diatoms) to a known amount of sediment to determine the concentration of diatoms. This also enables the concentrations of individual taxa to be determined, thus avoiding the distortion caused when individual species are expressed as proportions or percentages of the total count.

In the Department of Geography we use DVB (divinylbenzene) spheres with a mean diameter of 6.4m. Unfortunately they may not survive the digestion process, or if they do the difference in density between the spheres and diatoms results in the possibility of some spheres being lost in the washing process following digestion. For these reasons they have to be added to the samples just before the slides are prepared. It is important to use DVB spheres since other plastics dissolve in the toluene mountant. Although glass spheres have the advantage of being able to survive the digestion, they are less easy to count because they come in a much larger size range than the plastic spheres and are also more easily confused with chrysophyte cysts under the microscope.

The DVB spheres are bought as a concentrated suspension. This is sonicated to disperse the spheres before dilution with distilled water to make a concentrated stock suspension which is calibrated using a coulter counter. This is then diluted further to make 1 litre batches of working concentration at around 5x106 spheres per ml. The addition of a small amount of ammonia solution helps to keep the spheres from clumping. To prevent the growth of micro-organisms a very small amount of mercuric chloride is added (<3 mg/L) and the prepared suspension is kept at 4oC.


If diatom concentrations are required, the microsphere markers are added after the last wash just before the slides are made. Use the suspension (x106 concentration) stored in the 1 litre conical flask in the cold room. Do not use the stock solution kept in the white polythene bottle in the lab fridge. Because of the toxicity of mercuric chloride it is important to wear gloves when handling the microsphere suspension and to carefully clean up any spills. Initially add 1.5-2 mls of 5 x 106 suspension for each 0.1 grams of dry sediment digested, being careful to shake and sonicate the microsphere suspension before each use to disperse the spheres evenly. A suspension of 5x105 spheres per ml is easier to use for sample sizes of 0.01 gram dry weight when using the waterbath method. In this case it may be advisable to include some replicate samples since the smaller sample size makes this method less suitable for quantitative work. Make a test slide to check the diatom to microsphere ratio (ideally 1:1) and calculate the required amount of microsphere suspension before adding spheres to a whole batch of samples.

When making up slides containing DVB microspheres, care must be taken not to heat them above 130oC.