A Methods Manual for the Collection, Preparation and Analysis of ...

2.1 PreparationPrinciple: Slides should be free of contamination by other diatomaceous material and shoulddisplay an assemblage of diatoms that is as close as possible, in terms of composition, to thatof the original sample. For this reason strewn slides are used almost exclusively.Note: It is always necessary to keep the sample well mixed or shaken, as the larger diatomcells will tend to settle out of solution quicker than the smaller cells and thus the communitycounts will be skewed and unreliable (a mechanical vortex mixer is very useful for thispurpose).2.1.1 Slides and cover slips should be scrupulously cleaned with detergent soap and storedin ethanol until needed.2.1.2 Using a pipette, a portion is drawn from a well-shaken numbered vial of cleanedmaterial. The cleaned diatom suspension is diluted until it appears only slightlycloudy to the naked eye.Note: Obtaining the correct dilution is a matter of practice as the concentration ofdiatom cells in the solutions will always differ from sample to sample.2.1.3 A single drop of ammonium chloride (NH 4 Cl; 10% solution) is added for every 10 mlof diluted diatom suspension to neutralise electrostatic charges on the suspendedparticles and reduce aggregation. This is a very important step; if the diatoms andother particles clump together this destroys the random distribution of the cells on theslide which will in turn have an effect on the results of the analysis.2.1.4 Using a pipette ~1.5 ml of this suspension is placed on a clean, dry cover-slip (22 x32 mm). The amount of solution used will vary according to the size of the coverslip.2.1.5 After being placed on the cover slip the diatom suspension should be allowed to dryat room temperature in a dust free environment. It should not be disturbed until dry,because vibration causes clumping of the diatom valves.2.1.6 The drying of cover slips on a hot plate is not recommended because the resultantconvection currents form more or less concentric rings of diatoms, with consequentoverlying.2.1.7 After the water has evaporated, diatom-coated cover-slips are placed on a hot plate at~350ºC for 3 minutes to drive off the excess moisture and to sublimate the residualammonium chloride.2.1.8 After the cover slips have cooled, they can be briefly examined under 400 xmagnification to determine if the concentration of diatoms in the solution was correct.At least 10, but not more than 40, valves should be visible per field. When the sampleis finally viewed at 1000 x magnification there should ideally be between 5 and 15,but not more than 20, valves visible in each field. If the concentration is too high orlow, steps 1-7 need to be followed again, using a more or less dilute suspension,before proceeding further.36