Laboratory treatment

Pre-preparation examination

On return to the laboratory, a quick examination of unpreserved samples should be performed to assess whether they consist predominantly of live cells (dead cells will form part of the bio-film and are not washed away, under normal conditions). If the majority of the diatoms are dead cells (empty frustules with no chloroplasts) the sample should be discarded, as it will not be possible to obtain a true reflection of recent water quality at the particular sampling site from this sample (Bate et al., 2002).

Cleaning Technique

Frustules may be cleaned with either acids or hydrogen peroxide. These procedures are modified from the techniques of Hasle (1978), Welsh (1964), Lohman (1982), McBride (1988), Taylor et al., (2005) and Krammer and Lange Bertalot (2000).Optimum conditions for Light Microscopy (LM) and Scanning Electron Microscopy (SEM) must be achieved as most structures of a frustule, used for identification, are fine and difficult to resolve. The organic components of the cell must, therefore, be removed. Diatom slides should meet the following criteria (DARES, 2009):

• Complete removal of organic matter in the sample;
• Foreign matter should be either absent or insufficient to cause problems during the enumeration or identification of the specimens;
• The distribution of valves on the cover slip should not be clumped, but be evenly spread, over the whole area of the cover slip without edge effects;
• Ideally, there should be 5–15 valves, but not less than 1 and not more than 20 valves, per field of view when viewed at 1000 x magnification;
• The mountant should be properly cured, without air bubbles, and should evenly spread right up to the edge of the coverslip.

Contamination must be guarded against in all phases of preparation from the collection of the sample in the field to the final mounting of the sample on a glass slide. Only simple glassware, such as glass beakers, watch glasses and centrifuge tubes, are used as these are capable of being easily and thoroughly cleaned after each use, with distilled water. As it is impossible to clean a pipette tip, it should be used only once ¹⁰³. A cheap alternative to a pipette is a plastic drinking straw.

Necessary precautions should be taken with all cleaning methods to avoid health hazards. The chemicals used for preparation of samples may be carcinogenic and corrosive, adequate health and safety precautions should be taken in each step. Familiarise yourself with the materials safety data sheets for each chemical in question.

Acid oxidation is a common method of preparing diatoms slide. It effectively removes all organic parts of a cell, including the diatotepum covering membrane. It has the disadvantage that very delicate silica structures of the cell wall may be damaged. The acids dissolve one of the solid phases of the silicic acid of the cell wall so that, when studied at high magnification under SEM, the cell wall appears more or less jagged in structure. In LM studies such damage is of little significance (Krammer and Lange-Bertalot, 2000). A series of techniques, including both acid and non-acid techniques, are described below. When material is required for SEM techniques, the use of acid oxidation should be avoided, and the more gentle method using hydrogen peroxide should be employed (Round et al., 1990), or the material should be left untreated (Taylor, 2003). With the exception of material from calcium deficient water, it is almost always necessary to dissolve traces of calcium in the sample with hydrochloric acid and then to rinse the sample (Krammer and Lange-Bertalot, 2000). This is particularly important if further processing with sulphuric acid is needed, otherwise a calcium sulphate diatom precipitate will form, which will make subsequent identification of the valves difficult. In the absence of a fume cabinet, all methods employing boiling acids must be avoided.

In all these methods the original sample should be allowed to settle for 24 hours and subsequently cleared of supernatant water without losing any diatom materials. A portion of the original sample should be retained throughout the preparation stages until the slide has been prepared and checked under a microscope. After cleaning, the final rinsing of the samples is essential, to remove any remnant acid and also to prevent its reaction with the mounting medium when a slide is prepared (Round et al., 1990).

Decalcification

Decalcification is necessary if samples are to be later treated with Hot HNO₃/H₂SO₄ method, as in these methods both the acids combine with calcium causing the formation of an insoluble precipitate. This stage can be omitted if you are sure that the sample does not come from a site with any calcareous rock in the catchment or if using the Hot HCl and KMnO₄ method

1. Shake the sample well and pour 5 to 10ml (depending on the concentration of the material) of thick suspension into a heat-resistant beaker.
2. In a fume cabinet, add a few drops of dilute HCl (e.g. 1 M) and agitate gently the material should effervesce as the carbonates are reduced to CO₂. [If the sample does not effervesce on addition of HCl there is not a significant amount of Ca in the sample and it is not necessary to continue with decalcification];
3. Continue adding dilute HCl, and agitate the beaker gently until effervescence stops;
4. Pour the solution into a centrifuge tube (10 ml) and add distilled water to 1 cm below the rim of the centrifuge tube and centrifuge to remove the acid;
5. The samples are rinsed by centrifuging with distilled water at 2500 rpm for 10 minutes;
6. After centrifugation the supernatant is decanted and the washing is repeated a further 4 times until the sample is circumneutral.

Hot HCl and KMnO₄ method

This is based on that of Hasle (1978) and adapted by Round et al., (1990). It has proved suitable for samples from India, as demonstrated by the ongoing ecological research at Indian Institute of Science. The procedure is as follows:

1. Shake the sample well and pour 5 to 10ml (depending on the concentration of the material) of thick suspension into a heat-resistant beaker.
2. Mark the beaker clearly with the sample number in several places.
3. Add 10ml saturated potassium permanganate (KMnO₄) solution, mix well and leave it for at least 48 hours.
4. Add 10ml concentrated HCl (32%), taking care not to inhale the gasses released. Cover the beaker with a watch glass and heat on a hot plate at 90°C for 1 to 3 hours inside a fume cabinet, until the solution becomes clear and yellow in colour. Do not allow the sample to boil. Care should be taken to avoid cross contamination of samples during violent bubbling while heating with acid (Welsh, 1964).
5. After oxidation of organic material, carefully add 1ml of hydrogen peroxide, one drop at a time, to check if the oxidation process is complete. In the absence of organic material, hydrogen peroxide will not cause lasting foaming.
6. When oxidation is complete, allow the sample to cool and transfer to a 10ml centrifuge tube. Beakers must be vigorously swirled, to re-suspend the diatoms and for settling of stone and heavier sand particles before transferring to centrifuge tubes.
7. Rinse the samples by centrifuging with distilled water at 2500rpm for 10 minutes followed by washing.
8.  During washing, supernatant should be poured off in a single movement, while not losing any diatom material. Then, diatoms and small particles of sand at the bottom of the tube are loosened by means of a jet of distilled water from a wash bottle. More water is then added till it reaches the required volume in the centrifuge tube.
9. Decant the supernatant and repeat the centrifugation and further washing at least 4 times or until the sample is circumneutral.

After the last wash, the diatoms are again loosened by means of a jet of distilled water and then poured into a small glass storage vial bearing the necessary sample information. It is important to store diatom samples in glass as opposed to plastic vials, as glass releases silica, which counters the dissolution of diatom valves.

Hot HNO₃/H₂SO₄ method

1. Check the sample for the presence of calcium and decalcify the sample if necessary (as per the decalcification procedure mentioned above).
2. Mix the diatom suspension carefully and take a subsample (~10ml) into a beaker. The size of the sample depends on density, which is judged by the visible concentration of suspended material.
3. Mark the beaker clearly in several places with the sample number.
4. Add 5ml of the strong acid mixture (HNO₃ + H₂SO₄, 2:1) and place the beakers with a watch glass on a hot plate. Heat the samples at 90°C for 2–3 hours, depending on the amount of organic matter in the sample. Care should be taken to avoid mixing of samples during violent bubbling while boiling with acid.
5. Rinse the samples and test for organic material as in points 5–10 in the Hot  HCl and KMnO₄ method

Hydrogen Peroxide Methods

Hydrogen peroxide is much gentler than acid as it is not as corrosive as in the former described methods. It is best used with samples that require little cleaning, and where corrosion should be limited, as in SEM studies (Krammer and Lange-Bertalot, 2000). The choice of technique (either hot or cold) depends on the availability of a fume cabinet. If one is available the peroxide can be boiled and, if not, a cold method should be used, but only in a well ventilated room.

Hot H₂O₂

1.  Mix the diatom suspension and place 5 to 10ml of the suspension in a beaker.
2. Mark the beaker clearly in several places with the sample number.
3. Add 20ml H₂O₂ and heat on a hot plate at 90°C for 1 to 3 hours.
4. Add a few drops of HCl and leave to cool.
5. Rinse the samples as in Hot HCl and KMnO₄ method.

Cold H₂O₂

1.  Mix the diatom suspension and place 5 to 10ml of the suspension in a beaker.
2. Mark the beaker clearly (preferably in several places) with the sample number.
3. Add 20ml H₂O₂ to the beaker and leave for a minimum of four days.
4. Rinse the samples as in Hot HCl and KMnO₄ method.

Bleach Method

1. Rinse any preservative (e.g. ethanol) from the sample by centrifugation with distilled water (3 runs at 2500 rpm).
2. Mix the diatom suspension and place 5 to 10ml of the suspension in a beaker.
3. Mark the beaker clearly in several places with the sample number.
4. Add 5-10 g of commercially available bleach (5.25% sodium hypochlorite) to the beaker and leave for a minimum of one day (this depends on the amount of organic content in the sample).
5. Rinse the samples five times using distilled water.