Fixed sediment samples contain a mixture of formalin and sediment components such as silt, clay, sand grains and organic detritus, in addition to the fauna. Although the following processes are often referred to as 'extraction of the fauna', in reality what we are endeavouring to do is to extract various sediment components, so as to end up with the fauna from the original sample and as little else as possible. Figure 1 is a flow-diagram showing the order of the processes and the type of decisions which need to be made. Equipment and materials required for processing samples are listed in here

Meiofauna-free water supply Prior to processing samples it is important to check that the laboratory freshwater supply does not contain meiofauna. To do this run tapwater through a 63µm sieve for 5-10 minutes and check the contents of the sieve (if any) under a binocular microscope. If meiofauna is present it is necessary to make some arrangement to have it removed. Many of the following techniques involve washing and concentrating meiofauna on sieves with freshwater. Attaching a flexible tube to the freshwater tap is highly recommended, as it greatly increases one's control over the direction and strength of the flow. Meiofauna are small, so do not use a strong jet of water, and splashing must be avoided.

An initial washing of the sample with freshwater on a 63µm sieve removes the finer sediment components, silt and clay, and much of the formalin. Take care not to overload the sieve, larger muddier samples may need to be sieved in smaller amounts. Puddling may help if the sieve becomes clogged. Continue washing until the water passing through the sieve is relatively clear. For some fine sediments, especially if the initial samples are small, this is sufficient to reduce the sample to a quantity that can be extracted with Ludox (see section 1.4).

Some types of samples may contain significant quantities of larger material such as leaf or paper fragments. It may be helpful in such circumstances to remove this material by washing the sample through a 1mm sieve nested on top of the 63µm sieve. Extreme care is necessary not to clog the smaller mesh as it is not in direct view. The contents of the 1mm sieve should be checked under a binocular microscope to ensure that any meiofauna has been washed out of it.

If, after initial washing, the sample contains appreciable quantities of sand, this can be removed by a decantation extraction. Wash the remaining sample into a 1 litre widemouth stoppered measuring cylinder (no more than 150ml of sediment at a time) and fill the cylinder to above the 1 litre mark with fresh water. Put in the stopper and vigorously shake and invert the cylinder 5-10 times to distribute the sediment evenly throughout the volume. Allow to stand briefly, probably for no more than 5 seconds, until most of the dense particles (mainly sand) have dropped out, then carefully pour the supernatant onto a 63µm sieve. Repeat 3-6 times. As the next process is a flotation extraction with Ludox (see section 1.4) do not be too concerned if some fine sand appears on the 63µm sieve. It is good practice to check an aliquot of the sediment remaining in the cylinder for meiofauna before discarding it, particularly if the sample is one of the first to be processed from a new survey, to assess the efficiency of the extraction.

View Figure 1

Flow diagram illustrating the steps taken and decisions made during the initial processing of meiofaunal samples. The numbers in bold refer to sections in the text.

Ludox is a colloidal silica solution. Small spills can dry to silica dust which is harmful . Spills should be mopped and cleaned up immediately and care must be taken when using Ludox. An example of a safe working practice for Ludox is included here. Sieves, glassware, and other equipment such as washbottles, which have been used for Ludox should be soaked in dilute NaOH solution and rinsed in hot water.

The following methods are concerned with processing extracted samples to allow the quantification and identification of the meiofauna. Figure 2 is a flow diagram showing the order of the processes and the type of decisions, which need to be made. Equipment and materials required for processing meiofauna are listed in here.

2.2 Subsampling

Typically, sediment samples contain large numbers of meiofaunal organisms, and it would be impossible to count and identify all of them. Meiobenthic communities are, however, patchy at small spatial scales, so simply taking smaller samples is not the answer. This is why we take larger samples and then routinely identify a proportion, extracted at random from the whole sample, which we refer to as a subsample. As a general rule, a subsample which contains at least 200 specimens is adequate for standard community analyses. It is helpful to keep the subsample size (defined as a fraction of the whole sample) constant within a particular study.

A simple but reliable subsampling apparatus consists of a ladle and a wide-mouthed plastic container marked with a volume corresponding to a specific number of ladle volumes. Details of this simple subsampling apparatus are given in here. If the sample has been stored in formalin or alcohol after extraction wash the sample with freshwater on a 63µm sieve. Wash the extracted sample into the plastic container with freshwater and add water until the total volume is equivalent to a known number of ladle volumes. The key to efficient subsampling with this apparatus is in agitating the contents of the container in such a way as to distribute the sample homogeneously throughout the volume. Simply stirring the sample with a circular motion concentrates the meiofauna. The best method is to use the ladle to vigorously agitate the sample with a vertical up and down motion for 20-30 seconds, then to carefully remove a single ladle-full into a 63µm sieve. Carefully rinse the ladle, collecting the washings on the sieve. If further subsamples are required the remaining volume must be agitated again prior to each removal. Once subsamples have been removed, the remaining sample should be returned to alcohol or formalin, and the size of subsample removed should be recorded on the container.

View Figure 2

Flow diagram illustrating the steps taken and the decisions made while processing and mounting meiofaunal samples after initial processing. The numbers in bold refer to sections in the text.

2.4 Preparing slides

Microscope slides should be prepared with paraffin wax rings (rectangular or circular) of similar proportions to the margins of the coverslips to be used to cover the finished preparations. The ring of wax should be complete, and there must be sufficient wax in the ring to effectively surround the sample and to seal the coverslip. It is not possible to give an infallible guide to the production of these rings, as several factors affect the quality of the resulting rings. Details of a metal applicator for making rectangular wax rings on microscope slides are given in here. Clean 76×39mm glass microscope slides with a tissue to remove dust. Melt paraffin wax (melting point 57-60ºC) in a crystallising basin or glass petri dish and leave the applicator in the wax to warm up. Do not overheat the wax, as this degrades it. Pure paraffin wax may be too brittle for satisfactory results, so adding some beeswax or white petroleum jelly to the wax is sometimes recommended. Keeping it level, lift the applicator from the wax and place it briefly on the slide (3 seconds or so), then return the dipper to the wax. A ring of paraffin wax should remain on the slide. Some workers press the applicator briefly (1 second) onto absorbent paper towel before placing it on the slide. The following are some frequently encountered problems, their possible causes and remedies.

(i) The applicator sticks, or the wax does not adhere properly, to the slide. This happens when the wax begins to solidify while the applicator is still in contact with the slide. The wax, the applicator or the slide are not warm enough, or the applicator has been left on the slide for too long.

(ii) The wax ring is too thin. This happens when the depth of wax in the container from which the applicator has been removed is too shallow, so an insufficient quantity is transferred to the slide.

(iii) The wax ring is uneven. Failure to keep the applicator level while transferring it to the slide can result in splashes and uneven rings. Not leaving the applicator on the slide for long enough can also result in uneven or thin rings.

(iv) The wax ring is too thick, or spreads too far. This occurs when the wax in the dish is too deep, or because the wax, the applicator or the slide are too hot.

Once prepared, the slides should be kept free of dust prior to use. Care should also be taken not to damage the rings during storage.

If possible mount the sample whilst it is still warm from evaporaing to pure glycerol (section 2.3) as this makes the glycerol more fluid. Tilt the cavity block containing the sample material in glycerol and scrape the contents close to the lower edge with a flexible implement such as a quill. Carefully, but quickly, tip the material into the centre of the wax ring on the slide. If necessary, add a little more glycerol to the cavity block and repeat the process. Check the cavity block under a dissecting microscope to ensure that all the animals have been transferred to the slide. If there are still animals in the cavity block either add more glycerol and repeat the tipping process, or pick them out individually with a fine needle.

There is no substitute for experience in deciding how much material to put on a slide. If there is not enough material add an extra drop or two of glycerol. If it looks as though there is too much for one slide tip the material onto two or more slides. Carefully add a coverslip, making sure that it overlaps with the wax ring and that it makes contact with the glycerol. This helps to exclude air bubbles and also prevents melted wax from flowing over the sample. Transfer the slide to a warm hotplate (55-60°C) and carefully melt the wax. This seals the coverslip to the slide while retaining the sample underneath it, and must be a controlled process. Some workers prefer to melt the wax in sections, keeping the preparation close to the edge of the hotplate. As soon as the wax has melted gently remove the slide from the hotplate and place it on a level surface to allow the wax to solidify slowly. Once the wax has set record the details of the sample, or a code representing the relevant information, on the border of the slide with a diamond pencil. Excess wax can be removed using a sharp knife or scalpel. If the glycerol leaks out through imperfections in the wax ring this can be cleaned up using a tissue dipped in a small amount of acetone. The preparation is finished off by sealing it with two coats of a suitable xylene-resistant sealant (Fig. 3). Bioseal No. 2 mountant has been found to be very useful for this purpose.

View Figure 3

Generalised appearance of a completed slide. The sample is retained beneath the coverslip with a wax ring, the mount is sealed with a suitable compound, and the sample details are recorded near one edge.

As a general rule, problems encountered during the mounting process are irreversible, and another subsample will need to be taken. Some commonly encountered problems include:

• Pieces of detritus left in the sample prevent the coverslip from settling evenly, leading to an uneven mount and often disrupting the integrity of the wax ring.
• If there is too much glycerol on the slide, or the wax ring is too thin, the slide will leak. Although the fauna may still be analysed the mount can not be stored satisfactorily.
• Having insufficient glycerol, or too much wax in the ring, can lead to molten wax flowing over the glycerol, which obscures the sample from view when it solidifies. This problem is provoked by failing to ensure that the glycerol is in contact with the coverslip before melting the wax ring, by having the hotplate at too high a temperature so that the wax melts too quickly and the wax and glycerol overheat, or by not keeping the slide level until the wax solidifies (which takes longer if the mount is too hot).