Meiofauna extraction with Ludox

This information on sample processing has been provided by the Darwin Nematode Project, the site is maintained at the Plymouth Marine Labs, UK (posted with permission)

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.

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.

1 Removing silt/clay and large fragments
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 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.

2 Decantation
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 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.

3 Preparing Ludox
The objective of flotation extraction is to suspend the fauna in a fluid which has a specific gravity very close to that of the animals themselves, so that the animals are neutrally buoyant and remain in suspension but sediment components are negatively buoyant and slowly sink. A variety of dense fluids have been used to extract fauna from sediments in the past. The disadvantage of most of them, such as NaCl or sucrose, is that they have a very high osmotic potential and therefore can damage some of the fauna. Ludox is a colloidal silica solution, primarily developed for use in iron foundries, with properties which make it ideal for the extraction of meiofauna. It is available in a range of grades, but Ludox TM is most widely used. As supplied, Ludox TM has a specific gravity of 1.13 to 1.14. The specific gravity needed to extract meiofauna is approximately 1.15, so the stock solution must be diluted. Adding two parts fresh Ludox to three parts freshwater will give a solution of approximately the correct density, but the density should be measured with a hydrometer. Do not use seawater directly with Ludox, as this can cause the suspended silica to precipitate, rendering the sample useless.

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. 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.

4 Ludox flotation
A number of centrifugation techniques involving Ludox have been used, but the following method has been found to be simple and effective. After decantation, or after initial washing if the sample consists of a small amount of material, carefully wash the extracted portion of the sediment to one side of the sieve, then wash it into a tallform beaker using Ludox. Add at least 10 times the sample volume of Ludox, made up to S.G. 1.15. Stir vigorously to distribute the sample evenly throughout the volume and leave to settle for at least 40 minutes or until there is a clear separation of a scum containing meiofauna floating on top of the Ludox and the sediment at the bottom of the beaker. Some fine clay particles may remain in the sample even after washing and decantation and these will still remain in suspension even after several hours. They should be ignored as they will pass through a 63µm sieve. Carefully pour the supernatant through a 63µm sieve into a jug. Return the Ludox to the sample and repeat the flotation process 2-4 times. It may be necessary to check that the specific gravity of the Ludox has not been altered by water still in the sediment sample after its initial washing, if necessary add more concentrated ludox to restore the specific gravity to 1.15. After each flotation wash the extracted fauna thoroughly with fresh water. If the sample is not to be worked on immediately, preserve it with 70% alcohol with 5% glycerol or 4% formalin in a suitable container, such as a glass tube with an airtight plastic closure.