474 MICROPALAEONTOLOGICAL TECHNIQUES enhances their appearance for microscopical study The lignin found in peats, though resistant to acetylation, is susceptible to oxidation and becomes soluble in a hot alkali solution However, pollen and spores are also easily destroyed by the process of oxidation, so the effect on these grains has to be monitored and the process stopped before damage occurs Many methods of oxidation may be used, depending on the lithology of the specimen For example, silicates and argillaceous rocks will respond to 10% nitric acid to which is added 10% sodium hydroxide or Schulze’s solution (a mixture of potassium chlorate and concentrated nitric acid); carbonates require fuming nitric acid (95%); peats can be treated with glacial acetic acid, sodium chlorate, and sulphuric acid; and oxidation of coals can be achieved by using Schulze’s solution, fuming nitric acid, or hydrogen peroxide After these various chemical techniques have been completed, the final stages involve washing by centrifuge The resulting palynomorphs in the residue are finally washed in 95% alcohol before staining with compounds such as Safranine-O, Malachite green, or Bismarck brown in 0.1% solutions, and are then transferred to a storage phial in distilled water with a mould inhibitor To prepare slides for study, a small amount of the residue is pipetted onto a cover slip, and after the water has evaporated, a mounting medium, such as Elvacite 2044, Canada Balsam, or epoxy resin, is applied to the glass slide and the cover slip is placed on the slide and allowed to cure The usual method of observation is with a conventional biological microscope However, modern methods of illustration and study involve the use of confocal laser scanning microscopy (CLSM), which gives high-resolution, blur-free images with a greater depth of focus than is possible with conventional microscopy These images may also be reconstructed to give three-dimensional information and can be viewed as rotating computer animations Physical Separation and Concentration Methods Additional techniques are sometimes required after the initial processing has been completed and the sample is thoroughly disaggregated The purpose is to concentrate the sample to increase the ratio of microfossil specimens to rock particles such that time spent examining the residue is kept to a minimum Sieving The principal aim of sieving the sample is to remove particles of finer or larger sizes, compared to the fossils of interest Usually the finer particles are the deflocculated and dispersed clays; fragments larger than the microfossils may be stones, shelly material, or rock that has not broken down completely The choice of sizes of sieves is often dependent on the fossil group being studied Usual practice for the larger calcareous microfossils is to use a nest of sieves, of sizes ranging from (bottom to top) finest (75 mm), to medium (250 mm), to coarse (1 mm) If studying planktonic foraminifera, it is essential that the finest sieve is either 75 or 63 mm, because heterohelicids and similar-sized foraminifera will easily fall through coarser sizes It is worth noting that sieves are designed to separate sedimentary particles that are equant in shape; however, many microfossils are elongated in one direction and may pass through a sieve lengthways Therefore, the smallest microfossil dimension should be considered when choosing sieves to retain a certain element of the fauna Occasionally microfossils can become stuck in the mesh, and are then liable to be transferred to another sample as a contaminant In order to identify such errant specimens, the sieve is stained with a solution of methylene blue; once the surplus stain is washed off, any contaminating material takes the blue colour and is readily spotted in the residue Magnetic Separation Rock particles in residues often contain a proportion of iron, and calcareous or phosphatic microfossils not, thus it is possible to use differences in magnetic properties to separate the two fractions For example, this technique is particularly successful in concentrating calcareous microfossils in a residue containing glauconite This is achieved by using a Frantz Isodynamic Magnetic Separator, which consists of a very large electromagnet that can be tilted in two planes, a vessel into which the sample is placed, and a vibrating motor that moves the particles down a chute The chute divides into two sections so that separate collecting buckets can receive the magnetic and non-magnetic grains Flotation A common method for concentrating specimens in a residue is to use differences in the specific gravities of the fossil and the matrix A suitable heavy liquid can then be used to separate the two components, such that one floats and one sinks Each fraction can then be collected separately Toxic liquids such as carbon tetrachloride, bromoform, and tetrabromoethane, which have been used widely in the past, have now been superseded by safer compounds, such as sodium