MICROPALAEONTOLOGICAL TECHNIQUES 473 of monochloroacetic acid, although giving a rapid reaction, has been shown to damage conodont elements Similarly, the use of unbuffered acids, such as formic and acetic acids, can also cause dissolution However, when 10% formic acid (HCOOH) is buffered with calcium carbonate and tricalcium carbonate to a pH of less than 3.6, it becomes a suitable digesting medium Similarly, 10% glacial acetic acid (CH3COOH), when buffered with sodium acetate or calcium acetate to a pH of 3.5, can be used quite effectively The technique is similar with both acids and involves immersing the crushed rock into the acid in a fume cupboard and waiting for the effervescence to cease Formic acid may require up to day before the residue is washed with water and sieved, whereas acetic acid will require many regular changes of acid to dissolve the matrix, and this process can continue for several weeks For other rock types, such as shales, methods similar to those used for calcareous microfossils are generally used Once the residue has been sieved, conodonts may be further concentrated by either electromagnetic or heavy liquid separation Siliceous microfossils Several groups of microfossils have siliceous skeletons, including radiolaria, diatoms, silicoflagellates, and ebridians These may occur in both clastic and carbonate rock types, and by virtue of their chemistry are resistant to some acids that may be used successfully to extract them However, this is not always necessary, because diatoms may be found in unconsolidated sediments and can even form the majority of the rock, which is then termed a ‘diatomite’ Radiolarians may also be numerous enough to form radiolarian earth Various methods are used for loosely consolidated sediments, involving several stages: (1) removal of the carbonate component by using dilute acids, such as hydrochloric, formic, nitric, or acetic acids, (2) removal of organic matter using oxidizing agents, such as hydrogen peroxide or stronger nitric acid, and (3) dispersal of the clay component by using water softeners With more indurated material, the strength of the acid and the times of digestion are increased For extraction of radiolarians from mudstones, shales, and marls, a combination of 15% hydrogen peroxide and a degreasing agent such as MP 10 is effective Very often radiolarians occur in chert beds, siliceous shales, siltstones, or mudstones, which contain silica of the same composition as the fossils In these cases, 10% hydrofluoric acid (HF) has to be used with extreme caution to etch the radiolarians out of the rock The observation of siliceous fossils such as radiolaria is made more difficult because the refractive index of the shells is similar to that of the glass on which the specimens are mounted, rendering them invisible Techniques to overcome this involve selecting a mounting medium with a high refractive index to contrast with that of the glass, and observing in transmitted light with dark-field illumination, or by coating the specimens with silver nitrate, thus allowing oblique incident light to be used Larger specimens may be observed directly using a stereozoom microscope The electron microscope is also used for studies requiring observation of the finest detail and is invaluable in taxonomic work Organic microfossils As a group, organic microfossils include pollen and spores, dinoflagellates, acritarchs (see Microfossils: Acritarchs), tasmanitids, Chitinozoa (see Microfossils: Chitinozoa), and scolecodonts With any palynological methods, a clean working environment is essential to reduce the risks of sample contamination, because the specimens are only micrometres in size and are easily transferred between tools and vessels Although methods vary widely according to the fossil group and the lithology being treated, a broad working plan would follow several stages, including cleaning, disaggregation and dispersal, chemical extraction, and concentration Palynological preparations differ from other techniques in the chemical extraction stage, because the fossils are composed of sporopollenin or pseudochitin, which allows the use of strong acids; these methods should only be carried out in properly equipped laboratories Initial disaggregation is achieved by cleaning, crushing, and soaking or boiling in strong detergents such as ‘Quaternary O’ Other dispersal agents are used depending on the lithology, such as KOH, NaOH, or Na3PO for peat; KOH for coals; or NaOH for shales Chemical extraction takes several stages to complete and may consist of demineralization and maceration, depending on the rock type Demineralization is needed to remove the inorganic component of the rock, and different acids are used to achieve this Carbonates are removed by treatment with HCl; silicates are removed using HF If sulphates or sulphides are present, they are removed by treatment with HNO3 Other phases, such as heavy minerals, are removed using heavy liquids The second stage involves the removal of unwanted organic material; plant disintegration products in the residue may consist of cellulose, hemicellulose, and lignin Cellulose is broken down by the process of acetylation using a reagent that is a mixture of concentrated sulphuric acid (H2SO4) and glacial acetic acid The broken down cellulose is removed by centrifuging and washing in glacial acetic acid, and finally washing in distilled water Acetylation also has the beneficial effect of darkening spores and dinoflagellate cysts, which