Radiolaria Polycystina
Sample preparation and analysis

The following section offers some general comments on the preparation of whole samples for routine counting and identification procedures. It does not review the methods involved in special cytological and ultrastructural studies (see Anderson, 1983a, for a review of these topics), as well as those used for detailed taxonomic work, which can involve thin-sectioning, etching and polishing, etc. (Riedel and Sanfilippo, 1977; Boltovskoy et al., 1983; Petrushevskaya, 1986).

Pelagic surface sediments are usually clean enough as to require little treatment before preparation of the slides. Elimination of the organic matter and disaggregation of the materials is achieved boiling the sample (5-10 g) for a few minutes in a beaker with water to which hydrogen peroxyde (10%, 300 ml per liter) and tetrasodium pyrophosphate (10 g per liter) have been added. Disaggregation, cleaning and removal of clay coatings and infilling particles can be aided by treating the sample in a gentle ultrasonic bath. For further disaggregation of heavily indurated sediments various products, such as kerosene, paint thinner, or ammonia can be helpful (the sediment is dried, soaked in the solvent, and then immersed in water, upon which disaggregation usually occurs rapidly). If calcareous material is abundant it can be removed with a few drops of hydrochloric acid (after eliminating the hydrogen peroxyde by wet-sieving). The resulting clean material is then sieved with abundant water in order to eliminate the reagents and smaller particles. The mesh size used depends on the aims of the study; most surveys routinely employ 40-60 µm-meshes, yet these, as described above, miss many of the smaller species, as well as most developing forms. If precise abundance estimates are sought, mesh openings around 15 to 20 µm should be employed, although these will retain large numbers of unidentifiable skeletal fragments, as well as non-radiolarian material (especially diatoms), which can make subsequent observation more laborious. The clean residue in the sieve is pipetted onto glass microscope slides, dried, and soaked with a few drops of xylene; before the xylene has evaporated the mounting medium is added and covered with a cover glass. Canada Balsam is most often used for these preparations, although it takes longer to harden than some other synthetic materials, commercially known as Norland, Pleurax, Hyrax or Depex.

Moore (1973) proposed a convenient method which allows quantification of the number of radiolarian shells per unit weight of sediment. Before processing as described above, the sample is dried and weighed. This weighed sediment is then cleaned and sieved, and all the resulting residue is poured into a large (e.g., 5 l) beaker full of distilled water, on the bottom of which one or two cover gasses have been positioned. The water with the sediment in the beaker is then thoroughly stirred (avoiding rotational motion, which will result in centrifugal fractionation) for achieving a random distribution of the particles, and the sediment is allowed to settle. With the aid of a siphon all but 3-5 cm of water are removed, and the remainder is evaporated with an overhead infrared lamp. When the surface of the cover glasses is dry they are removed from the beaker and mounted as described above. The slide thus prepared will contain a fraction of the radiolarian shells present in the original sample, this fraction being equivalent to the proportion that the surface of the cover glass makes of that of the surface of the bottom of the beaker.

Preparation of plankton and sediment trap samples is somewhat more labororious due to the large amounts of organic material they contain. When both absolute radiolarian concentrations and specific inventories are sought, it is recommended that counting be performed separately from the identifications. Polycystines can be counted (although not identified) in whole, unprocessed samples in counting chambers under the inverted microscope (Hasle, 1978; Boltovskoy, 1981c; Villafañe and Reid, 1995). Subsequently, either the entire sample or a subsample can be treated in order to eliminate all organic matter leaving the clean siliceous skeletons that will be mounted as described above for sedimentary materials. It should be born in mind, however, that radiolarian cells are often very difficult to recognize in preserved, unprocessed plankton samples. The siliceous skeleton, usually the most conspicuous distinguishing feature, is obscured by the cytoplasm to such an extent that radiolarians are easily confused with other planktonic protists, fecal pellets, eggs, various organic aggregates, debris, etc. Adding a few drops of hydrogen peroxide and/or hydrochloric acid, which slowly digest the organic matter, and comparing the dubious particles before and after treatment can greatly help to pinpoint radiolarian cells (Alder, pers. comm.).

Several different methods have been used for eliminating organic material from water-column samples, including high- and low-temperature ashing, oxidizing with hydrogen peroxide and/or ultraviolet light, etc. (see review in Boltovskoy et al., 1983). One of the most widespread, however, is that proposed by Simonsen (1974) for cleaning diatom frustules. The plankton sample is rinsed with abundant fresh water (wet-sieving), and placed in a beaker to which an equal volume of saturated KMnO4 is added; it is then left for 24 hs. A volume of concentrated HCl equivalent to that already contained in the beaker is subsequently added to the sample; the dark brown liquid is gently heated until it becomes transparent or light yellow. Once the sample has cooled, it is sieved again thoroughly with fresh water and rinsed with distilled water. The residue is pipetted onto microscope glass slides as described above.

Analysis of the specimens is best performed in mounted slides, which by transparency permits observing the internal structures (such as medullary shells, spiral structures, etc.), and the wall-thickness. In addition, slight variations in the depth of field allow one to determine whether a shell of circular outline is a disc (in which case most of the surface is in focus simultaneously), or a sphere (either the central part or the periphery are in focus). Photographs taken in the light microscope have the advantage of being readily comparable to mounted specimens. The scanning electron microscope (SEM), on the other hand, is especially suitable for analyzing the surface morphology, but only in specimens with large openings in the outermost shell, or in those partially broken, can internal structures be observed. SEM photographs produce very appealing results, but their comparison with routine collections mounted in slides is tricky. Ideally, both techniques should complement each other (Boltovskoy et al., 1983, described a method which allows performing light and SEM observations and photographs of the same radiolarian specimens; electron- and light microsc. ;electron- and light microsc. 2;electron- and light microsc. 3).

Assessment of radiolarian species-specific absolute and relative abundances are based on identifications and counts. Since any given slide often contains thousands of polycystine shells, the researcher is forced to decide how many specimens should be identified and counted in order to achieve an adequate estimate of overall numbers and species proportions. Several methods have been proposed for the assessment of bias in sample-based particle counts (see reviews in Venrick, 1978a, 1995; Frontier, 1981), and in the appraisal of species proportions (Patterson and Fishbein, 1989; Buzas, 1990). Patterson and Fishbein (1989) concluded that for species representing >50% of the overall taxocoenosis at least 50 specimens should be counted in order to achieve reliable percentage data, 300 counts for species which comprise approximately 10% of a sample, 500-1000 counts for species that make up 5%, and counts of several thousands for those that comprise 1%. Unfortunately, in the case of the polycystines these efforts are unrealistic because in any given sample containing 100-150 species only 1-3 are above 10%, and 70-90 occur at levels below 1% (see RaPo. 5 Geographic patterns and RaPo. 6 Vertical profiles; quantitative radiolarian distribution). In terms of the amount of information attained, it is more profitable to analyze more samples at a lower resolution, than to examine fewer sites at these statistically more reliable levels. Thus, in practice proportions are estimated in bulk, regardless of the individual species abundances, usually scanning 300-600 specimens per sample. It is common practice to identify the first 300-600 individuals on the slide, and then check the rest of the slide or slides for the given sample in order to account for the rarer taxa. The relative abundances of the latter are estimated approximately, and they are usually excluded from subsequent general numerical analyses (e.g., multivariate techniques, such as cluster and factor analysis) because of the uncertainties associated with their assumed absences. It should be stressed, however, that the counting effort necessary for reliable estimates of the fractional abundance of the rare species is inversely proportional to the equitability of the assemblage. Thus, when the sample is strongly dominated by a single or only a few taxa, such as in polar areas (see changes in species richness), chances of recording the rare polycystines in random sequential counts are low because the observer repeatedly hits the dominant species. On the contrary, as equitability increases so does the probability of logging a so far unrecorded species with every new specimen scanned.