Natural samples often yield cells at approximately the same stage in the life cycle of the species in question. This is presumably due to a coordination of processes, such as sexual reproduction (auxospore formation in diatoms), caused by chemical and physical factors in the environment. For similar reasons, diatom resting spores are often formed only at certain times. For studies of morphology and taxonomy, it is desirable to know the whole range of morphological variation including the effect of size variation and resting spore formation. This information is normally not obtained by studying a few natural samples. Cultures in which auxospore and resting spore formation can be induced may increase the information, although a single clone does not necessarily reflect the whole range of variation of a species. It is important to note, however, that a single clonal culture does not necessarily reflect the whole possible range in variation of a population or species.
Dilution cultures (Sournia, 1978, p. 218) or crude cultures may serve as a base for unialgal cultures. The best universal method to establish unialgal diatom cultures is to isolate single cells by a micropipette (Stein, 1973, p. 53) into an appropriate synthetic or enriched seawater growth medium (Stein, 1973, p. 25).
It is important to keep in mind that dense unialgal cultures as well as dense natural populations may contain aberrant forms. This seems to be especially the case for Pseudo-tiitzschia spp. and Synedropsis hyperborea and related species (Grunow, 1884; Takano & Kikuchi, 1985; Subba Rao & Wohlgeschaffen, 1990; Hasle et al., 1994).
PRESERVATION AND STORAGE (Table 79)
If possible, diatom samples should be studied immediately after sampling for information on colony formation and chloroplasts. However, in most cases, due to practical reasons they have to be preserved for later studies. A pH lower than 7 is preferable to hinder dissolution of the siliceous structures. The most
TABLE 79 Preservation
Lugol's solution, acidic
Equal volumes of p.a. grade 40% HCHO and 100% acetic acid
Equal volumes of 40% HCHO and distilled water; to 1 liter solution 100 g hexamin Dissolve in 1 liter distilled water 100 g KI, then 50 g h, and finally add 100 ml glacial acetic acid
20 ml of the solution to 70 ml net sample; 2 ml of the solution to 100 ml water sample (= 0.4% HCHO) As described above
For water samples, 0.2-0.4 ml to 100 ml sample; for net samples, add to a weakly brown color
Note. Samples should be preserved immediately after collection and stored in glass bottles, preferably in darkness and at low temperature. Iodine will oxidize with time, and samples preserved with Lugol's solution need regular attention. Diatom frustules stored in alkaline solution may dissolve with time.
commonly used preservatives are formaldehyde neutralized with hexamethy-lenetetramine (hexamin) or acidified with acetic acid, and the Lugol's solution (Sournia, 1978, p. 69).
For storage of diatom samples over a longer period of time containers (bottles, jars) of moderate glass quality should be used. Silica dissolved from the containers apparently helps to keep the diatom siliceous wall intact. Metal caps and lids should be avoided. The samples should not be exposed to temperatures much higher than ca. 15°C and should be kept away from bright light, especially when preserved with Lugol's or other iodine solutions.
PREPARATION FOR LIGHT MICROSCOPY (Tables 80 and 81)
Examination of raw (not cleaned) material in a water mount or embedded in a resin may give sufficient information to identify a number of common planktonic diatoms, e.g., Chaetoceros spp. and Rhizosolenia spp. (Sournia, 1978, p. 137). These diatoms are identified by their gross morphology and/or special structures like the Chaetoceros setae and the shape of the Rhizosolenia valves and processes. This procedure is often ineffective for revealing the essential morphological structures of other genera, e.g., the areolation and processes of Coscinodiscus and Thalassiosira and the striation and raphe structure of Navicula and Pseudo-nitzschia. Cell content and the organic part of the cell wall obscure the image of the valve structures and have to be removed. The
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