Ciliophora
Sample preparation and analysis

The following are only some general comments for the determination and enumeration of ciliates. For accurate species identification, in vivo observations are necessary, preferably with interference contrast. Important characters, e.g., movement, cortical granules and their colour, presence and shape of extrusomes, are often not preserved or do not stain in fixed material. However, an experienced researcher should be able to identify many species in live samples, at least after some preliminary work in the area, which considerably shortens sample processing. Sometimes, particularly for taxonomic investigations, determinations have to be verified by silver impregnation. Several techniques are available for this, of which the protargol method is the most universal. These procedures and some modifications as well as preparation for scanning electron microscopy are described in detail by Foissner (1991) and in the 'Protocols in Protozoology' (Lee and Soldo, 1992).

Traditionally, however, a variety of fixatives is utilized in different concentrations in protozooplankton studies:
¥ Acid Lugol's iodine: 10 g iodine and 20 g potassium iodide dissolved in 200 ml distilled water, then add 20 ml glacial acetic acid; used in 0.4-10% final concentration, precludes most silver impregnation methods and epifluorescence microscopy;
¥ Bouin: 150 ml saturated aqueous picric acid, 50 ml formaldehyde, 10 ml glacial acetic acid; prepare immediately before use;
¥ Modified Bouin: buffered concentrated formaldehyde saturated with picric acid, add glacial acetic acid immediately before use so that the final concentration of acetic acid is 1% (v/v) after addition of the sample; use a fixative:sample ratio of 1:19 for brackish and of 1:10 for open ocean water (Lee et al., 1985);
¥ Glutaraldehyde: 1-6% final concentration; addition of 2% tannic acid reduces cell shrinkage (Choi and Stoecker, 1989);
¥ Formaldehyde: 0.4-2% final concentration, buffered with hexamethylenetetramine;
¥ Mercuric chloride: 380 ml saturated aqueous mercuric chloride (60 g mercuric chloride dissolved in 1 l of boiling distilled water), 100 ml formaldehyde, 30 ml glacial acetic acid; used in 2.5% final concentration.

Although widely used, formaldehyde is particularly inappropriate for preservation of marine ciliates because most specimens burst (up to 72% loss relative to Lugol), and many are strongly distorted (Pace and Orcutt, 1981; Revelante and Gilmartin, 1983; Leakey et al., 1994a; Stoecker et al., 1994a). Fewest losses occur with Lugol's iodine, higher concentrations, e.g., 10% or 20%, usually yielding more ciliates (Stoecker et al., 1994a). This fixative is followed by Bouin (up to 46% loss relative to Lugol), and glutaraldehyde (up to 66% loss; Revelante and Gilmartin, 1983; Leakey et al., 1994a; Stoecker et al., 1994a). Particularly less destructive is mercuric chloride but it is costly and highly toxic (Pace and Orcutt, 1981; Laybourn-Parry, 1992). In contrast to seawater, the most destructive fixative in freshwater is Lugol's solution, causing 58% loss after nine months, followed by glutaraldehyde (29%), formaldehyde (24%), and mercuric chloride (15%) (Sime-Ngando and Groliere, 1991).

From the above mentioned studies it may be concluded that the effects of fixatives vary with concentration, salinity of sample, ciliate species, perhaps the organism's nutritive state and apparently the duration of sample storage. A further problem with Lugol is that it stains ciliates and detritus the same colour, which may cause significant counting errors in the presence of abundant particulate organic matter (Pace and Orcutt, 1981).

There are several methods for concentration. But like fixatives they can severely reduce aloricate ciliate numbers (Sorokin, 1981). Filtration of samples, which is widely used, may decrease the abundance of aloricate ciliates by up to 95% (Sorokin, 1977; Gifford, 1985). Reverse filtration can be only slightly less destructive, and may cause losses of over 75% (Gifford, 1985; Laval-Peuto and Rassoulzadegan, 1988).

All fixatives change the volumes of ciliates; i.e. they usually shrink. Thus, the biomass derived from preserved samples is considerably lower than the standing stock if used without correction. Pierce and Turner (1992) concluded that the error caused by cell shrinkage and loss from fixation and handling may underestimate the true aloricate ciliate biomass by at least an order of magnitude. Compared with the live volume, formaldehyde shrinks cells the least, viz. 10-20% on average. Lugol's solution and glutaraldehyde decrease cell volumes by 13-45% (Choi and Stoecker, 1989; Ohman and Snyder, 1991; Leakey et al., 1994a). But mercuric-chloride-fixed cells are about 20% larger than those preserved in 2% acid Lugol (Sime-Ngando et al., 1992). Similarly, 5% Bouin usually shrinks cells less than 10% Lugol or the quantitative protargol stain (Jerome et al., 1993; Stoecker et al., 1994a). Consequently, the ratio of cell carbon content to volume depends on the fixative used and its concentration. Putt and Stoecker (1989) experimentally determined carbon contents in preserved ciliates at 0.19 pg C µmö-3 in 2% Lugol, and 0.14 pg C µmö-3 in 2% formaldehyde, whereas an average of 0.11 pg µmö-3 is suggested for living specimens (Turley et al., 1986).

Particular attention should be paid to the method of enumeration, since a formally adequate sampling procedure cannot replace an inappropriate counting technique. The most accurate method is live counting of unconcentrated water samples as demonstrated by comparative studies (Sorokin, 1977, 1981; Dale and Burkill, 1982; Sime-Ngando et al., 1990). The last study also found that live counting of filtered samples provided good results (but see above). For enumeration, the water sample (0.4-30 ml) is put in a Dolfuss or other shallow counting chamber or a Bogorov counting tray. The entire sample or a fraction is examined using a stereo microscope at 25-60x magnification and bright or dark field illumination (Dale and Burkill, 1982; Sorokin, 1982; Sime-Ngando et al., 1990). Shallow counting chambers with a lid are generally well suited for shipboard use, but the Bogorov tray may be used only on firm ground. There, and if conditions are fairly stable on a big ship, the simplest counting method is to transfer the sample dropwise onto a clean slide and inspect it under a compound microscope at 40x magnification.

Live counting is a quick, well tested, and cheap method for the enumeration of ciliates (Dale and Burkill, 1982; Sorokin, 1982; Foissner, 1983; Lüftenegger et al., 1988; Sime-Ngando et al., 1990). Counting of 1 or 5 ml samples takes 15-120 minutes (Dale and Burkill, 1982; Sime-Ngando et al., 1990). The volume of water investigated depends on the concentration of ciliates, i.e. a smaller volume (about 1 ml) is sufficient in near-shore rich waters, but if specimens are scarce (<5 ind./ml), as in the oligotrophic open ocean, a larger volume (about 5-15 ml) has to be counted. An advantage of live counting is that it allows species identification. This requires some experience, but in contrast to preserved samples important morphological characters (shape, size, colour, extrusomes, movement) are not lost or distorted, which makes correct determination much easier. However, a crucial drawback of live counting is that it has to take place immediately after sampling, i.e. at least on the same day. This limits considerably the number of samples that can be taken and processed.

More common than live counting of planktonic ciliates is enumeration of fixed and concentrated material using an inverted microscope following the procedure of Utermöhl (1958). However, this method is less satisfactory because almost all aloricate ciliates may have been lost during prior preservation and concentration (see above). Furthermore, as specimens are frequently shrunk, distorted, darkly stained, or masked by detritus in the settled samples, identification to genus or species level is usually impossible (Sorokin, 1977; Pace and Orcutt, 1981; Leakey et al., 1994a).

An alternative to counting fixed samples is the quantitative protargol stain of Montagnes and Lynn (1987), which also allows preliminary species identifications. The organisms are fixed with Bouin and concentrated on a Millipore cellulose filter (0.8-3.0 µm pore size; suction <100 mm Hg). The material on the filter is then embedded in agar, stained and counted. By this, a permanent record of the community is obtained. However, as in fixed samples, characters present solely in living individuals are lost and definitive species determinations are often impossible. Another disadvantage is that the procedure is costly, time consuming, and not suitable for routine treatment of many samples. However, the modification by Skibbe (1994) is distinctly quicker (4 hours/preparation) and works also with Lugol-fixed cells. The efficiency is similar to that of settling chambers but abundances are about 13% lower than with direct counting (Montagnes and Lynn, 1987).

Especially small ciliates are often counted using epifluorescence microscopy (Sherr and Sherr, 1983). Aliquots of preserved samples are filtered onto black polycarbonate filters and stained with DAPI, proflavin, fluorescein isothiocyanate (FITC), or acridine orange. This permits distinction between autotrophic and heterotrophic cells as specimens containing plant pigments fluoresce either red or orange depending on the type of pigment present (Stoecker et al. 1989b). But species identification is usually impossible. The use of electronic counters like the Coulter Counter requires considerable experience and thus often yields unrealistic figures (Sheldon, 1978).