The techniques to apply will depend on research needs, the life stage of the fish, otolith size and shape, and reading techniques. The increments are read with the light microscope or with the scanning electron microscope, both requiring special techniques.
Under normal viewing conditions and sample quality, the light microscope has a very short working distance and a resolution of 1 μm. Except for larval otoliths, otoliths are too thick and must therefore be sectioned for transparent reading.
The scanning electron microscope has very high resolution (a few Amstrongs depending on sample quality) but very little penetration power. Only the surface topography of the specimen is accessible for reading, and the otoliths must therefore be sectioned to read the internal structure. The chemical differences between the two units of the increments are what signals their presence on the surface of the otolith section (Fig. 2).
The scanning electron microscope should be used in otolith structural studies, in the verification of structures first read with the light microscope, and in species where the growth increments are too thin to be read under the light microscope.
The otoliths must be weighed and measured before preparation, in order to determine the fish size/otolith measurement ratio. First determine otolith orientation using the standard terminology (see Glossary) to describe the exact location of the measurements.
Fig. 2. Sagitta of Lutjanus kasmira showing the slow growth seasonal rings. a: read with binocular microscope and, b: read with scanning electron microscope (scale 10 μm).
The most frequent measurements are length (from rostrum to postrostrum) and maximum width, perpendicular to otolith length. When the central focus of the otolith is visible, other measurements are possible, such as radius (from the focus to the postrostrum) distance to the antirostrum, etc. Do not use broken or altered otoliths for morphometry.
Very small otoliths must be measured under the microscope using millimetre grids. Larger ones (> 1 cm) can be measured with calipers. All otoliths must be measured without changing their axial inclination: the small, fragile, edge markings which may be broken should not be counted.
Image analysis is a technique which provides objective data on otolith dimensions, allowing length, width and area to be calculated (Lombarte, 1990). The image is acquired through a high resolution television camera coupled to a computer, and transformed and analysed through image analysis. The stages of the process are:
The real image, caught by the camera, is transformed into an analog image which is digitalized by an analog transformer and real-time image processor. The digital image is composed of pixels: pixel size and light level determine image quality.
The real size of the digitalized sagitta is determined by calibration and calculation of the pixel equivalent in mm. Lastly, image analysis produces the biometric measurements of area, perimeter, length and height. Using these data as a basis, biodimensional morphometric measurements or the implied functions can be plotted to determine growth (Berman et al., 1984), differentiate populations or determine otolith biometry (Lombarte, 1990).
To avoid possible errors in weight caused by changes in moisture content (Pawson, 1990), the otoliths should be dried in a moderate oven (80°) until the weight remains constant. The otoliths are then kept in a dryer until it is time to weigh them.
Cross-sections are necessary to study increment sequence in the otolith. Sections across the various planes of the otolith (Fig. 3) can be cut or polished. All sections must be taken across the nucleus to avoid missing part of the increment sequence.
The orientation of the section is important as many otoliths grow asymmetrically. In sagitta with preferential growth in the internal or sulcal phase, transverse and diagonal cross-sections are recommended. In otoliths with preferential longitudinal growth, the transverse sections may contain many growth checks, and therefore the frontal sections are to be used. In beginning the study of a species, otolith sections representative of the size range must be prepared, and the most suitable section plane determined.
Fig. 3. Diagram showing the transverse section of the sagitta and its principal parts. r: rostrum, s: sulcus, c: collum, n: nucleus, pr: postrostrum, mr: dorsal crests, gr: annulae.
The otoliths are cut with precision saws (Isomet type) with fine diamond blades. The otoliths should be enclosed in a plastic resin in order to obtain exact sections, so that the blocks can be easily positioned in the saw (Bedford, 1977; Rauck, 1975).
The otoliths are embedded in moulds (ice cube trays, semi-rigid plastic or metal, etc.). The moulds should be sprayed lightly with a silicone spray to facilitate subsequent extraction of the block. Pour a small amount of plastic into the mould and wait until the resin is semi-hardened. The otoliths are placed on the resin, lined up so as to facilitate subsequent cutting along the plane selected. Lastly, the moulds are filled with resin, carefully ensuring that no air bubbles remain. Each otolith must have enough resin around it to avoid it breaking when cut. The moulds are placed in a dust-free place and left to harden for 24 hours. If the blocks are still rather soft when extracted, they may be hardened in a moderate oven.
First eliminate all irregularities from each block, then determine the position of the nucleus, marking this on the block with a fine-point marking pen or diamond point. Mark the best section plane on the surface of the block with a ruler, leaving a margin for the thickness of the cutting knife. Then place the block on the saw arm so that the edge of the blade is lined up with the line made on the surface. The section is sawed at low rpm to avoid breaking the otolith and so that the heat from the friction will not melt the resin covering the section, which would make a subsequent reading difficult.
To prepare a fine section bring the blade forward (forward distance = desired thickness of section + blade thickness) and proceed to the second cut. When two diamond blades separated by a metallic spacer of the proper thickness are used, the section is obtained in a single operation. The section is glued to a slide with the face nearest the nucleus downwards and then ground to the right thickness for reading the increments.
To obtain transverse sections of large otoliths, place them on a semi-hard surface and break them across the nucleus using a knife. With a little practice you can control the blade pressure so as to obtain precision sections. This kind of section is useful for SEM readings and for studying the annulae.
Preparing sections for polishing is a two-stage process. First the otolith is ground down to the desired plane and polished to eliminate any surface irregularities. This is a painstaking process, the grinding plane must be constantly checked to avoid overshooting the nucleus or wearing away the otolith edges.
Small otoliths can be embedded in plastic with the polishing face up. The resulting block is easy to handle and will keep the otolith from breaking while handled. The otoliths can also be stuck on to a slide or fingertip with two-sided tape. Both faces of the otolith can be polished if necessary.
Small otoliths with some opaque zones can be etched with a weak acid solution rather than polished. The process of etching, washing and drying is repeated under the dissecting microscope until the desired otolith thickness is obtained. The acid (HCl 0.1 N) is very carefully applied with a fine-tipped paintbrush. The otolith edge can be protected with a bit of nail varnish, which is then removed with acetone. This method is valid only for the light microscope, as the resulting surface will be too irregular for SEM reading.
The short working distance of the light microscope and the blow-up necessary to read growth increments (400–1 000 X), implies a very thin otolith section. Except for the small larval otoliths, which can be read directly (Fig. 4), all otoliths must be finely sectioned as described above.
The section is washed in water and dried under a gentle heat source such as a lamp or moderate oven (60°C) and mounted on a slide. Section slide mounting is identical to larval otolith mounting. The clarity of the growth increments increases after some time (2 to 3 weeks) in the mounting medium.
Fig. 4. Daily growth increments in an Engraulis encrasicholus larval otolith read under the optical microscope.
The otolith edge needs to be embedded in enough mounting medium to avoid optical deformations. The mounting medium shrinks as it dries, and so new medium must be added with a capillary tube. Leave the section to dry at a slant to allow air bubbles to escape.
Mounting media can be dissolved, if necessary, with a proper solvent such as toluene (for Pro-texx and Euparal) or xylene (for Flo-texx) and the otolith repolished and remounted.
Sections across the nucleus are prepared for viewing the internal structure of the otolith with the SEM. Reading is unaffected by the thickness of the section.
The sections are stuck to the SEM slide and ground with a fine polishing paste (0,3 μm) until the sample surface is mirror-like. The preparation is washed with distilled water (cleaning may be facilitated by using ultrasound), and etched with a weak HCI 0.1 N solution or with a 0.2 M EDTA solution to highlight the increment zones. Increment clarity and thickness depends on how and how long the sections are etched. The most appropriate interval must be determined for each specific case. First use a short interval (1 minute for EDTA, 30 seconds for HCI), if the prepared section is not sufficiently etched it can be re-polished and re-etched for a longer time.
Lastly, the otoliths are metallicized with palladium gold, the usual SEM technique, to increase conductivity.
It may be impossible to prepare thin sections of large thick otoliths. For such otoliths, acetate replicas of the surface of a cross-section may be the only way to read increments.
Prepare the otolith as for SEM viewing: the otolith is etched in acid to bring out increment contrasts. The etched otolith surface is washed with acetone and carefully dried with a blower (e.g. an aquarium compressor). Spray the section lightly with a silicone spray, air-blowing off the excess silicone. The otolith section is positioned horizontally on a glass slide with plastiline (children's moulding clay).
The acetate paper must be thin - some 0.3 mm thick (Wild, 1982; Fagade pers. comm.) and kept perfectly dry. There are two replication methods:
1. Cut a thin strip of acetate paper with scissors, a bit bigger than the otolith, and immerse the edge of the acetate paper in acetone with forceps (do not wet the forceps). Immersion time in the acetone is calculated by dissolving a bit of acetate paper. Three-quarters of the time needed to dissolve the paper is the proper replication interval (Wild, 1982). Acetate paper dissolving time is particularly affected by temperature, which should therefore be read before starting the replication.
Gently press the surface of the otolith to be replicated onto the acetone-dipped acetate paper strip, using a spatula to eliminate air bubbles. After drying for 3–4 minutes, carefully separate the strip and the otolith to avoid fracturing the otolith.
2. Place a somewhat larger strip of acetate paper over the otolith, carefully depositing 1–2 drops of glacial acetate acid on the acetate paper. Be sure the acid covers only the otolith area (Fagade pers. comm.). When the replica is dry, use forceps to remove it from the otolith.
Fig. 5. Photomicrograph (scanning electron microscope) showing growth increments in a continuous unit (in relief) and a check. Scale 75 μm.
While the replicas are drying, prepare slides by fixing a cover slip with a bit of transparent tape so that the slide and cover slip open like a book. Slip the replica into this and fix the cover slip with transparent tape, pressing gently.
The replicas can be read with the SEM like thin sections. Should the quality of the replica be poor because of errors in the preparation or poorly-done sections, the otolith can be re-polished and the process repeated.
