Otolith annuli

Preparation of Thin Sections

Preparation of Thin Sections

Thin sections are often the best form of preparation for otolith annuli. They may also be appropriate for examination of daily increments in large otoliths. Transverse sections through the core are the norm. Here we describe a good method for embedding and sectioning otoliths more than 3 mm in length, suitable for careful preparation of individual otoliths with an Isomet saw. Mass production techniques are described on a separate page.

Embedding

It is possible to section very large otoliths (>2 cm in length) with an Isomet saw with no prior embedding. It is also possible to embed large otoliths (>1 cm in length) in molten wax prior to sectioning. However for precise sectioning with an Isomet saw, it is best to embed the otolith in a hard epoxy. We use Araldite epoxy GY502 and hardener HY956 in a 5:1 weight ratio (available from Brenntag Canada Inc.). We also use Buehler Epo Thin resin (catalog # 20-8140-032) and Buehler Epo Thin hardener (catalog # 20-8142-016) ( Buehler catalog) in a 5:2 weight ratio. However, other epoxies and setting compounds, as long as they are transparent and very hard, will work.

To prepare the epoxy, add the epoxy and hardener together in a paper cup on a tared top-loading balance. Do not use a plastic cup. 20 g Araldite epoxy and 4 g hardener will stay soft for 45-60 mins, which is enough time to embed about 20 otoliths. Stir for a minimum of 3-4 mins, then sonify in the cup to remove bubbles for 5 mins or until clear (some froth will stay on top). If there are only a few otoliths to embed, let the epoxy sit for 5-15 mins to set a little.

Free Pour Procedure:

Pour a few mls of epoxy into a pear shape (~ 2 cm long and 1-2 cm wide) on a sheet of Parafilm

Pour a few mls of epoxy into a pear shape (~ 2 cm long and 1-2 cm wide) on a sheet of Parafilm

Pour a few mls of epoxy into a pear shape (~ 2 cm long and 1-2 cm wide) on a sheet of Parafilm; label a small piece of paper and stick the label into the narrow end of the epoxy pool. Prepare a separate preparatory pool of epoxy to be used for all otoliths. Push the otolith into the preparatory pool of epoxy, sulcus side up; use a fine probe to remove all bubbles from around the otolith, particularly in and around the sulcus. Use forceps to lift the otolith out of the preparatory epoxy and put it sulcus side down into the embedding pool of epoxy, with the long axis of the otolith parallel to the long axis of the epoxy pool. Push the otolith to the bottom, making sure there are no bubbles.

Let the epoxy solidify overnight, then wipe the bottom of the epoxy with 95% ethanol. Pour a few mls of freshly mixed epoxy over the bottom to encase the otolith on both sides. Allow to set 3 days before sectioning. Use 95% ethanol to remove spilled liquid epoxy.

Mold Procedure:

Prepare labels and coat otolith with epoxy as in the free pour procedure

Prepare labels and coat otolith with epoxy as in the free pour procedure

We use 12-well silicone EPDM (silcone combined with a synthetic rubber) molds, 19 x 11 cm. Individual wells are 4 x 2 x 0.5 cm and hold up to 4 ml of epoxy. Prepare molds by applying, with a small brush to each well, a light coat of release agent such as Frekote (Henkel/Loctite) and allow to dry. It is best to apply the release agent immediately following the last use of the mold.

Prepare labels and coat otolith with epoxy as in the free pour procedure. Allow to solidify overnight before removing the embedded otolith. Do not wipe with ethanol. There is usually no need to coat the otolith with additional epoxy to encase. Cure 3 days prior to sectioning.

Sectioning

Isomet low-speed diamond bladed saw

Isomet low-speed diamond bladed saw

The epoxy-embedded otolith is clamped perpendicular to the saw blade with a V-notch clamp, whereby the otolith is positioned on the lower surface of the epoxy block.

The epoxy-embedded otolith is clamped perpendicular to the saw blade with a V-notch clamp, whereby the otolith is positioned on the lower surface of the epoxy block.

We use an Isomet low-speed diamond bladed saw for preparing otolith sections, but any equivalent saw can be used. By fitting the saw with two blades separated by a spacer, a thin section be prepared from an epoxy-embedded otolith with a single cut in about 2 minutes.

The epoxy-embedded otolith is clamped perpendicular to the saw blade with a V-notch clamp, whereby the otolith is positioned on the lower surface of the epoxy block. The water tray for the saw is filled with 60% glycerin in water to a level where it just touches the tip of the blade. We use 4" blades between 3" flanges; with only 0.5" of free blade exposed, blade wobble is minimized. Where two blades are to be used, we separate them by a thin metal or plastic spacer with a diameter of 3" and a thickness appropriate to the section being prepared. Typically, a 500 µm spacer will be used, producing a 400-500 µm otolith thin section.

The saw can be run relatively quickly (which is still slow) with 100 g of weight on top until the blade is almost through the epoxy. Stop the saw immediately after 1 side of the epoxy block falls off. Remove the block from the clamp and use fingers or forceps to break off the section (which is probably attached to the block by very little). Rinse the thin section in 95% ethanol, then use a scalpel to cut off the broken block attachment point.

Polish one side of the section with 30 µm dry lapping film mounted on a smooth surface (eg- glass plate), then 3 µm lapping film, using a finger. The polish can be brief, and should be to remove coarse scratches only. In many cases, polishing is not necessary. The section can then be mounted onto a microscope slide with a mounting medium. We often use a cyanocrylate glue (Krazy Glue) or thermal-setting plastic (Crystalbond). However, a more durable bond can be made with epoxy. To do this, use an approach similar to that of making a blood smear. Use a microscope slide to spread a layer of epoxy on one half of the mounting slide, making the epoxy layer no thicker than the section (eg- 400 µm thick). Slide the section into the epoxy making sure that there are no bubbles underneath and no epoxy on top of the section. Press the section down onto the slide and allow to dry 2-3 days. Polish the top of the section if required.

Production Ageing

Production Ageing

Age determination is an important component of fish stock assessments. In our laboratory, tens of thousands of otoliths are sectioned and aged each year in support of stock assessments. We use production methods which involve embedding 50-100 otoliths in a polyester resin block, sectioning with a high-speed saw to produce transverse thin sections, coating with a photographic clear coat to enhance visibility, and ageing under an image analysis system. Using these methods, it is possible for one person to prepare on the order of 300-800 otoliths per day, or to age about 200 per day. Note that these methods are most appropriate for robust otoliths at least 1 cm in length; smaller or more fragile otoliths are best embedded in epoxy and sectioned individually with an Isomet saw, a process which is much more time-consuming.

We have prepared a 15-minute VIDEO DEMONSTRATION of production methods for embedding and sectioning otoliths embedded in polyester resin blocks, which provides an easy and complete overview of the process. The information below also describes the steps and materials used in mass producing and mounting transverse sections, but provides more detail on some of the materials and suppliers than does the video.

Embedding:

Aluminum embedding tray for epoxy resin

Aluminum embedding tray for epoxy resin

Otoliths positioned on reference lines in an embedding tray whose base coat of epoxy has been poured.

Otoliths positioned on reference lines in an embedding tray whose base coat of epoxy has been poured.

Aluminium trays (177 x 154 x 18 mm, outside dimensions) are used as moulds. The trays have grooved bottoms and sides to produce cutting guides; removable sides to assist in the release of the hardened resin block; and removable bolts which serve to make holes for the insertion of the positioning posts on the saw. To release the block from the mould and to prevent leakage of resin a thin layer of household paste wax is applied to the inside of the tray to seal all seams. To ensure correct sequencing of sections a piece of dry spaghetti is placed diagonally across the tray from upper left to lower right corners.

Clear casting resin (23189), pigmented black with 100 ml pigment: 4 litres of resin, and catalysed with the hardener Luperox DDM-9 BT300 to gel in 30 minutes is used as the embedding medium. All procedures involving the resin are carried out in a fume hood and any resin spills are removed with acetone. A layer of resin mixture is poured into the trays to cover the spaghetti strand and fill the corners.

Once the resin has set reference lines can be etched into the resin, using the grooved lines on the tray as guides. Otoliths are arranged with the core of the otolith positioned on the reference line. Taking care not to disturb the alignment of the otoliths, trays are filled with resin and left to cure for a minimum of 24 hours prior to sectioning. 100 to 150 otoliths (depending on size) may be placed in one tray.

Sectioning:

A surface grinder was adapted to accommodate a diamond-edged cutting wheel (UPC # 66260257622, shape 1AIR, 7 x .025 x 1 1/4) and modified to include a mounting bracket and cutting base for the resin block, a water outlet to provide cooling and lubrication to the cutting wheel, and a splash tray with water drainage.

Blocks (approximately 10 mm thick) are removed from the moulds, mounted in the saw on the positioning posts, and secured with the mounting bracket. Using the vertical, horizontal and lateral controls the edge of one of the block's cutting guides is positioned so it is in alignment with the saw's cutting wheel. Sections (1 mm thick) are removed, rinsed with water, and air-dried. Sections to 0.5 mm thickness can be produced without breakage.

Care is taken to ensure that individual otoliths, or otolith pairs, can be identified throughout the embedding and sectioning procedures. A soft pencil can be used to write otolith numbers on the cut resin strips, directly under the otolith.

Strips of sectioned otoliths and appropriate labels are mounted onto sheets of clear acrylic (185 x 95 x 2mm) with transparent spray coating (Krylon Crystal Clear 41303). After the plates have dried a topcoat of spray is applied to both protect the sections and enhance their clarity. The Krylon coating tends to hide scratches and grooves in the cut surface of the otolith.

High-speed cutting saw for mass-embedded otoliths in resin

High-speed cutting saw for mass-embedded otoliths in resin

Close-up of cutting wheel cutting through embedded resin mould mounted in high-speed saw

Close-up of cutting wheel cutting through embedded resin mould mounted in high-speed saw

Strips of sectioned otoliths mounted on acrylic sheet

Strips of sectioned otoliths mounted on acrylic sheet

Ageing:

Inexpensive image analysis system used for reading mass-embedded otolith sections

Inexpensive image analysis system used for reading mass-embedded otolith sections

Once the plates have dried otolith sections are aged using an Image Analysis System with reflected light and a magnification of 10 to 12x.

Otoliths prepared in this manner provide an efficient and cost effective means for ageing and storing large numbers of samples. Since the otolith plates are dry, they can be stored compactly (eg- vertically in filing cabinets) with an indefinite shelf life.

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