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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.
The information below briefly describes the steps and materials used in
producing and mounting transverse sections from otoliths embedded in polyester
resin blocks.
Embedding:
| 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. |
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Clear casting polyester resin solution in styrene
(F31-2125), pigmented black with 100 ml pigment: 4 litres
of resin, and catalysed with the hardener Luperox
DDM-9 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.
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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:
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A surface grinder
was
adapted to accommodate a diamond-edged cutting wheel
(UPC # 69014192103, 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.
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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. |
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Ageing:
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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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