OTOLITH PROCESSING LINE
OTOLITH PROCESSING LINE FOR FISH AGEING STUDIES
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Introduction
Information on age composition of fish catches is important in studies of fish populations and growth studies. Age determination in bony fish species can be made by studying the small calcic earstone called the Otolith. Each bony fish has 2 Otoliths which grow as the fish grows and develop a ring structure in which, in temperate waters, the number of rings corresponds to the number of years. By counting the number of rings in sample fish catches information is obtained on the dynamics of fish populations.
We are presenting here a routine Otolith preparation system which is based on work recently completed at RIVO-DLO in Holland. The RIVO-DLO work was developed from earlier work at the CEFAS laboratories in Lowestoft UK and originally proposed by BAKER (1983).
The methodology is based on the preparation of very thin smooth sections of embedded Otoliths. This leads to excellent image quality in age determination and makes automated analysis possible. The procedure involves the following steps:-
1) Preparation of resin blocks with embedded Otoliths (figures1,2,3 &4)
2) Sectioning the embedded blocks to give thin sections with smooth surfaces (figure 5)
3) Mounting and viewing of the thin sections (figures 6 & 7)
Moulds for preparing resin blocks containing embedded Otoliths.
The double moulds, illustrated in figure 1, are designed to produce 2 resin blocks
146 x 72mm. There is a quick release device to facilitate release of the hardened blocks. In order to align rows of mounted Otoliths, 6 fiduciary marks 16mm apart are cut into the 3 lateral strips. For identification purposes and for sectioning orientation each block has one 10° sloping side.
In the first step in the processing procedure up to 4 moulds are placed on a levelling table (figure 5) and filled with 2-3mm layer of black resin which is then hardened. This work should be carried out in a fume hood which is available for this purpose.
Mounting Jig
This is a device, shown in figure 4, which facilitates the mounting of Otoliths in rows, in the moulds which now have a base layer of hardened black resin. The moulds are mounted on a X-Y Table X-Y Table and positioned so that the resin surface can be observed with a CCD Camera and Monitor. The lateral mould markings are used as a guide to mounting 6 rows of Otoliths. The mould is subsequently filled with more resin which is then hardened to give a solid block with embedded Otoliths. The edges of the cured resin block can be ground to a smooth finish using a grinding fixture(figure 3).
Sectioning
The sectioning of the resin blocks is carried out with a modified Labcut 230 Cutting Machine fitted with a thin metal bonded diamond cutting wheel and sectioning fixture (figure 5). The blocks are held in a fixture so that they can be accurately positioned to ensure that thin sections can be cut through the centre of Otolith rows.
Mounting and Viewing
The cut sections are mounted on 75 x 50mm Glass Slides. The mounting is carried out on a levelling table with 16 mounting stations (figure 6). Clear mounting resin is used to fix
3 or 4 cut sections on one slide. This work should also be carried out in a fume hood. A mini fume hood is available for the mounting work.
The sections can be viewed in this state or a cover slip can be fixed for permanent storage. Uncovered slides are useful if, at a later stage, further analysis is required. The mounted sections can be viewed for age determination studies by incident or transmitted light with a Stereo Microscope. For easy viewing a CCD Camera and monitor can be mounted on the Microscope. Two examples of sectioned Otoliths are shown in figure 7.
Use of the Mini-fume Hood
All the steps in the protocol for preparing and mounting the thin sections, should be carried out in a Fume Hood. The model of fume hood available is sufficient large for both types of levelling tables and for the mounting jig.
A. MOUNTING
OTO 910 Levelling Unit for Casting Resins in Moulds
OTO 919 Mini-Fume Hood for OTO 910 Levelling Unit
OTO 914 Double Mould (4 units)
OTO 915 Grinding Jig for Resin Moulds
OTO 916 Quick Release system for Moulds
OTO 920 Mounting Station Complete
Comprising:
OTO- 919 Mini-Fume Hood w/extraction adaptor
OTO- 920 Otolith Mounting Jig w/X-Y positioning
OTO- 922 CCD Camera & 15in Monitor with fixing attachment to Fume Hood
B. SECTIONING
OTO-LABCUT 230B Bench Top Otolith Sectioning Machine
complete with:
Sectioning fixture
Diamond wafering blade.
ALTERNATIVE
OTO-LABCUT 230F Floor Standing Otolith Sectioning Machine
complete with:
Sectioning fixture
Diamond wafering blade.
C. SLIDE PREPARATION
OTO 926 Slide Mounting Kit
OTO 928 Slide Staining Kit
OTO 911 Levelling Station for Slide Preparation
OTO 919 Mini-Fume Hood for Slide Preparation
D. VIEWING
OTO 940 Stereo Viewing Station
Stereo Zoom binocular microscope transmitted illumination,
twin flexible fibre optics and power supply
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Figure 1: Double Mould (RIVO Type) with score marks to define Otolith rows |
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Figure 2: Levelling platform for double moulds. The resin casting should be carried out in a fume hood. A mini fume hood is available for this purpose. |
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Figure 6: Levelling platform for slide preparation. The mounting procedure should be carried out in a fume hood. A mini fume hood is available for this purpose. |
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Figure 3: Grinding Jig for Resin Blocks with embedded Otoliths |
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Figure 4:Mounting Jig showing double mould in positionThe CCD Camera is visible above the mould.(The fume hood is not shown) |
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Figure 5: Labcut 250 Cutting Machine with Sectioning Jig in position on the work table. The Diamond wafering blade is visible |
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Cod |
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Plaice |
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Figure 7: Otolith sections of different fish species |
