Otolith microstructure preparation

Learn a quick way to prepare otolith microstructures using cyanoacrylate glue or thermoplastic cement.

On this page

• Overview
• Mounting the otolith
• Flipping an otolith mounted in glue
• Polishing
• Examples

Overview

Preparing otoliths for microstructural examination takes practice, and initial attempts can be frustrating. However, consistently good results are possible after days or weeks of practice, depending on the size of the otolith and the clarity of the microstructure.

Mounting the otolith on a microscope slide with either cyanoacrylate glue or thermoplastic cement is a quick preparation method. This way you can easily flip and then polish one or both sides of a thin section.

This method is ideal for most larval and juvenile otoliths. For adult otoliths, it’s often preferable to prepare a thin section first, then to polish. Alternatively, you can use David Secor’s method for the preparation of epoxy-embedded thin sections for microstructural. Secor's method is time consuming, but produces good quality results with larger otoliths.

Mounting the otolith

For most otoliths greater than 50 to 100 micrometres in diameter, you’ll need to polish them to fully reveal the narrow growth increments encircling the core. If this is the case, the otolith will need to be mounted so that the plane of the growth increments is parallel to the microscope slide.

For some otoliths, this means that the sulcus side should face up on the slide so that the otolith lies flat. However, as long as the otolith lies flat, the side left up really doesn't matter. In some cases, it’s preferable to leave the side facing up which has the most otolith material between the core and the surface, so that this excess material can be removed with polishing.

Cyanoacrylate glue does an excellent job of holding the otolith to the slide, and at the same time, partially clearing the otolith. It’s easy and quick to apply and can be flipped over after polishing. However, it tends to peel off the slide after a year or more of storage, especially after exposure to immersion oil. For most studies, this isn’t a problem, since the otolith examination will have been completed early on. If archival properties are important however, glue shouldn’t be used.

How to mount with glue

To mount the otolith in glue:

1. place the otolith near the centre of the microscope slide
   • mount only 1 otolith per slide
2. place the slide on the stage of a dissecting microscope at low magnification
3. ensure that the otolith is lying flat
4. place a drop of glue beside the otolith, close to but not touching it
   • if the glue applicator touches the otolith, the otolith will likely stick to it
5. use the microscope to look at the glue drop and otolith, and put the end of a probe into the glue drop at a tilted angle and draw the glue around, then over, the otolith
   • surface tension will allow the glue to move with the probe (as long as the probe is held at about a 30 degree angle from the slide) without the probe touching the slide
   • it’s best that the probe not touch the otolith so it doesn't stick to the probe

It’s important that some of the glue be drawn over top of the otolith so that the glue has wetted the entire otolith. Once the otolith is covered, excess glue can be drawn to other parts of the slide. However, there is some advantage to leaving the otolith in a small dome of glue, with the top of the dome being the glue-wetted top of the otolith.

Leave the glue and otolith undisturbed while drying, anywhere from 15 minutes to 2 hours. You’ll see an obvious mark if you poke a probe tip into dry glue.

Thermoplastic cement

An alternative to using glue for mounting is thermoplastic cement. The cement does a good job of bonding the otolith to the slide, and can be heated to remove or flip an otolith. However, it’s a little messier during the polishing stage. It's the best choice for large otoliths, but it's awkward to use for small otoliths or for thin sections that need to be flipped.

To mount with thermoplastic cement:

1. heat the microscope slide to about 275 degrees Celsius on a hot plate
2. add a light touch of cement to the hot slide
3. place the otolith in the cement’s gummy liquid residue
   • it isn’t necessary to cover the otolith with the cement, as was the case with the glue
   • the cement is used merely to hold the bottom of the otolith onto the slide
4. adjust the orientation of the otolith while the slide is still hot

The cement sets quickly after removal from the hot plate. To move or remove the otolith from the slide, simply reheat the slide until the cement liquefies.

Flipping an otolith mounted in glue

It’s relatively easy to flip an otolith mounted in glue if you need to polish both sides to prepare a thin section. The resulting section is usually of excellent quality, and is thin enough to allow good light transmission and thus clear examination of the daily increment sequence.

When you polish the first side of the otolith, stop a little short of the plane of the increments. To flip:

1. soak the microscope slide (holding the otolith) in water for about 10 minutes
2. remove the slide from the water and place it on the stage of a dissecting microscope under reflected light
   • the bottom of the slide can be quickly dried on a paper towel first, but not the upper surface holding the otolith, which needs to remain wet
3. use a scalpel to cut a square in the glue containing the otolith, with the otolith in the centre
4. leave approximately 1 otolith diameter of glue on each side of the otolith
5. cut with the scalpel in a rocking motion
   • as you observe the cut through the microscope, you'll see the glue begin to lift off the slide

Usually, the square of glue containing the otolith will peel off the slide entirely in the last cut (the fourth side). If it doesn't, do not use the scalpel to try and cut under the otolith as this will almost always break the otolith.

Instead, repeat the 4 scalpel cuts a little bit closer to the otolith. If the otolith and glue have still not peeled off the slide, try soaking the slide in water for another 10 minutes and repeating the procedure.

Drying and flipping removed otolith

Peel off the square of glue containing the otolith, and use a probe to push the square off to the edge of the slide. Let it hang halfway off the slide to speed up drying.

To use the same slide for re-gluing the otolith, you may slough off the glue with your fingers, then dry it with a tissue.

Allow the slide and glue to dry for 5 to 10 minutes under the microscope light.

When dry, use forceps to flip over the glue square and dry for another 5 minutes. At this point, you may move the square back into the centre of the slide in preparation for re-gluing.

Re-gluing

Re-gluing the flipped otolith is a simpler variation of the original gluing procedure:

• place 1 drop of glue immediately beside, but not touching, the glue square containing the otolith
• use a probe to draw the glue around the square on all sides
  • surface tension will draw the glue under the otolith
• don’t draw glue over top of the square, since it will have to be polished off later anyways

Once the glue has dried (after about 1 hour), you may polish the preparation to the plane just short of the increment sequence.

Flipping cement-mounted otoliths

The advantage of using glue is that the square containing the otolith keeps the otolith rigid. This makes the flipped otolith lie exactly opposite to the original polishing plane, making it much easier to prepare a uniform thin section.

This isn’t the case with thermoplastic cement. When using cement, the otolith may be flipped after re-heating the cement, but it can be difficult to align the flipped otolith flat on the slide.

Polishing

You’ll need to polish most otoliths greater than 50 to 100 micrometres in diameter to remove otolith overburden and fully reveal any narrow growth increments encircling the core.

It’s a common misconception that if daily increments are clearly visible somewhere in the otolith, polishing isn’t necessary. However, the central region of the otolith is the thickest and the most likely to obscure any increments near the core. Therefore, clear increments elsewhere in the otolith aren’t necessarily an indication of a completely visible growth sequence.

Comparing increments visible before and after polishing is the only way to see whether or not polishing is required for otoliths from a given species and size.

Using metallurgical lapping film

It’s possible to polish an otolith mounted on a microscope slide with glue or thermoplastic cement. Attach adhesive-backed metallurgical lapping film to a scratch-free glass plate or other very smooth surface.

Surface smoothness is important, since nicks or cracks can chip the otolith during polishing. For many otoliths, 2 grades of lapping film mounted side by side will suffice (30 and 3 micrometres). This type of lapping film is dry, and thus easier to use than wet lapping films.

Polish process

To polish the otolith:

1. invert the slide on the lapping film
2. centre the otolith between your index and middle fingers, using the least possible pressure (to avoid fracturing the otolith)
3. move the slide in a rapid circular motion on the lapping film

Polishing time varies with the:

• otolith thickness
• grit size of the lapping film
• amount of glue covering the otolith

However, it’s often best to begin with only a few seconds of polishing with a fine grit lapping film, checking the level of the polish under the microscope to determine how much material has been removed.

Aggressive initial polishing isn’t recommended until the polisher has some familiarity with the speed at which polishing progresses. It’s possible to overpolish (and ruin) an otolith in the first 10 seconds.

Once you’ve completed your initial assessment, you may polish the otolith for up to 10 seconds on the 30 micrometre lapping film. Follow this with 2 to 10 seconds on the 3 micrometre film to smooth out the surface for microscopic examination.

Small otoliths will only be polished with the 3 micrometre film. Quickly rubbing your finger over the otolith will remove surface debris before you assess the state of the polish under a compound microscope.

Repeat polishing and re-examining the otolith under the microscope until the polishing plane is slightly above the plane of the growth increments. At this point, the otolith is ready for:

• counting
• interpretation
• measurement

If the otolith is particularly large, you may need to flip it over and polish it from the other side as well. This double polishing essentially prepares a thin section. If you need to double polish, stop polishing the first side slightly above the plane of the growth increments. Otherwise, they’ll become less clear if too much overlying material is removed.

Polishing plane and incremental plane

The key step in otolith polishing is checking where the polishing plane is in relation to the plane of the growth increments. Initially, you must do this under a compound microscope at low magnification (about 100x for all but the largest otoliths).

To start, the polishing surface of the glue is brought to focus under the microscope. This surface is readily determined by looking for surface scratches and pieces of debris. A 1-second polish on 3 micrometre lapping film helps make the surface clear on previously-unpolished otoliths.

At later stages of polishing, the polishing surface will be within the otolith itself instead of the surface of the glue.

Once you locate the polishing surface, re-focus the microscope on the plane of the growth increments or primordia. This plane has been located when the increments or primordia are in the best possible focus.

The visibility of this plane will be poor (or sometimes nonexistent) at an early stage of polishing. This is due to the considerable overburden of glue and otolith material between the surface and the increment plane.

This by itself indicates that considerably more polishing is required. In general, the amount of re-focusing required to move between the polishing plane and the incremental plane indicates how much more polishing is required.

Polish completion

As you continue the cycle of polishing and microscopic examination, the distance between the polishing surface and the incremental plane will become smaller and smaller.

Eventually, increments will become visible while the microscope is focused on the surface. When this happens, it indicates that the polishing is nearly completed. It’s also when you can increase the magnification to about 400x to achieve finer control of the polishing. You should only use fine-grit lapping film (3 micrometres) at this point. You can usually stop polishing when increments are visible while focused on the polishing plane at a magnification of 400x. In other words, polishing doesn’t proceed right to the incremental plane, but stops before it. Make this examination in the area of the otolith where the increments will eventually be counted.

The objective of the polishing procedure is to make the growth sequence clearly visible along a particular axis between core and edge. Since most otoliths aren’t completely planar, the optimal polish along 1 growth axis may well leave other growth axes underpolished, or grossly overpolished. This isn’t a concern as long as the growth axis selected provides a complete growth sequence.

The time required to completely polish an otolith from 1 side varies with the size of the otolith and the skill of the polisher. Typically, an experienced polisher can polish a 100 micrometre otolith in 1 to 5 minutes.

Examples

The following image pairs show a trout sagitta glued to a slide as it moves through the polishing process. The key to monitoring polishing progress is focusing first on the grinding surface (left images), then on the incremental plane (right images).

You can tell that you’ve correctly polished your otolith if it’s polished to a plane just above that of the growth increments. Many of the growth increments will be visible while focused on the grinding surface, but they shouldn't be as clear as when focused on the increments themselves.

You can tell if you've overpolished if the increments aren’t visible. Polishing beyond the plane of the increments, even slightly, usually makes the increments completely disappear. If increments in a particular area of the otolith were visible throughout polishing, but cease to be visible in later stages of polishing, it's likely they've been overpolished. This may happen if the incremental plane isn’t quite parallel to the plane of the slide, making some areas disappear due to overpolishing before other areas are ready. This isn’t a problem as long as 1 complete growth sequence is visible somewhere else.

An otolith polished right to the incremental plane is more difficult to interpret than is one where some otolith overburden is left unpolished. Subdaily increments and increment splitting tends to be most prominent in a growth sequence polished right to the increment plane.