Dear all,
currently we are trying to read daily growth increments of damselfish (Chrysiptera glauca) from Mauritius. However, we have yet to find a perfect methodology to make the increments more readable/visible.
For small otoliths the most promising method so far was to
1. Burn otolith (10-20 sec over bunsen burner)
2. store otolith in immersion oil
(Picture 1 and 2)
Other methods used include
1. glue otolith on a glass slide
2. grind otolith down (using 4000 sand paper)
3. polish otolith (using colloidal silica suspension)
4. burn otolith (over bunsen burner for 10-20 sec)
or
4. stain otolith with 0.1% toluidin blue
(Picture 3, rings seem too big)
I would be extremely happy about any suggestions regarding otolith processing to enhance readability.
Regards,
Julian
Hi, I think that, according to pictures, these otoliths are too thick for microscope observation. I recommend to ground and polish them (i.e. 5.5 um, 3 um, 1 um sand papers, and 0.3 um alumina... it depends on the species and age) to properly see the primordium area and then the daily increments will appear. You should make several proofs in order to decide the best plane (medial or distal) to have a well-polished continuous radius.
Hi. Have you tried to embed your otoliths in clear epoxy and then grind/polish on both sides (one side to your otolith core of course)? If your microscope penetrating light can go through your otolith sample without being much scattered or blocked, the ring structure should be more clear on your focal plane. I used to process otoliths of several reef fishes, including damselfish, using this way, and we got very good and clear results.
Based on the provided scale, those otoliths may represent a challenge for epoxy embedding, particularly if you want to cut epoxy with a lowspeed saw. Polishing/grinding should be a better option (with or without epoxy).
You may also consider the use of claryfing liquid. Glycerin could be provide surprising results, sometime better than immersion oil.
Sure thing, as some pointed out, the otolith thickness is still probably not helping you.
Finally, you could try to use to observe otolith using transmitted light with a black background. Sometime, this is offering best results (see the picture).
Thank you, guys! Now, what we actually did was to focus on the Lapilli instead of Sagittae. Otoliths were fixed on glass slides using 2-component adhesive (Araldite 2020/A and Araldite 2020/B) and ground sequentially on glass plates by silicon carbide (SiC) powder with grit sizes of 400 and 800. After polishing with waterproof silicon carbide grinding paper (grain size of 5µm), otoliths in immersion oil were examined with a digital microscope (Keyence VHX-5000) using transmitted light at a magnification of 400x – 900x. With the digital microscope it was possible to do depth stitchings of different focus levels.
(BTW: Scale on the original pictures was not calibrated and therefore wrong)
I think you did great with protocol optimization Julian. Whole-otolith and microstructure clarity are definitely better. Cheers and good luck.
Thanks again for helping me out, guys! Using age readings from lapillus otoliths we discovered that Chrysiptera glauca juveniles grow faster in a tide pool subjected to the influx of fresh acidic groundwater when compared to their conspecifics taken from a strictly marine rock pool.
https://www.frontiersin.org/articles/10.3389/fmars.2019.00613/full
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