1/22/98

I am being asked to fix and section fish otoliths for TEM. Does anyone

have a protocol? Or opinions? For instance do I have to decalcify? If so

is formic acid better than EDTA? How do I determine the end point of

decalcification. Can I use Epon or would another embedding media work

better? How much damage can I expect this to do to my diamond knife? I

did get a few tips from the University of Florida's web site but would

value input from anyone experienced in working with bone. Thanks very

much! Kim

-------------------

Kim DeRuyter

Histology and Electron Microscopy

PO Box 755780

University of Alaska

Fairbanks, Alaska 99775-5780

fnksd1@aurora.alaska.edu

(907)-474-5452

I have been working on ultrastructure and micro-chemistry of fish

otolith for many tears in Hawaii. I have developed a protocol especially

for ultramicrotoming of undecalcified otolith. The following is my method:

1) Double embedding: Pick up a small piece of otolith (<0.5mm),

embede it with Epon resin. Grind (#600 through #1200 sand paper). As soon

as the grinding plane reaches the otolith, stop. Put a drop of Spurr

resin on top and cure it. Then go grind-embed back and forth 3-5 times.

By doing this, the resin around otolith is compressed and stronger support

could be achieved.

2) Sectioning: I used Ultracut E for sectioning and it worked very well.

Use very low cutting speed, low level of water surface, and diamond knife.

Due to the high hardness of otolith, the otolith is acturally not

sectioned, but micro-broken. The enhanced embedding procedure can

prevent otolith from movement and turning, and hold the material of

otolith on wherever it was in otolith. You can see crystal-composed image

under TEM. The daily increment rings also can be seen in details under

x10K. The sections are suitable for EDS and EELS analysis as well as

element filter mapping.

3) Decalcification: Fish otolith is high calcified material with about

90% of inorganic. After decalcification, without support from inorganic

crystalls, the organic residure will be in form of pieces or dissolved. It

can not form a solid structure represent the structure of otolith.

Therefore, young otolith from few-day's larvae may have more organic

materials in otolith. After decalcification, you can see something left,

but it is in the form of gel. It is impossible to go through

fixation-dehydration and embedding procedure. I have never been

succesfull for decalcified otolith. You can decalcify otolith on surface

of sectioned block, then go through the fixation-dehydration and embedding

procedure on top of block. But for otolith research, the information

of micro-chemical components, location and distribution is much more

important than ultrastructure. The number of grow rings and its structure

can be better determined by light microscope with polarized ligh source

and SEM with SEI or BEI image models.

Hope it helps.

******************************************

Zhiyu Wang

Electron Microscope Lab and Imaging Center

Western Kentucky University(WKU)

Bowling Green KY 42101

Phone: (502)745-5993(office)

wangz@hera.wku.edu

For the uninitiated, otolith is a bone in fish's ear, now used to estimate

the age of fish. Otolith have growth rings somewhat like trees).

I doubt that there will be anything left after decalcification. Try it. I

suggest that you use say 10% ascorbic acid in buffer, it's milder but EDTA

certainly will do.

A diamond knife will cut undecalcified otolith better than bone, but use a

"hard" mixture of whatever your favourite embedding medium. Block size

should be very small, but it would be anyway unless you are working on

whales.

Microprobe analysis suggests that the rings are visible because of

differing crystalline conditions as no elemental variations is detectable

across the otolith. Since spatial resolution of X-rays is much better in

TEM, you may be able find elemental variation and certainly you should

obtain greater morphological details then is possible in SEM.

Cheers

Jim Darley

ProSciTech Microscopy PLUS

PO Box 111, Thuringowa QLD 4817 Australia

Phone +61 77 740 370 Fax: +61 77 892 313

Great microscopy catalogue, 500 Links, MSDS, User Notes

************************ http://www.proscitech.com.au

>

>I did my graduate work in Irwin Baird's lab at Penn State's medical school

>in Hershey. We used Na4-EDTA, exclusively. It worked great on every otic

>capsule we worked on. The feeling then was that the formic acid based

>decalcifiers were too vigorous for EM.

>

>Irwin worked on lizards, and each of the graduate students worked on

>different groups. I had birds, Jim White had amphibians, and Dave Jenkins

>had fish. I don't have the citations for Dave's work handy, but it would

>have been in the '70's and on catfish, Corydoras, if I remember right.

>Could have been in Amer. J. Anat.

>

>Irwin published a paper, in '63, I think, with Bill Winborn and Dale

>Bockman on the decalcification procedure. It's worth looking up, if you

>haven't already. Actually, any of Irwin's work is an example of clear and

>thorough, if dry, scientific writing.

>

>If you have trouble finding any of these papers, let me know, and I can

>help either with specific citations or, in some cases, reprints.

>

>John

>chandler@lamar.ColoState.EDU