Re: [Histonet] Razor Clams

From:Gayle Callis

Access this book:  Histological techniques for marine bivalve 
mollusks.  Howard DW , Smith CS. NOAA Technical memorandurm 
NMFS-F/NEC-25.  US Dept of Commerce, National Oceanic and Atmospheric 
Administration, Woods Hole MA, 1983.

I have heard this book has wonderful illustrations and includes techniques.

Another book is:   Harrison FW et al  Microscopic Anatomy of 
Inverterbrates.  Vol 5: Mollusca I and Vol. 6:  Mollusca II.  Wiley-Liss, 
New York NY 1991.

Also, a simple 0.5 to 0.1% Toludine blue stain applied to a polished edge 
may also help you out.  Any grooves caused by a diamond wafer blade can be 
gently sanded away with 600 grit paper, rinsed (ultrasonicator get rid of 
debris), dried and stained.  For better depth of stain on calcium bands, 
simply etch the calcium away for  30 sec to 1 min with 1% formic acid, 
rinse well with water, blot dry and apply toluidine blue stain for 2 min or 
so.  You will have to determine staining time.  IF the stain is too dark, 
polish it off with 600 grit and stain for shorter time and/or more dilute 
stain.  Diamond saws leave ski tracks - finer grit paper polishes saw marks 

This is similar to surface staining methylmethacrylate embedded thick bone 
sections that are acid etched and stained with 0.1% toluidine blue in pH 8 
0.1M phosphate buffer.  One could even take 2 mm thick slices of  shell, 
superglue it to a white plastic slide before polishing, this will stabilize 
section for all other handling - polishing, staining, and 
microscopy.   Etched sections will show darker bands where calcium resided 
before acid etching, so you will have darker and lighter areas.

For  microscopy, sit stained slice on slide (with a 1 1/2 thick coverslip 
on top of slice, and illuminate with the brightest microscope light 
possible (no filters), but make sure a clear glass slide has white paper 
under it to diffuse the light up and around the section rather than light 
passing through the sample.   Coverslip corrects for refractive index so 
you can view at higher power without a fuzzy image.  Viewing at low power 
is generally not a problem and you may be able to just view a stained edge 
at lower power and not mess with a coverslip.  Coverslipping media is not 
needed.   It may take some playing around to get the results you want, but 
T blue is simple and cheap.

Good luck

At 02:48 PM 3/2/2005, you wrote:
>Hello Histonetters,
>I have a researcher that is ageing (aging) Razor Clams and would like to
>better differentiate the conchiolin, a keratin like substance, versus the
>calcium carbonate of the shell.  The growth rings consist of alternating
>bands of calcium carbonate and conchiolin ( as far as I can find out
>conchiolin is keratin "like").  A smooth facet of the shell is produced
>after a diamond saw is used to slice it in two.   The growth bands are in
>the smooth facet but difficult to see.
>The bands in older animals are very close together and very difficult to
>see.  Does anyone have any suggestions re: stains or procedures that might
>help better differentiate the bands?
>Thanks in advance for any help.
>Bill Bennett
>Fisheries and Oceans Canada
>Pacific Biological Station
>Nanaimo, B.C.
>Histonet mailing list

Gayle Callis
Research Histopathology Supervisor
Veterinary Molecular Biology
Montana State University - Bozeman
PO Box 173610
Bozeman MT 59717-3610
406 994-6367 (lab with voice mail)
406 994-4303 (FAX)

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