EDTA endpoint testing, not an easy task

From:Gayle Callis

This is in reply to Barry Rittmans question on chemical test for EDTA.
Yes, there is a test, but it is tedious, and requires acidifying the used
EDTA solution so calcium is released from EDTA molecule, then the basic
chemical test is done to ppt out calcium and visualize the milky ppt. I
resisted using this method because it took too much time. 

I don't bother with chemical test for EDTA,  I use a weight loss/weight
gain originally published as a test for nitric acid decalcification. This
method  is also good for testing EDTA endpoint decalcification, no tedious
chemicals and waiting. I no longer having my beloved Faxitron for xray
testing which is the most sensitive and rapid way to do decalcification
endpoint testing. 

We use 14% EDTA tetrasodium salt, pH 7.6 in distilled water.  The pH is
adjusted with conc. acetic acid, and use 20:1 solution to size of tissue
ratio for decalcification.  No fixative is involved, the bone is totally
fixed with NBF prior to decalcification.  I like high concentrations of
EDTA (more molecules available to chelate calcium) and pH a bit higher
since EDTA decalcifies as a function of pH, lower pH being slower.   A long
chemical discussion, another time and also in Histonet Archives. 

Weight Loss/Weight Gain Decalcification Endpoint Check
1.	Weigh sample (blot excess fluid off, do not weigh in a cassette) in
milligrams, and record weight BEFORE decalcification.  You must weigh in
milligrams.  Use appropriate balance.
2.	Suspend bone in decalcifier of choice -  formic is slower, HCl is
faster.  More than 5% HCl is very harsh, fast.  Nitric acid should not be
more than 5%.  The method works with EDTA. 
3.	Next morning or after several hours, 4,6,8 hrs depending on decalfifier
used, rinse bone with tap water, blot, weigh in milligrams, record weight.
If weight is lost, calcium is still being removed, return to decalcifier,
(preferably fresh, be generous, 20 times decalcifying fluid to size of
bone.   EDTA is more effiecient if made up in larger volumes, then you can
return bone for several days before a change. 
4.	Repeat step 3 until weight begins to increase (a gain which means
calcium is removed and water is being taken on).  At this point, rinse bone
with running tap water for 30 min to 1 hour (overnight causes swelling)
store in NBF if you are waiting for other bones to finish OR in 70% ethanol
(this stops ionization of calcium from bone). 
5.	Process accordingly using vacuum/pressure, no added heat, infiltrate and
embed in Tissue Prep 2 (harder paraffin).  
Avoid overdecalcification or over exposure to acid, if the weight loss is
small, endpoint is near, take bone out of decal, put into NBF and resume
decalcification the next morning or after a weekend/holiday. 
Rinse bone between changes of fresh decal solution, calcium resides on
surface of bone, must be rinsed off, this is important if you do chemical
test, which is probably more sensitive than weight loss/weight gain method. 

When decalcifying many mouse skulls or verterbral columns, I have picked
the largest 2 or 3 samples, weighed them rather than each sample.  Since
sampling is the same for all these bones, a random few gives me ball park
figures that if the big ones are finished, the smaller ones are too.  EDTA
will not do much damage as long as the bone is TOTALLY FIXED.  A bit lazy?
or just more time efficient, I chose the latter. 

So far, perfect morphology and sectioning, but bone gets extended
processing, even larger mouse skulls or vertebrae. I had beautiful myelin
staining with solochrome cyanine R/neutral red. Bone was excellent, and
spinal cord perfect after 7 days in EDTA (my mixture). 


Gayle Callis
Research Histopathology Supervisor
Veterinary Molecular Biology - Marsh Lab
Montana State University - Bozeman
S. 19th and Lincoln St
Bozeman MT 59717-3610

406 994-6367 (lab with voice mail)
406 994-4303 (FAX)

email: gcallis@montana.edu

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