[Histonet] RE: Histonet Digest, Decalcifying sol'n recipe

From:"Jones, Lynne"

The concentrated 37% HCl (more or less 12N) is weight by weight, not weight by volume - I'm too tired to figure out how that affects the math, so I Googled for a recipe.  The folks at CSH are pretty reliable, and having a reference is always nice if people question the math.  Recommended concentrations for formic acid are pretty variable (5-15%) while some folks prefer buffered formic acid to the HCl mixture), but that may be due to misunderstanding the formulation.  No matter the technique, you should test to determine when it is complete (and make sure the samples are fixed properly before you start.)  The timetable may be altered, but the end result should be the same.

Remember - always add concentrated acids to water!

Good luck,



Cold Spring Harb. Protoc.; 2008; doi:10.1101/pdb.rec11329


Decalcifying solution

 100 mL formic acid stock (88%)

 80 mL HCl (concentrated)

820 mL H2O (distilled)



Formic acid

Formic acid HCOOH is highly toxic and extremely destructive to tissue of the mucous membranes, upper respiratory tract, eyes, and skin. It may be harmful by inhalation, ingestion, or skin absorption. Wear appropriate gloves and safety glasses (or face shield) and use in a chemical fume hood.



HCl (Hydrochloric acid, Hydrochloride)

HCl (hydrochloric acid, hydrochloride) is volatile and may be fatal if inhaled, ingested, or absorbed through the skin. It is extremely destructive to mucous membranes, upper respiratory tract, eyes, and skin. Wear appropriate gloves and safety glasses. Use with great care in a chemical fume hood. Wear goggles when handling large quantities.

Also from the 2001 archives:

Gayle Callis 

Sorry to disagree with disagreement. Interesting comments. Plus you can
"overdecalcify' with any of the common acids, even formic - hence necessity
for endpoint test.

I never had problems with swelling using the formic acid/HCl mixture
(Richman, Gelfand Hill, 1947 Arch Path 44:92. and have hundreds whole rat
knee tissue sections to prove it. This was decalcifying method passed on to
me from AFIP orthopedic histopathology aka bone and mineral laboratory
years ago. I saw huge bones from human and animal decalcified with this
mixture at their lab, and they were excellent preparations.  The important
thing is that the bone was COMPLETELY FIXED with NBF before any
decalcification was started and xray tested to completion, and I never left
knees (or other huge bones from bovine, sheep, goat or dog) in decalcifier
overnight, particularly near endpoint.  However, I may have reduced
swelling since I rinse bone briefly, then immerse into either 70% ethanol
or NBF to interupt the decalcification process near endpoint or at end of a
week. A whole rat knee was usually decalcified in 2 days with the
formic/HCl mixture.

I have colleagues who used formic/HCl decalcifier before bone IHC
controlled with testing. They also did not report swelling but did interupt
decalcification near endpoint by reimmersion into NBF. Our technics were
basically identical.

As for immunostaining considerations, some argue that the faster calcium is
removed by strong acids, the less damage there is to tissue antigens with
exception of immunoglobulins. However, endpoint testing is necesary. I
would be more inclined to stay with buffered formic acid.

For mouse feet/ankles, rat feet/ankles, with many tiny bones involved and
in my experience, they actually took longer to decalcify than whole femurs
or tibias.  The skin, toenails, connective tissues, and tight proximity of
the bones seems to slow things down (that was a surprise!) and goes for
fixation too.  It was very evident that endpoint testing on feet/ankles
samples is extremely important (xray saved the day!) or the final sections
were poor, crunchy things.   They also take some extended processing to
have good infiltration of paraffin. The problems occuring with your bone
preparation may be more than decalcification woes, it may be a combination
of factors stemming from fixation, decalcification or processing -
reevaluate carefully what has been done from fresh tissue to finished

This is a At 08:47 AM 5/15/01 -0500, you wrote:
>I prefer to use Kristensen's solution (1948). This is a mixture of
>sodium formate formic acid.
>340 gms sodium formate
>1700 ml. 90% w/v formic acid.
>Distilled water to 10 liters
>can be made up and stored at RT for several months.
>Need to use in a fume hood.
>Need to have specimen well fixed before starting decalcification.
>As Gayle pointed out this c an be used fro some IHC especially if
>decalcifying at cold temperatures.
>For large specimens such as entire dog jaws can use a  much lower
>concentration with approximately 1-2% concentration of formic acid and
>this allows some control. Need to stop maceration when carrying out
>longer term decalcification by refixing in NBF.
>I cannot agree with Gayle re adding hydrochloric acid to a formic acid
>mixture. While this may speed up the decalcification process, it has a
>tendency to swell and as she pointed out to overdecalcify. Hydrochloric
>acid alone has been used for some studies for IHC against some types of
>If you are looking for speed and routine histology can still use 1-5%
>nitric acid with meticulous attention to end point. I prefer using
>X-rays (if they are available to you).
>Barry R. J. Rittman, Ph.D.
>UTHHSC Dental Branch
>6156 John Freeman avenue
>Houston, TX. 77030
>>                 Hello Histonetters!  I promise, no more questions
>> concerning Elmer's Glue!:-)  My next quandry.......what kind of formic
>> acid do you use for bone decalcification?  I have looked in the Sigma
>> catalog and there are many to choose from. Also is the final pH a
>> concern or do you just prepare a 5% solution?  I did consult my
>> histology books but there isn't too much detail.  Thank you for any
>> suggestions!
>> Jennifer Hoover
>> Eli Lilly and Company
Gayle Callis
Histopathology Supervisor
Veterinary Molecular Biology
Montana State University - Bozeman
Bozeman MT 59717-3610

406 994-6367
404 994-4303 (FAX)

-----Original Message-----

Message: 1

Date: Wed, 8 Oct 2008 10:34:15 -0600

From: "Liz Chlipala" 

Subject: RE: [Histonet] Simple (?) equation question

To: "jstaruk" ,




Content-Type: text/plain;       charset="us-ascii"

This is the way I would do it.  Someone can correct me if I'm wrong.  I

consider the HCL to be 100% and the Formic acid to be just 88% at least

that's the way we make up our formic acid for decal here (our stock

formic acid is at 98%).  You just need to use the simple equation V1xC1

= V2xC2 and perform two equations.

Volume 1 - volume of stock reagent - this is what you want to figure out

Volume 2 - volume of diluted reagent 100mls

Concentration 1 - concentration of stock reagent - 88% formic acid ----

100% HCL

Concentration 2 - concentration of diluted reagent - 8% formic acid

----- 2% HCL


X x 100% = 100mls x 2%

X = 2 mls of HCL acid

FOR 8% Formic Acid

X x 88% = 100mls x 8%

X = 9.01 mls of Formic acid

So you would add 2 mls of HCL and 9.01 mls of Formic Acid and 88.99 mls

of dH20 (just subtracted from 100mls the final volume with the 2 amounts

of acid to get 88.99)


Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC


Premier Laboratory, LLC

P.O. Box 18592

Boulder, CO 80308

phone (303) 682-3949

fax (303) 682-9060



Ship to Address:

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Longmont, CO 80504

-----Original Message-----

From: histonet-bounces@lists.utsouthwestern.edu

[mailto:histonet-bounces@lists.utsouthwestern.edu] On Behalf Of jstaruk

Sent: Wednesday, October 08, 2008 10:08 AM

To: histonet@lists.utsouthwestern.edu

Subject: [Histonet] Simple (?) equation question


I want to make a 2% HCl and 8% formic acid decalcifying solution.  Since

straight HCl is 37%, do I treat this as 100% (like you do using

formalin), so the formula will simply be 98 water and 2 HCl or do I have

to fraction it out so a 2% solution will actually be 95 water to 5 HCl?

Also, the same question is for the formic acid, which is actually 88%

My choices to make a 2% HCl and 8% formic acid solutions is either:

A) 2 HCl, 8 formic acid and 90 water


B) 5 HCl, 8.8 formic acid and 86.2 water




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