Methods Of Decalcification | Histology |

What Is Decalcification

Decalcification is a process or Complete removal of calcium salt from the tissues like bone and teeth and other calcified tissues following fixation. Except if the tissues in totally decalcified the segments will be torn and battered and may harm the forefront of the microtome blade.

The steps of decalcification are the following:-

  1. To guarantee satisfactory obsession and complete evacuation of the calcium the cuts must be 4-5 mm thick. Calcified tissue needs 2-3 hours in particular, for complete decalcification to be accomplished so it is important to check the decalcification following 2-3 hours.
  2. Fixative of decision for bone or bone marrow is Zenker formal or Bouin’s liquid. Unfixed tissue will in general be harmed multiple times more prominent during decalcification than an appropriately fixed tissue.

Decalcification is effected by one of the following methods:-

  • Removal of calcium by use of dilute mineral and along with ion exchange resin to
  • keep the decalcifying fluid free of calcium.
  •  Using Chelating agents EDTA.
  • Electrolyticremoval of calcium ions from the tissue by use of electric current.

The criteria of good decalcifying agents area are:-

  • Complete removal of calcium.
  • Subsequent staining was not altered.
  • Short time required for decalcification.

Removal of Calcium by Mineral Acids

Strong acid eg. nitric and hydrochloric acid. Nitric acid 5-10% aqueous solution used. They decalcify very rapidly but if used for longer than 24-48 hrs. cause deterioration stainability especially of the nucleus.

Hydrochloric acid 5-10% aqueous solution decalcification slower than nitric still rapid. Fairly good nuclear staining. Corrosive however Weak corrosive model formic, acidic, and picric corrosive of these formic acids are widely utilized as corrosive decalcifier.5-10% the watery arrangement or with added substances like formalin.

Formic Acid

  • Brings Out fairly rapid decalcification.
  • Nuclear staining in better.
  • In any case, requires balance and exhaustive washing before parchedness.

Aqueous Nitric Acid

Procedure:-

  • Place calcified specimen in large quantities of nitric acid solution decalcification complete.
  • Washing running water for 30 minutes.
  • And magnesium carbonate has been added.
  • Washing running water overnight.

Perenyi’s Fluid

It is slow for decalcifying hard bone but excellent fluid for small deposits of calcium example calcified arteries, coin lesions, and calcified glands. Also good for the human globe contains calcium due to pathological conditions. There is the little hardening of tissue but excellent morphologic detail is preserved.


Formalin Nitric Acid

Nitric acid causes serious deterioration of nuclear stainability which partially inhibited aldehyde. Old nitric corrosive likewise will in general create yellow staining which might be forestalled by adjustment with 1% urea. 

Procedure:-

  • Spot calcified example in enormous amounts of formic corrosive sodium citrate arrangement until decalcification is finished. 
  • Wash in running water for 4-8 hours.
  • Get dried out, clear, and impregnate with paraffin or cycle as wanted. 
  • This procedure gives better-recoloring results then nitric corrosive strategy since Formicidae disodium citrate is less unforgiving on the phone properties. Consequently even with the overexposure of tissue in this arrangement after decalcification has been finished, causes little loss of recoloring characteristics. 
  • This strategy for decision for all orbital decalcification including the globe. 

Surface Decalcification

The outside of the square to be decalcified is managed with a surgical blade. The square is then positioned in a corrosive arrangement at 1%hydrochloric corrosive face downwards so corrosive washes the cut surface for 15-60 min. As infiltration and decalcification are just adequate for a couple of segments to be cut the square will be painstakingly situated in microtome to maintain a strategic distance from wastage of decalcified tissue. 

Decalcification of Bone Marrow Biopsy

Tissue after obsession in Bouin’s or Zenker’s fixative is decalcified for 2hours followed by an hour of washing, The tissue is then dried out start with liquor. 

Use of lon Exchange Resins

lon trade saps in decalcifying liquids are utilized to eliminate calcium particles from the liquid. In this manner guaranteeing a fast pace of dissolvability of calcium from tissue and decrease season of decalcification. The resins an ammoniated salt of sulfonated resin along various concentrations of formic acid are used. After use, the resin may be regenerated by washing twice with dilute N/10 HC followed by three washes in distilled water. Use of lon exchange resin has the advantage of  faster decalcification, tissue preservation and, cellular details better preserved

Chelating Agents

Chelating operators are natural mixes that have the intensity of restricting certain metals. Ethylene diamine-tetra-acetic acid, disodium salt called senate has the power of capturing metallic ions. This is a moderate cycle however has almost no impact on other tissue components. A few catalysts are as yet dynamic after EDTA decalcification.

Electrolytic Method: This depends on the standard of drawing in calcium particles to a negative anode notwithstanding the arrangement. 

Decalcifying Solution

Decalcify with electrolyte mechanical assembly with the previously mentioned decalcifying liquid. 


Neutralization: It has been said that following inundation in mineral acids, tissues ought to be deacidification or balance, before washing by treatment with salt. This may be effected treatment overnight in 5% lithium or sodium sulfate.

Washing:  Through the washing of the tissue before processing is essential to remove acid which would otherwise interfere with staining.

Treatment Of Hard Tissues

Keratin and chitin are relaxed by the utilization of concentrated sulphuric and with that guide of warmth keratin is totally disintegrated from the tissue segments. But much tissue distortion will also occur.

For softening of chitin foll procedure gives a satisfactory result:

  • Fix the specimen in fixative of choice.
  • Change the solution every two days for the best results.
  • Washing running water for 3 hours.
  • Dehydrate, clear and impregnate with paraffin.
Prenyi’s Fluid– Immersing hard tissues in these solutions for 12-24 hours will make sectioning easier and excellent preparation of calcified arteries, thyroid, and calcified glands is possible.

Lendrum’s Technique: It is very useful for tissues which became hard at the time of fixation. The following washing out of the fixative, the tissue is immersed in a 4% aqueous solution of phenol for 1- 3 days.


Wax Blocks: The treatment of wax embedded block of hard tissue may be done by soaking in soap water overnight.
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