 Decalcification is the process of removal of calcium from decalcified tissue and making suitable for section cutting.  In presence of calcium salts makes the tissue hard and brittle, which will cause difficulty in section cutting and damage to the microtome knife.

 Selection of tissue  Fixation  Decalcification  Detection of end point  Neutralization  Washing

 Thin sizes of bone and other calcified hard tissues are obtained by using fine toothed forceps or hack saw.Have to take thickness of tissue at 4-5 mm.

 Adequate fixation should be needed before decalcification otherwise tissue will be damaged in acid decalcification.  Bony tissue is fixed in 10% buffered neutral formalin for 2-4 days,  For bone marrow zenkers formalin is used.

 Different steps of decalcification are  By using dilute mineral acid  By using ion exchange resins  By chelating agent  Electrophoretic decalcification

 Complete removal of calcium  Minimal tissue damage  Shouldn't interfere the staining reaction  Speed of decalcification  The factors which influence the speed of decalcification are  Heat  Strength of acid  Agitation

 The acid which is used for decalcification should be simple solution or mixed with other reagents especially with fixative or buffered solution.  Different type of decalcifying fluid  Gooding and stewarts fluid  Formic acid(90%)-100ml  Formalin-50ml  Distilled water-850ml  By using formic acid gives a good routine decalcifying fluid it will give reasonable speed and minimum tissue damage.  Formaldehyde gives protection to the tissue from acids.  Decalcification by using this solution with in 2-4 days depends on the thickness and degree of decalcification

 Conc. HCL-15ml  Nacl-175gm  Distilled water-up to 1lt  0.5 % HCl should be added daily till decalcification is complete.This is a moderately rapid decalcification.

 Absolute alcohol-73ml  Chloroform-10ml  Acetic acid-3ml  HCl-4ml  Distilled water-10ml  More amount of fluid is needed that is times the volume of tissue  After decalcification the tissue is directly transfer to several changes of absolute alcohol till the acid is removed from the tissue.

 PH-4.5  7% citric acid monohydrate-5ml  7.54% anhydrous ammonium citrate-95ml  1% zinc sulphate-2ml  Chloroform-2 drops  calcium ions are soluble at PH 4.5  This is slower in action but there is no damage to the tissue.

 Nitric acid recause the formation of yellow dis coloration to the tissue and it will interfere with subsequent staining reaction  The formation of yellow dis coloration can be prevented by adding 1% urea to pure nitric acid. But it is having only a temporary effect 

 Concentrated HNO3-5-10ml  Distilled water-up to 100ml  It is a good protein decalcifying fluid  Rapid in action but it will cause damage to the tissue  It will give brilliant staining reaction

 Formalin-5ml  Conc. HNO3-7.5ml  Distilled water-up to 100ml  Formalin prevents the softening effect of nitric acid on the cell

 Conc. HNO3-10 ml  Phloroglucin-1gm  When bubbling stops at 100 ml of 10% nitric acid to this solution  Phloroglucin protect the tissue from softening and gives brilliant staining effect

10% HNO3-40ml Absolute alcohol-30ml 0.5% chromic acid-30ml It’s very slow in action for bones. But excellent for small deposits of calcium It will cause little hard to the tissue The end point detection is by x-ray

 It is use to remove calcium ions from the fluid that will make more rapid rate of solubility of calcium from the tissue and time taken for decalcification can be reduced  The resins commonly used as ammonium forms of suphonated poly styrene resins  It’s layered on the bottom of the container to a depth of a rod 1cm and the specimen is allowed to rest on it  The volume of fluid will be times the bulk of the specimen  Formic acid containing decalcifying fluid will be better results  After use with resins the tissue must be washed twice in diluted HCL and followed by washing in running tap water for 3 times

 These are organic compounds having capacity to bind with calcium metals  Tissue decalcified by this method showing minimum of artifacts and good staining results

 The fixative used is 10% neutral formal saline  After fixation the tissue is transffered to 50 times its bulk of 55% sequestrine buffer of ph 7.4 is prepared in phosphate buffer  The fluid is changed daily for determination of end point  After decalcification, the tissue is transffered to 70% alcohol for dehydration

 HILLEMANN’S AND LEE FLUID  EDTA disodium salts – 5.5 gm  Distilled water – 90ml  Formalin – 10ml  NEUTRAL EDTA  It is a cloudy solution it can be neutralised by adding 2.5 gm of NaOH

 The tissue is placed in electrophoretic tank containing 2 electrodes and electrolyte solutions  Equal parts of 8% HCL + 10% Formic acid is used as an electrolyte

 Tissue should be exposed for longer time in decalcifying fluid in which it will cause damage to the tissue  So the end point of decalcification should be determined to prevent tissue damage and to ensure the complete removal of calcium

 1) PHYSICAL METHOD  It is a crude method consists of probing the tissue with a needle and cutting using a scalpel or should check the flexibility of the tissue  2) CHEMICAL METHOD  5 ml decalcifying fluid is utilized by strong ammonia then add 5ml of ammonium oxalate solution  3) RADIO GRAPHIC METHOD  X-ray

 After decalcification the tissue should be neutralized with treating with alkali overnight.  5% Lithium carbonate or NaSO4 can be used for neutralization  Failure to do the neutralization property that will cause the swelling of the tissue

 Washing is necessary for removal of alkali otherwise that will interfere with the staining reaction  Washing can be done overnight in water or 70% alcohol for 3-5 hrs