Decalcification of bone and tooth
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Decalcification of bone and tooth
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Decalcification of bone and tooth
- 1. Presented by, Dr.D.Venkatesh kumar III year PG
- 2. Contents • Introduction • Classification of decalcifying agents • Factors affecting the rate of decalcification • Decalcification end tests • Processing & Staining of decalcified bone • Teeth decalcification • Advanced methods • Precautions • Conclusion • References
- 3. What is decalcification?? “Is the removal of calcium ions from the bone through histological process thereby making the bone flexible and easy for pathological investigation”. Bone Teeth Hard tissues
- 4. BONE
- 5. lamellar bone Woven bone Features Mature bone Woven bone Orientation of collagen fibres Collagen fibres in one lamella lies at right angles to other lamella Collagen fibres oriented in different directions Interfibrillar space Space less Space is more Deposition and mineralization Slow than woven bone,osteocytes lesser in number Faster, more number of osteocytes are present H & E staining Eosinophilic Basophilic
- 6. Choice of decalcifier Urgency of the case. Degree of mineraliz ation Extent of the investigat ion Staining technique Be fast Be goo Do goo Ideal decalcifying agent
- 7. Criteria for good decalcifying agent Complete removal of calcium Minimal damage to cells and tissue Non impairment of subsequent Reasonable speed
- 8. Classification Decalcifying agents Acids Strong inorganic – nitric acid, HCLWeak organic- formic, acetic, picric acid Chelating agents EDTA
- 9. Others • Perenys fluid • Formalin nitric acid • Gooding stewarts fluid • Trichloroacetic acid • Ebners fluid • Jenkins fluid • Ion exchange resins • Electrolytic decalcification • Ultrasonic decalcification
- 10. Nitric acid & HCL • Used as simple aqueous solutions - conc of 5-10%. • Decalcify rapidly. • Routinely used • Permit rapid diagnosis. Nitric acid- 5- 10ml Distilled water to -100ml
- 11. Disadvantages of nitric acid • Nitrous oxide - yellow color – may interfere with subsequent staining. • Addition of 0.1% urea obviates this color. • Tissue left for long time – damage. • Old nitric acid is particularly damaging & should be replaced with fresh stock.
- 12. Perenyi’s fluid Nitric acid containing….. Decalcification time – slower than nitric acid. Small tissue samples – which are not densely calcified Preserves cellular details & staining - good. often used as tissue softener Decalcified tissues can be directly placed in 70% alcohol. Disposing along with Hgcl2 -Ethanol + mercuric chloride + chromic acid =
- 13. Disadvantage • Routine chemical test cannot be performed to test end point of decalcification. • As precipitate is formed when ammonia is added to the fluid
- 14. Weak organic- Formic acid • Ideal - post-mortem & research specimen • Also used when IHC staining is needed. • Excellent staining results. • Most widely used decalcifying fluid. • It is gentle and slower.
- 15. • Due to its slow action - not suitable – rapid diagnosis. • At conc ›8% action may be rapid but cloudiness produced - interferes with chemical test. • X ray & chemical test are used to test for end point Formic acid (90%) – 100ml Formalin – 50ml Distilled water – 850ml
- 16. Ethylene diamine tetraacetic acid (EDTA) • Binds metallic ions - calcium & magnesium. • EDTA will not bind to calcium below PH 3 and EDTA, disodium salt – 5.5g Distilled water – 90ml 40% Formaldehyde – 10ml Hillema n and Lee (1953)
- 17. Mechanism - very slow process. does not damage tissue staining. Minimizes formation of histological artefacts. Dense cortical bone - 6-8weeks or longer. For small bone spicule – less than a week. Tissues decalcified - should not be placed - 70% alcohol Water rinse after decalcification or overnight storage in formal saline, NBF, or PBS
- 18. Others.... Ebners fluid : Ideal for teeth. Fairly rapid action. Decalcified tissue – directly transferred to 90% alcohol. Nuclear staining – slightly affected.
- 19. Jenkins fluid • Swelling effect of HCL counteracted by shrinkage effect of alcohol. • Human rib cross section are decalcified in 4-6 days Absolute alcohol – 73ml Distilled water – 10 ml Chloroform – 10 ml Glacial acetic acid – 3ml HCL – 4ml
- 20. Ion exchange resins Principle: • Calcium ions are removed from solution by resins which increases rate of solubility of Ca from the tissue. • Resin – ammonium form of sulphonated polystyrene • Ammonium ions from the resins are exchanged for the calcium ions, keeping the solutions free from calcium ions and speeding up the reactionResins: Cross-linked polystyrene Cross-linked polymethacrylate Phenol-formaldehyde
- 21. Technique Layer of resin about 13mm thick is spread over bottom of the vessel Specimen is allowed to rest on it Decalcifying fluid about 20 times volume of resin added. X ray method is used to test end point
- 22. Advantages • Well preserved. • Faster decalcification. • Elimination of the daily solution change. • Reclaimed for further use. • Excellent staining
- 23. Electrolytic decalcification Principle : Based on attraction of positively charged calcium ions to a negatively charged electrode. • Calcified tissue is placed - electrolytic solution (5% HCL & 5- 10% formic acid in equal parts) • Temperature is raised during electrolysis. • charring of the tissue & results in distortion - poor nuclear staining. • Therefore this method is not recommended
- 24. Ultrasonic decalcification • Ultrasonic waves - ultrasonic generator - metal jacket. This is known as ultrasonic bath. • Fluid -7.5% glacial acetic acid is placed in bath. • Faster than conventional acid treated controls. • Distortion and staining interference is minimal. Thorp e, bellam y & sharp.
- 25. Factors influencing the rate of decalcification Factors concentrat ion Temper ture Agitatio n Suspen on
- 27. Physical methods • Probing, needling, slicing, bending or squeezing. • Inaccurate and damages tissues - creates artefacts. E.g. 1. Needle tracks 2. False positive microfractures of fine trabeculae – a potential misdiagnosis.
- 28. Chemical methods • Most favourable, simple, reliable. • Depends on dissolved calcium in the decalcifying fluid. Calcium Oxalate test: (Clayden 1952) • Detection of calcium in acid solutions by precipitation of insoluble calcium hydroxide or calcium oxalate.
- 29. Method Allow solution to stand for 30min. Add 5ml of saturated ammonium oxalate & shake well. Add ammonium hydroxide drop by drop, Shaking after drop, until litmus indicates solution neutral. Add a piece of litmus paper Take 5ml of decalcifying fluid. Solutions: Ammonium hydroxide, Saturated aqueous ammonium oxalate.
- 30. Bubble test • Tiny bubbles indicate – less calcium is present. • It is subjective and unreliable. Acids calcium carbon te carbo n dioxid e
- 31. Radiography • Most sensitive test - detecting calcium in bone. • FAXITRON with a manual exposure setting of approx 1minute, 30kv, and Kodak X-OMAT X-ray film is used. • Possible to expose several specimens at same time.
- 32. Procedure Rinse acid from sample, carefully place specimen on waterproof polyethylene sheet on top of the X-ray film, expose to directions, and leave until film is developed and examined calcifications.
- 33. Metal dust particles Spicules of metal, metallic paint, or glass from saw blades forced deep into tissue by a traumatic injury. radio-opaque, sharply delineated fragments that never change in size, unaffected by decalcification, sharply delineated but cannot be removed without damaging tissue - appearing as gray specks on the bone surface and can be easily removed. care must be taken during microtomy to not damage the knife.
- 34. Treatment after decalcification • Acids - removed from tissues or neutralized. • Chemical neutralization - saturated Lico3 or 5–10% aqu NaHco3 solution for several hours. • Few authors - simply rinse -running tap water. • washing in two changes of 70% alcohol for 12–18 hours - to avoid contamination of dehydration solvents.
- 35. Surface decalcification • when partially decalcified bone or unsuspected mineral deposits in soft tissue - during paraffin sectioning. • Done to prevent knife damage & torn tissue sections.The exposed tissue surface in a paraffin block is placed. Face in 1% 10% formic or proprietar y acid solutions. Time: 15- 60minute . Rinsed to remove corrosive acids, and Resection d. After finding calcification,
- 36. Masson’s trichrome stain PAS Silver impregnation Schmorl’s picro-thionin Weigert – van gaeson H & E Frost's basic fuchsin stain Villanueva's bone stain Villanueva's tetrachrome bone stain Modified Villanueva-Goldner trichrome A modification of Movat's pentachrome stain Gordon and Sweet's method for cement lines Phosphotungstic acid hematoxylin UNDECALCIFIED BONE STAINS DECALCIFIED BONE STAINS Staining methods
- 37. Staining methods H&E: • No modification - properly decalcified tissues. • The routine haematoxylin staining time - doubled. • The acid differentiation step – shortened. • Bluing solutions should be mild bases.
- 38. VON KOSSA'S METHOD: PURPOSE: Abnormal deposits of calcium PRINCIPLE: Tissue sections are treated with silver nitrate solution, the reduced by the strong light and with silver deposits, visualized as silver. PROCEDURE: Dehydrate, clear and mount counterstain Wash well in distilled water Treat with sodium thiosulfate – 5min Wash in 3 changes of distilled water Siver nitrate and expose to strong light - 10-60 min Deparaffinize and hydrate to distilled water. SOLUTION: 1% aqueous Silver nitrate 2.5% sodium thiosulfate 1% saffranin O or van gieson picro fuchsin
- 39. RESULTS • Mineralized bone -black • Osteoid -red • Calcium salts -black • Nuclei -red • Cytoplasm -pink
- 40. Artefacts Factor – heat Physical test Under decalcification Over decalcification.
- 41. Teeth decalcification • Teeth - same treatment as bone prior to sectioning. • After fixation ,decalcification, processing , embed in paraffin, or cellodin to produce thin sections. Fixation • Neutral buffered formalin – choice of fixative • Adult teeth – 4days fixation • Younger teeth – 24hours
- 42. Decalcification: Enamel is almost impossible to preserve. Processing: Since the tooth consist mainly of very dense material, • processing times - extended similar to those used in bone methods.
- 43. Study Neutral EDTA - most considerate & 5% nitric acid - least considerate to the tooth structure. Sanjai K,et al; Evaluation and comparison of decalcification agents on the human teeth. Jomfp.2012;16(2). EDTA 5% nitric acid, formali n– nitric a cid, 5% trichlor acetic acid, 10% formic acid Perenyi 's fluid,
- 44. • Application of microwave energy in histotechnology. • Idea of using microwaves to decrease the time for decalcification of temporal bones - rat cochleas Maye rs (1970 ). Advanced methods Microwave decalcification Hard tissues - decalcifying agent - microwave oven - intermittent periods - regular changes of the solution. Decalcification significantly–from days to hours The temperature restriction between 42-45°C for best results 5% Formic Acid Solution
- 45. Procedure: • Fix for standard times in 10% NB Formalin • Bone biopsies - microwave-safe container with 5% Formic Acid • Microwave for 10 minutes at 55ºC • Repeat this procedure until desired softness is achieved • Rinse in running tap water for at least 10 minutes • Proceed with processing.
- 46. Precautions Acids - handled carefully as concentrated acid is hazardous. Nitric acid: Corrosive to skin mucous most metals, toxic by inhalation. Wear apron, gloves and goggles for handling however small the quantity.
- 47. CONCLUSION • Techniques for the demonstration of bone and its components are possibly more varied and difficult than for any other tissue. • For proper diagnosis of bone lesions there should be sufficient knowledge of decalcification procedure and decalcification agents to choose perfect decalcification agent for faster decalcification, good staining and lesser tissue damage.
- 48. References 1. Bancroft JD, Gamble M: Theory and practice of histological techniques: Churchill Livingstone Elsevier: 6th edition. 2. Culling C F A, Allison R T, Barr W T: Handbook of Histo pathological and histochemical techniques. Butterworth & Co Ltd: 4th edition 3. Praful B. Godkar;Text book of medical laboratory technology – 2nd edition. 4. Antonio Nanci ;Tencates Oral Histology –8th edition 5. Medical laboratory science – J ochei , A kolhatkar. 6. Sanjai K et al. Evaluation and comparison of decalcification agents on the human
- 49. 7. Sung-Eun Choi etal “Proposal of an Appropriate Decalcification Method of Bone Marrow Biopsy Specimens in the Era of Expanding Genetic Molecular Study” J Pathol Transl Med. 2015 May; 49(3): 236–242. 8. Rastogi V et al. Artefacts A Diagnostic dilemma – A Review. Journal of Clinical and Diagnostic Research. 2013;7(10): 2408-13. 9. Kapila SN et al. Driving the Mineral out Faster: Simple Modifications of the Decalcification Technique. J Clin Diagn Res. 2015 Sep; 9(9): ZC93–ZC97
Editor's Notes
- What is decalcification Is the removal of calcium ions from the bone through histological process thereby making the bone flexible and easy for pathological investigation”. Without the removal of calcium microtomy is virtually impossible using standard microtomy procedures.
- Bone tissue - dense mineralized connective tissue. Bone is highly vascular, living, constantly changing, mineralized chief supporting tissue of the body.
- Two types of bone can be identified microscopically
- Choice of decalcifier is influenced by 4 interdependent factors Urgency of the case Degree of mineralization Extent of the investigation Staining technique- Ideal decalcifying agent should be 1. Be fast 2. Be good 3. Do good
- These may be used as simple aqueous solutions with recommended concentrations of 5-10%. They decalcify rapidly. Permit rapid diagnosis, Aqueous nitric acid, 5-10% Nitric acid – 5-10ml Distilled water to – 100ml easy to purchase and use
- Disadvantages of nitric acid Due to the formation of nitrous oxide fluid usually imparts yellow color to the tissue which affects the staining reaction. Addition of 0.1% urea obviates this color. Tissue left for long time – damage the tissue. Old nitric acid is particularly damaging & should be replaced with fresh stock. Advantages Rapid in action Gives better nuclear staining Causes very little hydrolysis
- Perenyi’s fluid Slow than nitric acid Decalcification time- 2-10 days. Its Popular Especially For Small Specimens That Are Not Densely Decalcifed Preserves cellular details & subsequent staining good. More often used as tissue softener before dehydration. Decalcified tissues can be directly placed in 70% alcohol. When disposing of Perenyi's fluid: Never dispose of Perenyi’s fluid into a waste system containing mercuric chloride. Because ethanol, mercuric chloride and chromic acid can cause spontaneous combustion.
- Disadvantage Routine chemical test cannot be performed to test end point of decalcification. As precipitate is formed when ammonia is added to the fluid
- Weak organic acids Formic acid, acetic acid, picric acid – of these formic is the only weak acid used extensively as primary decalcifier. Picric acid, acetic acid- causes tissue swelling, not used alone as decalcifier, used in fixatives. Ideal - post-mortem & research specimen Suitable for most routine surgical specimens particularly when IHC staining is needed. Excellent staining results. Most widely used decalcifying fluid. Formic acid is gentler and slower.
- Due to its slow action - not suitable – rapid diagnosis. At conc ›8% action may be rapid but resultant cloudiness produced interferes with the chemical test for the end point. X ray & chemical test are used to test for end point . Gooding & stewarts fluid – also used for routine purpose. Advantgae – reasonable speed & min damage to the tissue can be obtained by use of formic acid
- Chelation is a type of bonding of ions and molecules to metal ions. Chelating agents – organic compounds which have the power of binding certain metals. EDTA-Most commonly used chelating agent for decalcification. Hilleman and Lee (1953) was the first to describe EDTA as decalcifying agent. EDTA sometimes called as sequestrene or versene. Although EDTA is nominally acid, it does not act like acids But binds metallic ions, notably calcium & magnesium. EDTA will not bind to calcium below PH 3 and Faster at PH 7-7.4; optimal binding, PH 8 and above damages alkali sensitive protein linkages
- It is very slow process & does not damage tissue staining EDTA binds to ionized calcium on the outside of the apatite crystal and as this layer becomes depleted more calcium ions; the crystal becomes progressively small during decalcification. Less artefacts. The time required totally decalcify dense cortical bone - 6-8weeks or longer For small bone spicule – less than a week. Tissues decalcified in EDTA solutions should not be placed directly into 70% alcohol, as this causes residual EDTA to precipitate in the alcohol and within the tissue. The precipitate does not appear to affect tissue staining since EDTA is washed out during these procedures, but may be noticeable during microtomy. A water rinse after decalcification or overnight storage in formal saline, NBF, or PBS should prevent this.
- Ebners fluid : Ideal for teeth. Fairly rapid action. Decalcified tissue – directly transferred to 90% alcohol. Nuclear staining – slightly affected.
- Jenkins fluid Quantity of fluid – 40 to 50 times bulk of the tissue. Swelling effect of HCL counteracted by shrinkage effect of alcohol. Human rib cross section are decalcified in 4-6 days
- Ion exchange resins Incorporation of ion exchange resins add little or no improvement to staining results. Principle: Calcium ions are removed from solution by resins which increases rate of solubility of Ca from the tissue. Resin – commonly an ammonium form of a sulphonated polystyrene resin Ammonium ions from the resins are exchanged for the calcium ions, keeping the solutions free from calcium ions and speeding up the reaction Resins: Cross-linked polystyrene Cross-linked polymethacrylate Phenol-formaldehyde
- Technique
- Advantages : Well preserved. Faster decalcification. Elimination of the daily solution change. Reclaimed for further use. Staining is excellent with this method Disadvantage: Slower then hydrochloric or other strong acids
- Electrolytic decalcification Principle; Based on attraction of positively charged calcium ions to a negatively charged electrode. Brass plate – negative electrode , platinum wire – positive electrode. Positive electrode wind around specimen. Calcified tissue is placed in a electrolytic solution composed of 5% HCL & 5-10% formic acid in equal parts. Speed of decalcification is increased by moving electrodes together and decreased by moving away. Temperature is raised during electrolysis & may or may not enhance the rate of decalcification. Rise in temp may also bring about charring of the tissue & definitely results in distortion & poor nuclear staining. Therefore this method is not recommended.
- Ultrasonic decalcification It was 1st described by Thorpe, bellamy and sharp. Ultrasonic waves are produced from an ultrasonic generator which is housed in a metal jacket. This is known as ultrasonic bath. Fluid consisting of 7.5% glacial acetic acid is placed in bath & then bone is placed in it. Faster than conventional acid treated controls. Distortion and staining interference is minimal.
- Factors influencing the rate of decalcification Concentration of decalcifying agent More concentrated acid solutions decalcify bone more rapidly but are more harmful to the tissue. Temperature Increased temperature accelerates decalcification, but also increases the damaging effects. 18-30 degrees centigrade is acceptable. Agitation Effect controversial. General acceptance – agitation influences fluid exchange within as well as around tissues with other reagents. Suspension The decalcifying fluid should be able to make contact with all surfaces of a specimen. Flat bone slabs should not touch the bottom of a container as this is enough to prevent by suspended the samples in the fluid with a thread or placed inside cloth bags tied with thread.
- Decalcification endpoint test Accurate determination of the end point of decalcification is therefore necessary. Specimen Radiography (the most accurate way) Chemical testing (accurate) Physical testing (less accurate and potentially damage of specimen)
- Physical methods Probing, needling, slicing, bending or squeezing. These are inaccurate and damages tissues and creates artifacts. E.g. 1. Needle tracks 2. False positive microfractures of fine trabeculae – a potential misdiagnosis.
- Chemical methods Most favourable, simple, reliable. Depends on dissolved calcium in the decalcifying fluid. Calcium Oxalate test: It involves detection of calcium in acid solutions by precipitation of insoluble calcium hydroxide or calcium oxalate.
- Solutions: Ammonium hydroxide, concentrated, Saturated aqueous ammonium oxalate. Method & Result Take 5ml of decalcifying fluid. Add a piece of litmus paper Add ammonium hydroxide drop by drop, Shaking after each drop, until litmus indicates solution neutral. If a white precipitate (calcium hydroxide) forms immediately after adding the ammonium hydroxide, a large quantity of calcium is present making it unnecessary to proceed further to step 3. If step 2 is negative or clear after adding ammonium hydroxide, then proceed to step 3 to add ammonium oxalate. If precipitation occurs after adding the ammonium oxalate, less calcium is present. When a smaller amount of calcium is present, it takes longer to form precipitate in the fluid, so, if the fluid remains clear after 30minutes, it is safe to assume decalcification is complete.
- Bubble test Acids react with calcium carbonate in bone to produce carbon dioxide, seen as a layer of bubbles on the bone surface. Tiny bubbles indicate – less calcium is present. It is subjective and unreliable.
- Radiography This is the most sensitive test for detecting calcium in bone or tissue calcification. In this FAXITRON with a manual exposure setting of approximately 1minute, 30kv, and Kodak X-OMAT X-ray film is used. Possible to expose several specimens at same time.
- Procedure This method is to rinse acid from sample, carefully place identified bones on waterproof polyethylene sheet on top of the X-ray film, expose according to directions, and leave bones in place until film is developed and examined for calcifications. Bones with irregular shapes and variable thickness can occasionally mislead workers on interpretation of results. This problem is resolved by comparing the test radiograph to the pre-decalcification specimen radiograph and correlate suspected calcified areas with specimen variation.
- Metal dust particles from saw blades are radio-opaque, sharply delineated fragments that never change in size. These are unaffected by decalcification, appearing as gray specks on the bone surface and can be easily removed. Spicules of metal, metallic paint, or glass forced deep into tissue by a traumatic injury are also sharply delineated but cannot be removed without damaging tissue. Radiography only indicates the presence of deeper foreign objects and care must be taken during microtomy to not damage the knife.
- Treatment after decalcification Acids can be removed from tissues or neutralized chemically after decalcification is complete. Chemical neutralization is accomplished by immersing decalcified bone into either saturated lithium carbonate solution or 5–10% aqueous sodium bicarbonate solution for several hours. Few authors suggest simply rinse the specimens with running tap water for a period of time. Culling (1974) recommended washing in two changes of 70% alcohol for 12–18 hours before continuing with dehydration in processing, a way to avoid contamination of dehydration solvents even though the dehydration process would remove the acid along with the water.
- Surface decalcification when partially decalcified bone or unsuspected mineral deposits in soft tissue are found during paraffin sectioning. Done to prevent knife damage and torn tissue sections. After finding calcification, The exposed tissue surface in a paraffin block is placed. Face down in 1% HCL, 10% formic or proprietary acid solutions. Time: 15-60minutes. Rinsed to remove corrosive acids, and Resectioned.
- Staining methods 1. H&E No modification to standard H&E for staining of properly decalcified tissues. The routine haematoxylin staining time can be doubled as nucleic acids are intolerant to acid decal The acid differentiation step after hematoxylin can be shortened. ( acid dyes eosin tend to stain more) Bluing solutions should be mild bases.
- VON KOSSA'S METHOD: PURPOSE: Abnormal deposits of calcium PRINCIPLE: Tissue sections are treated with silver nitrate solution, the calcium is reduced by the strong light and replaced with silver deposits, visualized as metallic silver.
- RESULTS Mineralized bone -black Osteoid -red Calcium salts -black Nuclei -red Cytoplasm -pink
- Artefacts Under decalcification • Inability to section • Incomplete infiltration of paraffin • Staining characteristics • Remedy- surface decal, redecal Over decalcification • Nuclear detail lost • Swelling of tissue, especially collagen
- Teeth decalcification Teeth generally receive the same treatment as bone prior to sectioning. After fixation and decalcification, processing and embedment in either paraffin, or cellodin, can be used to produce thin sections. Fixation Neutral buffered formalin – choice of fixative Adult teeth – 4days fixation Younger teeth – 24hours
- Decalcification: Enamel is almost impossible to preserve. Radiography is ideal for progress and end point testing of decalcification Processing- Since tooth consists mainly of very dense material, processing methods should be extended similar to bone methods.
- Sanjai K,et al; Evaluation and comparison of decalcification agents on the human teeth. Jomfp.2012;16(2). study was done to evaluate the rate of decalcification of six different decalcifying agents and also their effect on staining characteristics on dental hard tissues. namely, neutral ethylene diamine tetra acetic acid (EDTA) decalcifying solution, 5% nitric acid, Perenyi's fluid, formalin–nitric acid, 5% trichloracetic acid, 10% formic acid Neutral EDTA was the most considerate to the soft and hard tissues and 5% nitric acid was the least considerate to the tooth structure.
- Advanced methods Microwave decalcification The potential application of microwave energy in histotechnology was first recognized by Mayers (1970). The idea of using microwaves to decrease the time for decalcification of temporal bones was originally introduced by Hellstrom and Nilsson (1992) for rat cochleas. Microwave decalcification is a novel technique compared to the manual method. In this method, hard tissues are placed in the decalcifying agent in a microwave oven for intermittent periods with regular changes of the solution till the end point is reached. Microwave irradiation has been shown to speed up the process of decalcification significantly–from days to hours. The temperature restriction between 42-45°C for best results 5% Formic Acid Solution
- Procedure: Keep bone biopsies as thin as possible Fix for standard times in 10% NB Formalin Place the bone biopsies in a microwave-safe container with 5% Formic Acid Solution Microwave for 10 minutes at 55ºC Repeat this procedure until desired softness is achieved Rinse in running tap water for at least 10 minutes Proceed with either conventional or microwave processing.
- Precautions Acids used to make decalcifying solutions should be handled carefully, concentrated acid is very hazardous. Nitric acid: Corrosive to skin mucous membranes and most metals, toxic by inhalation. Wear apron, gloves and goggles for handling however small the quantity.
- CONCLUSION Techniques for the demonstration of bone and its components are possibly more varied and difficult than for any other tissue. For proper diagnosis of bone lesions there should be sufficient knowledge of decalcification procedure and decalcification agents to choose perfect decalcification agent for faster decalcification, good staining and lesser tissue damage.