In presence of calcium salts makes the tissue hard and brittle which will cause difficulty in section cutting and damage to the microtome knife DECALCIFICATION Selection of tissue Fixation Decalcification ID: 288471
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Slide1
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.
DECALCIFICATIONSlide2Slide3
Selection of tissueFixationDecalcificationDetection of end pointNeutralization
Washing
STEPSSlide4
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.
SELECTION OF TISSUESlide5
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.
FIXATIONSlide6
Different steps of decalcification are By using dilute mineral acid By using ion exchange resins By chelating agent
Electrophoretic decalcification
DECALCIFICATIONSlide7
Complete removal of calcium Minimal tissue damageShouldn't interfere the staining reactionSpeed of decalcification
The factors which influence the speed of decalcification are
Heat
Strength of acid
Agitation
CRITERIA OF GOOD DECALCIFYING AGENTSlide8
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
BY USING DILUTE MINEREAL ACIDSlide9
Conc. HCL-15mlNacl-175gmDistilled water-up to 1lt 0.5 % HCl should be added daily till decalcification is complete.This is a moderately rapid decalcification.
VON-EBNER’S FLUIDSlide10
Absolute alcohol-73mlChloroform-10mlAcetic acid-3mlHCl-4mlDistilled water-10ml
More amount of fluid is needed that is 40-50 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.
JENKER’S FLUIDSlide11
PH-4.57% citric acid monohydrate-5ml7.54% anhydrous ammonium citrate-95ml1% zinc sulphate-2mlChloroform-2 drops
calcium ions are soluble at PH 4.5
This is slower in action but there is no damage to the tissue.
CITRATE-CITRIC ACID BUFFERSlide12
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
FLUID CONTAINING NITRIC ACID Slide13
Concentrated HNO3-5-10mlDistilled water-up to 100ml It is a good protein decalcifying fluidRapid in action but it will cause damage to the tissue
It will give brilliant staining reaction
AQUEOUS NITRIC ACIDSlide14
Formalin-5mlConc. HNO3-7.5mlDistilled water-up to 100ml Formalin prevents the softening effect of nitric acid on the cell
FORMALIN NITRIC ACID Slide15
Conc. HNO3-10 mlPhloroglucin-1gm When bubbling stops at 100 ml of 10% nitric acid to this solutionPhloroglucin protect the tissue from softening and gives brilliant staining effect
PHLOROGLUCIN NITRIC ACIDSlide16
10% HNO3-40mlAbsolute alcohol-30ml0.5% 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
PERENYS FLUIDSlide17
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 reducedThe 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 20-30 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
BY USING ION EXCHANGE RESINSSlide18
These are organic compounds having capacity to bind with calcium metalsTissue decalcified by this method showing minimum of artifacts and good staining results
CHELATING AGENTSSlide19
The fixative used is 10% neutral formal salineAfter 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
PROCEDURESlide20
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
DIFFERENT TYPES OF CHELATING AGENTSSlide21
The tissue is placed in electrophoretic tank containing 2 electrodes and electrolyte solutionsEqual parts of 8% HCL + 10% Formic acid is used as an electrolyte
ELECTROPHORETIC DECALCIFICATIONSlide22
Tissue should be exposed for longer time in decalcifying fluid in which it will cause damage to the tissueSo the end point of decalcification should be determined to prevent tissue damage and to ensure the complete removal of calcium
DETECTION OF END POINTSlide23
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
DIFFERENT METHODS ARESlide24
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
NEUTRALISATION OF ACIDSlide25
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
WASHINGSlide26