DECALCIFICATION

Question 1

The procedure whereby __ are removed from tissues following fixation.
It should be done after fixation and before impregnation to ensure and facilitate the normal cutting of sections.

Answer 1

Decalcification
- calcium or lime salts

Question 2

Decalcification

Answer 2

Acids
Chelating Agents
Ion Exchange
Electrophoresis

Question 3

Purpose of decalcification

Answer 3

To ensure and facilitate the normal cutting of sections
To prevent obscuring the microanatomic detail of sections

Question 4

Most widely used agents for routine decalcification. Stable, easily available and relatively inexpensive

Answer 4

Acid decalcifying agent

Question 5

Acid decalcifying agent

Answer 5

Chromic Acid
Nitric Acid
Hydrochloric acid
Formic acid
Trichloroacetic acid
Sulfurous acid
Citric acid

Question 6

ü Prolonged decalcification may lead to tissue distortion
ü Produces __ and __
ü Seriously damage tissue stainability
ü Imparts __ which will impair
staining reaction

Answer 6

10% Aquaeous Nitric acid Solution
- minimum distortion of tissues and good nuclear staining
- yellow color

Question 7

Decalcification Time __
Most common and fastest decalcifying agent
Rapid in action
easily removed by _

Answer 7

10% Aquaeous Nitric acid Solution
12-24 hours
70% alcohol

Question 8

• Produces less tissue destruction than 10% aqueous nitric acid
• The solution should be used inside the fumehood

Answer 8

Formol- Nitric Acid

Question 9

Decalcification time: __
reco for urgent biopsies
__ is relatively good

Answer 9

Formol- Nitric acid
1-3 days
nuclear staining

Question 10

• _ and __ is good
  ü Maceration is avoided due to the presence of __ and __
  ü Can not be determined by chemical test

Answer 10

Perenyi’s Fluid
- Nuclear and cytoplasmic staining
- chromic acid and alcohol

Question 11

Decalcification Time __
Relatively slow decalcifying agent for dense bones
Recommended for routine purposes
Decalcifies and softens tissue at the same time

Answer 11

Perenyi’s Fluid
2-7 days

Question 12

Decalcification Time __
Most Rapid decalcifying agent so far
Poor Nuclear staining
Recommended for urgent works

Answer 12

Phloroglucin- Nitric acid
12-24 hrs

Question 13

Slower action
Greater distortion of Tissues
ü Inferior to __ in its role as a decalcifying agent
ü Produces ___
ü ___ - recommend for surface decalcification of the tissue block

Answer 13

Hydrochloric acid
- nitric acid
- good nuclear staining
- 1% solution in 70% alcohol

Question 14

Permits relatively_
Does not require washing out before dehydration
Moderately rapid decalcifying agent
Recommended for __ and _

Answer 14

Von Ebner’s Fluid
- good cytologic staining
- teeth and small pieces of bone

Question 15

• Moderate acting decalcifying agent
  ü Safer to handle than __ or __
  ü Recommended for routine decalcification of __
  ü Suitable for routine surgical specimens, when __staining is needed

Answer 15

Formic Acid
- nitric acid or hydrochloric acid
– post mortem research studies
- immuno histochemical

Question 16

Decalcification time __
May be used as a fixative and decalcifying agent
Relatively slow not for urgent works
Recommended for __ and __
It permits _ and _
Requires neutralization with __ and _

Answer 16

Formic Acid
- 2-7 days
- teeth and small pieces of bone
excellent nuclear and cytoplasmic staining
5% sodium sulfate and washing out

Question 17

Decalcification time __
Relatively slow; not recommended for routine purposes
Requires neutralization with __

Answer 17

Formic Acid- Sodium Citrate Solution
- 3-14 days
- 5% sodium sulfate

Question 18

ü Permits better nuclear staining than nitric acid method
ü Recommended for __,__,__ and __ studied for research purposes

Answer 18

Formic Acid- Sodium Citrate Solution
- autopsy materials, bone marrow, cartilage and tissues

Question 19

Decalcification Time __
Very slow-acting, not recommended for urgent works
Permits good nuclear staining
Suitable only for small spicules of bone

Answer 19

Trichloroacetic acid
4-8 days

Question 21

Does not require washing out
Weak decalcifying agent; not used for dense tissues

Answer 21

Trichloroacetic acid

Question 22

Very Weak Decalcifying Agent
Suitable only for minute pieces of bone

Answer 22

Sulfurous acid

Question 23

May be used as a fixative and a decalcifying agent
Used to decalcify minute bone spicules
Nuclear staining with __ is inhibited

Answer 23

Chromic acid ( Flemmings Fluid)
- hematoxylin

Question 24

Decalcification Time___
Too slow action for routine purposes
Permits excellent nuclear and cytoplasmic staining
Does not produce cell or tissue distortion

Answer 24

Citric acid citrate buffer solution
6 days

Question 25

Chelating agent

Answer 25

Edta

Question 26

Cellular detail is well-preserved Daily washing of solution is eliminated Permits excellent statining results The tissue may stay for __ The degree of decalcification may be measured by __ or _

Answer 26

Ion exchange resin - 1-14 days physical or X-ray method

Question 27

ü Ammonium form of polystyrene resin that hastens decalcification by removing calcium ions from formic acid-containing decalcifying solutions. ü A layer (1/2 thick) is spread over the bottom of the container and the specimen is placed on top of it. Then the decalcifying agent is added, usually 20-30X the volume of the tissue.

Answer 27

Ion exchange resin

Question 28

Commercial name: _ Recommended for detailed microscopic studies Very slow Decalcifying Agent Permits excellent staining results

Answer 28

EDTA - Versene

Question 29

Combines with calcium ions and other salts to form weakly dissociated complexes and facilitate removal of calcium salts ü For small specimen, __ ü For dense cortical bone, it will take __to decalcify - is an excellent bone decalcifier for __ , __ and __ ü inactivates alkaline phosphatase activity, which can be restored by adding __

Answer 29

EDTA 1-3 weeks 6-8 weeks - immunohistochemical, enzyme staining and electron microscopy magnesium chloride

Question 30

A process whereby positively charged calcium ions are attracted to a negative electrode and subsequently removed from the decalcifying solution ü D.T. short due to the _ and_ ü Same principles with Chelating agent: ü this process utilizes electricity and is dependent upon a supply of direct current to remove the calcium deposits. ü This method is satisfactory for ___, processing only a limited number of specimens at a time ü Good cytologic and histologic details are not always preserved.

Answer 30

Electrophoresis - heat and electrolytic reaction - small bone fragments

Question 31

factors influencing rate of decalcification

Answer 31

concentration volume time temperature

Question 32

__ and __ will increase the speed of the process

Answer 32

High concentration and greater amount of fluid

Question 33

- _ impaired nuclear staining of_ for collagen fibers ü __ tissue will undergo complete digestion within 24-48 hours ü RT range of __

Answer 33

Temperature 37oC Van Gieson’s stain 55oC 18-30 C

Question 34

Increase in size and consistency of tissue require longer period.

Answer 34

Ratio 20:1 Volume

Question 35

ü __ ideal timefor decalcification ü Dense bone tissue–__

Answer 35

TIME 24-48 hours 14dayslonger

Question 36

Measuring the extent of decalcification

Answer 36

physical or mechanical test xray or radiological method chemical method ( calcium oxalate test)

Question 37

- Done by touching with fingers to determine the consistency of tissue. ü Bending, needling or by use of scalpel if it bends easily that means decalcification is complete. ü __, causes damage and distortion of tissue.

Answer 37

physical or mechanical test Pricking

Question 38

Best method for determining complete decalcification. Not recommended on Tissue fixed in __ . (radio opacity)

Answer 38

xray or radiological method - mercuric chloride

Question 39

Simple, reliable and convenient method for routine purposes. Detect Calcium in the decalcifying solution by precipitation of insoluble __ or __

Answer 39

chemical method ( calcium oxalate test) - calcium hydroxide or calcium oxalates

Question 40

The removal of acid from tissue or neutralized chemical by immersing the specimen either __ or__ for several hours. ü Simply rinse the decalcified specimen with running tap water.

Answer 40

post decalcification saturated lithium carbonate solution or 5-10% aqueous sodium bicarbonate soln

Question 41

How to soften unduly Hard Tissues?

Answer 41

Selected portions are left in the fluid for 12-24 hours and dehydrated in the same or manner Submerge the cut surface of the block in the fluid for 1-2 hours before sectioning, to facilitate easier cutting of tissues

Question 42

may act both as a decalcifying agent and tissue softener

Answer 42

perenyis fluid

Question 43

Washing out and immersion of fixed tissues + 4% Aqueous Phenol Solution for 1-3 days =

Answer 43

- Considerable Tissue Softening - Easier Block Sectioning without producing marked delerious effects & tissue distortion

Question 44

Other substances which may be used as Tissue Softeners:

Answer 44

Molliflex 2% Hydrochloric Acid 1% Hydrocloric Acid in 70% Alcohol

Question 45

good nuclear staining

Answer 45

10% Aquaeous Nitric acid Solution formol-nitric acid hydrochloric acid formic acid- scs trichloroacetic acid

Question 46

good nuclear and cytoplasmic staining

Answer 46

citric acid- cbs formic acid perenyis fluid

Question 47

good cytologic staining

Answer 47

von ebner

Question 48

poor nuclear staining

Answer 48

phloroglucin nitric acid

Question 49

reco for routine purposes

Answer 49

perenyis fluid citirc acid cbs (too slow)

Question 50

cannot be determined by chemical test

Answer 50

perenyis fluid phloroglucin chromc acid- flemming fluid

Question 51

recommended for teeth and small pieces of bone

Answer 51

von ebner formic acid

Question 52

May be used as a fixative and decalcifying agen

Answer 52

formic acid chromic acid flemmings fluid