DECALCIFYING & CLEARING OF BONES

Question 1

Define decalcification.

Answer 1

Decalcification is a routine procedure done with the purpose of making a calcified bone tissue compatible with embedding media to cut micro slides and subsequent staining

Question 2

Why is decalcification necessary?

Answer 2

To section hard mineralized tissue decalcification is necessary.

Question 3

Define bone biopsy.

Answer 3

Bone biopsy is a procedure where by a small sample of the bone is taken from the body and looked under the microscope for cancer, infection and other bone disorders

Question 4

Describe calcium and state what it does.

Answer 4

Calcium is an inorganic mineral stored in bones. It hardens the bone structure by forming crystal deposits around the fibrous element

Question 5

What needs to happen to be able to study the histological structure?

Answer 5

To study the histological structure, the tissue should be properly prepared for microscopic examination. Tissue specimen must be thin enough to permit passage of light

Question 6

Criteria for good decalcification:

Answer 6

 Complete removal of calcium
 Absence of damage to tissue cells or fibers
 Non impairment of subsequent staining technique
 Reasonable speed of decalcification

Question 7

Factors influencing the rate of decalcification

Concentration of decalcifying agent and temperature:

Answer 7

 Concentration of decalcifying agent – Increased volume of decalcifier
increases calcium removal

 Temperature – Increased temperature speeds up decalcification

Question 8

Factors influencing the rate of decalcification:

Answer 8

 Agitation – Gentle agitation may increase the rate slightly by influencing the fluid exchange within as well as around tissues

 Fluid renewal – Suspension in fresh decalcifier enhances diffusion and penetration into the specimen increasing ionization and removal of
calcium

Question 9

Electrophoretic decalcification:

Advantage & Disadvantage:

Answer 9

 Attraction of calcium ions to a negative electrode in addition to the solution of the
calcium in the electrolyte.
 Advantage : Shorter decalcifying time and better preservation of soft tissue detail
 Disadvantage : Limited number of specimens processed at a time.

Question 10

Electrophoretic decalcification

Materials

Answer 10

A glass containing acid decalcifying solution in which is the electrode assembly and bone
specimen, bone specimen is suspended by a platinum wire anode in a jar

Question 11

Decalcifying solution components:

Answer 11

 88% Formic acid 100ml
 Hydrochloric acid 80ml
 Distilled water 820ml

Question 12

Electrophoretic decalcification

Temperature:

Answer 12

Temperature of the reaction is between 30 – 40 degrees Celsius

Question 13

Electrophoretic decalcification

Last 3 steps:

Answer 13

 Solution changed after 8 hours of use to ensue max. speed of decalcification

 Tissues are rinsed well in alkaline water and immersed in lithium carbonate before staining

 Lithium carbonate treatment of a cut section will neutralize any remaining acid in the
tissue

Question 14

Microtomy

Materials:

Answer 14

Base sledge microtome and wedge shaped steel or tungsten carbide
edged knife is used.

Question 15

Microtomy steps:

Answer 15

 Optimal section thickness is 4 up to 7μm. Bone marrow biopsies should be cut at 2 - 3 for marrow cell identification

 The floating water bath may need to be hotter for bones than for soft tissue as bone has the tendency to crinkle when cut

 Lift onto the chrome-gelatin coated slide

Question 16

Staining methods:

Answer 16

H & E stain compact bone

Silver stain compact bone