DECALCIFICATION

Question 1

A procedure whereby calcium or lime salts are removed from tissues (most especially bones and teeth) following fixation

Answer 1

Decalcification

Question 2

This is done after FIXATION and before
IMPREGNATION, to ensure and facilitate the normal cutting
of sections and to prevent obscuring the microanatomic
detail of such sections by bone dust and other cellular
debris

Answer 2

Decalcification

Question 3

When the small calcified areas of paraffin-embedded tissue blocks cause resistance and a rough feeling when cutting sections with a microtome knife, what should you do?

Answer 3

The block can be temporarily removed and soaked face down in a 10% hydrochloric acid solution on a cotton or gauze pad for about an hour.

Question 4

This produces a deep, uniform red stain without shading differences

Answer 4

Eosin stain

Question 5

A good decalcifying agent must be capable of

Answer 5

Removing calcium salts from tissues completely without producing considerable destruction of cells and tissue components and without adversely affecting the staining capacity of the cell, particularly the nucleus.

Question 6

Calcium may be removed by any one of the following agents:

Answer 6

1. Acids
2. Chelating agents
3. lon exchange resins
4. Electrical ionization (electrophoresis)

Question 7

This is the most common and the fastest decalcifying agent used so far, utilized both as a simple solution or combined with other reagents.

Answer 7

Nitric acid

Question 8

Enumerate the decalcifying agents under nitric acid

Answer 8

• Aqueous Nitric Add Solution 10%
• Formol-Nitric Acid
• Perenyi’s Fluid
• Phloroglucin-Nitric Acid

Question 9

This is inferior compared to nitric acid in its role as a decalcifying agent because of its slower action and greater distortion of tissue produced on the section decalcified

Answer 9

Hydrochloric acid

Question 10

It produce good nuclear staining and if used in 1% solution with 70% alcohol, may be recommended for surface decalcification of the tissue blocks.

Answer 10

Hydrochloric acid

Question 11

The decalcifying agent under Hydrochloric acid

Answer 11

Von Ebner’s fluid

Question 12

A moderate-acting decalcifying agent which produces better nuclear staining with less tissue distortion, and is safer to handle than nitric acid or hydrochloric acid

Answer 12

Formic acid

Question 13

It is recommended for routine decalcification of postmortem research tissues, although not suitable for urgent examinations. It is the only weak acid used extensively as a primary decalcitying agent.

Answer 13

Formic acid

Question 14

The decalcifying agent under Formic acid

Answer 14

Formic Acid-Sodium Citrate Solution

Question 15

Permits good nuclear staining. It does not require washing out; the excess acid may be removed by several changes of 90% alcohol, thus improving tissue dehydration.

Answer 15

Trichloroacetic acid

Question 16

A very weak decalcifying solution suitable only for minute pieces of bone.

Answer 16

Sulfurous acid

Question 17

It may be used both as a fixative and decalcifying agent.
It may be used for decalcifying minute bone spicules.

Answer 17

Chromic acid (flemming’s fluid)

Question 18

It permits excellent nuclear and cytoplasmic staining. It
does not produce cell or tissue distortion.

Answer 18

Citric acid-Citrate Buffer solution (pH 4.5)

Question 19

Enumerate the mineral acids that may be used for decalcification

Answer 19

I. Nitric acid
II. Hydrochloric acid
III. Formic acid
IV. Trichloroacetic acid
V. Sulfurous acid
VI. Chromic acid
VII. Citric acid

Question 20

Substances which combine with calcium ions and other salts (e.g. iron and magnesium deposits) to form weakly dissociated complexes and facilitate removal of calcium salt.

Answer 20

Chelating agents

Question 21

The most common chelating agent in the market is?

Answer 21

ethylene diamine tetraacetic acid (EDTA) salt

Question 22

What is the commercial name of EDTA?

Answer 22

Versene

Question 23

This is recommended only for detailed microscopic studies.

Answer 23

EDTA or Versene

Question 24

EDTA is traditionally referred as

Answer 24

acid

Question 25

EDTA will not bind to calcium below _____ and is faster at _______

Answer 25

pH 3 and pH 7-7.4, respectively

Question 26

Often used in decalcification, accelerates the removal of calcium ions from formic acid-based solutions, making the tissue more soluble

Answer 26

Ammonium form ion exchange resin

Question 27

To reuse the resin, it can be reactivated by immersing it in ______________ then washing it with
distilled water three times

Answer 27

N/10 hydrochloric acid twice

Question 28

A process whereby positively charged
calcium ions are attracted to a negative electrode and subsequently removed from the decalcifying solution

Answer 28

Electrophoresis

Question 29

The recommended ratio of fluid to tissue volume for decalcification is

Answer 29

20 to 1 or 20:1

Question 30

It serves to hasten decalcification, but it also
increases the damaging effects of acids on tissue

Answer 30

Heat

Question 31

At 37°C, there will be impaired nuclear staining of ______________ for collagen fibers

Answer 31

Van Gieson’s stain

Question 32

At ______, the tissue will
undergo complete digestion within 24-48 hours.

Answer 32

55°C

Question 33

These produce heat and must be carefully monitored to prevent excessive temperatures that damage tissue

Answer 33

Microwave, sonication and electrolytic methods

Question 34

The optimum temperature so far recommended is the room temperature range of

Answer 34

18°C — 30°C

Question 35

There are three ways by which the extent of
decalcification may be measured

Answer 35

1. Physical or Mechanical Test
2. X-Ray or Radiological Method
3. Chemical Method (Calcium Oxalate Test)

Question 36

This is done by touching or bending the tissue
with the fingers to determine the consistency of tissues

Answer 36

Physical or Mechanical Test

Question 37

This is a very expensive although the most ideal,most sensitive and most reliable method of determining extent of decalcification due to its
ability to detect even the smallest focus of calcium which appears opaque in an X-ray plate.

Answer 37

X-Ray or Radiological Method

Question 38

It is not recommended for mercuric
chloride-fixed tissues due to the latter’s
characteristic radio-opacity which will interfere
with the correct interpretation of the plate.

Answer 38

X-Ray or Radiological Method

Question 39

This is a simple, reliable and convenient method recommended for routine purposes, to detect the presence of calcium in the decalcifying solution

Answer 39

Chemical Method (Calcium Oxalate Test)

Question 40

This method involves the detection of calcium in
acid solutions by precipitation of insoluble calcium hydroxide or calcium oxalate

Answer 40

Chemical Method (Calcium Oxalate Test)

Question 41

Many laboratories simply rinse the decalcified specimens with

Answer 41

running tap water

Question 42

Adequate water rinsing can usually be accomplished in ______ for small samples and ________ for larger specimen

Answer 42

30 minutes and 1-4 hours, respectively

Question 43

May act both as a decalcifying agent and
tissue softener.

Answer 43

Perenyi’s fluid

Question 44

Other substances which may be used as tissue softeners are:

Answer 44

Molliflex, 2% hydrochloric acid, or 1% hydrochloric
acid in 70% alcohol

Question 45

Tissues immersed in this fluid may appear swollen and soapy. This does not, however, affect the normal processing and subsequent staining of tissue sections.

Answer 45

Molliflex

Question 46

Washing out and immersion of fixed tissues in____ for 1-3 days may also cause
considerable tissue softening and easier sectioning of blocks without producing marked deleterious effects and tissue distortion.

Answer 46

4% aqueous phenol solution