CHAPTER 8 DECALCIFICATION

Question 1

Strong Mineral Acids:

Answer 1

I. NITRIC ACID

II. HYDROCHLORIC ACID

III. FORMIC ACID

IV. TRICHLOROACETIC ACID

V. CHROMIC ACID (FLEMMING’S FLUID)

VI. CITRIC ACID-CITRATE BUFFER SOLUTION (pH 4.5)

Question 2

I. NITRIC ACID:

Question 3

II. HYDROCHLORIC ACID:

Question 4

III. FORMIC ACID:

Question 5

IV. TRICHLOROACETIC ACID:

Question 6

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

Answer 6

NITRIC ACID

Question 7

This may be used as simple aqueous solutions with recommended concentrations of 5- 10%.

Answer 7

NITRIC ACID

Question 8

It is a very rapid decalcifying agent, producing minimal distortion and is, therefore, recommended for routine purposes.

Answer 8

NITRIC ACID

Question 9

It has, however, the disadvantage of inhibiting nuclear stains and destroying tissues, especially in concentrated solutions.

Answer 9

NITRIC ACID

Question 10

The endpoint of decalcification must be carefully watched for, to prevent progressive tissue damage and impaired staining. This may be prevented by combining nitric acid with formaldehyde or alcohol.

Answer 10

NITRIC ACID

Question 11

DECALCIFICATION TIME: 12-24 hours

Answer 11

Aqueous Nitric Acid Solution 10%

Question 12

1. It is rapid in action.

Answer 12

Aqueous Nitric Acid Solution 10%

Question 13

2. It produces minimum distortion of tissues.

Answer 13

Aqueous Nitric Acid Solution 10%

Question 14

3. It produces good nuclear staining (although less than in slower​ acting agents).

Answer 14

Aqueous Nitric Acid Solution 10%

Question 15

4. The acid may be easily removed by 70% alcohol.

Answer 15

Aqueous Nitric Acid Solution 10%

Question 16

5. It is recommended for urgent biopsy, and for needle and small biopsy specimens to permit rapid diagnosis within 24 hours or less.

Answer 16

Aqueous Nitric Acid Solution 10%

Question 17

6. It can be used for large or heavily mineralized cortical bone specimen if decalcification progress is carefully monitored by a decalcification endpoint test.

Answer 17

Aqueous Nitric Acid Solution 10%

Question 18

1 Prolonged decalcification may lead to tissue distortion.

Answer 18

Aqueous Nitric Acid Solution 10%

Question 19

2. It can seriously damage tissue stainability.

Answer 19

Aqueous Nitric Acid Solution 10%

Question 20

3. It imparts a yellow color with nitrous acid, thereby impairing the staining reaction of the tissue.

Answer 20

Aqueous Nitric Acid Solution 10%

Question 21

4. Old nitric acid solution is particularly damaging and should be replaced with fresh stock solution.

Answer 21

Aqueous Nitric Acid Solution 10%

Question 22

5. Strong acids tend to be more damaging to tissue antigens for immunohistochemical staining, and enzymes may be totally lost.

Answer 22

Aqueous Nitric Acid Solution 10%

Question 23

DECALCIFICATION TIME: 1-3 days

Question 24

1. It is rapid-acting; hence, is recommended for urgent biopsies.

Answer 24

Formol-Nitric Acid

Question 25

2. Nuclear staining is relatively good.

Answer 25

Formol-Nitric Acid

Question 26

3. It produces less tissue destruction than 10% aqueous nitric acid.

Answer 26

Formol-Nitric Acid

Question 27

1. The yellow color imparted by nitrous acid formation will impair staining reaction of the cell. This may be prevented by neutralizing the tissue with 5% sodium sulfate and washing in running tap water for at least 12 hours. Addition of 0.1% urea to pure concentrated nitric acid will also make discoloration disappear without considerably affecting the efficiency of the decalcifying solution.

Answer 27

Formol-Nitric Acid

Question 28

2. The solution should be used inside a fume hood.

Answer 28

Formol-Nitric Acid

Question 29

DECALCIFICATION TIME: 2 - 7 days

Answer 29

Perenyi’s Fluid

FORMIC ACID

Question 30

1. It is recommended for routine purposes.

Answer 30

Perenyi’s Fluid

Question 31

2. It decalcifies and softens tissues at the same time.

Answer 31

Perenyi’s Fluid

Question 32

3. Nuclear and cytoplasmic staining is good.

Answer 32

Perenyi’s Fluid

Question 33

4. Maceration is avoided due to the presence of chromic acid and alcohol.

Answer 33

Perenyi’s Fluid

Question 34

1. It is a slow decalcifying agent for dense bones; hence, is not recommended for urgent diagnosis.

Answer 34

Perenyi’s Fluid

Question 35

2. Complete decalcification cannot be determined by chemical test because a precipitate is formed upon the addition of ammonia to Perenyi’s fluid even in the absence of calcium ion.

Answer 35

Perenyi’s Fluid

Question 36

This may be dissolved by adding glacial acetic acid drop by drop. About 0.5 ml. of saturated aqueous ammonium oxalate is then added to the solution. Reappearance of a white precipitate within 30 minutes will reaffirm the presence of calcium in the agent, signifying that decalcification is still incomplete.

Answer 36

Perenyi’s Fluid

Question 37

DECALCIFICATION TIME: 12-24 hours

Answer 37

Phloroglucin-Nitric Acid

Question 38

It is the most rapid decalcifying agent so far, recommended for urgent cases.

Answer 38

Phloroglucin-Nitric Acid

Question 39

1. Nuclear staining is poor.

Answer 39

Phloroglucin-Nitric Acid

Question 40

2. Prolonged decalcification produces extreme tissue distortion.

Answer 40

Phloroglucin-Nitric Acid

Question 41

3. Yellow color must be neutralized with 5% sodium sulfate and thoroughly washed with running tap water for at least 24 hours.

Answer 41

Phloroglucin-Nitric Acid

Question 42

4. Complete decalcification cannot be determined by chemical means.

Answer 42

Phloroglucin-Nitric Acid

Question 43

When decalcification is complete, the acid must be removed by three changes of 70% to 90% ethanol, since washing in watery solutions will lead to excessive swelling and deterioration of tissue. When the sections are cut, the slides are brought to water and placed in 1% aqueous lithium carbonate for I hour, washed in later for 15 minutes, and then stained.

Answer 43

Phloroglucin-Nitric Acid

Question 44

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 decalcified section.

Answer 44

HYDROCHLORIC ACID

Question 45

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

Answer 45

HYDROCHLORIC ACID

Question 46

Rapid proprietary solutions usually contain hydrochloric acid, whereas slow proprietary mixtures contain buffered formic acid or formalin/formic acid.

Answer 46

HYDROCHLORIC ACID

Question 47

Dilution of a proprietary [?] is not deleterious for effective decalcification or staining, and this is an option if a strong mixture is considered too concentrated.

Answer 47

HYDROCHLORIC ACID

Question 48

1. It permits relatively good cytologic staining.

Answer 48

Von Ebner’s Fluid

Question 49

2. It is a moderately rapid decalcifying agent.

Answer 49

Von Ebner’s Fluid

Question 50

3. It does not require washing out before dehydration.

Answer 50

Von Ebner’s Fluid

Question 51

4. It is recommended for teeth and small pieces of bone.

Answer 51

Von Ebner’s Fluid

Question 52

The extent of decalcification cannot be measured by a chemical test.

Answer 52

Von Ebner’s Fluid

Question 53

is 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 53

FORMIC ACID

Question 54

It is recommended for routine decalcification of postmortem research tissues, although not suitable for urgent examinations.

Answer 54

FORMIC ACID

Question 55

can be used as a simple 10% aqueous solution or combined with formalin or with a buffer.

Answer 55

FORMIC ACID

Question 56

It is slower than the strong acid agents, but it is much gentler in action and less likely to interfere with nuclear staining.

Answer 56

FORMIC ACID

Question 57

[?] in a 10% concentration is the best all-around decalcifier.

Answer 57

FORMIC ACID

Question 58

Some commercial solutions are available that combine formic acid with formalin to fix and decalcify tissues at the same time.

Answer 58

FORMIC ACID

Question 59

1. It may be used both as a fixative and decalcifying agent.

Answer 59

FORMIC ACID

Question 60

2. It permits excellent nuclear and cytoplasmic staining.

Answer 60

FORMIC ACID

Question 61

3. It is recommended for small pieces of bones and teeth.

Answer 61

FORMIC ACID

Question 62

4. It is suitable for most routine surgical specimens, particularly when immunohistochemical staining is needed.

Answer 62

FORMIC ACID

Question 63

1. It is relatively slow; hence, is not suitable for urgent specimens. Decalcification may be hastened by increasing the proportion of formic acid to 25 ml. However, such concentration may make the solution opaque, thereby interfering with the staining results.

Answer 63

FORMIC ACID

Question 64

2. It requires neutralization with 5% sodium sulfate, and washing out to remove the acid from the tissue.

Answer 64

FORMIC ACID

Question 65

DECALCIFICATlON TIME: 3 -14 days

Answer 65

Formic Acid-Sodium Citrate Solution

Question 66

1. It permits better nuclear staining than nitric acid method.

Answer 66

Formic Acid-Sodium Citrate Solution

Question 67

2. It is recommended for autopsy materials, bone marrow, cartilage and tissues studied for research purposes.

Answer 67

Formic Acid-Sodium Citrate Solution

Question 68

1. It is relatively slow; hence, is not recommended for routine purposes and for dense tissues.

Answer 68

Formic Acid-Sodium Citrate Solution

Question 69

2. It requires neutralization with 5% sodium sulfate.

Answer 69

Formic Acid-Sodium Citrate Solution

Question 70

DECALCIFICATION TIME: 4- 8 days

Answer 70

TRICHLOROACETIC ACID

Question 71

1. It permits good nuclear staining.

Answer 71

TRICHLOROACETIC ACID

Question 72

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

Answer 72

TRICHLOROACETIC ACID

Question 73

1. It is a weak decalcifying agent, not used for dense tissues, and is suitable only for small spicules of bone.

Answer 73

TRICHLOROACETIC ACID

Question 74

2. It is very slow-acting; hence, is not recommended for urgent examinations.

Answer 74

TRICHLOROACETIC ACID

Question 75

-is a very weak decalcifying solution suitable only for minute pieces of bone.

Answer 75

SULFUROUS ACID

Question 76

1. It may be used both as a fixative and decalcifying agent.

Answer 76

CHROMIC ACID (FLEMMING’S FLUID)

Question 77

2. It may be used for decalcifying minute bone spicules.

Answer 77

CHROMIC ACID (FLEMMING’S FLUID)

Question 78

1. Nuclear staining with hematoxylin is inhibited.

Answer 78

CHROMIC ACID (FLEMMING’S FLUID)

Question 79

2. It tends to undergo reduction and forms precipitates at the bottom of the container thus requiring frequent changes of solution.

Answer 79

CHROMIC ACID (FLEMMING’S FLUID)

Question 80

3. Insoluble pigments are formed when decalcified tissue is dehydrated with alcohol; hence, tissues must be washed out prior to dehydration.

Answer 80

CHROMIC ACID (FLEMMING’S FLUID)

Question 81

1. Chromic acid is highly corrosive to skin and mucous membranes.

Answer 81

CHROMIC ACID (FLEMMING’S FLUID)

Question 82

2. It is carcinogenic.

Answer 82

CHROMIC ACID (FLEMMING’S FLUID)

Question 83

3. Suitable protective material is not readily available or practical for laboratory use.

Answer 83

CHROMIC ACID (FLEMMING’S FLUID)

Question 84

4. Drain disposal is not a legitimate option for any solution containing chromium, including subsequent processing of fluids following fixation or rinses following staining procedures involving chromium.

Answer 84

CHROMIC ACID (FLEMMING’S FLUID)

Question 85

DECALCIFICATION TIME: 6 days

Answer 85

CITRIC ACID-CITRATE BUFFER SOLUTION (pH 4.5)

Question 86

1 It permits excellent nuclear and cytoplasmic staining.

Answer 86

CITRIC ACID-CITRATE BUFFER SOLUTION (pH 4.5)

Question 87

2. It does not produce cell or tissue distortion.

Answer 87

CITRIC ACID-CITRATE BUFFER SOLUTION (pH 4.5)

Question 88

Its action is too slow for routine purposes.

Answer 88

CITRIC ACID-CITRATE BUFFER SOLUTION (pH 4.5)

Question 89

acts slowly but causes little tissue damage. Conventional stains are largely unaffected.

Answer 89

Neutral EDTA

Question 90

1. It permits excellent staining results.

Answer 90

Neutral EDTA

Question 91

2. It produces minimal cell and tissue distortion.

Answer 91

Neutral EDTA

Question 92

3. It forms minimal histological artifacts, usually caused by production of CO2 bubbles.

Answer 92

Neutral EDTA

Question 93

4. Extent of decalcification can be measured by routine chemical test.

Answer 93

Neutral EDTA

Question 94

5. EDTA is an excellent bone decalcifier for enzyme or immuno-histochemical staining, and for electron microscopy.

Answer 94

Neutral EDTA

Question 95

6. Enzymes require specific pH conditions in order to maintain
   activity, and EDTA solutions can be adjusted to a specific pH for
   enzyme staining.

Answer 95

Neutral EDTA

Question 96

1. It is very slow, and is therefore not recommended for urgent and routine purposes.

Answer 96

Neutral EDTA

Question 97

2. It causes slight tissue hardening.

Answer 97

Neutral EDTA

Question 98

3. EDTA inactivates alkaline phosphatase activity, which can be restored by addition of magnesium chloride.

Answer 98

Neutral EDTA

Question 99

1. Cellular detail is well-preserved.

Answer 99

ION EXCHANGE RESIN

Question 100

2. Daily washing of solutions is eliminated.

Answer 100

ION EXCHANGE RESIN

Question 101

3. It permits excellent staining results.

Answer 101

ION EXCHANGE RESIN

Question 102

4. Decalcification is hastened.

Answer 102

ION EXCHANGE RESIN

Question 103

5. It produces minimal cell and tissue distortion.

Answer 103

ION EXCHANGE RESIN

Question 104

6. It forms minimal histological artifacts, usually caused by production of CO2 bubbles.

Answer 104

ION EXCHANGE RESIN

Question 105

7. Extent of decalcification can be measured by routine chemical test.

Answer 105

ION EXCHANGE RESIN

Question 106

1. The degree of decalcification cannot be measured by chemical means.

Answer 106

ION EXCHANGE RESIN

Question 107

2. It is very slow, and is therefore not recommended for urgent and routine purposes.

Answer 107

ION EXCHANGE RESIN

Question 108

3. It causes slight tissue hardening.

Answer 108

ION EXCHANGE RESIN

Question 109

hastens decalcification by removing calcium ions from formic acid-containing decalcifying solutions, thereby increasing solubility from the tissue.

Answer 109

ION EXCHANGE RESIN

Question 110

It is not recommended for fluids containing mineral acids such as nitric acid or hydrochloric acid.

Answer 110

ION EXCHANGE RESIN

Question 111

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

Answer 111

ELECTROPHORESIS (ELECTRICAL IONIZATION)

Question 112

This method is satisfactory for small bone fragments, processing only alimited number of specimens at a time. Good cytologic and histologic details are, however, not always preserved in tissues that have been electrically decalcified.

Answer 112

ELECTROPHORESIS (ELECTRICAL IONIZATION)

Question 113

is faster than routine decalcification irrespective of the decalcifying agents used

Answer 113

MICROWAVE OVEN DECALCIFICATION

Question 114

has been used quite often for tissue processing, but there are very few studies describing its use in decalcification of bone or teeth

Answer 114

microwave oven

Question 115

Factors influencing the rate of decalcification

Answer 115

Concentration

Fluid access

Size and consistency

Agitation

Temperature

Question 116

The recommended ratio of fluid to tissue volume for decalcification is

Answer 116

20 to 1.

Question 117

Dense bone tissues usually require up to [?] or longer in order to complete the process.

Answer 117

14 days

Question 118

At [?], there will be impaired nuclear staining of Van Gieson’s stain for collagen fibers.

Answer 118

37°C

Question 119

At [?], the tissue will undergo complete digestion within 24-48 hours.

Answer 119

55°C

Question 120

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

Answer 120

18°C -30°C.

Question 121

The decalcifying fluid is usually changed every [?] and the chemical test is performed on the discarded fluid.

Answer 121

24 -48 hours

Question 122

If the solution remains clear after [?], decalcification is considered to be complete.

Answer 122

30 minutes

Question 123

Adequate water rinsing can usually be accomplished in [?] for small samples and [?] for larger specimens.

Answer 123

30 minutes

1-4 hours

Question 124

Acid decalcified tissues for frozen sections must be thoroughly washed in water or stored in formol-saline containing 15% sucrose or phosphate-buffered saline (PBS) with 15-20% sucrose at [?] before freezing.

Answer 124

4°C

Question 125

To soften unduly hard tissues, selected portions are left in the fluid for [?] and dehydrated in the usual manner; or the cut surface of the block may be submerged in the fluid for [?] before sectioning, to facilitate easier cutting of tissues.

Answer 125

12-24 hours

1-2 hours