Clearing

Question 1

process whereby alcohol or a dehydrating agent is removed from the tissue and replaced with a substance that will dissolve the wax with which the tissue is to be impregnated (e.g. paraffin) or used as the medium on which the tissue is to be mounted (e.g. Canada balsam).

Answer 1

Clearing

Question 2

most commonly used clearing agent

Answer 2

xylene

Question 3

are used when the tissue is to be cleared directly from water, as in
a frozen section.

Answer 3

Glycerin and gum syrup

Question 4

has a lower boiling point than xylene but takes longer than the latter to clear

Answer 4

chloroform

Question 5

affects the speed of penetration of the clearing agent

Answer 5

Viscosity

Question 6

What will happen to prolonged exposure of most clearing agents?

Answer 6

tissue become brittle therefore more difficult to cut

Question 7

• colorless clearing agent that is most commonly used in histology
  laboratories.
• Clearing time is usually 1/2 to 1 hour
• used for clearing, both for embedding and mounting procedures

Answer 7

Xylene (Xylol)

Question 8

example of toxicities believed to be caused by intermediate product of xylene metabolism

Answer 8

methyl benzaldehyde

Question 9

What will happen if xylene is used longer than 3 hours?

Answer 9

makes tissue excessively hard and brittle

Question 10

What happens to xylene when an incomplete dehydrated tissue is immersed?

Answer 10

milky

Question 11

better at preserving tissue structure and is more tolerant of small amounts of water left behind in the tissues than xylene

Answer 11

Toluene

Question 12

Why is xylene more commonly used than toluene

Answer 12

Toluene is expensive and more toxic

Question 13

What is the time recommended for clearing Toluene?

Answer 13

1-2 hours

Question 14

What are the different Clearing Agents?

Answer 14

1. Xylene
2. Toluene
3. Benzene
4. Chloroform
5. Cedarwood Oil
6. Aniline Oil
7. Clove Oil
8. Carbon Tetrachloride
9. Tetrahydrofuran
10. Dioxane

Question 15

What are the other xylene substitutes?

Answer 15

1. Terpenes
2. Limonene
3. Orange oil based clearing agents
4. Chlorinated hydrocarbons
5. Coconut oil
6. Bleached palm oil

Question 16

• preferred by some as clearing agent in the embedding process of
  tissues because it penetrates and clears tissues rapidly
• used to be a popular routine clearing agent until recently when its highly carcinogenic properties were recognized.

Answer 16

Benzene

Question 17

How many minutes does benzene clear the tissue in urgent biopsies?

Answer 17

15-60 minutes

Question 18

Benzene is carcinogenic and may damage the bone marrow resulting in?

Answer 18

Aplastic anemia

Question 19

• slower in action than xylene, but causes less brittleness
• Thicker tissue blocks, even those up to 1 cm in thickness, can be processed.
• tissues do not become translucent

Answer 19

Chloroform

Question 20

How to avoid tissues floating in chloroform?

Answer 20

wrapping the tissues with absorbent cotton gauze to facilitate sinking of the section in solution

Question 21

• used to clear both paraffin and celloidin sections during the embedding process
• recommended for central nervous system
  tissues and cytological studies, particularly of smooth muscles and skin.
• requires two changes in clearing solution

Answer 21

Cedarwood oil

Question 22

How long does it usually take to complete the clearing process in Cedarwood oil?

Answer 22

2-3 days

Question 23

How long does Cedarwood oil clears celloidin?

Answer 23

5-6 days

Question 24

How to improve or hasten the slow process of impregnation in using Cedarwood oil?

Answer 24

transferring the specimen from oil to benzene for 1/2 hour before finally placing the tissue in wax

Question 25

Tissues cleared in cedarwood oil initially float before gradually staying to the bottom as clearing proceeds hence tissue may dry out before it is completely cleared. How to prevent this?

Answer 25

superimposing absolute alcohol on the surface of the clearing agent, once saturated, the specimen should then be transferred to a fresh solution of cedarwood oil.

Question 26

What happens to Cedarwood oil if prolonged storage?

Answer 26

milky

Question 27

Cedarwood oil that has been previously used to clear acetic-alcohol fixed tissues may produce crystals with a melting point of approximately 35°C and therefore interfere with adequate clearing of tissue. What can you do to restore the solution to its normal state?

Answer 27

The solution must be heated to 200°C in order to dissolve the crystals

Question 28

not normally utilized as a routine clearing agent but it is recommended for clearing embryos, insects and very delicate specimens, due to its ability to clear 70% alcohol without excessive tissue shrinkage and hardening.

Answer 28

Aniline oil

Question 29

- causes minimum shrinkage of tissues - its quality is not guaranteed due to its tendency to become adulterated. - Wax impregnation after clearing is slow and difficult - Tissues become brittle, aniline dyes are removed, and celloidin is dissolved

Answer 29

Clove oil

Question 30

- its properties are very similar to that of chloroform although it is relatively cheaper - its disadvantage is the same as that of chloroform - produces considerable tissue hardening, and is dangerous to inhale on prolonged exposure due to its highly toxic effects.

Answer 30

Carbon tetrachloride

Question 31

- superior to ordinary dehydrating and clearing agents due to its ability to perform two processes at the same time, thereby shortening the total processing time and allowing more time for fixation - It is non-toxic but has offensive odor and should be used in a well-ventilated room.

Answer 31

Tetrahydrofuran

Question 32

- miscible both with water and paraffin - used primarily when time is important because the tissues may be embedded with paraffin within 4 hours after fixation - is changed 3 times within 4 hours and the tissues are transferred directly to paraffin (3 changes are made in a total of 90 minutes). - causes greater shrinkage than xylene does - dangerous (fumes are toxic to human especially to the liver)

Answer 32

Dioxane

Question 33

- are isoprene polymers found in essential oils originally derived from plants, though some are now synthesized. - clear tissues and celloidin sections from 80%-95% alcohol, render tissues transparent and have a slow gentle non-hardening action - safe but have strong odors - moderately effective solvents, but considered toxic - dry slowly, leave an oily residue on slides, and expensive

Answer 33

Terpenes

Question 34

- one of the recommended xylene substitutes from the terpene family - a volatile oil found in citrus peels - natural oil found in the skins of citrus fruits, such as lemons or oranges, and in cooking is usually referred to as lemon or orange zest - obtained industrially by the steam distillation of orange peel - a clear, colorless fluid with a distinctly citrus aroma - does not mix well with mounting medium dissolved in xylene or toluene

Answer 34

Limonene

Question 35

- offer the clearing action with the lowest hazard rating of all xylene alternatives - excellent for preserving fine tissue structure, and can often be used in place of xylene with no alteration of protocol - if neither pure nor stable can break down to produce compounds which will interfere with staining procedures

Answer 35

Orange oil based clearing agents

Question 36

- can be effective solvents, but they are considered toxic chemicals, posing serious health risks - Government regulations have restricted most of the effective solvents in this class

Answer 36

Chlorinated hydrocarbons

Question 37

- an efficient substitute for xylene, as it is non-hazardous, less expensive and causes less shrinkage of the tissue - can be used as a dealcoholization agent without losing the quality - have the tendency to get solidified at a lower temperature but can be overcome by performing cleaning procedure in an incubator, maintaining the required temperature

Answer 37

Coconut oil

Question 38

- gives good tissues, sections and histological slides - nontoxic, nonhazardous, nonflammable, bio-degradable, economic, easy to handle, and readily available

Answer 38

Bleached palm oil