LESSON 5: CLEARING

Question 1

otherwise referred to as fluidity

Answer 1

VISCOSITY

Question 2

benign overgrowth usually composed of

muscle tissue and fibers

Answer 2

FIBROID

Question 3

benzene ring with two methyl groups

Answer 3

XYLENE

Question 4

methyl benzene

Answer 4

TOLUENE

Question 5

CHCl4

Answer 5

CHLOROFORM

Question 6

CCl4

Answer 6

CARBONTETRACHLORIDE

Question 7

C6H6

Answer 7

BENZENE

Question 8

substances that cause malignancy

Answer 8

CARCINOGENIC

Question 9

combustible

Answer 9

FLAMMABLE

Question 10

cyclic organic compounds

Answer 10

AROMATIC

Question 11

✓ Removal of dehydrating agent from the tissues and replacing it by a
solvent → transparent & translucent tissue
✓ Not all dealcoholizing agents act as clearing agents
✓ Clearing agents only: glycerin, gum syrup and Brun’s solution
✓ Dealcoholizing agents only: chloroform and carbon tetrachloride

Answer 11

CLEARING

Question 12

➢ Done after dehydration & before infiltration
➢ Solvent: dealcoholize and act as solvent of paraffin
➢ Agents: xylene, toluene, dioxane and chloroform

Answer 12

Clearing in Embedding (TISSUE PROCESSING)

Question 13

➢ Done after staining & before mounting
➢ microscopic preparations transparent (use of solvents with high refractive
index)
➢ Agents must be solvents of the Mounting media: xylene, toluene, terpineol,
carbol-xylene

Answer 13

Clearing in Mounting

Question 14

Refractive index of clearing agents: approximately equal to that of the tissues

Answer 14

For the purpose of making the tissues transparent so that their internal structure is demonstrable to the naked eye.

Question 15

✓ Should be miscible with dehydrating agent and either infiltrating medium or mounting
medium
✓ Should remove alcohol quickly & clear quickly without overhardening the tissues
✓ Should not dissolve out aniline dyes
✓ Should not evaporate quickly in the water baths
✓ Evaporates quickly in paraffin oven
✓ Could be used in amounts at least 10Xthe volume of tissue

Answer 15

Characteristics of an Ideal Clearing Agent

Question 16

Agents with low BP are readily replaced by

Answer 16

paraffin (Except chloroform)

Question 17

leads to slow penetration

Answer 17

Higher viscosity

Question 18

does not affect the rate but affects the quality of cleared tissue

Answer 18

Refractive Index

Question 19

▪ Special considerations should be noted depending on the clearing agent used
▪ Some reagents will not clear tissues

Answer 19

Reagent Used

Question 20

✓ Most rapid (15 - 30 mins/ 30 min – 1 hr)
✓ Excellent clearing agent but tends to make tissues excessively hard & brittle.
✓ Turns milky when dehydration is not complete

Answer 20

Xylene/Xylol

Question 21

✓ Rapid agent (15 – 60 mins)

✓ Carcinogenic, causes aplastic anemia

Answer 21

Benzene

Question 22

✓ Similar to xylene but does NOT harden tissues nearly so much
✓ Slower than xylene or benzene (1 – 2 hrs)
✓ Not carcinogenic but emits toxic fumes

Answer 22

Toluene/Toluol

Question 23

✓ For nervous tissues, lymph nodes & embryos
✓ Tissue do not become translucent
✓ Best for large specimens (up to 1cm thick) and tough tissues
✓ Toxic to the liver on prolonged inhalation
✓ Tissues tend to float: remedy → wrap tissues with absorbent cotton gauze

Answer 23

Chloroform

Question 24

✓ Recommended for CNS, smooth muscles & skin
✓ Slow (2 – 3 days); minimal shrinkage
✓ For both celloidin and paraffin sections
✓ Tissue floats – use Absolute alcohol to prevent drying out of tissues
✓ Must be followed by immersion in xylene or benzene to remove oil from tissues
✓ Turns milky on prolonged storage

Answer 24

Cedarwood oil

Question 25

✓ Clears 70% alcohol | ✓ Recommended for embryos, insects and delicate specimens

Answer 25

Aniline oil

Question 26

✓ Slow; may be adulterated; removes aniline dyes

Answer 26

Clove Oil

Question 27

✓ Similar to chloroform

Answer 27

CCl4

Question 28

Both dehydrating and clearing

Answer 28

Dioxane

Question 29

For large pieces of tissues & embryonic materials

Answer 29

Amyl acetate

Question 30

Substitute for cedarwood oil

Answer 30

Terpineol

Question 31

Used in double embedding.

Answer 31

Methyl | Benzoate/Salicylate

Question 32

when the tissue is to be cleared directly from water (no dealcoholization, it merely improves the RI)

Answer 32

Glycerin, Gum syrup & Brun’s solution

Question 33

✓ For materials that are difficult to clear (eg. Thick mucinous Papanicolaou smears) ✓ Should be thoroughly rinsed in xylene prior to mounting

Answer 33

Carbo - Xylene

Question 34

✓ Oil of bergamot ✓ Phenol in alcohol ✓ Creosote

Answer 34

Others USED FOR CLEARING

Question 35

✓ Based on limonene: a volatile oil found in citrus peels | ✓ Clearite - Long chain aliphatic HC

Answer 35

Newer Clearing agents

Question 36

Most commonly used and most rapid clearing agent

Answer 36

Xylene

Question 37

- Rapid-acting and clears tissues without causing hardness or brittleness - Causes aplastic anemia

Answer 37

Benzene

Question 38

Used as a substitute for xylene and benzene

Answer 38

Toluene

Question 39

- Versatile reagents as they can be used for different types of tissues - Used for delicate as well as tough tissue samples

Answer 39

CHCl3 and CCl4

Question 40

Recommended for CNS, smooth muscle and skin tissues

Answer 40

Cedarwood oil

Question 41

organ affected by chloroform toxicity

Answer 41

LIVER

Question 42

factor that does not affect clearing rate

Answer 42

R.I.

Question 43

turns milky if dehydration is incomplete

Answer 43

XYLENE

Question 44

clearing in _______; reagent should be | miscible in the stain

Answer 44

MOUNTING

Question 45

BP of chloroform compared to xylene

Answer 45

LOWER

Question 46

anemia caused by benzene

Answer 46

APLASTIC

Question 47

clearing agent that is prone to adulteration

Answer 47

CLOVE OIL

Question 48

also known as artificial oil of lilac

Answer 48

TERPINEOL

Question 49

turns milky on prolonged storage

Answer 49

CEDARWOOD OIL

Question 50

Aromatic oil used for insects

Answer 50

ANILINE