LECTURE 5: Clearing

CLEARING

3rd histotechnology step and AKA dealcoholization.

DEALCOHOLIZATION

Clearing is also known as?

CLEARING

It is the 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 or the medium on which the tissue is to be mounted.

translucent

Once these clearing reagents mix with the alcohol, the alcohol is removed, and the tissue has a _____ appearance.

high index of refraction

If the clearing agent is used after the tissue has been stained, the clearing agent will make microscopic tissue preparations transparent due to their ________.

XYLENE

Most commonly used clearing agent for this purpose is ___.

1. Miscible with alcohol to promote rapid removal of the dehydrating agent from the tissue.

2. Miscible with and easily removed by melted paraffin wax or by mounting medium to facilitate impregnation and mounting of sections.

3. Not produce shrinkage, hardening, and damage to tissues

4. Not dissolve out aniline dyes.

5. Not evaporate quickly in a water bath.

6. Make tissues transparent!

Characteristics of a Good Clearing Agent:

Because it is the first step to ensure that the tissues can be read in the microscope.

Why is it important that these tissues should be made transparent?

1.518

The refractive index of the glass slide is?

1. Xylene

2. Toluene

3. Benzene

4. Chloroform

5. Cedarwood oil

6. Citrus fruit oil

7. Carbon tetrachloride

8. Methyl benzoate

9. Methyl salicylate

Common Clearing Agents Used in the Laboratory:

XYLOL

Xylene is also known as?

XYLENE

Best clearing agent; most rapid clearing agent

XYLENE

Suitable for urgent biopsies which it clears within 15-30 minutes.

urgent biopsies

Xylene is suitable for ______ which it clears within 15-30 minutes.

15-30 minutes

Xylene is suitable for urgent biopsies which it clears within ______.

XYLENE

A colorless clearing agent that is most commonly used in histology laboratories; routinely used.

histology laboratories ; routinely used

Xylene is a colorless clearing agent that is most commonly used in ________; ______.

XYLENE

Clearing time: 1/2 to 1 hour.

1/2 to 1 hour

Clearing time of Xylene

XYLENE

Used for clearing both for embedding and mounting procedure.

XYLENE

Generally suitable in most routine histologic processing schedules of less than 24 hours and when the tissue block size is less than 5m in thickness.

routine histologic processing schedules of less than 24 hours

Xylene is generally suitable in most _____ and when the tissue block size is less than 5m in thickness.

less than 5mm

Generally suitable in most routine histologic processing schedules of less than 24 hours and when the tissue block size is _____ in thickness.

XYLENE

ADVANTAGES:

• Clears within 15-30 mins.

• Make tissues transparent.

• Miscible with absolute alcohol & paraffin.

• Does not extract out aniline dyes.

• Does not dissolve celloidin.

• Evaporates quickly in paraffin oven.

• Readily replaced by wax during impregnation and embedding.

• Affordable or reasonably cost-effective.

• Works well in short-term clearing small tissue blocks.

XYLENE

DISADVANTAGES:

• Highly inflammable

• Prolonged usage makes tissues hard and brittle.

• Not suitable for nervous tissues & lymph nodes.

• Becomes milky when an incompletely dehydrated tissue is immersed in it.

  • Complete dehydration is needed before submitting tissue specimen to avoid the clearing agent becoming milky.

XYLENE

Not suitable for nervous tissues & lymph nodes.

nervous tissues & lymph nodes

Xylene is not suitable for _______.

XYLENE

Becomes milky when an incompletely dehydrated tissue is immersed in it.

XYLENE

Works well in short-term clearing small tissue blocks.

hard and brittle

Prolonged usage of xylene makes tissues _____ .

milky

Xylene becomes ___ when an incompletely dehydrated tissue is immersed in it

TOLUENE

May be used as a substitute for xylene and benzene for clearing both during embedding and mounting procedures

xylene and benzene

Toluene may used as a substitute for ________ for clearing both during embedding and mounting procedures

TOLUENE

Time Recommended for Clearing: 1 to 2 hours.

1 to 2 hours

Time Recommended for Clearing for Toluene

TOLUENE

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

TOLUENE

ADVANTAGES:

• Miscible with absolute alcohol and paraffin.

• Fairly rapid.

• Recommended for routine purposes

• Tissues do not become hard and brittle even if left for 24 hours.

• Not carcinogenic.

TOLUENE

Fairly rapid.

TOLUENE

Recommended for routine purposes

routine purposes

Toluene is recommended for ____.

TOLUENE

Tissues do not become hard and brittle even if left for 24 hours.

Not carcinogenic

Is toluene carcinogenic or not?

TOLUENE

DISADVANTAGES:

• Slower than Xylene & Benzene.

• Tends to acidify.

• Highly concentrated solutions emit fumes that are toxic.

• More expensive than Xylene.

• Least commonly used.

TOLUENE

Slower than Xylene & Benzene.

TOLUENE

Tends to acidify.

TOLUENE

Highly concentrated solutions emit fumes that are toxic.

TOLUENE

More expensive than Xylene.

TOLUENE

Least commonly used.

BENZENE

Excellent but hazardous.

BENZENE

Preferred by some as a clearing agent in the embedding process in tissues because it penetrates and clears the tissues rapidly.

BENZENE

Before, it was used routinely and preferred by some; not until recently, when its highly carcinogenic property was recognized.

It is highly carcinogenic.

Is benzene carcinogenic or not?

BENZENE

Excessive exposure to this may be extremely toxic to man or may cause damage to the bone marrow, resulting in aplastic anemia.

aplastic anemia

Excessive exposure to benzene may be extremely toxic to man or may cause damage to the bone marrow, resulting in ______.

BENZENE

If used for clearing, the laboratory should always be ventilated

BENZENE

ADVANTAGES:

• Recommended for urgent biopsies (15-60 mins).

• Volatilizes rapidly in paraffin oven, easily eliminated from tissue.

• Miscible with absolute alcohol.

• Does not make tissues hard and brittle.

• Causes minimal shrinkage.

• Make tissues transparent.

BENZENE

Recommended for urgent biopsies (15-60 mins).

BENZENE

Volatilizes rapidly in paraffin oven, easily eliminated from tissue.

BENZENE

Does not make tissues hard and brittle.

BENZENE

Causes minimal shrinkage.

urgent biopsies (15-60 mins)

Benzene is recommende for _____.

BENZENE

DISADVANTAGES:

• Highly inflammable.

• Extremely toxic to man.

• Carcinogenic (acute myeloid leukemia/AML/aplastic anemia).

BENZENE

Extremely toxic to man

BENZENE

Carcinogenic (acute myeloid leukemia/AML/aplastic anemia).

CHLOROFORM

Not a true clearing agent because tissues do not become translucent or transparent after submerging in this clearing agent.

CHLOROFORM

Complete clearing with the use of this is actually difficult to evaluate.

CHLOROFORM

There should be adequate room ventilation and proper caution when handling specimens with this as clearing agent.

CHLOROFORM

ADVANTAGE:

• Recommended for routine work (6-24 hours).

• Miscible with absolute alcohol.

• Recommended for tough tissues.

• Suitable for large tissue specimens.

• Not inflammable.

• Causes less brittleness than Xylene.

• Tissue blocks up into 1 cm thickness can be processed.

• It is recommended for tough tissues (e.g.,skin, fibroid, decalcified tissues) for nervous tissues, lymph nodes, and embryos because it causes minimum shrinkage and hardening of tissues.

CHLOROFORM

Recommended for routine work (6-24 hours).

CHLOROFORM

Recommended for tough tissues.

CHLOROFORM

Suitable for large tissue specimens.

CHLOROFORM

Not inflammable.

CHLOROFORM

Causes less brittleness than Xylene.

CHLOROFORM

Tissue blocks up into 1 cm thickness can be processed.

CHLOROFORM

It is recommended for tough tissues (e.g., skin, fibroid, decalcified tissues) for nervous tissues, lymph nodes, and embryos because it causes minimum shrinkage and hardening of tissues.

routine work (6-24 hours)

Chloroform is recommended for _____.

CHLOROFORM

DISADVANTAGE:

• Hepatotoxic, after prolonged inhalation.

• Slower in action than xylene; relatively slow.

• Does not make tissues transparent.

• Not very volatile in paraffin oven; difficult to remove from paraffin sections.

• It evaporates quickly from a water bath.

• Produces considerable deterioration of the wax.

CHLOROFORM

Hepatotoxic, after prolonged inhalation.

CHLOROFORM

Slower in action than xylene; relatively slow.

CHLOROFORM

Does not make tissues transparent.

CHLOROFORM

Not very volatile in paraffin oven; difficult to remove from paraffin sections.

CHLOROFORM

It evaporates quickly from a water bath.

CHLOROFORM

Produces considerable deterioration of the wax.

1 cm

In chloroform, tissue blocks up into ___ thickness can be processed.

CHLOROFORM

It is recommended for tough tissues (e.g.,skin, fibroid, decalcified tissues) for nervous tissues, lymph nodes, and embryos because it causes minimum shrinkage and hardening of tissues.

CEDARWOOD OIL

Especially recommended for central nervous system tissues and cytological studies, particularly of smooth muscles and skin.

central nervous system tissues; cytological studies; smooth muscles and skin

Cedarwood oil is especially recommended for ________ and _____, particularly of ____.

CEDARWOOD OIL

It requires two changes in clearing solution.

2 changes

How many changes in clearing solution does cedarwood oil require?

CEDARWOOD OIL

Clearing is usually complete in 2 to 3 days; quite slow and not recommended for routine processes.

2 to 3 days

Clearing in cedarwood oil is usually complete in ___; quite slow and not recommended for routine processes.

CEDARWOOD OIL

Tissues may be left in this for an indefinitely without causing considerable tissue damage and distortion.

CEDARWOOD OIL

Used to clear both paraffin and celloidin sections during the embedding process.

CEDARWOOD OIL

ADVANTAGES:

• Very penetrating.

• Miscible with 96% alcohol.

• Clears celloidin in 5-6 days.

• Minimum shrinkage & hardening.

• Without considerable damage & distortion.

• Does not dissolve out aniline dyes.

• Makes tissues transparent.

• Improves cutting of the sections.

CEDARWOOD OIL

Very penetrating.

CEDARWOOD OIL

Miscible with 96% alcohol.

CEDARWOOD OIL

Clears celloidin in 5-6 days.

CEDARWOOD OIL

Improves cutting of the sections.

CEDARWOOD OIL

Without considerable damage & distortion.

96%

Cedarwood oil is miscible with how many percent alcohol?