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HCT > Lec 3: Clearing & infiltration > Flashcards

Lec 3: Clearing & infiltration Flashcards

1
Q

It is the appearance of tissue after it has been treated with clearing solutions to remove the dehydrating agent

A

Clearing

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2
Q

A process of tissue preparation wherein the dehydrating agent is replaced with a clearing agent

A

Clearing

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3
Q

Characteristics of a clearing agent (6)

A
  • removes alcohol
  • high refractive index
  • miscible with wax & mounting medium
  • miscible with ethanol & wax
  • removes fat
  • NOT less than 10 times the volume of the tissue
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4
Q

The clearing agent must be _____ times the volume of the tissue

A

NOT be less than 10 times

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5
Q

ADVANTAGE:
• most rapid
• makes tissue transparent
• miscible with abs. alcohol & paraffin
• does NOT extract aniline dyes
• doesn NOT dissolve celloidin (mounting)
• evaporates quickly in paraffin oven

DISADVANTAGE:
• flammable
• toxic & narcotic (high conc.)

A

XYLENE

  • most common
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6
Q

XYLENE
1. What causes it to become milky?
2. How long does it take to brittle and harden the tissue?
3. Clearing time.
4. TRUE or FALSE: Xylene can be used to replace wax.
5. TRUE or FALSE: Xylene can be used for celloidin sections because it dissolves celloidin.

A
  1. When an incompletely dehydrated tissue is immersed in Xylene.
  2. Longer than 3 hours
  3. 15 to 30 minutes
  4. TRUE
  5. FALSE
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7
Q

ADVANTAGE
• miscible with absolute alcohol and paraffin
• NOT carcinogenic
• clears overnight
• for routine purposes

DISADVANTAGES
• tends to acidify (partially filled vessel)
• emit toxic fumes at high conc.

A

Toluene

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8
Q

ADVANTAGES
• miscible with absolute alcohol
• makes tissues transparent
• does NOT brittle & harden

DISADVANTAGE
• flammable
• toxic & carcinogenic

A

Benzene

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9
Q

BENZENE
1. Clearing time
2. Why is it easily eliminated from the tissue?

A
  1. 15 to 60 minutes
  2. Benzene volatilizes rapidly in paraffin oven
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10
Q

• miscible with absolute alcohol
• for tough tissues
• for nervous tissues, lymph nodes, embryos
• for large tissue specimen
• NOT flammable
• tissues tend to float

A

Chloroform

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11
Q

CHLOROFORM
1. Clearing time
2. It is toxic to what organ after prolonged inhalation?
3. How to avoid tissues from floating?
4. TRUE or FALSE: Chloroform is volatile in paraffin oven.
5. TRUE or FALSE: Chloroform evaporates quickly in a water bath

A
  1. 6 to 24 hours
  2. Toxic to Liver
  3. Wrap tissue with absorbent cotton gauze to facilitate sinking.
  4. FALSE.
    » Chloroform is NOT volatile in paraffin oven.
  5. TRUE.
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12
Q

• miscible with 96% absolute alcohol
• does NOT dissolve aniline dyes
• extremely slow
• very expensive
• milky
• hard to eliminate in paraffin bath
• causes drying out of tissue
• produces crystals at 35°C

A

Cedarwood Oil

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13
Q

CEDARWOOD OIL
1. How long does it take to clear celloidin?
2. What to do when Cedarwood oil is hard to eliminate in paraffin bath?
3. How to prevent drying out of tissue?
4. What to do when crystals are produced?
5. The reasons it becomes milky.

A
  1. 5 to 6 days
  2. Transfer tissue from oil to benzene for 1½ hour, then place tissue in wax.
  3. Superimpose absolute alcohol on clearing agent’s surface. Once saturated, transfer specimen to fresh Cedarwood.
  4. Heat solution up to 200°C to dissolve crystals
  5. a. Cedarwood was not filtered before used
    b. Prolonged storage
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14
Q
  • aniline dyes are removed
  • celloidin is dissolved
A

Clove oil

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15
Q
  • similar to Chloroform
  • tissue hardening
  • dangerous to inhale & prolonged exposure
A

Carbon Tetrachloride

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16
Q
  • dehydrating and clearing agent
  • non toxic
  • offensive odor
A

Tetrahydrofuran

17
Q
  • miscible with water & paraffin
  • changed 3 times within 4 hours
  • toxic to liver
  • maybe embedded with paraffin within 4 hours after fixation
  • maybe transferred to dioxane straight from Bouin’s fluid or a formalin fixative
A

Dioxane

18
Q

The complete removal of clearing agent by substitution of impregnating solution

A

Impregnation/Infiltration

19
Q

Characteristics of an Impregnating Reagent

A
  • empty spaces of tissue and cells
  • hardens tissue
  • at least 25 times the volume of the tissue
20
Q

Types of Tissue Impregnation

A
  • paraffin wax
  • celloidin wax
  • gelatin
21
Q
  • cut with ease without distortion
  • nucleic acids may be recovered decades after fixation
  • many staining procedures are permitted
  • overheating makes specimen brittle
  • inadequate impregnation makes tissue soft & shrunken
  • not recommended for fatty tissues
A

Paraffin wax
• simplest, most common, best routinely used

22
Q

Ways of paraffin wax impregnation (3)

A
  • manual processing
  • automatic processing
  • vacuum embedding
23
Q

PARAFFIN WAX IMPREGNATION
1. Automatic processing example
2. Vacuum embedding example

A
  1. Autotechnicon
  2. Vacuum oven, vacuum fume
24
Q

PARAFFIN WAX IMPREGNATION
1. in vacuum embedding, complete impregnation is reduced by ___%.
2. This hastens the removal of air bubbles & clearing agent
3. Used for the complete removal of solvents, and for urgent biopsies & delicate tissues.

A
  1. 25% to 75%
  2. Vacuum
  3. Vacuum embedding
25
Q

An automated system to process tissue specimen from fixation down to infiltration

A

Autotechnicon

26
Q

A purified form of nitrocellulose

A

Celloidin

27
Q
  • for specimens with large hollow cavities
  • for hard and dense tissues (bones and teeth)
  • for large tissue sections of whole embryos
  • for neurological tissues
A

Celloidin

28
Q
  • does not require heat
  • very slow
  • unable to cut tissue into very thin sections
  • flammable vapor
  • very volatile
A

Celloidin

29
Q
  • recommended when dehydration is to be avoided
  • water soluble
  • for delicate specimens & frozen tissue sections
  • does NOT require clearing
A

Gelatin

30
Q

GELATIN
1. Function of phenol
2. Tissue should not be ____ thick.

A
  1. Prevents mold formation
  2. NOT be more than 2-3 mm
31
Q

GELATIN: Steps

After the fixative has been washed out, the tissue is placed in:

A
  1. 10% gelatin with 1% phenol for 24 hours
  2. Transfer it to 20% gelatin with 1% phenol for 12 hours
  3. Transfer it to 20% gelatin with 1% phenol
  4. Cooled in refrigerator until impregnation and embedding are completed
32
Q
  • Recommended for embryos, insects, and very delicate specimens
  • can clear 70% alcohol
A

Aniline Oil

HCT (9 decks)

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