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Fixation Lec Flashcards

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1
Q
A
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2
Q

What is the primary goal of fixation in histotechnology?

A

Preserve morphologic and chemical integrity of the cell in as life-like manner as possible

The secondary goal is to harden and protect the tissue for easier handling.

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3
Q

What is the ideal time to perform fixation after interruption of blood supply?

A

20 – 30 minutes

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4
Q

What is the recommended fixative to tissue ratio?

A

20:1

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5
Q

What are the two main mechanisms involved in fixation?

A
  • Additive fixation
  • Non-additive fixation
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6
Q

What is additive fixation?

A

The chemical constituent of the fixative is taken in and becomes part of the tissue by forming cross-links or molecular complexes.

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7
Q

What is non-additive fixation?

A

Tissue composition is altered and stabilized through removal of bound water attached to H-bonds within the protein molecule.

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8
Q

What are microanatomical fixatives used for?

A

Permit the general microscopic study of tissue structures without altering the structural pattern.

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9
Q

What are cytological fixatives used for?

A

Preserve specific parts and particular microscopic elements of the cell.

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10
Q

What is the purpose of secondary fixation?

A

To facilitate and improve the demonstration of particular substances and make special staining techniques possible.

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11
Q

What is ‘washing out’ in the context of fixation?

A

Process of removing excess fixative from the tissue after fixation to improve staining.

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12
Q

What type of fixative is recommended for demonstrating lipids in tissues?

A

Cryostat of frozen sections using fixatives containing mercuric chloride and potassium dichromate.

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13
Q

What is the most commonly used fixative for amino acid histochemistry?

A

Neutral buffered formal saline

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14
Q

What are the two types of fixation for electron microscopy?

A
  • Osmium tetroxide
  • Glutaraldehyde
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15
Q

What is the advantage of using formalin as a fixative?

A

Cheap, easy to prepare, and readily available.

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16
Q

What is the disadvantage of using 10% formal-saline?

A

Slow fixative and reduced metachromatic reaction of amyloid.

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17
Q

What is the formula for 10% neutral-buffered formalin?

A
  • Sodium Dihydrogen phosphate, anhydrous: 6.5 gm
  • Sodium Dihydrogen Phosphate * H20: 4 gm
  • Distilled water: 900 mL
  • 40% formaldehyde: 100 mL
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18
Q

What does glutaraldehyde preserve in tissues?

A

Cellular structures with less tissue shrinkage.

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19
Q

What is the application of Karnovsky’s fixative?

A

For light microscopy in resin embedding and sectioning, and for electron microscopy.

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20
Q

What is the disadvantage of mercuric chloride as a fixative?

A

May produce black granular deposits on tissues.

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21
Q

Recommended for trichome staining and congested specimens

A

ZENKER’S

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22
Q

What is the application of Heidenhain’s Susa solution?

A

For tumor biopsies, especially of the skin.

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23
Q

What is the fixation time for formalin?

A

5-10 minutes

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24
Q

What is the fixation time for glutaraldehyde for larger tissues?

A

6-8 hours up to 24 hours

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25
True or False: Formalin pigment is produced by using 10% neutral-buffered formalin.
False
26
Fill in the blank: The primary component of nuclear fixatives is _______.
glacial acetic acid
27
What pH should cytoplasmic fixatives maintain?
> 4.6
28
What is the purpose of acetone fixation?
Fix cells in -20°C acetone for 5-10 minutes without a permeabilization step.
29
What is the fixation time for methanol-acetone mix fixation?
5-10 minutes at -20°C
30
What is the application of 4% paraformaldehyde?
For immunocytochemical analysis.
31
What are the two categories of fixatives?
* Physical Fixatives * Chemical Fixatives
32
What is the formula for Excellent cytologic fixative?
Mercuric Chloride: 45 gm, Sodium chloride: 5 gm, Trichloroacetic acid: 20 gm, Glacial acetic acid: 40 ml, 40% Acid formaldehyde: 200 ml, 40% Distilled water: 800 ml
33
What are the advantages of Excellent cytologic fixative?
* Penetrates and fixes tissues rapidly and evenly * Produces minimum shrinkage and hardening of tissues * Permits easier sectioning of large blocks of fibrous connective tissues * Reduces processing time
34
What are the disadvantages of Excellent cytologic fixative?
* RBC preservation is poor * Some cytoplasmic granules are dissolved * Mercuric chloride deposits formation
35
What precaution should be taken after using Excellent cytologic fixative?
Tissue should be transferred directly to a high-grade alcohol (96% or absolute alcohol)
36
What is the fixation time for LILLIE’S B-5 FIXATIVE?
4-8 hours
37
What is the formula for LILLIE’S B-5 FIXATIVE?
Mercuric chloride: 12 g, Sodium acetate anhydrous: 2.5 g, Distilled water: 200 ml, Working solution: B5 stock solution: 20 ml, 40% formaldehyde: 2 ml
38
What are the advantages of LILLIE’S B-5 FIXATIVE?
* Rapid fixation can be achieved in 1 ½ - 2 hours * Produces excellent nuclear detail
39
What is a disadvantage of LILLIE’S B-5 FIXATIVE?
Overfixation hardens the tissue and makes cutting difficult
40
What is the role of CHROMATE in fixation?
Preserves chromatin
41
What is the use of CHROMIC ACID?
Used in 1-2% solution to preserve carbohydrates
42
What does POTASSIUM DICHROMATE preserve?
Mitochondria and lipids
43
What are the advantages of REGAUD’S (MULLER’S) fixative?
* Recommended for chromatin, mitochondria, golgi apparatus & RBC
44
What is a disadvantage of REGAUD’S (MULLER’S) fixative?
Prolonged fixation blackens tissue and produces precipitates of suboxide
45
What is the fixation time for ORTH’S FLUID?
36-72 hours
46
What is the formula for LEAD FIXATIVE?
4% aqueous solution
47
What is an advantage of LEAD FIXATIVE?
Fixes connective tissue mucin
48
What is a disadvantage of LEAD FIXATIVE?
Forms insoluble lead carbonate
49
What is ALCIAN’S BLUE used for?
For acid mucopolysaccharides
50
What is the formula for BRASIL’S ALCOHOLIC PICROFOMOL?
37% Formaldehyde + Picric acid + Ethanol or Isopropyl alcohol + Trichloroacetic acid
51
What is the freezing point of GLACIAL ACETIC ACID?
Freezes and solidifies at 16C
52
What is a key disadvantage of GLACIAL ACETIC ACID?
Destroys mitochondria and golgi elements
53
What is the fixation time for BOUIN’S SOLUTION?
4-18 hours
54
What are the advantages of BOUIN’S SOLUTION?
* Excellent for preserving soft and delicate tissues * Suitable for gastrointestinal tract biopsies and animal embryos
55
What is a disadvantage of BOUIN’S SOLUTION?
Penetrates large tissues poorly and causes hemolysis
56
What is the fixation time for GENDRE’S SOLUTION?
Not specified, but noted for soft and delicate tissues
57
What is the formula for CARNOY’S FIXATIVE?
Absolute alcohol + Chloroform + Glacial acetic acid
58
What is a disadvantage of CARNOY’S FIXATIVE?
Produces RBC hemolysis and dissolves lipids
59
What is the fixation time for NEWCOMER’S FLUID?
12-18 hours at 3C
60
What is the main use of ISOPROPYL ALCOHOL?
Used for fixing touch preparation
61
What is the fixation time for ALCOHOL FIXATIVE?
18-24 hours
62
What is a disadvantage of ALCOHOL FIXATIVE?
Strong reducing agent and should not be mixed with oxidizing agents
63
What is the purpose of FLEMMING’S SOLUTION?
Recommended for nuclear preparation of such section
64
What are the advantages of FLEMMING’S SOLUTION?
* Excellent fixative for nuclear structures * Permanently fixes fat
65
What is a disadvantage of FLEMMING’S SOLUTION?
Poor penetrating agent and solution deteriorates rapidly
66
What is the formula for FLEMMING’S SOLUTION?
Aqueous chromic acid + 1% Aqueous osmium tetraoxide + 2% Glacial acetic acid
67
What is the fixation time for TRICHLOROACETIC ACID?
1-6 hours
68
What is the fixation time for GENDRE’S FIXATIVE?
Not specifically mentioned, but involves rapid diagnosis
69
What is the formula for ACETONE?
For fixation of cryostat sections or for tissues which enzymes must be preserved
70
What is MICHEL’S SOLUTION used for?
Provide stable medium for transport of fresh unfixed tissue
71
What are the temperature ranges used for HEAT FIXATION?
37C – 45C commonly employed temperature
72
What is the optimum temperature for MICROWAVE FIXATION?
45-55C

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