FIXATION

Question 1

• The first and most critical step in histotechnology. prevents degeneration ,decomposition, putrefaction and distortion of tissues after removal from the body.

Answer 1

Fixation

Question 2

• Primary aim of fixation:

Answer 2

– to preserve the morphologic and chemical integrity of the cell in as life- like manner as possible

Question 3

• Secondary goal of fixation:

Answer 3

– to harden and protect the tissue from the trauma of further handling, – so it is easier to cut during gross examination

Question 5

• To preserve the tissue
– _ stops all cellular activities
• To prevent breakdown of cellular elements
– prevents autolysis by inactivating the __ or by chemically altering, stabilizing and making the tissue components insoluble
– protects the tissue from further decomposition after death due to bacterial or fungal colonization
- • To coagulate or precipitate __ substances
– Fixation renders insoluble certain tissue components that may otherwise leak out during subsequent histologic handling

Answer 5

Fixation
Fixatives
lysosomal enzymes
protoplasmic

Question 6

2 Basic Mechanisms

Answer 6

Additive fixation
Non-additive fixation

Question 7

chemical constituent of the fixative is taken in and becomes part of the tissue by forming cross-links or molecular complexes and giving stability to the protein.
Ex. ___,___,__

Answer 7

Additive fixation
Formalin, mercury, osmium tetroxide

Question 8

fixative is not incorporated into the tissue, but alters the tissue composition and stabilizes the tissue by removing the bound water within the protein molecule.
Ex. ____

Answer 8

Non-additive fixation
Alcoholic fixatives

Question 9

Effects of Fixatives

Answer 9

• Preserve the morphologic and chemical integrity of the cell • Harden soft & friable tissues
• Inhibit bacterial decomposition
• Act as mordants or accentuators

Question 10

• Osmolality

Answer 10

– Slightly hypertonic solutions 400-450 mOsm
- Isotonic – 340 mOsm

Question 11

• Concentration

Answer 11

– Formaldehyde – 10%
– Glutaraldehyde – 3%
• 0.25% ideal conc in Immuno EM

Question 12

Duration of fixation

Answer 12

– Usually for 2-6 hours
– Formalin can be washed after fixation for 24 hours

Question 13

Hydrogen ion concentration –

Answer 13

pH 6and 8 satisfactory

Question 14

• Temperature

Answer 14

– Room temp
– T. Processors: 400C,
– EM & Histochemistry: 0-40C
– Formalin Heated to 600C:rapid fixation
– Formalin at 1000C - fix tissues with tuberculosis

Question 15

• Thickness of section

Answer 15

– 1-2 mm2 for electron microscopy – 2 cm2 for light microscopy
– Large solid tissue
• Uterus
• Brain (suspended whole in 10% buffered formalin for 2-3 weeks)

Question 16

Practical Considerations of Fixation

Answer 16

• Speed
– prevent autolysis and putrefaction
• Penetration
– Formalin diffuses at 1 mm/hr
• Volume
– Amount of fixative – 10-25x the volume of tissue to be fixed
• DurationofFixation
– Fibrous organs take longer to fix than biopsies or scrapings
– Can be cut down using heat, vacuum, agitation or microwave

Question 17

Characteristics of fixation
• ____,___,__
• Must be ___
• Inhibit ___
• Must permit ___ and ___
• Must make cellular components insoluble to __ solutions.

Answer 17

• Cheap, stable, safe to handle
• isotonic
• bacterial decomposition
• rapid and even penetration of tissues
• hypotonic

Question 18

Types of Fixatives

Answer 18

According to COMPOSITION
1. Simple Fixatives: one component
2. Compound Fixatives: 2 or more components
• According to ACTION
– Microanatomical fixatives
– Cytological fixatives
– Histochemical fixatives

Question 19

Aldehyde

Answer 19

Formaldehyde, Glutaraldehyde

Question 20

Metallic Fixatives

Answer 20

Ø Mercuric chloride
Ø Chromate fixatives
> Potassium dichromate
>Chromic acid

Question 21

Lead fixatives

Answer 21

Ø Acetone
Ø Alcohol
ØPicric acid
ØAcetic acid
ØOsmium tetroxide (Osmic acid)

Question 22

Simple Fixatives

Answer 22

1. Aldehydes
2. Metallic Fixatives
   3 Lead Fixative
   4 Heat

Question 23

made up of two or more fixatives which have been added.

Answer 23

Compound Fixatives

Question 24

• Permit general microscopic study of tissue structures

Answer 24

Microanatomical Fixatives

Question 25

- Preserve specific parts

Answer 25

Cytological Fixatives Nuclear Cytoplasmic

Question 26

- Preserve the chemical constituents of cells and tissues

Answer 26

Histochemical Fixatives

Question 27

Microanatomical Fixatives

Answer 27

1. 10% Formol Saline 2. 10% Neutral Buffered Formalin 3. Heidenhein’s susa 4. Zenker’s solution 5. Zenker’s formol (Helly’s solution) 6. Bouin’s solution 7. Brasil soln

Question 28

– preserve nuclear structure (chromosomes). Contain glacial aa, pH 4.6 or less

Answer 28

Nuclear fixatives 1. Flemming’sFluid 2. Carnoy’sFluid 3. Bouin’sFluid 4. Newcomer’sFluid 5. Heidenhainsusa

Question 29

– preserve cytoplasmic structure, no glacial aa, pH > 4.6

Answer 29

Cytoplasmic Fixatives 1. Flemming’s Fluid without acetic acid 2. Helly’s Fluid 3. Regaud’s Fluid (Muller’s fluid) 4. Orth’s Fluid

Question 30

1. Formol Saline 10% 2. Absolute Ethyl Alcohol 3. Acetone 4. Newcomer’s Fluid

Answer 30

Histochemical Fixatives

Question 31

• 10% - most widely used • A gas produced by the oxidation of __ • ___ - unsatisfactory for routine fixation • Dilution – _ • Usual fixation time – __ • Buffered to

Answer 31

Formaldehyde (formalin) - methyl alcohol - Pure stock solution of 40% - 1:10 or 1:20 - 24 hours - ph 7 with phosphate buffer

Question 32

Formaldehyde (formalin) ADVANTAGES

Answer 32

- Cheap, readily available, easy to prepare, relatively stable - Compatible with most stains - Preserves fats, glycogen and mucin - Allows tissue enzymes to be studied because it does not precipitate proteins - Recommended for nervous tissue preservation - Allows natural tissue colors to be restored; recommended for colored tissue photography - Tolerant fixative used for mailing specimen

Question 33

DISADVANTAGES of formaldehyde ( Formalin)

Answer 33

- May cause sinusitis, allergic rhinitis, excessive lacrimation or allergic dermatitis - May produce considerable shrinkage of tissues - A soft fixative and does not harden some cytoplasmic structures adequately enough for paraffin embedding

Question 34

Ø Microanatomical fixative Ø Recommended for fixation of central nervous tissues and general postmortem tissues for histochemical examination Ø Fixation time: ___ Ø 48 hours at __ Ø Preserves __ and __ Ø Demonstrates ___ and __

Answer 34

10% Formol Saline - 24 hours at 35C / 95F - 20-25 °C / 65-77 °C - enzymes and nucleoproteins - fats and mucins

Question 35

Slow fixative Metachromatic reaction of amyloid is reduced

Answer 35

10% Formol Saline

Question 36

• Recommended for preservation and storage of surgical, post-mortem and research specimen • Fixation time: _ • Prevents precipitation of __ • Best fixative for tissues containing __ and__

Answer 36

10% Neutral buffered formalin or Phosphate-buffered formalin 4 -24 hours acid formalin pigments iron pigments and for elastic fibers

Question 37

• Longer to prepare • Positivity of mucin to __ is reduced • Reactivity of myelin to ___ is reduced • Inert towards lipids, especially ___

Answer 37

10% Neutral buffered formalin or Phosphate-buffered formalin disadv PAS Weigert’s iron hematoxylin neutral fats and phospholipids