LESSON 2: FIXATION Flashcards
(87 cards)
1
Q
✓ first and most critical step in tissue processing
✓ fixing or preserving fresh tissue for examination
✓ should be done immediately to preserve cellular and tissue morphology
A
FIXATION
2
Q
primary purpose of fixation
A
Preservation
3
Q
secondary purpose of fixation
A
Protection
4
Q
✓ capable of forming cross-links between proteins
➢ stabilizes tissue components making them insoluble to lysosomal enzymes
✓ capable of inactivating lysosomes
A
Fixative agents
5
Q
Types of Fixative that becomes part of the cross-link itself
A
Additive
6
Q
Types of Fixative that facilitates the removal of water in order for cross-links to form
A
Non-Additive
7
Q
FIXATIVE AGENTS ARE RETARDED BY:
A
- Increase in size and thickness
- Presence of mucus
- Presence of fats
- Presence of blood
- Decrease in temperature
8
Q
FIXATIVE AGENTS ARE ENHANCED BY:
A
✓ Decrease in size and thickness
✓ Presence of agitation
✓ Presence of heat
9
Q
pH considered in FIXATIVE AGENTS
A
6 to 8
10
Q
Temperature considered in Routine Manual Fixation
A
Room Temp (20 to 22oC)
11
Q
Temperature considered in Routine Automated Fixation
A
40 oC
12
Q
Temperature considered in Electron Microscopy Fixation
A
0 to 4oC
13
Q
Temperature considered in Formalin at very urgent biopsies
A
Formalin at 60 oC or 60 oC
14
Q
Temperature considered in Formalin for the diagnosis of tuberculosis
A
Formalin at 100 oC or 100 oC
15
Q
Temperature considered in DNA Fixation
A
65 oC
16
Q
Temperature considered in RNA Fixation
A
45 oC
17
Q
Size and Thickness in Light Microscopy
A
2cm2 by 0.4cm thick
18
Q
Size and Thickness in Electron Microscopy
A
1 to 2 mm2
19
Q
Size and Thickness in Lung Edema
A
1 to 2 cm thick
20
Q
Osmolality in Light microscopy
A
slightly
hypertonic (400-450 mOsm)
21
Q
Osmolality in Electron Microscopy
A
more or less isotonic (340 mOsm)
22
Q
Concentration of Formaldehyde
A
10%
23
Q
Concentration of Glutaraldehyde
A
3%
24
Q
Volume in Routine Fixation
A
10 to 25 times the
volume of the specimen
25
Volume in Museum Fixation
50 to 100 times the
| volume of the specimen
26
On Tissue/Organ Preparations an MT must cover this with several layers of gauze
Air-filled Lungs
27
On Tissue/Organ Preparations an MT must: Dilate this with cotton soaked in fixative
and Completely open specimen
Hollow Organs
28
On Tissue/Organ Preparations an MT must: ✓ Fix this first before sampling
✓ Suspend it by a cord tied under the Circle of Willis
✓ Intravascular perfusion using Ringer’s lactate
Brain
29
On Tissue/Organ Preparations an MT must: ✓ Fixed it whole
| ✓ Inject it formol-alcohol
Eyes
30
On Tissue/Organ Preparations an MT must:
| Do Lendrum’s Method: washed out with running water overnight and immersed in 4% aq. phenol solution for 1-3 days
Hard Tissues
31
On Tissue/Organ Preparations an MT must:
✓ Stretch this with sutures on each end
✓ Lay it in flat moist filter paper
Muscles
32
1. Cheap & economical
2. Stable and safe to handle
3. Fast acting, permits rapid an even penetration
4. Inhibits bacterial decomposition & autolysis
5. Must harden tissues
6. At least isotonic
7. Must render tissues insensitive to subsequent processing
8. Must be compatible with many staining procedures
CHARACTERISTICS OF A GOOD FIXATIVE
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A TYPE OF FIXATIVES According to Composition made up of only one component substance
Simple Fixatives
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A TYPE OF FIXATIVES According to Composition made up of two or more fixatives
Compound Fixatives
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A TYPE OF FIXATIVES According to Action: permit general microscopic study of tissue structures and normal intercellular relationship of tissues
Microanatomical Fixatives:
36
A TYPE OF FIXATIVES According to Action: preserve specific cellular components
Cytological Fixatives
37
Under Cytological Fixatives this
✓ preserve nuclear structures
✓ contain glacial HAc
✓ pH 4.6 or less
Nuclear Fixatives
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▪ high affinity to nuclear chromatin
| ▪ destroys mitochondria and Golgi bodies
glacial HAc
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Under Cytological Fixatives this
✓ preserve cytoplasmic structures
✓ do not contain glacial HAc
✓ pH of more than 4.6
Cytoplasmic Fixatives
40
preserve chemical constituents
Histochemical Fixatives
41
Histochemical Fixatives for Lipids
mercuric chloride or potassium dichromate
42
Histochemical Fixatives for Phospholipids
Baker’s formol-calcium
43
Histochemical Fixatives for Cholesterol
digitonin
44
Histochemical Fixatives for Carbohydrates
alcoholic fixatives
45
Histochemical Fixatives for Glycogen
Rossman’s fluid or cold absolute alcohol
46
Histochemical Fixatives for Proteins
neutral buffered formol saline or formaldehyde vapor
47
Histochemical Fixatives for Electron microscopy
double fixation
48
Mixture of Fixatives for Electron Cytochemistry
Karnovsky’s paraformaldehydeglutaraldehyde solution
49
Mixture of Fixatives for Acrolein
mixture of glutaraldehyde or fomaldehyde
50
✓ rapid penetration, preserves morphology and enzyme
activity at low concentrations
✓ immersion fixation of surgical biopsies
Acrolein
51
```
• 10% Formol saline
• 10% Neutral buffered
formalin
• Heidenhain’s SuSa
• Formol sublimate/corrosive
• Zenker’s solution
• Zenker-formol/Helly’s
solution
• Bouin’s solution
• Brasil’s solution
```
MICROANATOMICAL
52
* Heidenhain’s SuSa
* Newcomer’s
* Bouin’s
* Flemming’s
* Carnoy’s
CYTOLOGIC - NUCLEAR
53
```
• Helly’s
• Orth’s
• Regaud’s/Muller’s
• Flemming’s
• Formalin with
post-chroming
```
CYTOLOGIC - CYTOPLASMIC
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* 10% Formol saline
* Absolute ethanol
* Newcomer’s
* Acetone
HISTOCHEMICAL
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for routine paraffin sections, electron microscopy, histochemistry and
enzyme studies
ALDEHYDE FIXATIVES
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▪ gas produced by the oxidation of methyl alcohol
▪ buffered at ph 7 to 8
→ hypoxia in tissues leads to acidity which favors the formation of
Formalin heme pigments (black, polarizable deposits)
▪ Pure Stock: 40% formalin
▪ Dilution: 1:10 (10% solution); 1:20 (5% solution)
▪ Concentration for fixation: 10% formalin
▪ Paraformaldehyde: white precipitate due to prolonged storage
→ may be removed by filtration or addition of 10% methanol
maldehyde (Formalin)
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▪ central nervous tissues and general post-mortem tissues for histochemical
examination
▪ ideal with most stains including silver impregnation
▪ duration of fixation: more than 24 hours (slow fixative)
10% Formol-Saline
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▪ preservation and storage of surgical, post-mortem and research specimens
▪ best fixative for frozen sections
▪ best fixative for iron pigments and elastic fibers
10% Neutral Buffered Formalin
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▪ routine post-mortem tissues
▪ for lipids, especially neutral fats and phospholipids
▪ no need for washing-out
Formol-Corrosive
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▪ used to fix sputum
| ▪ for the demonstration of immunoperoxidase activity
Alcoholic Formalin
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▪ made up of two formaldehyde residues linked by three carbon chains
▪ used in conjunction with osmium tetroxide
▪ Fixation time: ½ hour to 2 hours
Glutaraldehyde
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✓ most common metallic fixative
✓ tissue photography
✓ tissues contain black precipitates of mercury (except Susa)
Mercuric Chloride
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✓ fixing small pieces of liver, spleen, connective tissues fibers and nuclei
Zenker’s Fluid
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removal of mercuric deposits in tissues
De-zenkerization
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✓ pituitary gland, bone marrow and blood containing organs (spleen and
liver)
Zenker-formol
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✓ for tumor biopsies
Heidenhain’s Susa Solution
67
✓ for bone marrow biopsies
B-5 Fixative
68
✓ preserves carbohydrates
Chromic Acid
69
✓ preserves lipids and mitochondria
Potassium Dichromate
70
✓ demonstration of chromatin, mitochondria, mitotic figures, Golgi bodies, RBC and colloid-containing tissues
Regaud’s Fluid
71
✓ study of early degenerative processes and tissue necrosis
| ✓ demonstrates Rickettsia and other bacteria
Orth’s Fluid
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✓ demonstration of acid mucopolysaccharides
Lead Fixatives
73
Picrates are soluble in water, therefore, tissues should not be washed in running tap
water (use 70% alcohol instead)
PICRIC ACID FIXATIVES
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✓ fixation of embryos and pituitary biopsies
Bouin’s Solution
75
✓ fixative for glycogen
Brasil’s Alcoholic Picroformol Fixative
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✓ fixation of nucleoproteins
GLACIAL ACETIC ACID
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Preserves but does not “fix” glycogen granules
95 % Ethanol
78
For dry and wet smears, blood and bone marrow samples
Methanol
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for touch preparations
Isopropyl alcohol (95%)
80
▪ most rapid fixative
▪ for chromosomes, lymph glands, urgent biopsies and brain tissue for the
diagnosis of rabies
Carnoy’s fluid
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demonstration of mucopolysaccharides and nucleoproteins
Newcomer’s
82
Permanently fixes fats and recommended for fixing nuclear structures
Flemming’s (chrome-osmium HAc)
83
For cytoplasmic structures
Flemming’s w/o acetic acid
84
may also be used as a weak decalcifying agent
TRICHLOROACETIC ACID
85
✓ use at cold temperature (-5 to 4oC)
| ✓ fixation of brain tissues for rabies diagnosis
ACETONE
86
Under ALDEHYDE FIXATIVES this is a Routinely used fixative
Formaldehyde
87
Used for electron microscopy
Glutaraldehyde
