Fixation Flashcards
The killing, penetration and hardening of tissues
Fixation
First and most crucial step in tissue processing
Fixation
Fixative of Choice
10% Neutral Buffered Formalin
Tissue to Fixative Ratio
1:10 or 1:20
Optimal pH for Fixative
6-8
Optimal Temp for routine fixative
Room Temp to 45C
Types of Fixative based on Composition
Simple & Compound
Type based on composition:
made of only 1 component
simple
Type based on composition:
consists of 2 or more components of fixatives
compound
Types of Fixative based on Action
Microanatomical
Cytological
Histochemical
Type of fixative based on action:
General study of tissue w/o structure alteration
Microanatomical
2 types of Cytological Fixative
Nuclear
Cytoplasmic
Aldehyde Fixative:
- Saturated Formaldehyde + 10% NaCl
- For fixation of CNS tissues and general post mortem tissues for histochemical examination
10% Formol-Saline
Aldehyde Fixative:
- Best general tissue fixative
10% Neutral Buffered Formalin
Aldehyde Fixative:
- Saturated aq. Mercuric chloride + 40% Formaldehyde
- For routine post mortem tissues and Silver Reticulum staining methods
Formol-Corrosive
Aldehyde Fixative:
- Has 95% ETOH, Picric Acid, and GHAc
Gendre’s
Aldehyde Fixative:
- For GI tissues, prostate biopsies, and bone marrow
Hollande’s
Aldehyde Fixative:
- Made up of 2 formaldehyde residues linked by 3 carbon chains
- For enzyme histochemistry and electron microscopy
Glutaraldehyde
Glutaraldehyde concentration for immune electron microscopy
0.25%
Glutaraldehyde concentration for small tissue fragments
2.5%
Most common glutaraldehyde concentration
3%
Glutaraldehyde concentration for large tissue fragments
4%
Aldehyde Fixative:
-Polymer of Formalin
- For ultrathin and electron microscopy
Paraformaldehyde
Aldehyde Fixative:
- Acrolein in glutaraldehyde or formalin
- for electron microscopy and electron immunocytochemistry
- Karnovsky’s Paraformaldehyde
- Karnovsky’s Glutaraldehyde
3 types of Metallic Fixatives
Mercuric Chloride
Chromates
Lead
Most common type of metallic fixative
Mercuric Chloride
Mercuric Chloride Fixative:
- HgCl2 + Potassium dichromate + glacial acetic acid
- Good general fixative for adequate preservation of all kinds of tissues
Zenker’s Fluid
Mercuric Chloride Fixative:
- HgCl2 + Potassium dichromate + strong formalin (40%)
- For pituitary gland, BM, & blood-containing organs
Zenker-Formol (Helly’s)
Mercuric Chloride Fixative:
- HgCl2 + NaCl + TCA + glacial acetic acid + formalin
- for skin tumor biopsy
Heidenhain’s Susa
Mercuric Chloride Fixative:
- recommended for hematopoietic and lymphoid tissue
B-5 Fixative
Mercuric Chloride Fixative:
- for making smears of loose cells on slides
Shaudinn’s Fluid
Chromate Fixatives:
- preserves carbohydrates; precipitates all protein
Chromic Acid 1-2%
Chromate Fixative:
- for chromatin, mitochondria, mitotic figures, golgi bodies, RBC and colloid containing tissues
Regaud’s / Muller’s Fluid
Chromate Fixative:
- study of early degenerative processes and necrosis; Rickettsia and other bacteria
- preserves myelin
Orth’s Fluid
Chromate Fixative:
- preserves lipids & mitochondria at pH 4.5 to 5.2; cytoplasm, chromatin, and chromosome are fixed
Potassium Dichromate
Metallic Fixative:
Preserves acid mucopolysaccharide
Lead
Normally used in strong aqueous solution
Picric Acid Fixatives
Picric Acid Fixative:
- for embryo and pituitary biopsies, and tissue to be stained with Masson’s Trichrome
Bouin’s
Picric Acid Fixative:
- better and less messy than Bouin’s
Brasil’s Alcoholic Picroformol
Picric Acid Fixative:
- incorporated in compound fixatives
- solidifies at 17C
- important for nuclear fixatives
- destroys mitochondira and golgi elements
Glacial Acetic Acid
Fixative that causes polarization of glycogen
Alcohol Fixative
Alcohol Fixative:
-most rapid tissue fixative
- fixing brain tissues for rabies dx
- fixes nissl granules and cytoplasmic granules
Carnoy’s Fluid
Alchohol Fixative:
- enzyme studies; does not fix but preserves glycogen
Ethanol
Alcohol Fixative:
- for dry and wet smears, bone marrow smears, and bacterial smears
Methanol / Wood Alcohol
Alcohol Fixative:
- for touch prep smears to be Wright-stained
Isopropanol
Alcohol Fixative:
- for mucopolysaccharide
Newcomer’s Fluid
Alcohol Fixative:
- for sputum
Gendre’s (Alcoholic Formalin)
Tissue-to-fixative ratio of Osmic Acid Fixatives
1:5
Osmic Acid Fixative:
- nuclear fixative
- most common osmic acid fixative
Flemming’s Solution
Osmic Acid Fixative:
- cytoplasmic fixative
Flemming’s Solution w/o Glacial acetic acid
Osmic Acid Fixative:
- incorporated into compound fixatives
- marked swelling effect on tissues
- poor penetration thus for small pieces of tissues or bones
- weak decalcifying agent, has softening effect on dense fibrous tissues
Trichloroacetic acid
Osmic Acid Fixative:
- use at ice cold temps (-5 to 4C)
- for water diffusible enzymes
- fixes brain tissue (for rabies)
Acetone
Involves thermal coagulation of tissue proteins for rapid diagnosis
Heat Fixation
Optimum temp for Heat Fixation
45 - 55C
To ensure further and complete hardening and preservation of tissues
Secondary Fixation
Technique whereby a primary fixed tissue is placed in aq. solution of 2.5% to 3% potassium dichromate for 24hrs
Post-Chromatization
Process of removing excess fixatives
Washing Out
used for washing out excess chromate, formalin, and osmic acid
Tap water
used for washing out of picric acid
50% - 70% Alcohol
used for washing out of Mercuric Fixatives
Alcoholic Iodine
