LESSON 2: FIXATION Flashcards
✓ 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
FIXATION
primary purpose of fixation
Preservation
secondary purpose of fixation
Protection
✓ capable of forming cross-links between proteins
➢ stabilizes tissue components making them insoluble to lysosomal enzymes
✓ capable of inactivating lysosomes
Fixative agents
Types of Fixative that becomes part of the cross-link itself
Additive
Types of Fixative that facilitates the removal of water in order for cross-links to form
Non-Additive
FIXATIVE AGENTS ARE RETARDED BY:
- Increase in size and thickness
- Presence of mucus
- Presence of fats
- Presence of blood
- Decrease in temperature
FIXATIVE AGENTS ARE ENHANCED BY:
✓ Decrease in size and thickness
✓ Presence of agitation
✓ Presence of heat
pH considered in FIXATIVE AGENTS
6 to 8
Temperature considered in Routine Manual Fixation
Room Temp (20 to 22oC)
Temperature considered in Routine Automated Fixation
40 oC
Temperature considered in Electron Microscopy Fixation
0 to 4oC
Temperature considered in Formalin at very urgent biopsies
Formalin at 60 oC or 60 oC
Temperature considered in Formalin for the diagnosis of tuberculosis
Formalin at 100 oC or 100 oC
Temperature considered in DNA Fixation
65 oC
Temperature considered in RNA Fixation
45 oC
Size and Thickness in Light Microscopy
2cm2 by 0.4cm thick
Size and Thickness in Electron Microscopy
1 to 2 mm2
Size and Thickness in Lung Edema
1 to 2 cm thick
Osmolality in Light microscopy
slightly
hypertonic (400-450 mOsm)
Osmolality in Electron Microscopy
more or less isotonic (340 mOsm)
Concentration of Formaldehyde
10%
Concentration of Glutaraldehyde
3%
Volume in Routine Fixation
10 to 25 times the
volume of the specimen
Volume in Museum Fixation
50 to 100 times the
volume of the specimen
On Tissue/Organ Preparations an MT must cover this with several layers of gauze
Air-filled Lungs
On Tissue/Organ Preparations an MT must: Dilate this with cotton soaked in fixative
and Completely open specimen
Hollow Organs
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
On Tissue/Organ Preparations an MT must: ✓ Fixed it whole
✓ Inject it formol-alcohol
Eyes
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
On Tissue/Organ Preparations an MT must:
✓ Stretch this with sutures on each end
✓ Lay it in flat moist filter paper
Muscles
- Cheap & economical
- Stable and safe to handle
- Fast acting, permits rapid an even penetration
- Inhibits bacterial decomposition & autolysis
- Must harden tissues
- At least isotonic
- Must render tissues insensitive to subsequent processing
- Must be compatible with many staining procedures
CHARACTERISTICS OF A GOOD FIXATIVE
A TYPE OF FIXATIVES According to Composition made up of only one component substance
Simple Fixatives
A TYPE OF FIXATIVES According to Composition made up of two or more fixatives
Compound Fixatives
A TYPE OF FIXATIVES According to Action: permit general microscopic study of tissue structures and normal intercellular relationship of tissues
Microanatomical Fixatives:
A TYPE OF FIXATIVES According to Action: preserve specific cellular components
Cytological Fixatives
Under Cytological Fixatives this
✓ preserve nuclear structures
✓ contain glacial HAc
✓ pH 4.6 or less
Nuclear Fixatives
▪ high affinity to nuclear chromatin
▪ destroys mitochondria and Golgi bodies
glacial HAc
Under Cytological Fixatives this
✓ preserve cytoplasmic structures
✓ do not contain glacial HAc
✓ pH of more than 4.6
Cytoplasmic Fixatives
preserve chemical constituents
Histochemical Fixatives
Histochemical Fixatives for Lipids
mercuric chloride or potassium dichromate
Histochemical Fixatives for Phospholipids
Baker’s formol-calcium
Histochemical Fixatives for Cholesterol
digitonin
Histochemical Fixatives for Carbohydrates
alcoholic fixatives
Histochemical Fixatives for Glycogen
Rossman’s fluid or cold absolute alcohol
Histochemical Fixatives for Proteins
neutral buffered formol saline or formaldehyde vapor
Histochemical Fixatives for Electron microscopy
double fixation
Mixture of Fixatives for Electron Cytochemistry
Karnovsky’s paraformaldehydeglutaraldehyde solution
Mixture of Fixatives for Acrolein
mixture of glutaraldehyde or fomaldehyde
✓ rapid penetration, preserves morphology and enzyme
activity at low concentrations
✓ immersion fixation of surgical biopsies
Acrolein
• 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
- Heidenhain’s SuSa
- Newcomer’s
- Bouin’s
- Flemming’s
- Carnoy’s
CYTOLOGIC - NUCLEAR
• Helly’s • Orth’s • Regaud’s/Muller’s • Flemming’s • Formalin with post-chroming
CYTOLOGIC - CYTOPLASMIC
- 10% Formol saline
- Absolute ethanol
- Newcomer’s
- Acetone
HISTOCHEMICAL
for routine paraffin sections, electron microscopy, histochemistry and
enzyme studies
ALDEHYDE FIXATIVES
▪ 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)
▪ 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
▪ 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
▪ routine post-mortem tissues
▪ for lipids, especially neutral fats and phospholipids
▪ no need for washing-out
Formol-Corrosive
▪ used to fix sputum
▪ for the demonstration of immunoperoxidase activity
Alcoholic Formalin
▪ 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
✓ most common metallic fixative
✓ tissue photography
✓ tissues contain black precipitates of mercury (except Susa)
Mercuric Chloride
✓ fixing small pieces of liver, spleen, connective tissues fibers and nuclei
Zenker’s Fluid
removal of mercuric deposits in tissues
De-zenkerization
✓ pituitary gland, bone marrow and blood containing organs (spleen and
liver)
Zenker-formol
✓ for tumor biopsies
Heidenhain’s Susa Solution
✓ for bone marrow biopsies
B-5 Fixative
✓ preserves carbohydrates
Chromic Acid
✓ preserves lipids and mitochondria
Potassium Dichromate
✓ demonstration of chromatin, mitochondria, mitotic figures, Golgi bodies, RBC and colloid-containing tissues
Regaud’s Fluid
✓ study of early degenerative processes and tissue necrosis
✓ demonstrates Rickettsia and other bacteria
Orth’s Fluid
✓ demonstration of acid mucopolysaccharides
Lead Fixatives
Picrates are soluble in water, therefore, tissues should not be washed in running tap
water (use 70% alcohol instead)
PICRIC ACID FIXATIVES
✓ fixation of embryos and pituitary biopsies
Bouin’s Solution
✓ fixative for glycogen
Brasil’s Alcoholic Picroformol Fixative
✓ fixation of nucleoproteins
GLACIAL ACETIC ACID
Preserves but does not “fix” glycogen granules
95 % Ethanol
For dry and wet smears, blood and bone marrow samples
Methanol
for touch preparations
Isopropyl alcohol (95%)
▪ most rapid fixative
▪ for chromosomes, lymph glands, urgent biopsies and brain tissue for the
diagnosis of rabies
Carnoy’s fluid
demonstration of mucopolysaccharides and nucleoproteins
Newcomer’s
Permanently fixes fats and recommended for fixing nuclear structures
Flemming’s (chrome-osmium HAc)
For cytoplasmic structures
Flemming’s w/o acetic acid
may also be used as a weak decalcifying agent
TRICHLOROACETIC ACID
✓ use at cold temperature (-5 to 4oC)
✓ fixation of brain tissues for rabies diagnosis
ACETONE
Under ALDEHYDE FIXATIVES this is a Routinely used fixative
Formaldehyde
Used for electron microscopy
Glutaraldehyde
