UNIT 5: FIXATION

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Definition of Fixation
Procedure to kill, harden and preserve materials for microscopic study.
Process by which cell constituents are fixed in a physiocal, and partly in a
chemical state for the tissue to withstand subsequent treatment with various
reagents with a minimum loss, significant distortion, or decomposition.
FUNCTION OF FIXATION
Prevent
Due to:
decomposition -anoxia (oxygen deprivation)
-ischemia (blood supply deprivation)
-CO2 accumulation
Fixation :
denature/precipitate proteins, form sponge/meshwork, hold
other cell constituents.
Prevent
- Lysosomal enzymes inactivation
autolysis
- chemical alteration of tissue components
Protect from
Fresh tissue in water/air at ambient temp is very suscep to
putrefaction
bacterial/fungal infection.
Fixation : Protect tissue from microorganism damage
Protect from
-Dehydration
damage
-Embedding
-Sectioning
-Mounting
Stabilizes protein skeleton of cell, give some structural support
to resist deformation/crushing.
Insolubilization Insolubilize tissue components
of tissue
FACTORS THAT AFFECT FIXATION OF TISSUES
Buffers and
pH = 6 8
Hydrogen ion
Buffer must not react with fixative (may reduce buffering power
concentration
and fixation ability)
Buffer should not inhibit enzymes or react with the incubation
medium.
Temperature
Room Temperature
-Electron mic, histochemistry = 0 4 C
-Mast cells/ enhancement media = room temp
-Cold temperature retards fixation (inactivate enzyme)
-moderate heat accelerates fixation, hastens autolytic changes,
enzyme destruction
Tissue
Fixation depends on the rate of reaction with tissue components
penetration
and the reversibility of the reaction.
FIXATION MAY BE RETARDED BY:
Size and thickness
Larger tissue, longer fixation time
Presence of Mucus
Prevent complete penetration of fixative (slow and poor)
Presence of Fat
Fatty tissues should be cut thin and fixed longer
Presence of Blood
Tissues with blood should be flushed out with saline by
arterial cannulization before fixation.
Cold temperature
Inactivates enzyme
FIXATION ENHANCER
Size & thickness
Agitation
Moderate heat
37-56
Substances added
to the vehicle
Use of detergent
Concentration of
fixatives
Duration of fixation

Smaller, thinner = less fixative & time

Accelerate fixation
Accelerate fixation, hasten autolytic changes and enzyme
destruction
Enhance fixation
Allows entry of large molecules for immunofluorescent tech
without too great damage to the cell.
Cost effectiveness, solubility.
Staining can alter concentration of fixatives.

PRINCIPLES OF FIXATION
1. Autopsy should be fixed as soon as death.
2. Surgical specimen should be fixed soon, to prevent drying of surface layers and
ultimate tissue distortion.
3. Label and identify
4. IF ref, avoid slow freezing near 0 C, can cause ice crystal artifacts.
Dont repeat thawing and freezing of tissue, may destroy cellular organelles.
5. Tissue should be taken deep enough from the organ to show the normal
anatomic components
6. < 5mm thick with minimum squeezing and handling.

7. Mark and keep purulent materials, exudate/transudate.

8. Adequate amount of fixative. (10-20x)
9. Exceeding the required period for fixation may cause hardening and brittleness.
10. Avoid drying to prevent shrinkage and distortion of tissue with loss of cellular
details.
11. Adequate supply of fixative
12. Inject solid organ with enough fixative to ensure complete penetration &
fixation
13. Hollow organs (stomach, intestine) shoule be packed with cotton soaked in
fixative.
14. Air filled organs (lungs) float on fixative. Avoid by covering with gauze layers
15. Dense tissues are poorly penetrated, require longer fixation
16. Dont dissect eyes. Inject formol-alcohol
17. Frozen section may lead to formation of ice crystals artifacts.
DIFFICULTIES DURING FIXATION
Early cell autolysis
Removal of substances soluble in fixing
agents
Artifact pigments
Soft and feather like tissue
Inactivated enzymes
Shrinkage/ swelling of cells
Brittle and hard tissue blocks

Failure to mix immediately, insufficient

fixative
Wrong fixative
Incomplete fixation
Incomplete fixative
Wrong fixative
Over fixation
Prolonged fixation

POST MORDANTING (SECONDARY FIXATION)

Placing already fixed tissue in a second fixative
Purpose:
-improve demonstration of a particular subs
-make special staining tech (2nd = mordant)
-ensure complete hardening and preservation
1) Hellys fluids
2) Formol-mercuric chloride
GENERAL EFFECTS OF FIXATIVES
-Harden the soft and friable tissues
-facilitates easy handling and cutting
-make cell insensitive to damage and distortion by hyper/hypotonic solution
-inhibit bacte decomposition
-increase optical differentiation of cells and tissue
-act as mordant/accentuators to promote and hasten staining
-reduce risk of infection during handling and actual processing
CHARACTERISTICS OF GOOD FIXATIVE
-Cheap
-stable
-safe to handle
-kill cell quickly
-minimum distortion -inhibit bacte dcomposition & autolysis
-minimum shrinkage -penetrate rapidly & evenly
-harden tissue
-isotonic
-preserve volume
-nonallergenic or nontoxic
COMPOSITIONS OF FIXATIVE
1. Simple Fixative ( made up of only 1 component subs )
-aldehydes
(formaldehyde, glutaraldehyde)
-metallic fixative
(mercuric chloride, chromate fixative <K dichromate, chromic acid>, lead fixative)
-picric acid
-acetic acid
-acetone
-alcohol
-osmium tetroxide
-heat
2. Compound fixative (2/more fixatives, optimal combined effect)
ACTIONS OF FIXATIVE
Microanatomical Fixative
-microscopic study
-dont alter structural pattern

Cytological fixatives

-10%formol saline
-10%neutral buffered formalin
-heidenhains susa
-formol sublimate
-zenkers solution
-zenkers formol (helly)
-bousins solution
-brasils solution
a) Nuclear fixative pH 4.6

-preserve specific parts of cell

Histochemical fixatives
-preserve chemical constituents of cells
and tissue

-preserve nuclear structure

-flemmings fluid
-carnoys fluid
-bouins fluid
-newcomers fluid
-heidenhains susa
b) Cytoplasmic fixatives pH > 4.6
-preserve cytoplasmic structure
-no glacial acetic acid
-flemming + acetic acid
-helly
-post chroming formalin
-regaud/moller
-orths fluid
-formol saline 10%
-absolute ethyl alcohol
-acetone
-newcomers fluid

I. ALDEHYDE FIXATIVE = ROUTINE PARAFFIN SECTION

FORMALDEHYDE (volatile gas by methyl alcohol oxidation)
-water soluble
-30-40% formalin
-pure formalin = overharden outer layer.
-diluted in water/buffer (1:10 or 1:20)
Fixation time : 24 hours
ADVANTAGE
DISADVANTAGE
10%
-Cheap
-irritating fumes
Formalin
-Easy to prepare
-acidity forms formic
-Relatively stable
acid, brown pigment
-Readily available
granules.
-Compatible with
-prolonged causes:
most stain
bleaching, fat
-penetrates tissue
dispersal
well
-study nervous tissue
-preserves glycogen
Formal
-Ideal for most
-slow fixative > 24hr
saline
staining including
-reduces
silver impregnation
metachromatic rx
Netural
Best fixative for tissue Inert towards lipids
buffered
with iron pigments
formalin
Formol
-Minimum shrinkage
Forms mercuric
corrosive/
-Excellent for silver
chloride deposits
sublimate
reticulum
Removal of formalin pigments:
Kardesewitch, lillie, picric acid

PRECAUTIONS
-10% methanol
(PRSV
formaldehyde) =
prevents formic
acid

GLUTARALDEHYDE: 2 formaldehyde residues + 3 C chains

ADVANTAGE
DISADVANTAGE
Doesnt cause dermatitis
More expensive
Preserve plasma protein better
Less stable

Mercuric Chloride
(common, used in
saturated aq 5-7%)
Chromate Fixations

II. METALLIC FIXATIVE

Zenkers fluid
Zenker-formol/ helly
Heidenhains susa
Chromic Acid
-ppt all protein
-adequately preserves carbs
Potassium dichromate
-preserves lipids
-preserves mitochondria
Regauds/Mollers
-prolonged fixation blackens tissue pigments
Orths fluid
-demonstrates rickettsiae and other bacteria

Lead fixatives
-for acid mucopolysaccharides
-fixes conn.tissue mucin
III. PICRIC ACID FIXATIVES (trinitrophenol : protein & glycogen fixative
Bouins solution
For fixation of embryos
Brails alcoholic picroformol Excellent fixative for glycogen

Rossmans fluid
Best fixative for glycogen retention & preservation
IV. GLACIAL ACETIC ACID (forms compound solution, solidifies at 17C)
V. ALCOHOL FIXATIVES
Ethyl alcohol
Fixes blood, tissue films, smear
Methyl alcohol
Excellent for fixing dry and wet smear, blood, bone marrow
Carnoys fluid
Fixing chromosomes, lymph gland, urgent biopsies.
THE MOST RAPID FIXATIVE
Alcoholic
Useful for sputum, coagulates mucus
formalin/ gendre
Clarks fixative
Newcomer
Fixing mucopolysaccharides and nuclear proteins
VI. OSMIUM TETROXIDE/ OSMIC ACID : Pale yellow powder, dissolves in water to
for strong oxidizing solution
-Fixes conjugated fats & lipids permanently, making them insoluble during
treatment with alcohol and xylene.
-Prolonged exposure to acid vapor can irritate eye, produce conjuctivitis, cause
deposition of black osmic axide in cornea, blindness
-should be kept in dark-colored, chemically clean bottle to prevent evaporation and
reduction by sunlight
Flemmings solution
Excellent fixative for nuclear structure
Permanently fixes fat
Flemmings solution
Chromic acid + osmic acid
without acetic acid
For cytoplasmic structures (mitochondria)
VII. TRICHLOROACETIC ACID : sometimes incorporated into compound fixatives
-May be used as a weak decalcifying agent
VIII. ACETONE (used at ice cold temp -5C 4 C
-used in fixing brain tissues for diagnosis of rabies
IX. HEAT FIXATION : involves thermal coagulation of tissue proteins for rapid dx.

UNIT VII DEHYDRATION

Dehydration = removal of intercellular and extracellular water from tissue after
fixation, before impregnation. Removal of water to be replaced by wax (paraffin or
celloidin, which are immiscible with water)
Dehydrating agents = Reagents that mix with and have affinity with water
COMOMNLY USED:
Alcohol, acetone, dioxane, cellosolve, triethyl phosphate, tetrahydrofuran
ALCOHOL
Ethyl alcohol
Best dehydrating agent, fast acting
Methyl alcohol
Toxic dehydrating agent, for blood and
tissue films
Butyl alcohol
For plants and animal microtechniques
Isopropyl alcohol
Cheaper
-Tissue should not be transferred directly to highter grades of alcohol
-for delicate tissues (brain, embryos, insects, dehydration should be more gradual,
beginning with 50% alcohol)
*Complete dehydration = Add a layer of anhydrous copper sulfate to accelerate
dehydration by removing water from dehydrating fluid. Blue discoloration =
saturation.
ACETONE
-Cheap, rapid, for most biopsies
-2-3 changes should be used
-change frequently
DIOXANE/ DIETHYLENE DIOXIDE = EXCELLENT DA
-readily miscible in water
-expensive
CELLOSOLVE / ETHYLENE GLYCOL MONOETHYL ETHER
-tissues can be stored in solution for months without hardening or distortion
TRIETHYL PHOSPHATE
-removes water rapidly
TETRAHYDROFURAN/THF
-dehydrates and clears
DIMETHOXYPROPANE / DMP
-reacts with water to form methanol and acetone

-rapid and endothermic reaction

UNIT VIII CLEARING
Clearing = de-alcoholization = removal of alcohol, and prep for impregnation
Clearing agent = antimedium. Miscible,can make tissue prep transparent due to
high index of refraction
XYLOL/XYLENE
-most common used today
-1/2 1 hour
-for clearing and embedding and mounting
-most rapid CA
-miscible with absolute alcohol and paraffin
-can be used for celloidin section
-cheap
-highly inflammable
-hardening and shrinkage or tissue
-milky when incompletely dehydrated tissue is immersed
TOLUENE
-tissues dont become excessively hard and brittle even if left 24hr
Relatively slower than xylene and benzene
More expensive
BENZENE
-CA in embedding, penetrates and clears rapidly
-highly inflammable
-excessive exposure to benzene = toxic, carcinogenic, damage BM, aplastic anemia
CHLOROFORM
-for tough & large tissue
-Toxic to liver
-tissue tends to float in chloroform
CEDARWOOD OIL
-clears paraffin and celloidin section
-tissues may be left in cedarwood oild indefinitely without considerable damage
and distortion
Extremely slow
Hard to eliminate from paraffin bath tissue
ANILINE OIL
CLOVE OIL
CARBON TETRACHLORIDE
TETRAHYDROFURAN/THF
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