Histotechnology– LECTURE (1st SEMESTER)

Instructor: Sir CHARLES ANDRE VLILACERAN <3

Transcribed by: FRANCIS ANDREI H. MIRA

LECTURE 2: FIXATION AND FIXATIVES

NOTE: It is very important to understand how to properly handle MAIN FACTORS INVOLVED IN FIXATION
tissue specimens as in some instances, prior to fixation, there • Hydrogen Ion Concentration (pH)
are already artifacts in tissue sample: commonly, due to • Temperature
ischemia (restricted blood flow in a certain part of the body, o Tissue processors usually work at 40°C (except for
deoxygenation of an organ) whether it is warm or cold Cryostats – it is used in cold temperatures).
ischemia, it can produce poor-tissue fixation. It cannot be o Electron microscopy workouts and Histochemistry
overemphasized that a tissue removed for Histopathologic Examination ideal temperature is 0-4°C.
examination should be fixed right away. The earlier the tissue o Formalin heated to 60°C is sometimes used for rapid
is fixed, the better the fixation → more resistant to artifacts. fixation of very urgent biopsy specimens.
These artifacts would affect or hinder the proper diagnosis of the o DISADVANTAGE:
conditions. In addition, improperly preserved/fixed tissues will ▪ Risk of tissue distortion is increased.
not process cut and stain well. ▪ Formalin at 100°C for fixing tissues with
Tuberculosis.
FIXATION • Thickness of Section
• FIRST and MOST critical step in Histotechnology • Osmolality
o Affects the proceeding steps, so it should be done o Best results are usually obtained using slightly
right. hypertonic solutions.
o Mostly, it is irreversible. • Concentration
o Sometimes, larger tissues (uterus) are divided into o Formaldehyde: Usually used at 10% Solution
sections. o Glutaraldehyde: Used at 3% Solution
• Preserve the morphologic and chemical integrity of the • Duration of Fixation
cell in as life-like a manner as possible. o Primary fixation in buffered formalin is usually carried
• Prevents degeneration, decomposition, putrefaction, for 2-6 hours.
and distortion of tissues after removal from the body. o Most of the formalin can be washed out after fixation
o Ischemia is likely upon removal of a tissue from the for 24 hours.
body it should be fixed immediately. o Prolonged fixation = shrinkage and hardening of
• Harden and Protect the tissue from the trauma of further tissues and may severely inhibit enzyme activity and
handling. immunological reactions.
o Washing of tissues in running water can
NOTE: Fixatives or Fixative Solution is to stabilize structures considerably restore the activity of some enzymes.
to maintain a proper relationship of cells and their stroma o Depends on the sample as some specimens take
(collagen, elastin fibers, reticulin fibers, or any amorphous longer time to completely fixate.
substances present in that cellular element).
PRACTICAL CONSIDERATIONS OF FIXATION
2 BASIC MECHANISMS INVOLVED IN FIXATION • Speed
o The specimen should be placed in a fixative as soon
1. ADDITIVE FIXATION as it is removed from the body.
• Chemical constituent of the fixative is taken in and ▪ Prevents putrefaction and autolysis.
becomes part of the tissue by forming cross-links or • Penetration
molecular complexes and giving stability to the protein. o There are some tissues that are easier to fixate as
• Examples: the rate of penetration is faster, some tissues have
o Formalin slow penetration, and those with variable rate of
o Mercury penetration.
o Osmium Tetroxide • Volume
o 10-25x the volume of tissues to be fixed.
2. NON-ADDITIVE FIXATION o Recently, the maximum effectiveness of fixation is
noted to be 20x the tissue volume.
• Fixing agent is not incorporated into the tissue, but alters
• Duration of Fixation
the tissue composition and stabilizes the tissue by removing
o Depends on the tissue type.
the bound water attached to H-bonds of certain groups
within the protein molecule.
EFFECTS OF THE FIXATIVES
• Examples:
o Alcoholic Fixatives • Harden soft and friable tissues
▪ Ethanol o Make handling and cutting of tissue sections easier
▪ 100% Methanol • Make cells resistant to damage and distortion caused by
▪ 95% Isopropyl Alcohol hypotonic and hypertonic solutions used during tissue
▪ Carnoy’s Solution processing.
▪ Newcomer’s FLuid • They inhibit bacterial decomposition.
• Increase the optical differentiation of cells and tissue
components thereby rendering them more readily visible
during examination.

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• They act as mordants or accentuators to promote and ▪ Trichloroacetic Acid

hasten staining, or they may inhibit certain dyes in favor of ▪ Glacial Acetic Acid
another. ▪ Formaldehyde
o Mordants serve as a link between dye and o C. Bouin’s Solution
substrate. Aluminum Salts, Iron, Lead, Tungsten are ▪ Picric Acid
mordants for the promotion of the hastening of ▪ Formaldehyde
Hematoxylin staining process. ▪ Glacial Acetic Acid
• They reduce the risk of infections during handling and
actual processing of tissues. B. ACCORDING TO ACTION
1. MICROANATOMICAL FIXATIVES
CHARACTERISTICS OF A GOOD FIXATIVE • Permit the general microscopic study of tissue structures
• Cheap and accessible without altering the structural pattern and normal
• Stable intercellular relationship of tissues.
• Safe to handle – not hazardous, not irritating, does not
induce allergic reaction. 2. CYTOLOGICAL FIXATIVES
• Kill the cell quickly to produce minimum distortion of cell
• Preserve specific parts and particular microscopic elements
constituents – minimal distortion is acceptable.
of the cell itself.
• Inhibit bacterial decomposition and autolysis.
• Minimal shrinkage of tissue
2.1.1 NUCLEAR FIXATIVES
• Permit rapid and even penetration of tissues – whole
specimen must be penetrated by the fixative. • Preserve nuclear structures (e.g. chromosomes) in
• Hardens tissues particular
• MUST BE ISOTONIC – not always recommended but it is • Usually contain glacial acetic acid as their primary
only IDEAL. Hypotonic Fixatives causes tissue/cell burst. component due to its affinity for nuclear chromatin
Hypertonic Fixatives may cause tissue/shrinkage. • pH of 4.6 or less (acidic environment)
• Must make cellular components insoluble to hypotonic • Examples:
solutions and render them insensitive to subsequent o Flemming’s
processing. o Carnoy’s
• Permit the subsequent application of many staining o Bouin’s
procedures to facilitate easier and more profitable o Newcomer’s
examination. o Heidenhain Susa

TYPES OF FIXATION ACCORDING TO ACTION AND 2.1.2. CYTOPLASMIC FIXATIVES

COMPOSITION • Preserves cytoplasmic structures in particular.
A. SIMPLE FIXATIVES o SER, RER
• Made up of only one component substance. o Lysosomes
o Golgi Body
o Mitochondria
1. ALDEHYDE
• Must never contain glacial acetic acid since it destroys
• a. FORMALDEHYDE mitochondria and golgi bodies.
• b. GLUTARALDEHYDE • pH of more than 4.6
• Examples:
2. METALLIC FIXATIVES o Flemming’s without acetic acid
• a. Mercuric Chloride o Kelly’s Fluid
• b. Chromative Fixatives o Regaud (Muller’s Fluid)
o Potassium Dichromate o Orth’s Fluid
o Chromic Acid
• c. Lead Fixatives 2.1.3. PRECIPITANT FIXATIVES
o Picric Acid • For RNA, the precipitant fixatives Ethanol and Acetone
o Acetic Acid give the best quantitative results using frozen tissues as
o Acetone the standard.
o Alcohol
o Osmium Tetroxide (Osmic Acid) 2.1.4. HISTOCHEMICAL FIXATIVES
• d. Heat
• Preserve the chemical constituents of cells and tissues.
3. COMPOUND FIXATIVE • Examples:
o Formol Saline 10%
• Those that are made up of two or more fixatives which o Absolute Ethyl Alcohol
have been added together to obtain the optimal combined o Acetone
effect of their individual actions upon the cells and tissue o Newcomer’s Fluid
constituents.
o A. Carnoy’s Fixative
▪ Alcohol
▪ Chloroform
▪ Glacial Acetic Acid
o B. Heidenhain’s Susa Fixative
▪ Mercuric Chloride
▪ Sodium Chloride

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LIPID FIXATION PROS (ADVANTAGES)

• Lipids are largely removed during preparation of tissues. • Cheap, readily available, easy to prepare and stable
• Cryostat or Frozen Sections should be used for especially if stored in buffered solutions
demonstrating lipid in tissues, followed by a general lipid • Compatible with many stains
stain. • Does not overharden tissues (as long as not 40% Formalin)
• Fixatives containing mercuric chloride and potassium • Preserves fat and mucin
dichromate can be effective for lipid preservation in • Preserves glycogen
cryostat sections. • Preserves tissue well
• Phospholipids which contain amino groups are fixed by • Preserves but does not precipitate proteins
aldehydes.
• Does not make tissue brittle (recommended for nervous
• Improved ultrastructural demonstration of lipids has tissue preparation)
been achieved by post-fixing in imidazole osmium
• Allows natural tissue colors to be restored
tetroxide.
• Cholesterol may be fixed with digitonin for ultrastructural
CONS (DISADVANTAGES)
demonstration.
• Can cause allergic dermatitis
CARBOHYDRATE FIXATION • May produce considerable shrinkage of tissues
• Alcoholic Fixatives are generally recommended for • Soft fixative and does not harden some cytoplasmic
glycogen fixation. structures adequately enough for paraffin embedding
• Alcoholic formaldehyde is a better fixative in human skin • IF unbuffered:
compared with neutral buffered formaldehyde. o Reduces both basophilic and eosinophilic staining of
cells thereby reducing quality of routine cytologic
PROTEIN FIXATION staining.
• Neutral Buffered Formol Saline or Formaldehyde Vapor o Also forms abundant brown pigment granules on blood
are the most commonly used fixative for amino acid containing tissues due to blackening of hemoglobin.
histochemistry. • Prolonged fixation may cause bleaching of the specimen
and may lose natural tissue colors. Dispersal of fat from the
tissues into the fluid, and loss of glycogen, biurate of sodium
GLYCOGEN FIXATION
crystals and uric acid.
• Most useful fixative for preserving glycogen are alcohol-
based such as Rossman’s Fluid or Cold Absolute
Alcohol. B. 10% FORMOL SALINE
• Essential when processing tissues from patients with • Simple microanatomical fixative made up of saturated
glycogen storage diseases. formaldehyde (40% by weight volume) diluted to 10% with
Sodium Chloride.
• Celloidin coating can enhance better retention of
glycogen. • Recommended fixative for fixation of central nervous
tissues and post-mortem tissues (cold autopsy) for
histochemical examination.
MIXTURE OF FIXATIVES
• COMPONENTS:
• Two aldehyde fixative mixtures have been particularly
o 40% Formaldehyde + NaCl + Distilled Water
useful for electron cytochemistry:
• FIXATION TIME:
o Karnovsky Paraformaldehyde-Glutaraldehyde
o 24 hours at 35°C / 48 hours at 20-25°C
▪ Fast tissue penetrator
▪ Optimal in preservation
▪ Stabilize cellular proteins PROS (ADVANTAGES)
• Acrolein – mixture with Glutaraldehyde or Formaldehyde • Penetrates and fixes tissues evenly
o Penetrates the tissues rapidly, preserves morphology • Preserves microanatomic and cytologic details with minimal
and enzyme activity at low concentrations. shrinkage and distortion
o Useful for immersion fixation of surgical biopsies. • Large specimens may be fixed for a long time provided that
solution is changed every three months.
o To prevent precipitation of proteins.
ALDEHYDE FIXATIVES ▪ If not prevented, lead to artifacts, hindering the
• Satisfactory for routine paraffin sections, electron results.
microscopy, and histochemical and enzyme studies. • Preserves enzymes and nucleoproteins
• Demonstrates fats and mucin
A. FORMALDEHYDE • Does not overharden tissues
• Most widely used fixative is 10% Formalin • Ideal for most staining techniques
• Pure stock solution of 40% Formalin is unsatisfactory for • Allows natural tissue color to be restored
routine fixation since formaldehyde concentrates tend to
overharden the outer layer of the tissue and affect CONS (DISADVANTAGES)
staining adversely. • Slow fixative, requires 24 hours or longer (e.g. 48hrs)
• Commonly used as 4% Solution to yield 10% Formalin • Tissues tend to shrink during alcohol dehydration
Solution for tissue fixation. • Metachromatic Reaction of amyloid is reduced
• 1:10 or 1:20 dilution to make a 10% or 5% Solution • Acid dye stains less brightly causing us to miss certain
• Fixation Time = 24 Hours elements.
• Formaldehyde is usually buffered to pH 7 with Phosphate
Buffer.

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C. 10% NEUTRAL BUFFERED FORMALIN OR CONS (DISADVANTAGES)

PHOSPHATE BUFFERED FORMALIN • Produces gross hardening of tissues
• Recommended for preservation and storage of surgical, • Causes partial lysis of RBC
post-mortem, and research specimens. • Preservation of iron-containing pigments is poor.
• COMPONENTS:
o Anhydrous Sodium Dihydrogen Phosphate + F. GLUTARALDEHYDE
Anhydrous Disodium Hydrogen Phosphate + 40% • Made up of two formaldehyde residues linked by three
Formaldehyde + Distilled Water carbon chains
▪ Anhydrous = Water-free, No Water • Buffered glutaraldehyde followed by secondary fixation in
• FIXATION TIME: osmium tetroxide is satisfactory for electron microscopy.
o 4-24 hours • 2.5% solution is used for small fragments and needle
PROS (ADVANTAGES) biopsies fixed in 2-4 hours at room temperature.
• 4% solution for larger tissues fixe in 6-8 hours up to 24
• Prevents precipitation of acid formalin pigments
hours
• Best tissue fixatives for tissues containing iron pigments
(liver, spleen tissues) and for elastic fibers that does not
stain well after Susa, Zenker, or Chromate Fixation.
PROS (OVER FORMALIN)
• Requires no post-treatment after fixation. • More stable effect on tissues, firmer textures
• Preserves plasma proteins better
CONS (DISADVANTAGES) • Less tissue shrinkage
• Longer to prepare, time-consuming • Preserves Cellular Structures better
• More pleasant and less irritating to the nose
• Positivity of mucin to PAS (Periodic-Acid Schiff) is
reduced • Does not cause dermatitis
• May produce gradual loss in basophilic staining of cells
• Reactivity of Myelin to Weigert’s Iron Hematoxylin Stain CONS (DISADVANTAGES)
is reduced. • More expensive.
• Inert towards lipids, especially neutral fats and • Less stable.
phospholipids. • Penetrates tissues more slowly.
o Chemically inactive or no chemical reactions with lipids • Tends to make tissue more brittle.
at all. o Due to the longer penetration and longer fixation time.

D. FORMAL-CORROSIVE SOLUTION NOTE: SPECIMEN VIAL MUST BE KEPT REFRIGERATED

• Recommended for routine post-mortem tissues. DURING FIXATION PROCESS
• COMPONENTS:
o Mercuric Chloride + Formaldehyde METALLIC FIXATIVES:
▪ Mercuric Chloride can produce mercuric chloride
MERCURIC, CHROMATE, LEAD
deposits/precipitates.
• FIXATION TIME:
o 3 - 24 hours
I. MERCURIC CHLORIDE
• MOST COMMON METALLIC FIXATIVE
PROS (ADVANTAGES) • Also a widely used secondary fixative reacting with a
• Minimal shrinkage and hardening number of amino acid residues and accompanied by
spectroscopic changes, probably due to Histidine
• Excellent for many staining techniques
residues
• Brightens cytoplasmic and metachromatic stains
• Penetrates poorly and produce shrinkage of tissues, usually
• Cytological and blood cell structures are well-preserved
combined with other fixative agents.
• Fixes lipids, especially neutral fats and phospholipids
• Mercury deposits can be treated by 0.5% Iodine Solution
in 70% Ethanol for 5-10 minutes. Sections are rinsed in
CONS (DISADVANTAGES) water, decolorized for 5 minutes in 5% Sodium
• Slow penetrations Thiosulfate and washed in running water (artifacts are
• Forms mercuric chloride deposits removed/eliminated).
o Removal of these deposits is termed Dezenkerization.
• Does not allow frozen tissue sections to be made PROS
• Nuclear components are shown in fine detail.
E. ALCOHOLIC FORMALIN (GENDRE’S) FIXATIVE
• Precipitates all proteins
• COMPONENTS:
• Greater affinity to acid dyes
o 95% Ethyl Alcohol Saturated with Picric Acid
o Strong Formaldehyde Solution (40%) • Trichome staining is excellent
o Glacial Acetic Acid • Routine fixative of choice for preservation of cell detail in
tissue photography.
PROS (ADVANTAGES) • Permits brilliant metachromatic staining of cells.
• Faster fixation CONS (DISADVANTAGES)
• Used for rapid-diagnosis because it fixes and
dehydrates at the same time. • May cause shrinkage of cells
• Good for preservation of glycogen and for • Rapidly hardens the outer layer of the tissue with
micro-incineration techniques. incomplete fixation of the center.
• Used to fix sputum since it coagulates mucus. • Penetration beyond first 2-3mm is slow.

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• Tissue becomes hard and brittle if left in fixative for more B. ZENKER-FORMOL (HELLY’S SOLUTION)
than 1-2 days. • COMPONENT:
• Formation of black granular deposits. o MC Stock Solution + Strong Formaldehyde
o Mercuric Chloride deposits • FIXATION TIME:
• Extremely corrosive to metals. o 12 – 24 hours
• Reduces amount of demonstrable glycogen.
• Inert to fats and lipids. PROS
• Excellent microanatomic fixative for pituitary gland, bone
MERCURIC CHLORIDE STOCK SOLUTION FORMULA marrow, and blood-containing organs such as spleen and
• Mercuric Chloride 5g liver.
• Potassium Dichromate 2.5g • Penetrates and fixes tissues well.
• Sodium Sulfate 1g • Nuclear fixation and staining is better than Zenker’s
• Distilled Water 100mL • Preserves cytoplasmic granules

A. ZENKER’S FLUID CONS (DISADVANTAGES)

• COMPOSITION: • Similar to Zenker’s except brown pigments are produced
• Mercuric Chloride Stock Solution + Glacial Acetic Acid due to prolonged fixation and RBC lysis.
• Glacial Acetic Acid must be added before use to prevent o Can be removed by immersion in Saturated Alcoholic
turbidity and formation of dark precipitates (mercuric Picric Acid or Sodium Hydroxide.
chloride deposits).
• Recommended for fixing small pieces of liver, spleen, C. HEIDENHAIN’S SUSA
connective tissue fibers and nuclei. • Main fixative for tumor biopsies especially the skin.
• FIXATION TIME: • Excellent cytologic fixative.
o 12-24 hours • COMPONENT:
o Mercuric Chloride
PROS (OVER FORMALIN) o Sodium Chloride
• Fairly rapid and even fixation o Trichloroacetic Acid
• Stock solutions keep well without disintegrations o Glacial Acetic Acid
• Recommended for trichome staining o 40% Formaldehyde
• Permits brilliant staining of nuclear and connective tissue • FIXATION TIME
fibers. o 3-12 hours
• Compatible with most stains.
• Act as a mordant PROS (ADVANTAGES)
• Rapid and even fixation/penetration
CONS (DISADVANTAGES) • Minimal shrinkage
• Poor penetration • Easier sectioning of large blocks of fibrous connective
• Not stable after addition of acetic acid tissues
• Prolonged fixation will make tissues brittle and hard
• Can cause lysis of RBCs CONS (DISADVANTAGES)
• Does not permit cutting of frozen sections • RBC Preservation is poor
• Tendency to form mercuric pigment deposits or precipitates • Cytoplasmic granules are dissolved
• Tissue must be washed with running water for several hours • May form mercuric chloride deposits
before processing. • Weigert’s Method of staining elastic fibers is not possible in
Susa-fixed tissues.
HOW TO DEZENKERIZE
• Removal of black precipitates (mercuric chloride NOTE: After using Heidenhain’s Susa Fixative, tissue should be
precipitates/deposits) transferred directly to a high-grade alcohol (e.g. 96% or
1. Bring slides to water Absolute Alcohol) to avoid undue swelling of tissues
2. Immerse in Lugol’s Iodine 5 min caused by treatment with a low-grade alcohol or water.
3. Wash in running water 5 min
4. Immerse in 5% Na Thiosulfate 5 min D. B-5 FIXATIVE
5. Wash in running water 5 min • Used for bone marrow biopsies
6. Proceed with required water soluble stain • COMPONENT:
o Distilled water
NOTE: Reagents needed for Dezenkerization: (1) Lugol’s o Mercuric Chloride
Iodine & (2) 5% Sodium Thiosulfate. o Anhydrous Sodium Acetate
• FIXATION TIME
o 1-2 Hours
NOTE: In prior use, add 1mL 40% Formaldehyde for every
10mL of B-5 Solution.

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PROS (ADVANTAGES) III. LEAD FIXATIVES

• Rapid fixation • Used in 4% Aqueous solution of basic lead acetate

CONS (DISADVANTAGES) PROS (ADVANTAGES)

• Overfixation hardens the tissue and makes cutting difficult • Recommended for acid mucopolysaccharides
• May form precipitates on standing • Fixes connective tissue mucin

II. CHROMATE FIXATIVES CONS (DISADVANTAGES)

• CHROMIC ACID • Take up carbon dioxide to form insoluble lead carbonate
o Used in 1-2% Aqueous solution especially on prolonged staining.
o Precipitates all proteins and adequately preserves o May be removed by adding acetic acid, drop by drop,
carbohydrates to lower pH level and dissolve the residue.
o Strong oxidizing and strong reducing agent
• POTASSIUM DICHROMATE PICRIC ACID FIXATIVE
o Used in 3% Aqueous solution • Normally used in strong saturated aqueous solutions
o Fixes but does not precipitate cytoplasmic structures • This fixative also dyes tissues producing yellow color.
o Preserves lipids o It can be removed by treatment with another acid
o Preserves mitochondria dye or lithium carbonate.
• Preserves glycogen but causes considerable shrinkage of
A. REGAUD’S (MULLER) FLUID tissue.
• COMPONENT: • Washing with 50% and 70% Ethanol will remove most of
o Potassium Dichromate 3% the yellow color.
o 40% Strong Formaldehyde
• FIXATION TIME PROS (ADVANTAGES)
o 12-48 Hours
• Excellent for glycogen demonstration
• Rapid fixation and penetration
PROS (ADVANTAGES)
• Allows brilliant staining with Trichome Method
• Penetrates the tissue well • Suitable for Aniline Stains
• Hardens tissue better and more rapidly than Orth’s fluid • Precipitates all proteins
• Recommended for the demonstration of chromatin, • STABLE Fixatives
mitochondria, mitotic figures, Golgi bodies, RBC and
Colloid-containing structures CONS (DISADVANTAGES)
• Causes RBC hemolysis
CONS (DISADVANTAGES)
• Not suitable for frozen tissues
• Deteriorates and darkens on standing (must always be • Picrates are formed upon protein
freshly prepared) • Picric acid may produce excessive staining of tissues
• Slow penetration • Highly explosive when dry.
• Prolonged fixation blackens tissue pigments o Cannot be touched with Metal since it is highly
• Glycogen penetration is poor, generally contradicted for explosive.
carbohydrates • Alters and dissolves lipids
• Poor nuclear staining • Interferes with Azure-Eosin Method
• Does not preserve fats
A. BOUIN’S SOLUTION
B. ORTH’S FLUID • Recommended for fixation of embryos and pituitary
• COMPONENT biopsies
o Potassium Dichromate • COMPONENT:
o Sodium Sulfate o Saturated Solution of Picric Acid
o Strong Formaldehyde (40%) o Strong Formaldehyde (40%)
• FIXATION TIME o Glacial Acetic Acid
o 36-72 hours • FIXATION TIME:
o 6-24 hours
PROS (ADVANTAGES)
• Recommended for early degenerative process and tissue PROS (ADVANTAGES)
necrosis • Minimal distortion
• Demonstrates Rickettsiae and other Bacteria • Excellent fixative for preserving soft and delicate structures
o Causes Rocky-Mountain-Spotted Fever
• Preserves myelin better than buffered formalin CONS (DISADVANTAGES)
CONS (DISADVANTAGES) • Penetrates large tissues poorly, hence, very limited to small
fragments of tissue
• Same as Regaud’s (Muller) Fluid • Not suitable for fixing kidney structures, lipids, and mucus
• Destroys cytoplasmic structures
• Produce RBC Hemolysis

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B. BRASIL’S ALCOHOLIC PICROFORMOL FIXATIVE B. 95% ISOPROPYL ALCOHOL

• COMPONENT: • Used for fixing touch preparations
o 37% Formaldehyde
o Picric Acid C. ETHYL ALCOHOL
o Ethanol/Isopropyl Alcohol • Used at concentrations 70-100%
o Trichloroacetic Acid • Lower concentrations can cause RBC hemolysis and
inadequate preservation of WBCs
PROS (ADVANTAGES) • May be used as a simple fixative, however, more frequently
• Better and less messy than Bouin’s incorporated into compound fixatives for better results.
• Excellent fixative for Glycogen • FIXATION TIME
o 18-24 hours
GLACIAL ACETIC ACID
NOTE: Associated/correlated with Compound Fixatives: (1) D. CARNOY’S FLUID
Carnoy’s; (2) Susa; (3) Bouin’s, etc. • Recommended for fixing chromosomes, lymph glands, and
urgent biopsies.
• Normally used in conjunction with other fixatives to form a
• MOST RAPID FIXATIVE
compound solution
• Used to fix brain tissue for diagnosis of rabies
• Solidifies at 17°C
• COMPONENT:
o Absolute alcohol
PROS (ADVANTAGES) o Chloroform
• Fixes and precipitates nucleoproteins o Glacial Acetic Acid
• Precipitates chromosomes and chromatin materials, very • FIXATION TIME
useful for studying nuclear components of the cell o 1-3 Hours
• Essential constituent of most compound nuclear fixatives
• Causes tissues containing collagen to swell, better PROS (ADVANTAGES)
visualization. • Fixes and dehydrates at the same time.
• Permits good nuclear staining and differentiation.
CONS (DISADVANTAGES) • Preserves Nissl Granules and Cytoplasmic Granules
• Contradicted for cytoplasmic fixation since it destroys • Excellent fixative for glycogen
mitochondria and Golgi elements of the cells.
CONS (DISADVANTAGES)
ALCOHOLIC FIXATIVES • Produces RBC hemolysis
• Denatures and precipitates proteins by destroying
• Causes considerable tissue shrinkage
hydrogen and other bonds.
• Suitable only for small pieces of tissue due to slow
• Must be used in concentrations 70-100% because less
penetration
concentrated solutions will produce lysis of cells.
E. NEWCOMER’S FLUID
PROS (ADVANTAGES)
• COMPONENT:
• Ideal for small tissue fragments o Isopropyl Alcohol
• May be used both as fixative and dehydrating agents o Propionic Acid
• Excellent for glycogen preservation o Petroleum Ether
• Preserves nuclear stains o Acetone
o Dioxane
CONS (DISADVANTAGES) • FIXATION TIME
• Low concentrations may cause RBC hemolysis and o 12-18 hours at 3°C (Refrigerated)
inadequately preserve leukocytes.
• Dissolves fats and lipids. PROS (ADVANTAGES)
• Causes glycogen granules to move towards the poles or • Recommended for fixing mucopolysaccharides and
ends of the cells (polarization). nuclear proteins.
• Tissue left in alcohol too long will shrink, making it difficult • Produces better reaction in Feulgen Stain than Carnoy’s
or impossible to cut. Fluid
• Act as Nuclear and Histochemical Fixative
A. METHYL ALCOHOL 100%
• Excellent for fixing dry and wet smears, blood smears, and
bone marrow tissues.
• Fixes and dehydrates at the same time.

CONS
• Slow penetrations
• Prolonged fixation for more than 48 hours may overharden
tissue and make it difficult to cut.

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OSMIUM TETROXIDE (OSMIC ACID) TRICHLOROACETIC ACID

• Pale yellow powder which dissolves in water to form a • Incorporated into compound fixatives
strong oxidizing solution.
• Fixation with this solution causes complete denaturation PROS (ADVANTAGES)
of proteins. • Precipitates proteins
• Marked swelling effect on tissues serves to counteract
PROS (ADVANTAGES) shrinkage produced by other fixatives
• Fixes conjugated fats and lipids permanently • May be used as a weak decalcifying agent.
• Preserves cytoplasmic structures well • Softening effect on dense fibrous tissues
• Fixes myelin and peripheral nerves well
• Used extensively for neurological tissues CONS (DISADVANTAGES)
• Produces brilliant nuclear staining with Safranin • Poor penetrating agent
• Adequately fixes materials for ultrathin sectioning
ACETONE
CONS (DISADVANTAGES) • Used at ice cold temperature (-5°C to 4°C)
• Very expensive
• Poor penetrating agent PROS (ADVANTAGES)
• Readily reduced by contact with organic matter and • Recommended for study of water diffusible enzymes
exposure to sunlight, forming a black precipitate especially phosphatases and lipases
• Prolonged exposure may cause blindness • Used in fixing brain tissues for diagnosis of rabies
• Inhibits hematoxylin and makes counterstaining difficult • Used as solvent for certain metallic salts to be used in
• Extremely volatile freeze substitution.

A. FLEMMING’S SOLUTION CONS (DISADVANTAGES)

• MOST COMMON CHROME-OSMIUM ACETIC ACID
• Produce inevitable shrinkage and distortion
FIXATIVE USED
• Dissolves fats
• COMPONENT:
• Preserves glycogen poorly
o Aqueous Chromic Acid 1%
o Aqueous Osmium Tetroxide 2% • Evaporates rapidly (High volatility)
o Glacial Acetic Acid
• FIXING TIME: HEAT FIXATION
o 24-48 hours • Involves thermal coagulation of tissue proteins for rapid
diagnosis.
PROS (ADVANTAGES) • Usually for frozen tissue sections and preparation of
bacteriologic smears
• Excellent fixative for nuclear structures
• Permanently fixes fat PROS (ADVANTAGES)
CONS (DISADVANTAGES) • Fixation is better
• Preserves nuclear and cytoplasmic detail
• Poor penetrating agent • Suitable for frozen tissue preparation
• Deteriorates rapidly
• Depress the staining power of Hematoxylin CONS (DISADVANTAGES)
• Tendency to form artifact pigments
• Very expensive • May produce tissue shrinkage and distortion
• Destroys RBC
B. FLEMMING’S SOLUTION WITHOUT ACETIC ACID • Dissolves starch and glycogen
• COMPONENT:
o Chromic Acid
o Osmic ACid
• FIXING TIME:
o 24-48 hours
• Recommended for cytoplasmic structures
• Removal of acetic acid = improves cytoplasmic detail
of the cell

PROS & CONS

• Same as Flemming’s Solution

THE HOMIES <3 | MLS-2E 8

 CLINICAL CHEMISTRY – LECTURE

SECONDARY FIXATION NOTE:

• Process of placing an already fixed tissue in a second REMEMBER:
fixative mainly to: • The pros and cons don’t have to be memorized, just
o 1. Facilitate and improve the demonstration of familiarized.
particular substances. • Memorize the specifics of the fixatives: (1) Where it is
o 2. Make special staining techniques possible. commonly used; (2) Components; (3) Fixation Times.
o 3. Ensure further, complete hardening and • Read the Gregorios’s Histopathologic Techniques.
preservation of tissues.

POST-CHROMATIZATION
• Form of secondary fixation whereby a primarily fixed tissue
is placed in aqueous solution of 2.5-3% Potassium
Dichromate for 24 hours to act as mordant for better
staining effects and to aid in cytologic preservation of
tissues.

WASHING OUT
• Process of removing excess fixative from the tissue after
fixation in order to improve staining and remove artifacts
from the tissues.
• 1. Tap Water – removes excess:
o Chromates
o Formalin
o Osmic Acid
• 2. 50-70% Alcohol – washout excess amount of:
o Bouin’s Solution
• 3. Alcoholic Iodine – used to remove excessive:
o Mercuric Fixatives

FACTORS AFFECTING FIXATION

OF TISSUES

SLOWED DOWN BY:

1. Size and Thickness of tissue specimen
o Larger Tissues = More Fixative = Longer Fixation Time
2. Presence of Mucus
o Prevents complete penetration of fixative.
o Excess mucus should be washed away by NSS
o Coagulates and forms precipitates.
3. Presence of Fat
o Fatty tissues should be cut in thin sections and fixed
longer.
4. Presence of Blood
o Tissues containing large amount of blood should be
flushed out with saline (NSS) before fixing.
5. Cold Temperature
o Inactivates enzymes.

ENHANCED BY:
1. Size and Thickness of tissue specimen
o Smaller and thinner tissues require less fixative and
shorter fixation time.
2. Agitation
o Fixation is accelerated when automatic or mechanical
tissue processing is used.
o Moderate heat (37-56°C) accelerates fixation but
hastens autolytic changes and enzyme destruction.
▪ Distort tissues and cellular elements.

THE HOMIES <3 | MLS-2E 9