Source: Mr. Gamaliel Issamar S.

de Vera, RMT, MSMT

- Primary fixation in buffered formalin is usually carried
FIXATION
out for 2-6 hours during the day the specimen is
o Fixation is the first “technical” step in histotechnology after
obtained
Numbering
- Prolonged fixation may cause shrinkage and hardening
o Process of preserving cells and tissue constituents in a condition
of the tissue and may severely inhibit enzyme activity
identical to that existing during life
and immunological reaction
GOALS OF FIXATION
PRACTICAL CONSIDERATIONS OF FIXATION
o Preserve the morphological and chemical integrity of the cell by
1. Speed
preventing degeneration, decomposition, putrefaction
- Place in a fixative solution as soon as it is removed
o Harden and preserve tissue for further handling
from the body to prevent autolysis and putrefaction
MECHANISM OF ACTION
1) Additive fixation 2. Penetration
- Non coagulant cross linking fixatives - Most fixatives diffuse into the tissue at the rate of 1 mm
- Chemical constituent taken into the cell, forming per hour and slows down as it goes deeper in the
molecular complexes and stabilizing proteins tissue

2) Non-additive fixation 3. Volume

- Dehydrant coagulant fixatives - 10–25 times the volume of the tissue to be fixed
- Alteration of tissue composition by removing bound - The maximum effectiveness of fixation is noted to be
water molecule at H bonds within protein molecules 20 times the tissue volume
- Stabilizes proteins by forming crosslinks after water * Fixation time can be cut down by using heat, vacuum, agitation or
molecule removal microwave.
MAIN FACTORS INVOLVED IN FIXATION CLASSIFICATION OF FIXATIVES
1. Hydrogen Concentration
COMPOSITION
- Satisfactory fixation occurs between pH 6 and 8
- High acidity decreases effectiveness A. SIMPLE FIXATIVES – made up only of one substance
1) Formaldehyde
2. Temperature 2) Glutaraldehyde
- Fixation of surgical specimens are done at room 3) Metallic Fixatives
temperature • Mercuric Chloride, Chromate Fixatives, Lead
- For electron microscopy: 0–4°C Fixatives
- Except for mast cells – room temperature 4) Picric Acid
- Nucleic acids do not react with fixatives at room 5) Acetic Acid
temperature 6) Acetone
- Chemical reactions are rapid at high temperature (60 – 7) Alcohol
65°C) 8) Osmium Tetroxide
9) Heat
3. Thickness of Section
- 1–2 mm2 for electron microscopy B. COMPOUND FIXATIVES – 2 or more fixatives to obtain
- 2 cm2 for light microscopy optimal combined effect
- It is done to obtain full penetration and satisfactory
fixation ACTION
- Brain is usually suspended whole in 10% buffered 1. MICROANATOMICAL – General microscopic study of tissue
formalin for 2–3 weeks to ensure fixation and some structures
hardening prior to sectioning • 10% Formol Saline
4. Osmolality • 10% Neutral Buffered Formalin
- Hypertonic solutions give rise to cell shrinkage • Heidenhain’s Susa
- Isotonic and hypotonic fixatives cause swelling and • Formol Sublimate (Formol Corrosive)
poor fixation • Zenker’s Solution
- The best result is usually obtained using slightly • Zenker-Formol (Helly’s Solution)
hypertonic solutions (400–450 mOsm) • Bouin’s Solution
• Brasil’s Solution
5. Concentration
- Formaldehyde: 10% 2. CYTOLOGIC FIXATIVES – Preserve specific parts and particular
- Glutaraldehyde: 3% microscopic elements
- 0.25% Glutaraldehyde for immunoelectrochemistry 2.1 NUCLEAR FIXATIVES
6. Duration of Fixation - Preserve nuclear structures
- Glacial acetic acid as primary component
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 • Flemming’s Fluid 1.3 10% NEUTRAL BUFFERED FORMALIN
• Carnoy’s Fluid - Preservation and storage of surgical, post-mortem
• Bouin’s Fluid and research specimens
• Newcomer’s Fluid - Fixation time: 4–24 hrs
• Heidenhain’s Susa
1.4 FORMOL–CORROSIVE (Formol-Sublimate)
2.2 CYTOLOGICAL FIXATIVES - Sat. Aq. Mercuric chloride as diluent
- Preserve cytoplasmic structures in particular - Fixation time: 3–24 hrs
• Flemming’s Fluid without Acetic Acid - Recommended for routine post-mortem tissues
• Helly’s Fluid - Brightens cytoplasmic and metachromatic stains
• Formalin with Post-Chroming - Slow penetration
• Regaud’s Fluid (Moller’s Fluid)
2. GLUTARALDEHYDE
• Orth’s Fluid
- 2.5% SOLUTION – small tissue fragments and needle
• RNA: ethanol and acetone
aspirates
3. HISTOCHEMICAL FIXATIVES – Preserves chemical constituents of - 4% SOLUTION – larger tissues
cells and tissues - For routine light microscopy, also for EM
• 10% Formol Saline - Preserves cellular structures better
• Absolute Ethyl Alcohol - Less irritating to nose and skin
• Acetone - More expensive
• Newcomer’s Fluid - Less stable
- Slow penetration
4. LIPID FIXATION
- Largely removed during processing METALLIC FIXATIVES
- Use frozen sections and special stains
MECHANISM: Mercuric Chloride and Lead Fixatives’ Mercuric salts
- Aldehydes can be used in general
bind with sulfhydryl groups in acidic solutions. Chromate Fixatives in
5. CARBOHYDRATE FIXATION water form Cr-O-Cr complexes that have an affinity for –COOH and –
- Alcoholic fixatives for better retention OH groups of proteins, so that complexes between adjacent
molecules are formed.
* Fixatives in order of decreasing speed of penetration:
Formaldehyde → Acetic acid → Mercuric chloride → Ethyl/Methyl
1. MERCURIC CHLORIDE
alcohol → Osmium tetroxide → Picric acid
1.1 5 – 7% MERCURIC CHLORIDE
- Included in most compound fixatives
ALDEHYDE FIXATIVES
- Nuclear components are shown in fine detail
MECHANISM: The aldehydes form cross-links between proteins, - Excellent trichome staining
creating a gel, thus retaining cellular constituents in their in vivo - Preservation of cell detail in tissue photography
relationships to each other. - Pigments removed by treatment with iodine
1. FORMALDEHYDE solution in 95% alcohol
- Produced from oxidation of methyl alcohol 1.2 ZENKER’S FLUID
- Fixation time: 24 hrs - With glacial acetic acid for affinity to nuclear
1.1 10% FORMALIN chromatin
- Cheap, readily available, easy to prepare - Permits brilliant staining of nuclear and connective
- Relatively stable tissue fibers
- Compatible with many stains - For liver, spleen, connective tissue fibers, and
- Fumes irritating to nose and eyes nuclei
- Solution is irritating to skin - Fixation time: 12-24 hrs
- For routine paraffin sections, electron microscopy, - Lyses RBC and can make tissues brittle
histochemistry & enzyme studies 1.3 ZENKER–FORMOL (Helly’s Solution)
- Pigments removed by treatment with alcoholic - With 40% formaldehyde (5 mL)
picric acid - For pituitary gland, bone marrow and blood
1.2 10% FORMOL–SALINE containing organs (e.g. Spleen and liver)
- 10% NaCl solution as diluent - Preserves cytoplasmic granules well
- Fixation of central nervous tissues 1.4 HEIDENHAIN’S SUSA SOLUTION
- Post-mortem tissues for histochemical - With trichloroacetic acid, glacial acetic acid, 40%
examinations aldehyde
- Fixation time: 24 hrs at 35 degrees / 48 hrs at RT - Recommended for tumor biopsies of skin
- Excellent cytologic fixative

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 - Fixation time 3-12 hrs BRASIL’S ALCOHOLIC PICROFORMOL FIXATIVE
- Penetrates and fixes tissue rapidly and evenly - With 37% formaldehyde, ethanol or isopropyl alcohol, and
trichloroacetic acid
1.5 B–5
- Less messy than Bouin’s solution
- Commonly used for bone marrow biopsies
- Excellent for glycogen
- Fixation time: 1-2 hrs
- Unstable GLACIAL ACETIC ACID
- Can make tissues brittle - Normally used in conjunction with other fixatives to form a
compound solution
2. CHROMATE FIXATIVES
- Fixes and precipitates nucleoproteins, chromosomes and
2.1 1–2% CHROMIC ACID
chromatin materials for nuclear component studies
- Usually constituent of a compound fixative
- Preserves carbohydrates and precipitates all ALCOHOL FIXATIVES
proteins
MECHANISM: Rapidly denatures and precipitates proteins by
- May produce sub oxide precipitates
destroying hydrogen bonds to stabilize the tertiary structure of
2.2 3% POTASSIUM DICHROMATE proteins; used both as fixative and dehydrating agent
- Preserves lipids
100% METHANOL
- Preserves mitochondria
- Recommended for fixing dry and wet smears, blood smears
2.3 REGAUD’S FLUID (Moller’s Fluid) and bone marrow tissues
- Fixation time: 12-48 hrs
70-100% ETHANOL
- Penetrates well
- May be used as simple fixative
- Recommended for demonstration of chromaffin
- Usually incorporated into compound fixatives for better
tissues (phaeochromocytoma), mitochondria,
results
mitotic figures, golgi bodies, RBC, and colloid-
- Fixes blood, tissue films and smears
containing tissues
- Preserves nucleoproteins and nucleic acids for
- Unstable
histochemistry and enzyme studies
- Poor nuclear staining
- Dissolves fat and lipids
2.4 ORTH’S FLUID - May shrink tissues
2.5 2.5% Potassium Dichromate; 40% Formaldehyde
- Fixation time: 36-72 hrs CARNOY’S FLUID
- Recommended for study of early degenerative - Most rapid fixative (1-3 hrs only)
processes and tissue necrosis - Fixes and dehydrates at the same time
- Used to fix brain for diagnosis of rabies
LEAD FIXATIVES - May cause RBC lysis
4% LEAD ACETATE - Shrinks and may harden tissues excessively
- Recommended for acid mucopolysaccharides - Slow penetration
- Fixes connective tissue mucin
ALCOHOLIC FORMALIN (Gendre’s Fixative)
- Takes up CO2 to form insoluble lead carbonates on
- 95% ethanol sat. with picric acid
standing
- 40% formaldehyde
PICRIC ACID FIXATIVES - glacial acetic acid
- Yellow color which can be removed by lithium carbonate or - Useful for sputum – it coagulates mucus
washing with 50-70% ethanol
- Picrates are soluble in water NEWCOMER’S FLUID
- Isopropanol, propionic acid, petroleum ether, acetone,
1% PICRIC ACID SOLUTION dioxane
- Penetrates and fixes small tissue rapidly - Acts both as nuclear and histochemical fixative
- Excellent for glycogen demonstration - Produces better reaction in Feulgen stain
- Suitable for aniline stains - Recommended for fixing mucopolysaccharides
- Causes RBC hemolysis
- Not suitable for frozen sections – causes it to crumble when OSMIUM TETROXIDE
cut - MECHANISM: Various hypotheses of lipid stabilization have
- Prolonged fixation makes tissue hard been postulated
- Oxidation of double bonds between adjacent carbon atoms
BOUIN’S SOLUTION to form monoesters and diesters
- Yellow stain is useful for fragmented biopsies - Binding of lipid to protein
- Minimal distortion of microanatomical structures - Conversion of unsaturated fatty acids to stable glycol
- Excellent for soft and delicate tissues osmates
- Preferred fixative for connective tissue staining
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6% OSMIUM TETROXIDE o Does not remove stains
- Fixes conjugated fats and lipids permanently by making o Non-toxic and not a fire hazard
them insoluble to alcohol and xylene during dehydration
and clearing ALCOHOL
- Preserves cytoplasmic structures well A. Ethyl alcohol (ethanol)
- Fixes materials for ultrathin sectioning in electron - It is recommended for routine dehydration of tissues
microscopy and considered to be the best dehydrating agent.
- Very expensive - Characteristics: clear, colorless, flammable fluid
- Penetrates poorly - Advantages: fast-acting, mixes with water and many
- Forms black precipitates upon exposure to sunlight inorganic solvents, penetrates tissue easily, not
poisonous and expensive
FLEMMING’S SOLUTION
- With glacial acetic acid for nuclear affinity B. Methyl alcohol (methanol)
- With 1% chromic acid as diluent - It is primarily used for blood and tissue films, and for
- Fixation time: 24-48 hrs smear preparations
- Recommended for nuclear preparation of such sections - Disadvantage: toxic to the body
- Permanently fixes fat
- Excellent fixative for nuclear structures e.g. Chromosomes C. Butyl alcohol
- Expensive - It is utilized in plant and animal micro-techniques.
- Advantage: Slow dehydrating agent producing less
FLEMMING’S SOLUTION w/o HOAc shrinkage and hardening than ethanol
- Recommended for cytoplasmic structures particularly the - Disadvantage: Slow dehydrating agent thus, it is not
mitochondria suitable for rapid tissue processing

TRICHLOROACETIC ACID ACETONE

- Sometimes incorporated into compound fixatives o Characteristics
- It precipitates proteins - Clear, colorless fluid that mixes with water, ethanol
- Softening effect on dense fibrous tissues - Most organic solvent
- May be used as a weak decalcifying agent
- Poor penetration o Advantages
- Cheap
ACETONE - Rapid-acting dehydrating agent which dehydrate in ½
- Used at ice cold temperature (-5 to 4°C) to 2 hours
- Recommended for the study of water diffusible enzymes i.e. - More miscible with epoxy resins than alcohol
Phosphatases and lipases
- Used in fixing brain tissues for diagnosis of rabies o Disadvantages
- Volatile - Highly flammable
- Dissolves fat - Penetrates tissues poorly
- Shrinks tissues excessively - Causes brittleness (tissues placed for prolonged period
of time)
HEAT FIXATION - Most lipids are removed from tissues
MECHANISM: Involves thermal coagulation of tissue proteins - Extremely volatile and inflammable (limited to small
- For rapid diagnosis pieces of tissue)
- Employed for frozen tissue sections and bacteriologic * Because of considerable tissues shrinkage produced, acetone is not
smears recommended for routine dehydration purposed

DEHYDRATION DIOXANE (DIETHLYENE DIOXIDE)

o It is the step-in tissue processing by which the intercellular and
o Characteristics
extracellular water from the tissue are removed after fixation and
- An excellent dehydrating and clearing agent
prior to wax infiltration
- Readily miscible in water, melted paraffin, alcohol and
o Dehydrating agents/dehydrants
xylol
o Solvents utilized in the removal of water, (e.g. Graded strengths
of alcohol) o Advantages
- Produces less tissue shrinkage
CHARACTERISTICS OF AN IDEAL DEHYDRATING AGENT - Tissues can be left in this reagent for long period of
o Dehydrates rapidly, producing no considerable shrinkage or time without affecting the consistency or staining
distortion of tissues properties of the specimen
o Does not evaporate very fast - Tissues may be placed directly into the solution after
o Ability to dehydrate fatty tissues washing out
o Does not harden tissues excessively
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o Disadvantages o May be used for demixing, clearing and dehydrating paraffin
- Expensive extremely dangerous section
- Tissues sections tend to ribbon poorly o Most staining procedures give improved results with
- Its vapor produce a cumulative tetrahydrofuran
- Highly toxic o Toxic
- It should not be used routinely (laboratory room should
o Should be in well ventilated room
be properly ventilated, and all residues should be
o Should be avoided if possible
washed down in the sink)
- Should not be recycled as the risk of creating explosive CLEARING (DEALCOHOLIZATION)
peroxides increases greatly o Process whereby alcohol or a dehydrating agent is removed
from the tissue
The following is an example of a time schedule for dehydration with o “Clearing Agent”
dioxane: o Tissue has a translucent appearance when the dehydrating agent
has been replaced by the solvent
GRAUPNER’S METHOD
(1st) Pure dioxane solution 1 hour FUNCTIONS OF THE CLEARING AGENT
(2nd) Pure dioxane solution 1 hour o When used after alcohol dehydration
(3rd) Pure dioxane solution 2 hours - Serves to mix with alcohol and remove it from the
(1st) Paraffin wax 15 minutes tissue
(2nd) Paraffin wax 45 minutes o When used after tissue section has been stained
(3rd) Paraffin wax 2 minutes - Will make microscopic tissue preparation transparent
Embed in mold and cool in water o When the tissue is to be cleared directly from water
- Will merely improve refractive index of the tissue,
WEISEBERG’S METHOD (glycerin and gum syrup)
- The tissues wrapped in a qauze bag and suspended in - No dealcoholization involved
a bottle containing dioxane and a little anhydrous
calcium oxide CHARACTERISTICS OF A GOOD CLEARING AGENT
SHOULD SHOULD NOT
- Water displaced from the tissues by dioxane and in
turn absorbed by calcium oxide or quicklime ▪ Be miscible with alcohol ▪ Produce excessive
- Dehydration period range from 3-24 hours ▪ Be miscible with and easily shrinkage,
removed by paraffin wax – to hardening
* Tissues which have been treated with a chromate fixative, e.g. facilitate penetration in the ▪ Dissolve out
Regaud’s or Moller’s fluid, should be thoroughly washed in running embedding process aniline dyes
tap water prior to treatment with dioxane in order to remove the ▪ Mounting medium – to facilitate ▪ Evaporate quickly
chromate mounting of sections in a water bath
▪ Make tissues transparent – not all
CELLOSOLVE clearing agents exhibit this
o Ethylene glycol monoethyl ether property
o Cellosolve dehydrates rapidly
FACTORS AFFECTING CLEARING
o Stored in it for months without producing hardening or
o Boiling point
distortion
o Those with low boiling point are more readily replaced by melted
o CAUTION
paraffin
- Combustible at 110-120°F
o Viscosity
- Toxic
o Affects the speed of penetration of the clearing agent
- If cannot be avoided, propylene-based glycol ether
o Prolonged exposure to clearing agent causes tissue to become
should be used instead of ethylene-based glycol ethers
brittle
TRIETHYL PHOSPHATE COMMONLY USED CLEARING AGENTS
o Removes water very readily and produces very little distortion XYLENE (XYLOL)
and hardening
o Colorless
o Used to dehydrate sections and smears following certain stains o Most commonly used
o Produce minimum shrinkage o Clearing time: ½-1hour used for clearing, both for embedding
and mounting procedures
TETRAHYDROFURAN (THF) o Generally suitable for routine histologic processing schedules of
o Both dehydrates and clears tissues less than 24hours and when tissue block is less than 5mm in
o Dissolve many substances including fats thickness
o Miscible with lower alcohols, ether, chloroforms, acetone, o Advantages
benzene - Most rapid clearing – for urgent biopsies (15-30mins)
- Makes tissues transparent
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 - Miscible with absolute alcohol and paraffin o Advantages
- Does not extract out aniline dyes - For routine work (6-24 hours)
- For mounting procedures: does not dissolve celloidin - Miscible with absolute alcohol
- During embedding and impregnation: evaporates - Causes minimum shrinkage
quickly in paraffin oven - For tough tissues (e.g. skin, fibroid, decalcified tissues)
- For nervous tissues, lymph nodes and embryos
- Can be readily replaced by wax cheap
- For large tissue specimens not inflammable
o Disadvantages
o Disadvantages
- Highly inflammable
- Toxic to the liver after prolonged inhalation
- Makes tissue excessively hard if used over 3 hours
- Wax impregnation after chloroform clearing relatively
- Not suitable for nervous tissues and lymph nodes
slow
- Causes considerable hardening and shrinkage
- Does not make tissues transparent
- Not very volatile in paraffin oven
TOLUENE
- Vapor may attack rubber seal used in vacuum
o May be used as substitute for xylene or benzene for clearing
impregnating bath
both during embedding and mounting processes
- Complete clearing is difficult to evaluate
o Clearing time: 1-2 hrs
- Tissues tend to float in chloroform
o Advantages - Evaporates quickly from a water bath
- Miscible with both absolute alcohol and paraffin
- Acts fairly rapidly CEDARWOOD OIL
- Recommended for routine purposes o Used to clear both paraffin and celloidin sections during the
- Does not make tissues excessively hard and brittle if left embedding process
for 24 hours o Especially recommended for CNS tissues and cytological studies
(smooth muscles and skin)
o Disadvantages
o Requires two changes in clearing solution
- Slower than xylene and benzene
o Clearing time: 2-3 days
- Acidifies in a partially filled vessel
- Highly conc solutions will emit fumes that are toxic o Advantages
upon prolonged exposure - Very penetrating
- More expensive - Miscible with 96% alcohol which it removes readily
- Clears celloidin in 5-6 days
BENZENE - Causes minimal shrinkage and hardening
o Preferred by some in the embedding process because it - Tissues may be left indefinitely without causing
penetrates and clears tissues rapidly considerable damage and distortion
- Makes tissues transparent
o Advantages - Improves cutting of sections
- Rapid acting – for urgent biopsies (15-60 mins) and
routine purposes o Disadvantages
- Volatilizes rapidly in paraffin oven - Extremely slow – not for routine purposes
- Easily eliminated from the tissue - Hard to be eliminated from tissues in paraffin bath
- Does not make tissues hard and brittle - Quality not always uniform and good
- Causes minimum shrinkage - Cedarwood oil becomes milky upon prolonged
- Makes tissues transparent storage, should be filtered before use
- Cedarwood oil previously used to clear acetic-alcohol
o Disadvantages fixed tissues may produce crystals
- Highly inflammable - Very expensive
- Causes considerable tissue shrinkage if left in benzene
for a long time ANILINE OIL
- Excessive exposure may be extremely toxic to man
o Not normally utilized as a routine clearing agent
- May damage bone marrow
o Recommended for clearing embryos, insects, and very delicate
- Aplastic anemia
specimens
o Ability to clear 70% alcohol w/o excessive shrinkage and
CHLOROFORM
hardening
o When used for clearing in embedding process, slower than
xylene CLOVE OIL
o Causes less brittleness
o Causes minimum shrinkage
o Can be used for thicker tissue blocks (up to 1 cm)
o Quality not guaranteed – tendency to become adulterated
o Tissues do not become translucent
o Wax impregnation slow and difficult

6
o Tissues become brittle, aniline dyes removed, celloidin is EMBEDDING MEDIUM
dissolved PARAFFIN
o Expensive
o Rapidly converted from solid to liquid form on heating
o Unsuitable for routine clearing purposes
o Permeates the tissue in a liquid state
CARBON TETRACHLORIDE o Solidifies relatively quickly on cooling
o Becomes fluid on heating to a temperature which will not
o Used in clearing tissues for embedding
damage the tissue
o Properties similar to chloroform
o When the paraffin solidifies it becomes firm enough to section at
o Produces considerable tissue hardening
room temperature
o Highly toxic- dangerous to inhale on prolonged exposure
o Types of Paraffin Waxes
METHYL BENZOATE AND METHYL SALICYLATE - Paraplast
o Slow-acting clearing agents - Fibrowax
o Can be used when double embedding techniques are required - Bioloid Embeddol
- Ester Wax
IMPREGNATION AND EMBEDDING - Highly purified paraffin waxes with DMSO
EMBEDDING (dimethylsulfoxide) – elastic & resilient
o Upon completion of infiltration, the tissue is embedded in an
embedding medium o Advantages
o A suitable embedding mold is filled with the molten wax, the - Time of infiltration and subsequent embedding are
tissue is placed in it and oriented so it is sectioned in the proper relatively short for small pieces of tissue
plane - Thin sections can be cut with the rotary microtome and
o Wax to be used must contain no trace of clearing agent, dust sections will adhere to each other to form a ribbon
particles, and must be rapidly cooled to reduce the wax crystal - Tissue once infiltrated and embedded can be stored in
size a dry condition indefinitely without damage to the
o A variety of molds can be used depending on the technician’s tissue
preference o Disadvantages
TECHNIQUES FOR EMBEDDING TISSUES - Distortion of the histology of the tissue due to
A. Transfer the tissue with warm forceps to a small container of shrinkage may occur, especially when sections are
freshly melted paraffin (tips of forceps are heated in an alcohol being attached to glass slides (paraffin artifact)
lamp or in forceps warmer, tips should be hot enough so - Sectioning of paraffin is difficult at high temperatures
paraffin does not solidify, but not so hot as to cause paraffin to - Time for infiltration of large blocks of tissue is excessive
smoke).
B. Fill the bottom of the mold with a small amount of paraffin. The CELLOIDIN
depth of the mold should be at least twice the thickness of the o Amorphous, slightly yellowish substance
tissue. o Purified form of collodion or nitro-cellulose
C. Pick up tissue, and place into the mold. Manipulation of the o For hard tissue specimens
tissue in the mold must be quick, so paraffin does not begin to
o Advantages
harden.
- Does not require heat
D. After tissue is in the mold, fill mold entirely with the paraffin. As
- Has a rubbery consistency
the paraffin begins to harden insert a code number label; the
- Minimal distortion of specimen
label should not go down to the bottom of the paraffin.
E. Allow the surface of the paraffin block to harden, then immerse o Disadvantages
the mold into a shallow, cool (10°C) water bath for about 10-15 - Difficult to cut thin sections
min to hasten solidification of the paraffin. - Serial sections are difficult to prepare
F. When paraffin is completely hardened, remove it from the mold. - Slow process
- Blocks and sections must be stored in 70% alcohol
Cooling Temperature otherwise they become discolored, dry, and shrunken
- 10°C temperature prevents cracking of the tissue block
- If paraffin is properly cooled, the crystals of paraffin are LOW VISCOSITY NITROCELLULOSE (LVN)
small and contiguous with each other o Advantages
- The paraffin will appear clear and homogeneous and - Low viscosity; allows higher concentration to be used
there is no layering of the paraffin - Greater speed of impregnation
- Final block is harder, allowing thinner sections to be cut
- Paraffin demonstrating these conditions is best for
- Has a greater water tolerance than celloidin
sectioning.

7
o Disadvantages
- Have a tendency to crack down during handling and DISPOSABLE EMBEDDING MOLD
staining 1. PEEL-A-WAY
- Use 0.5% oleum ricini (castor oil) to minimize this - Once the wax has solidified, the plastic walls are peeled
tendency off one at a time, giving perfect blocks that require no
- Highly explosive trimming
- They can be placed directly in the chuck of the
DOUBLE EMBEDDING microtome
o Tissue is first impregnated with celloidin, and subsequently
2. PAPER BOAT
blocked in paraffin wax
- Made from thick paper or cardboard paper
o Used in dealing with hard tissues
- Cheap to make and allow blocks to be stored without
o For maintenance of the morphological appearance of the tissue
being removed
o Serial sections are easily prepared
- Provide easy and accurate identification of specimens,
o Extra degree of resilience is given when cutting hard tissues
thereby avoiding confusion and interchange of tissue
AGAR blocks

o Main use is in double embedding technique with ester wax or PLASTIC ICE TRAY
paraffin wax
o Convenient molds for busy routine laboratory, one block being
o Cohesive agent for multiple fragments or friable tissue
embedded in each compartment
GELATIN o Blocks are easily removed by flexing the plastic trays and by
smearing the inside of the mold with glycerin or liquid paraffin
o Has a lower melting point than agar
o Main use in the production of whole organ sections WATCH GLASSES
o For friable tissues
o Ideal for embedding fragmentary biopsies
WATER-SOLUBLE WAXES o Not essential to smear them with glycerin
o Blocks are hard to remove
o Tissue can be embedded directly from water
- However, it is restricted, due to the violent diffusion TEST TUBES
currents which can lead to the complete fragmentation
o Used for small fragments which have been processed (e.g. Bone
of the section
marrow) which concentrates them without the damage caused
PLASTIC EMBEDDING MEDIUM by orientation with forceps
o Disadvantage: often necessary to break the tube to remove the
Classified into:
block
EPOXY Reduces antigenicity, toxic, and damages tissue
POLYESTER Not often used METHACRYLATE PLASTIC RESIN (EPON RESIN)
ACRYLIC Used extensively for light microscopy
o Used for embedding tissue intended
o For EM microscopy
MOLDS FOR EMBEDDING
LEUCKHART’S EMBEDDING MOLD STAINING
o Molds for routine work and are widely used MAJOR GROUPS OF TISSUE STAINING
o Consist of 2 l-shaped pieces of metal 1. HISTOLOGICAL STAINING
o Arranged on a glass metal plate to form a mold of desired size - The process whereby the tissue constituents are
demonstrated in sections by direct interaction with a
COMPOUND EMBEDDING UNIT dye or staining solution
- Active tissue component is colored
o Consist of a series of interlocking plates resting on a flat metal
- E.g. micro-anatomical stains, bacterial stains, specific
base, forming several compartments
tissue stains (e.g. muscles, connective tissue and
o Has the advantage of embedding more specimens at a time
neurologic stains)
PLASTIC EMBEDDING RING AND BASE MOLD 2. HISTOCHEMICAL STAINING (HISTOCHEMISTRY)
o Used in positioning histological tissues accurately in base molds - The process whereby various constituents of tissues are
o Compatible with most commonly-used processing and storage studied thru chemical reactions that permits
systems - Microscopic localization of specific tissue substances
o Rings are precision-molded from premium-grade, chemically- - E.g. Perl’s Prussian Blue reaction for hemoglobin and
inert, high impact polystyrene for dimensional rigidity and Periodic Acid Schiff staining for carbohydrates
sturdiness
* Enzyme histochemistry
POP-OUT EMBEDDING MOLD - Active reagent: substrate
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 - Tissue: enzymes o Alcohol
- The final opacity or coloration is produced from the - Differentiator for both acidic and basic dyes by
substrate rather than the tissue dissolving excess dye

3. IMMUNOHISTOCHEMICAL STAINING o Mordant (e.g. iron alum)

- A combination of immunologic and histochemical - A differentiating agent
techniques that allow phenotypic markers to be - Can oxidize hematoxylin to a soluble, colorless
detected by antibodies (e. g. polyclonal, monoclonal, compound
enzyme-labeled or fluorescent-labeled - Disadvantage: if a mordant stained section is allowed
to remain in a differentiating agent such as 1% or 2%
METHODS OF STAINING alcohol, all of the dye will be removed
1. DIRECT STAINING - Restaining faded slides
- Uses aqueous or alcoholic dye solutions (e.g.
methylene blue, eosin) to produce a color METACHROMATIC STAINING
o Makes use of specific dyes which differentiate particular
2. INDIRECT STAINING substances by staining it with a color that is different from that of
- Uses a mordant or another agent to intensify the action the stain itself (metachromasia)
of the dye used o Usually employed in staining cartilage, connective tissue,
epithelial mucins, amyloid and mast cell granules.
MORDANT o Metachromatic dyes (basic) – belongs to thiazine and
o Serves as a link or bridge between the tissue and the dye triphenylmethane groups
o The dye may stain weakly by itself, therefore the mordant 1. Methyl violet or crystal violet
combines with the dye forming a colored “lake” which would 2. Cresyl blue (for reticulocytes)
combine with the tissue forming an insoluble “tissue-mordant- 3. Safranin
dye-complex”, which would allow subsequent 4. Bismarck brown
o Counterstaining and dehydration 5. Basic fuchsin
o E.g. Potassium alum with hematoxylin in Ehrlich’s hematoxylin. 6. Methylene blue
Iron in Weigert’s hematoxylin 7. Thionine
8. Toluidine blue
ACCENTUATOR 9. Azure A, B, C
o Not essential and does not participate to the chemical reaction o Water is necessary for most metachromatic staining techniques
of the tissue and dye o Metachromasia is usually lost if section is dehydrated in alcohol
o Accelerates the speed of the staining reaction by increasing the after staining
staining power and selectivity of the dye o Metachromasia is satisfactorily seen in formalin-fixed tissues
o E.g. Potassium hydroxide in Loeffler’s methylene blue, Phenol in
Carbol thionine and Carbol fuchsin COUNTERSTAINING
o Application of a different color or stain to provide contrast and
PROGRESSIVE STAINING background to the staining of the structural components to be
o Tissue elements are stained in definite sequence demonstrated
o The staining with specific periods of time or until desired color is CYTOPLASMIC STAINS
attained Red Yellow Green
o Not washed or decolorized ▪ Eosin Y ▪ Picric acid ▪ Lt. Green SF
o The distinction of tissue detail relies solely on the selective affinity ▪ Eosin B ▪ Orange G ▪ Lissamine
of the dye for various cellular elements ▪ Phloxine B ▪ Rose Bengal Green

REGRESSIVE STAINING NUCLEAR STAINS

Red Blue
o First over-stain the tissue to obliterate cellular details
▪ Red ▪ Methylene Blue
o Excess stain is removed or decolorized from unwanted parts of
▪ Neutral Red ▪ Toluidine Blue
the tissue and until the desired color is obtained
▪ Safranin O ▪ Celestine Blue
DIFFERENTIATION / DECOLORIZATION ▪ Carmine ▪ Hematoxylin

o The selective removal of excess stain from the tissue during

METALLIC IMPREGNATION
regressive staining so that a specific substance may stain
distinctly from the surrounding tissue o The process where specific tissue elements are demonstrated not
o Usually done by washing the section in simple solution (e.g. by stains but by colorless solutions of metallic salts which are
water or alcohol) or use of acids and oxidizing agents deposited on the surface of the tissue
o Primary stain = basic dye; Differentation = acidic solution o It is not absorbed by the tissues, could be a precipitate or a
o Primary stain = acidic dye; Differentation = alkaline solution reduction product on certain tissues
o E.g. gold chloride, silver nitrate
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VITAL STAINING H and E RESULT
o The selective staining of living cell constituents o Nuclei – blue to blue black
o Demonstrates cytoplasmic structures o Karyosome – dark blue
o By engulfment of the dye particle o Cytoplasm – pale pink
o By staining of pre-existing cellular components o RBCs, eosinophilic granules, keratin – bright-orange red
o Nucleus is resistant to vital stains
o Calcium and decalcified bone –purplish blue
o Two types
o Decalcified bone matrix, collage and osteoid – pink
1. INTRAVITAL STAINING
o Muscle fibers – deep pink
• By injecting the dye into any part of the
animal body
STAINS AND STAINING SOLUTIONS
• E.g. lithium, carmine and India ink
o Divided into 2 categories:
2. SUPRAVITAL STAINING
- Natural dyes
• Used immediately after removal of cells from
- Synthetic (Artificial) dyes
the living body
• E.g. Neutral red (best), Janus green
NATURAL DYES
(mitochondria), Trypan blue, Nile blue,
Thionine and Toluidine Blue Routine o Derived from plants and animals
Hematoxylin and Eosin (H & E) o E.g. Hematoxylin, Cochineal dyes, Orcein, Saffron
• Most common method utilized for 1. HEMATOXYLIN
microanatomical studies of tissues - Hematoxylin campechianum
• Uses the regressive staining which consists of - It is not a stain
a. overstating the nuclei - Active coloring agent – hematin
b. removal of superfluous and - Oxidation of hematoxylin through the process of
excessive color of the tissue “ripening”
constituent by acid differentiation - Used in combination with a mordant such as alum,
iron, chromium and copper salts
H and E PROCEDURE
1. Clear paraffin embedded sections in first xylene bath for 3 RIPENING ARTIFICIAL RIPENING
minutes ▪ Natural ripening ▪ Chemical oxidation
2. Transfer to second xylene bath for 2 to 3 minutes. ▪ Expose the substance to ▪ Hydrogen peroxide,
3. Immerse in first bath of absolute ethyl alcohol for 2 minutes. air and sunlight mercuric oxide, potassium
4. Transfer to a bath of 95% ethyl alcohol for 1 to 2 minutes. ▪ A slow process, 3-4 permanganate
5. Rinse in running water for a minute. months ▪ Sodium perborate, sodium
iodate
6. Stain with Harris Alum Hematoxylin for 5 minutes (Ehrlich’s
▪ Over-ripening
hematoxylin requires 15-30 minutes).
▪ Excessive oxidation
7. Wash in running tap water to remove excess stain.
8. Differentiate in 1% acid-alcohol (1mL conc. HCl to 99 mL of 80% 2. COCHINEAL DYES
ethyl alcohol) for 10-30 secs. Until the nuclei are stained. - Extracted from Coccus cacti
9. Rinse in tap water. - With alum
10. Use ammonia water (average of 5 minutes) or 1% aqueous - Carmine dye
lithium carbonate until the section appears blue (about 30 - Chromatin and nuclear stain for fresh and smear
preparation
seconds)
- With picric acid
11. Wash in running water for 5 minutes.
12. Counterstain with 5% aqueous eosin for 5 minutes. If alcoholic Picrocarmine
eosin is used, the time can be reduced to 30 seconds or 1 - Neuropathological stain
minute. - With aluminum chloride
13. If aqueous eosin is used, wash and differentiate in tap water - Best’s carmine
under microscopic control until the nuclei appear sharp blue to - Demonstration of glycogen
blue black and the rest is in shades of pink. If alcoholic solution is 3. ORCEIN
used, differentiate with 70% alcohol. - Vegetable dye
14. Dehydrate, clear and mount. - Extracted from lichens
- Colorless, treated with ammonia, exposed to air to
produce a blue or violet color
- Weak acid, soluble in alkali
- Used for staining elastic fibers

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SYNTHETIC DYES D. COLE’S HEMATOXYLIN
o Also known as “coal tar dyes” - Ripened with alcoholic iodine
o Derived from hydrocarbon benzene
E. MAYER’S HEMATOXYLIN
o Collectively known as “aniline dye”
- Ripened with sodium iodate
o Chromophore
- Nuclear counterstain
- Substances that are capable of producing visible color
but is not permanent and can be easily removed IRON HEMATOXYLIN
o Auxochrome
o Used for regressive staining
- Substances that are added to a chromogen, which
o Blue black lakes
alters the property of the chromogen by altering its
o Iron is an active oxidizing agent
shade, enabling it to form salts with another compound
and enables it to retain its color in the tissue A. WEIGERT’S HEMATOXYLIN
- Standard iron hematoxylin
Classified into 3 groups based on where the coloring substance is
- Used in demonstrating connective tissue
found:
1. ACID DYES B. HEIDENHAIN’S HEMATOXYLIN
- The coloring substance is found in the acid component - For nuclear and cytoplasmic inclusions
and the inactive base is usually the sodium salt of a
sulfonate of rosaniline C. PHOSPHOTUNGSTIC ACID HEMATOXYLIN
- Basic cell structures have an affinity for acid dye ions - Natural ripening achieved with light and air
and are called acidophilic - Color: reddish brown to purple
- Progressive stain
2. BASIC DYES
- The coloring substance is found in the basic EOSIN
component that combines with the acid radical o A red acid dye
- Acidic structures have an affinity for basic dyes and are o Routinely used as a counterstain after hematoxylin and before
called basophilic methylene blue
3. NEUTRAL DYES o Stains connective tissues and cytoplasm differentially
- Formed by combining aqueous solutions of acid and o 3 forms:
basic dyes 1. Yellow (Eosin Y) – most commonly used; green yellow
- Stains the cytoplasm and nucleus simultaneously and fluorescence
differentially 2. Eosin B, Erythrosin B – deeper red color
- Insoluble to barely soluble in water 3. Eosin S, Eosin alcohol-soluble – Ethyl eosin
- Soluble in alcohol
MOUNTING AND LABELLING
COMMON STAINING SOLUTIONS o Process that involves the use of a medium and a coverslip to
facilitate the ease of handling and storage of the slide and to
HEMATOXYLIN prevent damage to the section
o Most commonly used for histologic studies o Syrupy fluid applied between the section and the coverslip after
staining, setting the section firmly, preventing the movement of
A. ALUMINUM HEMATOXYLIN
the coverslip
- Progressive and regressive staining
o Protects the stained section from getting scratched, slides for
- 2 main alum hematoxylin (ripening agent)
permanent keeping, to facilitate easy handling and storage
- Erlich’s – sodium iodate
o Serves to prevent the distortion of image during microscopic
- Harris – mercuric chloride
examination
- Forms blue lakes
CHARACTERISTICS OF A GOOD MOUNTING MEDIUM
B. ERLICH’S HEMATOXYLIN o A refractive index which is near to that of the glass (1.518)
- Natural ripening hematoxylin (2 months) o Miscible with xylene and toluene
- Regressive staining o Does not dry quickly
- Sodium Iodate shortens lifespan o Does not produce artifacts on the slides
- Stains mucopolysaccharide substances (e.g. cartilages, o Does not dissolve out or fade tissue sections
cement lines of bones), tissues subjected in acid o Does not cause shrinkage and distortion of tissues
decalcification, tissues stored in formalin o Does not leach out any stain or affect staining
- Not an ideal stain for frozen sections o Does not change in color or pH
C. HARRIS HEMATOXYLIN o Sets hard and produces permanent mounting of sections
- Dark purple color when ripened with mercuric chloride
- Glacial acid for better nuclear staining
- Routine stain

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KINDS AND USES OF MOUNTING MEDIA
1. AQUEOUS MOUNTING MEDIA
- Designed to mount water-miscible preparations directly
from water in cases where the stain is removed or
decolorized with alcohol or xylene, or for
metachromatic staining of amyloid
- Usually made up of gelatin, glycerin jelly or gum arabic,
glycerol, sugar and a preservative solution

Water Temporary medium

Glycerin Preservative actions, set hard;
Keeps sections for years after ringing
Farrant’s medium Dissolved gum Arabic;
Takes long to harder and requires ringing
Apathy’s medium Used for methylene blue stained nerve
preparations;
Set hard and does not require ringing
Brun’s fluid Recommended for mounting frozen sections

2. RESINOUS MOUNTING MEDIA

- Used for preparations that have been dehydrated and
cleared in xylene or toluene
- Recommended for majority of staining methods
- Divided into natural and synthetic

Canada Balsam
- Transparent and colorless resin that adheres to glass
- Darkens with age and oxidizes xylene to become acidic
- Recommended for whole mounts and thick sections

DPX®
- For small tissue sections due to shrinkage produced upon
drying
- Dries rapidly

XAM
- Synthetic mixture in xylene
- Dries quickly without retraction

CLARITE ®
HISTOCLAD
EUKITT
PERMOUNT
ENTALLAN

RINGING
o Process of sealing the margins of the cover-slip to prevent the
escape of fluid or semi-fluid mounts and evaporation of
mountant, to immobilize the coverslip, and to prevent sticking of
the slides upon storage

1. KRONIG CEMENT
• Made up of two parts paraffin was mixed with
4-9 parts powdered colophonium resin,
heated and filtered
2. DUROFIX
• Cellulose adhesives

LABELLING
o Process of indicating the year and specimen number on one end
of the prepared slide for proper identification

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