HISTOPATHOLOGIC

TECHNIQUES
 GROSS EXAMINATION
• “Grossing”
• From Middle English
“gross” – whole, entire.
• The pathologist describes
and obtains measurements
of the specimen.
• Small pieces of tissues are
cut for tissue processing.
• The medical technologist
assists in this process.
 TWO MAJOR PROCESSES:
• Fresh Tissue Examination
• Preserved Tissue Examination

Fresh Tissue Preserved Tissue

 FRESH TISSUE
EXAMINATION
Fresh tissues have the advantage of
being examined in the living state, allowing
protoplasmic activities (motion, mitosis,
phagocytosis, pinocytosis) to be observed.
 METHODS OF FRESH TISSUE
EXAMINATION

Teasing or Dissociation – process where a

tissue specimen is immersed in a watch glass
containing isotonic salt solution either stained or
unstained (viewed using a Phase Contrast or
Bright Field Microscope).
 METHODS OF FRESH TISSUE
EXAMINATION

Squash Preparation or Crushing

• Tissues less than or equal to 1mm in diameter are
pressed between 2 slides or between a slide and
a cover glass.
• Vital stain is applied between the slides
• Viewed using a Phase Contrast or Bright Field
microscope
 METHODS OF FRESH TISSUE
EXAMINATION
Smear Preparation
• Process of examining sections or sediments
where cellular materials are spread lightly over
a slide by means of a wire loop, applicator stick
or another slide.
• Includes streaking, spreading, pull-apart, and
touch preparation.
• This technique is especially useful in
cytological examinations especially for cancer
diagnosis.
 METHODS OF FRESH TISSUE
EXAMINATION

A. Streaking: rapidly and gently applied in a direct

or zigzag line throughout the slide.

B. Spreading: gently spread material into a

moderately thick film; recommended for fresh
sputum, bronchial aspirates and thick mucoid
secretions.
 METHODS OF FRESH TISSUE
EXAMINATION
C. Pull – Apart: a drop or sediment is placed on a
slide, faced into another slide and is pulled apart
in a single, uninterrupted motion. This is
recommended for thick secretions including
serous fluids, concentrated sputum, enzymatic
lavage samples from GI tract and blood smears.

D. Touch preparation or Impression smear:

surface of a freshly cut piece of tissue is brought
into contact and pressed on the surface of a
clean glass slide. The cells are examined without
destroying their actual intercellular relationship.
 METHODS OF FRESH TISSUE
EXAMINATION

Frozen section – is normally used when a rapid

diagnosis of tissues is required.

Uses a Cryostat: a cold chamber kept at an

atmospheric temperature of -10 to -20 degree
Celsius which contains a modified Rotary
microtome.
 METHODS OF FRESH TISSUE
EXAMINATION

Frozen section applications:

1. Rapid pathologic diagnosis during surgery,
2. Enzyme histochemistry,
3. Demonstration or soluble substances such as
lipids and carbohydrates,
4. Immunofluorescent and immunocytochemical
staining, and
5. Some specialized silver stains, particularly in
neuropathology.
 PRESERVED TISSUE
EXAMINATION
After gross examination,
tissues are placed in
plastic cassettes and are
subjected to subsequent
tissue processing.
 STEPS IN PRESERVED TISSUE EXAMINATION
1. Labeling (Numbering)
Minor Steps:
2. Fixation
Washing Out
3. Decalcification
Orientation
4. Dehydration
Trimming
5. Clearing
Floating Out
6. Impregnation
Adhesion
7. Embedding
Ringing
8. Section Cutting
Tissue processing
9. Staining
starts with labeling
10.Mounting and ends with labeling.
11.Labeling (Numbering)
 FIXATION
• One of the most critical step in histotechnology.
• Process that preserves tissues from decay,
thereby preventing autolysis and putrefaction.
• The process should be carried out as soon as
possible after removal of tissue from the body.

Goals of fixation:
• Preserve the morphologic and chemical integrity of
the cell in as life – like manner as possible.
• Harden and protect the tissue from the trauma of
further handling.
 FIXATION
Objectives of fixation:
• To preserve the tissue (stops all cellular
activities),
• To prevent breakdown of cellular elements
(prevent autolysis and putrefaction), and
• To coagulate or precipitate protoplasmic
substances (renders tissue components
insoluble).
 MAIN FACTORS INVOLVED IN
FIXATION
1. Hydrogen Ion Concentration (pH 6 to 8)
2. Temperature – for routine surgical specimens,
use room temperature.
3. Thickness of the section (block):
• Electron microscopy: 1 to 2 mm2
• Light microscopy: 2 cm2
• Measurements should not be compromised to
obtain full penetration and satisfactory fixation.
• Large tissues like uterus should be opened or
sliced thinly.
• Brain is suspended whole for 2-3 weeks.
 MAIN FACTORS INVOLVED IN
FIXATION
4. Osmolality
• Hypertonic – causes cell shrinkage.
• Isotonic and Hypotonic – causes cell swelling and
poor fixation.
• For best results, use slightly hypertonic solutions
with an osmolality of 400 – 450 mOsm (isotonic
solutions are 340 mOsm).
5. Concentration of solutions – Formaldehyde is
most commonly used as 10% solution while
glutaraldehyde is used as 3% solution. Low
concentration (0.25%) of glutaraldehyde is ideal for
immunoelectron microscopy.
 MAIN FACTORS INVOLVED IN
FIXATION

6. Duration of fixation
• Prolonged fixation: may cause cell shrinkage and
hardening of tissues.
• Incomplete fixation: softening of tissues, very
hard to cut during section – cutting.
 PRACTICAL CONSIDERATIONS OF
FIXATION

1. Speed – specimens are placed in fixative as soon

as it is removed from the body. This is done to
prevent autolysis and putrefaction.
2. Penetration – formalin diffuses into tissues at a
rate of approximately 1 mm per hour and slows
down as it goes deeper. However, this varies with
different types of tissues.
3. Volume – the ideal fixative to tissue ratio is 20:1
4. Duration of fixation – the usual length of time for
fixation is 24 hours but some tissues take longer
times to fix than others.
TYPES OF FIXATIVES
I. According to Composition
A. Simple Fixatives
B. Compound Fixatives

II. According to Action

A. Microanatomical Fixatives
B. Cytological Fixatives
a. Nuclear
b. Cytoplasmic
c. Histochemical
A. Microanatomical fixatives – for general
microscopic study of tissue structures.

Examples:
10% Formol Saline Formol sublimate
10% BNF Zenker's solution
Heidenhain's SuSa Zenker-formol
Bouin's Brasil's
B. Cytological – specific parts and particular
microscopic elements of the cell itself.

1. Nuclear fixatives - preserves nuclear structures of

the cell. They usually contains glacial acetic acid
(pH 4.6 or less) which fixes nucleoprotein.
2. Cytoplasmic fixatives – Useful for preservation of
cytoplasmic details or structures (pH greater than
4.6). This must never contain glacial acetic
acid because it destroys mitochondria and
Golgi bodies.
3. Histochemical fixatives – preserves the chemical
constituents of the cell.
 WASHING OUT
• The process of removing excess fixative from
the tissue after fixation in order to improve
staining and remove artefacts from the tissues.
• Several solutions may be used: tap water, 50 –
70% alcohol and alcoholic iodine.
 STEPS IN PRESERVED TISSUE EXAMINATION
1. Labeling (Numbering)
Minor Steps:
2. Fixation
Washing Out
3. Decalcification
Orientation
4. Dehydration
Trimming
5. Clearing
Floating Out
6. Impregnation
Adhesion
7. Embedding
Ringing
8. Section Cutting
Tissue processing
9. Staining
starts with labeling
10.Mounting and ends with labeling.
11.Labeling (Numbering)
DECALCIFICATION
• It is the process of removing calcium ions from a
bone or calcified tissue through a histological
process that makes them flexible and easier to cut.

• Principle: Strong mineral acids or chelating agents

form soluble calcium salts in an ion exchange
process that moves calcium into decalcifying
solution.

• Poorly – fixed specimens become macerated during

decalcification and stain poorly afterwards.
 Considerations:
1. More concentrated acid solutions decalcify bone
more rapidly but are more harmful to the tissue.
2. High concentrations and greater amount of fluid
will increase the speed of the process.
3. The recommended ratio of fluid to tissue volume:
20 is to 1.
4. Heat will serve to hasten decalcification but also
increases the damaging effects on tissues.
5. Optimal temperature is room temperature.
6. Ideal time required for complete decalcification is
24 to 48 hours but also varies with tissues and
the type of agent used.
DECALCIFICATION
7. Dense bone tissues usually require up to 14 days
or longer in order to complete the process
8. The extent of decalcification may be measured
with various methods.
9. Examples of decalcifying agents:
• Acids
• Chelating agents
• Ion exchange resin
• Electrical ionization
 MEASURING EXTENT OF
DECALCIFICATION
1. Physical or Mechanical Test – this is done by
touching or bending the tissue with fingers to
determine consistency of tissues. Another
method is by pricking with fine needle or probe.

2. X-ray or Radiological Method – considered to

be the most ideal, most sensitive, most reliable
method. It has the ability to detect even the
smallest focus of calcium which appears opaque
on x-ray plate.
 MEASURING EXTENT OF
DECALCIFICATION
3. Chemical (Calcium Oxalate) Method –
simple, reliable and the most convenient
method. It is the one recommended for routine
purposes.

If chemical method of decalcification is to

be done, the decalcifying agent should be
prepared with distilled water since false positive
readings may be produced by calcium ions
present in tap water.
 TISSUE SOFTENERS
They are used for unduly hard tissues that may
damage the microtome knives. Examples are:
• 4% aqueous phenol
• Molliflex
• 2% Hydrochloric acid
• 1% HCl in 70% alcohol
 STEPS IN PRESERVED TISSUE EXAMINATION
1. Labeling (Numbering)
Minor Steps:
2. Fixation
Washing Out
3. Decalcification
Orientation
4. Dehydration
Trimming
5. Clearing
Floating Out
6. Impregnation
Adhesion
7. Embedding
Ringing
8. Section Cutting
Tissue processing
9. Staining
starts with labeling
10.Mounting and ends with labeling.
11.Labeling (Numbering)
DEHYDRATION
• The process of removing intercellular and
extracellular water from the tissue after fixation and
prior to impregnation is called dehydration.
• It is necessary to remove the fixative and water
from the tissue and replace them with dehydrating
fluid in preparation for impregnation.
• General rule: the amount in each stage should not
be less than 10x the tissue volume.
• Dehydrating fluids are generally used in increasing
strengths (provides little disruption to the tissue due
to diffusion currents).
DEHYDRATION
Characteristics of an ideal dehydrating solution:
• It should dehydrate rapidly without producing
considerable shrinkage and distortion of tissues
• It should not evaporate very fast
• It should be able to dehydrate even fatty tissues
• It should not harden tissues excessively
• It should not remove stains
• It should not be toxic to the body
• It should not be a fire hazard
 DEHYDRATION
Commonly used dehydrating agents:
• Ethanol - for routine dehydration and
considered as the best dehydrating agent.
• Methyl Alcohol - employed for blood and
tissue films.
• Acetone - both fixative and dehydrating
agent
 DEHYDRATION
Commonly used dehydrating agents:
• Dioxane (Diethylene dioxide) - both
dehydrating and clearing agent
• Cellosolve (Ethylene glycol monoethyl ether)
• THF (Terahyrofuran) - both dehydrating and
clearing agent
• Triethyl phosphate
INDICATORS OF INCOMPLETE
DEHYDRATION
1. Anhydrous Copper Sulfate – turns blue
upon contact with water.
2. Xylene – If it turns milky, there is incomplete
dehydration (insoluble).
 STEPS IN PRESERVED TISSUE EXAMINATION
1. Labeling (Numbering)
Minor Steps:
2. Fixation
Washing Out
3. Decalcification
Orientation
4. Dehydration
Trimming
5. Clearing
Floating Out
6. Impregnation
Adhesion
7. Embedding
Ringing
8. Section Cutting
Tissue processing
9. Staining
starts with labeling
10.Mounting and ends with labeling.
11.Labeling (Numbering)
 CLEARING
• Also known as de alcoholization.
• The process by which the alcohol or a dehydrating
agent is removed from the tissue and replaced with
a substance that will dissolve the wax with which
the tissue is to be impregnated and or the medium
with which it is to be mounted.
• When the dehydrating agent has been entirely
replaced by the solvent, the tissue will have a
translucent appearance, hence the term clearing
agent.
 CLEARING
Characteristics of a good clearing agent:
• It should be miscible with alcohol to promote rapid
removal of the dehydrating agent from the tissue
• It should be miscible with, and easily removed by
melted paraffin wax and or by mounting medium to
facilitate impregnation and mounting of sections
• It should not produce excessive shrinkage,
hardening or damage of tissue
• It should not dissolve out aniline dyes
• It should evaporate quickly in a water bath
• It should make tissues transparent
CLEARING
Most commonly used clearing agents:
1. Xylene (routine)
2. Clove oil
3. Chloroform
4. Cedarwood
5. Toluene
6. Benzene
7. Cedarwood oil
8. Aniline oil
9. Carbon tetrachloride
 STEPS IN PRESERVED TISSUE EXAMINATION
1. Labeling (Numbering)
Minor Steps:
2. Fixation
Washing Out
3. Decalcification
Orientation
4. Dehydration
Trimming
5. Clearing
Floating Out
6. Impregnation
Adhesion
7. Embedding
Ringing
8. Section Cutting
Tissue processing
9. Staining
starts with labeling
10.Mounting and ends with labeling.
11.Labeling (Numbering)
 IMPREGNATION
• Also known as infiltration.
• Process whereby the clearing agent is completely
removed from the tissue and replaced by a medium
that will fill all tissue cavities thereby giving a firm
consistency to the specimen.
• This also allows easier handling and cutting of
suitably thin sections without any damage or
distortion to the tissue and its cellular components.
• The infiltrating medium is usually the same with the
embedding medium.
PARAFFIN WAX
Completely cleared tissues are submerged in 2 or
more changes of melted paraffin wax.

Oven Temp: 55-60°C or 2-5°C above the

melting point of wax

Melting points:
✓56°C – used for routine work
✓54-58°C – used in lab with 20-24°C temp
✓50-54°C – used in lab with 15-18°C temp
 STEPS IN PRESERVED TISSUE EXAMINATION
1. Labeling (Numbering)
Minor Steps:
2. Fixation
Washing Out
3. Decalcification
Orientation
4. Dehydration
Trimming
5. Clearing
Floating Out
6. Impregnation
Adhesion
7. Embedding
Ringing
8. Section Cutting
Tissue processing
9. Staining
starts with labeling
10.Mounting and ends with labeling.
11.Labeling (Numbering)
EMBEDDING
• Also known as casting or blocking.

• Process by which the impregnated tissue is placed into

a precisely arranged position in a mold containing a
medium which is then allowed to solidify.

• This process uses embedding molds such as

Leuckhart’s L–shaped mold or paper boat molds.

• Commonly uses melted paraffin at a temp between 5 –

10 C above its melting point and tissues are immersed
immediately freezing temperature (– 5 C).
EMBEDDING
ORIENTATION
• The process by which a tissue is arranged in
precise positions in the mold during
embedding, on the microtome before cutting,
and on the slide before staining.
 STEPS IN PRESERVED TISSUE EXAMINATION
1. Labeling (Numbering)
Minor Steps:
2. Fixation
Washing Out
3. Decalcification
Orientation
4. Dehydration
Trimming
5. Clearing
Floating Out
6. Impregnation
Adhesion
7. Embedding
Ringing
8. Section Cutting
Tissue processing
9. Staining
starts with labeling
10.Mounting and ends with labeling.
11.Labeling (Numbering)
 TRIMMING
• The process of removing excess wax after
embedding where sides, top and bottom are
trimmed perfectly.
• At least 2mm of paraffin wax must surround the
tissue block.
• Ideal shape of a tissue block: truncated pyramid or
four – sided prism .
SECTIONING
Sectioning/Microtomy – process by which
processed tissue is trimmed and cut into
uniformly thin slices or “sections”.

Microtome – instrument capable of cutting a

section at a predetermined thickness by sliding
the block into a cutting tool like blade or knife
which is attached to the machine.
Recommended thickness:
• Paraffin sections: 4-6 um
• Celloidin sections: 10-15 um
• Frozen sections: 0.5 um
MICROTOMES
3 Essential Parts of a Microtome:
1. Chuck = Block Holder
2. Knife carrier and knife
3. Pawl, rachet feed wheel and adjustment
screws
MICROTOME INVENTOR PURPOSE
Rocking/ Paldwell Trefall, SIMPLEST
Cambridge 1881 For large paraffin blocks

Rotary/ Minot Minot, 1885-1886 MOST COMMON

For paraffin-embedded
sections
Sliding Adams, 1789 MOST DANGEROUS
For celloidin-embedded
sections
Freezing Queckett, 1848 For unembedded tissue
sections; CO2 is used as a
propellant
Ultrathin For plastic-embedded
Note: uses fragments of sections to be examined
broken plate glass as knife under the ELECTRON
MICROSCOPE
Rocking Microtome
Rotary Microtome
Two types of Sliding Microtome:
1. Base-sledge – knife is stationary; the block
moves
2. Standard sliding – the knife is moving; block is
stationary. MORE DANGEROUS!

Base-sledge Standard sliding

 TYPES OF PURPOSE LENGTH
KNIVES
For frozen and extremely
hard and tough tissues
Plane wedge 100 mm
Used in a base-sledge or
sliding microtome
Plane side is for celloidin-
embedded tissue blocks
Plane
Concave side is for 25 mm
concave
paraffin-embedded
sections
For paraffin-embedded
Biconcave sections on a rotary 120 mm
microtome
 ANGLES ° DESCRIPTION
-Formed between the cutting
edges of the knife
Bevel angle 27-32° -Maintained for each knife by
means of a “knife back”
-Formed between the knife
and cutting plane
Clearance -Prevents uneven sections
0-15°
angle and provides perfect and
optimum cutting angle
Optimum for maximum
Cutting angle 15° penetration of tissue with less
distortion
 HONING STROPPING
(HARD SHARPENING) (POLISHING)
REMOVAL OF NICKS REMOVAL OF BURRS
1. Coarse Honing Use horse leather
-blemish removal and paddle strop;
grinding of cutting EDGE LAST,
edge TOE TO HEEL direction
2. Honing Proper 40-120 strokes
-to get an even edge
EDGE FIRST,
HEEL TO TOE direction
10-20 strokes
 HONES
(natural sharpening PURPOSE/ DESCRIPTION
stone)
Belgium Yellow Gives the best results
Arkansas Gives more polishing effect.
For badly nicked knives;
Fine carborundum Much coarse and followed
by either of the two knives.
 STEPS IN PRESERVED TISSUE EXAMINATION
1. Labeling (Numbering)
Minor Steps:
2. Fixation
Washing Out
3. Decalcification
Orientation
4. Dehydration
Trimming
5. Clearing
Floating Out
6. Impregnation
Adhesion
7. Embedding
Ringing
8. Section Cutting
Tissue processing
9. Staining
starts with labeling
10.Mounting and ends with labeling.
11.Labeling (Numbering)
 FLOATING OUT
• Process by which the sections are floated out on a
water bath set at 45 to 50oC, approximately 6 to
10oC lower than the melting point of the wax used
for embedding the tissue.
• This is to flatten the sections and prepare them for
placing in the slides.
• Adhesives: these are used to promote adhesion
of sections, adhesives may be spread thinly and
evenly on a clean grease-free slide which is then
approximated to the end of the ribbon and drawn
upwards in a near vertical motion.
• Essential for methods that require acid or alkali
exposure of tissue sections.
Mayer’s Egg Albumin
• Most commonly used adhesive.
• It is very easy to make, convenient and relatively
inexpensive.
• It is made up of egg whites, glycerin and thymol
crystals (prevent mold growth).
Other adhesives:
1. Dried albumin
2. 1% Gelatin
3. Gelatin-formaldehyde mixture
4. Poly-L-lysine
5. APES (3-amino propylthriethoxysilane)
6. Starch Paste
7. Plasma
 STEPS IN PRESERVED TISSUE EXAMINATION
1. Labeling (Numbering)
Minor Steps:
2. Fixation
Washing Out
3. Decalcification
Orientation
4. Dehydration
Trimming
5. Clearing
Floating Out
6. Impregnation
Adhesion
7. Embedding
Ringing
8. Section Cutting
Tissue processing
9. Staining
starts with labeling
10.Mounting and ends with labeling.
11.Labeling (Numbering)
 STAINING
• The process of applying dyes to tissues.
• This facilitates better viewing and examination of
the architectural pattern of the tissue and
physical characteristics of the cells.
 Purposes of Staining:
• To render the different tissue constituents more
visible through variations in colors
• To promote easier optical differentiation for
identification of cells and tissue components
• To display varying affinities of tissues and cells for
most dyes
• Physical characteristics and structural relationship
of tissues and cells are better studied and
evaluated.
 Stains and Staining Solutions
• Chromophores- substances with definite atomic
grouping and are capable of producing visible colors.
• Chromogens- benzene compounds which contain a
chromophore. These are different from dyes in that the
color that they impart to the tissue is not permanent and
can be easily removed. For a substance to be considered
a dye, it must first have the property of retaining its color
in the tissue.
• Auxochrome- an auxilliary radical substance which
imparts to the compound the property of electrolytic
dissociation, thereby altering the shade of the dye,
enabling it to form salts with another compound, and
ultimately retaining its color.
 DYE
• It is a substance that is consists of a
chromophore and an auxochrome attached to a
hydrocarbon benzene ring; the coloring property
attributed to the chromophore and the dyeing
property to the salt forming auxochrome.
 MAJOR GROUPS DESCRIPTION/PURPOSE
OF STAINING
1. Histological Tissue constituents are demonstrated in
(Micro-anatomical) sections by direct interaction with a dye
Staining or staining solution
Used to demonstrate the general
relationship of tissues and cells with
differentiation of nucleus and cytoplasm.

2. Histochemical Tissue constituents are studied through

staining chemical reactions.
(Histochemistry)
3. Immuno- Allow phenotypic markers to be
histochemical demonstrated using a wide range of
staining polyclonal or monoclonal, fluorescent-
labeled or enzyme labeled antibodies.
 METHODS
OF DESCRIPTION/PURPOSE
STAINING
Gives color to the sections by using
1. Direct
aqueous or alcoholic dye solutions.
Action of the dye is intensified by a
mordant: link or bridge between
2. Indirect tissue and dye; it combines with
the dye to form a colored lake (
tissue-mordant-dye complex)
 MORDANTS ACCENTUATOR
Link or bridge between Accelerates or hastens
the tissue and dye. only the speed of
Combines with the dye staining by increasing
to form a colored “lake” the staining power and
and finally a tissue- selectivity of the dye
mordant-dye complex
Examples: Examples:
• Potassium alum with • Potassium hydroxide in
hematoxylin in Loeffler’s methylene
Ehrlich’s hematoxylin blue
• Iron in Weigert’s • Phenol in carbol
hematoxylin thionine and carbol
fuchsin
METHODS OF
DESCRIPTION/PURPOSE
STAINING
Staining in a definite sequence
Staining solution is applied for
3. Progressive
specific periods of time or until
desire intensity of color is attained.
Process whereby tissue is first
4. Regressive overstained and the excess stain is
removed or decolorized.
 METHODS OF
DESCRIPTION/PURPOSE
STAINING
Selective removal of excess
stain from tissue during
5. Differentiation/ regressive staining.
decolorization Aims to stain a specific
substance distinctly from
surrounding tissue.
Differentiates particularly in
substances by staining them
6. Metachromatic with a color that is different from
that of the stain itself
(metachromasia)
 METHODS OF
DESCRIPTION/PURPOSE
STAINING
Application of a different color to
provide contrast and background
7. Counterstaining
to the staining of the structural
components to be demonstrated
Tissue elements are
demonstrated, not by stains, but
8. Metallic by colorless solutions of metallic
Impregnation salts which are reduced by the
tissues, producing an opaque or
black deposits
 METHODS OF
DESCRIPTION/PURPOSE
STAINING
9. Vital staining Selective staining of living cell
constituents by cytoplasmic
phagocytosis.
Nucleus of a living cell is resistant,
therefore is not demonstrated.

a. Intravital Done by injecting the dye into any

part of the animal body.

b. Supravital Stain living cells immediately after

removal from the body.
 Two Categories of Dyes
NATURAL DYES SYNTHETIC DYES
Hematoxylin Acid dyes
Cochineal dyes Basic dyes
Orcein Neutral dyes
Saffron
 HEMATOXYLIN
• Most valuable staining agent
• Derived from the heartwood of Mexican tree
Hematoxylin campechianum
Hematin
• Active coloring agent
• Formed by the oxidation of hematoxylin or a
process called “ripening”.
• Natural ripening: sunlight or air exposure
• Artificial ripening: hydrogen peroxide, mercuric
oxide, potassium permanganate, sodium
perborate or sodium iodate.
COCHINEAL DYES
• Came from the cochineal bug Coccus cacti

Formulations:
• Carmine = cochineal dye + alum (powerful
chromatin and nuclear stain)
• Picrocarmine = carmine + picric acid (for
neuropathological studies)
• Best’s carmine = carmine + aluminum
chloride (for demonstration of glycogen)
ORCEIN
• Vegetable dye extracted from lichens
• Mainly used for staining elastic fibers
HEMATOXYLIN & EOSIN STAINING
(H & E)
Xylene (2 changes)
Descending grades of alcohol
(tissue hydration)
Wash with water
Stain with Harris/Ehrlich’s/Delafield’s
(Primary stain-nucleus: light blue)
Wash with water
Acid alcohol
(nucleus: red)
Wash with water
Ammonia water (Blueing agent)
Wash with water
Eosin Y
(Secondary stain)
Ascending grades of alcohol
(tissue dehydration)
Xylene
Mount and label
H & E RESULTS
• Nuclei : Blue to black
• Karyosome : Dark blue
: Pale pink
• Calcium & decalcified bones: Purplish blue
• RBCs
• Eosinophilic granules Bright orange-red
• Keratin
• Decalcified bone matrix
• Collagen Pink
• Osteoid
 EOSIN
• Red acid dye
• Counterstain after hematoxylin and before
methylene blue

• Has 3 forms:
1. Eosin Y-Yellow, MOST COMMONLY USED
-produces green-yellow fluorescence
2. Eosin B (Erythrosin B) – deeper-red color
3. Eosin S (Eosin alcohol-soluble/Ethyl eosin)
PAPANICOLAU STAINING
(PAP STAIN)
Fix w/ 95% ETOH
Harris hematoxylin
Acid alcohol
Blueing step
Orange green 6 (OG6)
70-95% ETOH
Eosin azure 36 or 50
(EA36/50)
Dehyrate tissue
Xylene
Mount and label
 STAINS USES
Van Gieson’s Connective tissue
Acridine orange DNA (green); RNA (red)
Alcian blue Epithelial mucin
Aniline blue Cytoplasmic stain
Benzidine Stains hemoglobin
Bismarck brown For Diphtheria organisms
Carmine Chromatin and fresh tissue
Congo red For axis cylinders in embryos,
elastic tissues, amyloid and myelin
Janus green Mitochondria
Iodine OLDEST STAIN
For amyloid, cellulose, starch,
carotenes and glycogen
 STAINS USES
Malachite green Ascaris eggs, RBCs, bacterial spore
Methyl green Chromatin
Neutral red For cell granules and vacuoles
Night blue Substitute for carbol fuchsin in AFS
Orcein For elastic fibers
Victoria blue Neuroglia in frozen sections
Toluidine blue For Nissl granules
Sudan black B MOST SENSITIVE LIPID STAIN
For staining of phospholipids
Sudan IV aka Scharlach R
For staining triglycerides
Sudan III FIRST TO BE INTRODUCED
For staining CNS fats
 STEPS IN PRESERVED TISSUE EXAMINATION
1. Labeling (Numbering)
Minor Steps:
2. Fixation
Washing Out
3. Decalcification
Orientation
4. Dehydration
Trimming
5. Clearing
Floating Out
6. Impregnation
Adhesion
7. Embedding
Ringing
8. Section Cutting
Tissue processing
9. Staining
starts with labeling
10.Mounting and ends with labeling.
11.Labeling (Numbering)
• Mounting agent: syrupy fluid applied between the
section and cover slip after staining
• Purposes:
1. Sets the section firmly
2. Prevents movement of the coverslip
3. Protects the stained section from scratches,
bleach and deterioration due to oxidation
4. Preserves the slide for permanent keeping
5. Facilitates easy handling and storage
6. Prevents distortion of image during
microscopy
Notes to remember!
• Excess xylene must be wiped off first from around
the section and the slide.
• Excess xylene may cause improper setting of the
medium and bubble formation
• Slide may be incubated at 37°C for 12-20 hours
after mounting to harden the medium
• Setting may be hastened in a hot oven at 50°C
for 2 hours
• Excess mountant should be wiped of w/ a fine
cloth moistened with xylene
Notes to remember!
•Refractive index of the
mountant should be as near
as possible to that of the
glass which is 1.518.
Two Groups of Mounting Media

1. Aqueous Mounting Media

• for water-miscible preparations

2. Resinous Mounting Media

• For preparations that have been
dehydrated and cleared in xylene or toluene
• Recommended for majority of staining
methods
 Aqueous Mounting Media
MEDIUM RI USES
Water - Good only for temporary mounting
Glycerin 1.46 Gives greater visibility for moist sections
Semi-permanent and has preservative action
best when sealed with paraffin wax

Standard mounting medium when

•Glycerin Jelly dehydration and clearing with xylene cannot
be made; requires “ringing”
Farrant’s 1.43 Does not need to be heated before use and
medium takes longer time to harden
Apathy’s 1.52 General-purpose aqueous mountant
medium
Brun’s fluid - For frozen sections from water
 Resinous Mounting Media
MEDIUM RI USES
From Abus balsamea
Recommended for whole mounts
Canada Balsam 1.524
and thick sections
Miscible with xylene
DPX 1.532 For small tissue sections
Pale yellow in color
XAM 1.520 Dries quickly without retraction and
preserves stains well
Clarite 1.544 Preferred over DPX
 RINGING
• Process of sealing the margins of the cover slip
to:
-prevent escape of fluid or semi-fluid mounts -
evaporation of mountant
-immobilize the cover slip
-prevent sticking of slides upon storage
Ex: 1. DUROFIX
2. KRONIG CEMENT (made up of 2 parts
paraffin + 4-9 parts powdered colophonium resin,
heated and filtered.