HISTOPATHOLOGIC TECHNIQUES REVIEWER

- Mark Raymund G. Nava, RMT, MPA, MSMT

BASIC HISTOPATHOLOGY
INFLAMMATION
- From Latin word “inflammare” – to set afire
- Protective response of the tissues of the body to irritation and injury
- Composed of a series of physiologic and morphologic changes in the blood
vessels, blood components and surrounding connective tissues for the
purpose of protecting the body against injury

CARDINAL SIGNS OF INFLAMMATION

Latin English Cause
Due to arteriolar and capillary dilatation with increased rate of
Rubor Redness
blood flow towards the site of injury
Due to increased capillary permeability  causing extravasation
Tumor Swelling
of body fluid
Due to transfer of internal heat to the surface or site of the
Calor Heat
injury, brought about by increased blood content
Dolor Pain Due to pressure upon the sensory nerve by the exudate/tumor
Diminished
Functio Laesa Destruction of the functioning units of the tissue
function

CLASSIFICATION OF INFLAMMATION
ACCORDING TO DURATION
ACUTE SUBCHRONIC CHRONIC
Usually, but not necessarily of Persistence of the injuring agent
sudden onset An intergrade between acute for weeks/years
Vascular and exudative and chronic Vascular and fibroblastic
Predominantly PMNs Predominantly mononuclears

ACCORDING TO EXUDATIVE
SEROUS Secretions - secretions from mesothelial cells
- characterized by increased amounts
FIBRINOUS Fibrin
of fibrinogen
CATARRHAL Mucus - hypersecretion of the mucosa
- admixture of blood & other elements
HEMORRHAGIC Blood
of exudates
SUPPURATIVE/PURULENT Pus - large amount of pus/purulent exudate

1
CHANGES IN CELLULAR GROWTH PATTERNS
RETROGRESSIVE Aplasia -incomplete devt of a tissue/organ
Agenesia -non-appearance of an organ

organ
Atresia -failure of an organ to form an opening
smaller Hypoplasia -failure of an organ to reach full maturity
than normal Atrophy -decrease in size of a normal mature tissue/organ
Physiologic – occurs as natural consequence of maturation
e.g. Atrophy of thymus during puberty
Atrophy of brain & sexual organs at 50 yrs old
Pathologic – occurs as consequence of disease
 Vascular Atrophy  Atrophy of Disuse
 Pressure Atrophy  Exhaustion Atrophy
 Hunger/Starvation Atrophy  Endocrine Atrophy
PROGRESSIVE Hypertrophy -increase of size due to increase of size of individual cells
organ LARGER -increase of tissue size due to increase in the number of cells
than normal Hyperplasia making up the tissue
DEGENERATIVE Metaplasia reversible One type of adult cell  Another adult cell
Dysplasia reversible Adult cell changes in structural component
tissues have
Anaplasia irreversible Adult cell  Primitive cell
abnormalities
Neoplasia Irreversible Abnormal proliferation of cells w/o control

NOMENCLATURE OF NEOPLASTIC CELLS

BENIGN MALIGNANT
-does not produce death -produce death
MESENCHYMAL/CT Tumors suffix “OMA” suffix “SARCOMA”
Fibrous tissue Fibroma Fibrosarcoma
Adipose/Fatty Lipoma Liposarcoma
Cartilage Chondroma Chondrosarcoma
Bone Osteoma Osteogenic sarcoma
Blood vessels Hemangioma Hemangiosarcoma
Hematopoietic cells * Leukemia
Lymphoid tissue* Lymphoma
Smooth muscle Leiomyoma Leiomyosarcoma
Striated muscle Rhabdomyoma Rhabdomyosarcoma
EPITHELIAL TISSUE Tumors suffix “OMA” suffix “CARCINOMA”
Stratified squamous cells Squamous cell papilloma Squamous cell carcinoma
Glands & ducts Adenoma Adenocarcinoma
Renal Epithelium Renal tubular adenoma Renal cell carcinoma
Liver cells Liver cell adenoma Hepatocarcinoma (Hepatoma)
Neuroectoderm* Nevus/Mole Melanoma (Melanocarcinoma)
Testicular epithelium* Seminoma
* exceptions to the rule

2
GRADING AND STAGING OF TUMOR
“GRADING” “STAGING”
BASED ON: BASED ON:
- cytologic differentiation of tumor cells - size of primary lesion
- number of mitoses within the tumor - extent of spread to regional lymph nodes
- presence or absence of metastases
Broder’s Classification TNM System of Cancer Staging
DIFFERENTIATED UNDIFFERENTIATED - developed by UICC
resemble resemble (International Union Against Cancer)
normal cells abnormal cells - applicable to all forms of neoplasia
Lower grades: amenable to SURGERY T N M
GRADE I 100-75% 0-25% size lymph node metastases
GRADE II 75-50% 25-50% T1,T2,T3,T4 N0,N1, N2,N3 M0,M1
Higher grades: amenable to RADIATION -with -indicates -whether
GRADE III 50-25% 50-75% increasing progressively there are
GRADE IV 25-0% 75-100% size of advancing distant
primary lesion nodal disease metastases

TERATOMAS
- Type of neoplasm
- Compound tumors
- “Monstrous tumors”
- Tumor with normal tissue or organ components that are
inappropriate to surrounding tissues
- May contain hair, teeth, bones, and very rarely eyeballs, torso,
and hands

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 DEATH

CELLULAR DEATH – death at the cellular level

Apoptosis programmed cell death
Necrobiosis physiologic death of cells (e.g. shredding off of skin cells)
Necrosis pathologic death of cells
most common seen in: myocardium, lungs,
Coagulation
tombstone formation kidney, spleen
Liquefaction/ pus formation seen in: brain, spinal cords
Colliquative
Caseous/ yellow, cheesy, crumbly materials seen in: TB, Syphilis, Tularemia,
Caseation Lymphogranuloma inguinale
sulphide gas production Dry Gangrene – arterial
Coagulation & Liquefaction occlusions/blockages
Gangrenous
combination Wet Gangrene – venous
occlusions/blockages
Fat Necrosis chalky, white precipitates seen in: pancreatic degeneration
SOMATIC DEATH – death of the entire organism
Circulatory failure loss of heartbeat
Primary Changes
Respiratory failure loss of breathing
- during somatic death
CNS failure loss of responses
first demonstrable change observed
Algor Mortis cooling of the body
rate: 7°C per hour
stiffening of skeletal muscles after death
Rigor Mortis 2-3 hours after death
first occurs: head & neck muscles
post-mortem lividity
Livor Mortis purplish discoloration/lividity of the skin
Secondary Changes
after 10-12 hours
- after somatic death
settling of RBCs from plasma
Postmortem Clot
rubbery consistency
drying & wrinkling of the anterior chamber of the eye &
Dessication
cornea
invasion of intestinal microorganism
Putrefaction
greenish discoloration of the abdomen
self-digestion of cells
Autolysis
by lysosomes (suicide sac)

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FRESH TISSUE EXAMINATION

METHODS
selected tissue is immersed in watch glass containing NSS then carefully
Teasing/ Dissociation dissected/ separated and examined under the microscope
Squash Preparation/ small pieces not more than 1 mm are placed in a slide and forcibly
Crushing compressed with another slide or with a coverglass
examining sections of sediments, whereby cellular materials are spread lightly
over a slide by means of wire loop or applicator.
Streaking using an applicator stick by direct or zigzag spread
teasing by applicator stick and spread in circular spread.
Smear Spreading FOR: sputum, bronchial aspirates, thick mucoid secretions
Preparation thick secretions are dispersed evenly on two slide surfaces
Pull-Apart
FOR: gastric lavage, serous fluids, blood
Touch/ freshly cut tissue surface is brought into contact to slide;
Impression cells are examined in their actual intercellular relationship
tissue is frozen with liquid nitrogen and a section is examined under the
Frozen Section microscope; for rapid diagnosis during surgery, and demonstration of lipids and
nervous tissue elements

FIXED TISSUE EXAMINATION

F ixation Gross Examination

D ehydration
C learing/ Dealcoholization Decalcification (OPTIONAL)
I impregnation/ Infiltration
E mbedding/ Casting/ Blocking
T rimming
S ectioning/ Cutting/ Microtomy
Fishing Out
Deparaffinization
S taining
M ounting
Ringing
L abeling

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TISSUE PROCESSING

FIXATION 
 Preserving fresh tissue for examination
Types of Fixatives
 First and most critical step in
histotechnology According to Composition
 Primary aim: to preserve the A. Simple – one component
morphologic & chemical integrity of the B. Compound – 2 or more fixatives
cell in as life-like manner as possible
 Secondary aim: to harden and protect According to Action
the tissue from trauma of further A. Microanatomical – for general
handling microscopic study of tissue structures
 Fixatives have the property of forming B. Cytological – specific parts and
cross-links between proteins particular microscopic element of the
 Stabilization of proteins cell
a. Nuclear – contain gAc
Usual fixation time: 24 hours
o Bouin’s
Usual fixation temp: Room temperature
o Flemmings
Practical Consideration of Fixation: o Newcomer’s
1. Speed - almost 1 mm/hour o Carnoy’s
2. Penetration o Heidenhain’s Susa
3. Volume – 20:1 b. Cytoplasmic – do not contain gAc
4. Duration of Fixation o Helly’s
o Orth’s
Two Mechanisms Involved in Fixation:
o Moller’s (Regaud’s)
1. Additive Fixation – whereby the
o Flemming’s w/o Acetic Acid
chemical constituent of the fixative is
o Formalin with post-chroming
taken in and becomes part of the tissue.
c. Histochemical
2. Non-additive (Negative) Fixation –
o 10% Formol-saline
whereby the fixative is NOT taken in,
o Absolute ETOH
but changes the tissue composition and
o Acetone
stabilizes the tissue by removing the
o Newcomer’s Fluid
bound water attached to hydrogen
bonds of certain groups within the
protein molecule. LIPID FIXATION - use mercuric chloride and
Main Factors Involved in Fixation: potassium dichromate
1. Hydrogen ion concentration (pH) – CARBOHYDRATE FIXATION – use alcoholic
satisfactory at pH 6-8 formaldehyde
2. Temperature – room temperature
(surgical specimen); EM & IHC – 0-4°C PROTEIN FIXATION – use Neutral buffered
3. Thickness of section formol saline or formaldehyde
4. Osmolality GLYCOGEN FIXATION – Rossman’s fluid or
5. Concentration absolute alcohol
6. Duration of fixation – 24 hours (usual)

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FIXATIVES
MAJOR
EXAMPLE/S COMPOSITION CHARACTERISTIC/USE
COMPONENT
- Gas produced by the - Cheap, readily available,
oxidation of Methyl easy to prepare & stable
40% Formaldehyde Alcohol, soluble in H2O to - Compatible with many
(Formalin) the extent of 37-40% w/v stains
- Must be diluted 1:10/1:20 - Does not over harden
to make 10% or 5% solution - Penetrates tissue well
- 40% Formalin
10% Formol-Saline - NaCl - for CNS tissues
- Distilled H2O
- 40% Formalin - best fixative for tissues
10% Buffered Neutral - Distilled H2O containing iron pigments
ALDEHYDE Formalin - Na Dihydrogen Phosphate - BEST GENERAL TISSUE
- Disodium H Phosphate FIXATIVE
Formol-Corrosive/ - Saturated Aq HgCl - for routine post-mortem
Formol Sublimate - 40% Formalin tissues
- made up of 2 formaldehyde - stable, more expensive
2.5% Glutaraldehyde residues linked by 3 carbon - preserves plasma protein
chains better
Karnovsky’s - Paraformaldehyde, - for electron
Paraformalydehyde Glutaraldehyde, NaOH cytochemistry
- for plants, enzyme
Acrolein/Acrylic Aldehyde
histochemistry
- Mercuric Chloride stock
- for liver, spleen,
Zenker’s Fluid - Glacial Acetic Acid
Mercuric connective tissue
(added before use)
Chloride - Mercuric Chloride stock
- most Zenker’s Formol - for pituitary gland, bone
- 40% Formalin
common /Helly’s Solution marrow
(added before use)
metallic - Mercuric Chloride, NaCl,
fixative Heidenhain’s
TCA, glacial Acetic Acid, - for biopsies of the skin
- may Susa Formalin, Distilled H2O
produce
Schaudinn’s - Mercuric chloride, NaCl, - for protozoa and
black
Fluid Alcohol, glacial Acetic Acid helminths
deposits
- Mercuric chloride, Sodium - commonly used for bone
B-5 Fixative
acetate, Distilled H2O marrow, lymph nodes
METALLIC
Chromic Acid - 1-2% Aqueous solution - preserves carbohydrates
FIXATIVES Potassium - preserves, lipids,
- 3% Aqueous solution
Dichromate mitochondria
Chromates
- for chromatin, golgi
- may Regaud’s/ - 3% Aqueous K dichromate
bodies, mitochondria,
produce Moller’s Fluid - 40% Formalin
yellow- mitotic figures, RBC
brown - for early degenerative
deposits - 2.5% K dichromate processes & tissue
Orth’s Fluid - Na sulphate necrosis
- 40% Formalin - For Rickettsia & other
bacteria
- recommended for Acid
Lead Lead Acetate - 4% Aqueous Lead Acetate MPS e.g. Umbilical
cord/Wharton’s Jelly

7
 MAJOR
EXAMPLE/S COMPOSITION CHARACTERISTIC/USE
COMPONENT
- Saturated Picric Acid
PICRIC ACID/ Bouin’s Solution
- 40% Formalin
- Fixatives of embryo
PICRATE
FIXATIVES - 37% Formalin,
- Excellent fixative of
(1% Aqueous solution) Brasil’s Alcoholic - ETOH
glycogen
Picroformol Fixative - Picric Acid
- Less messy than Bouin’s
 HIGHLY EXPLOSIVE - TCA
WHEN DRY
- For fixing dry & wet smear,
blood & bone marrow smear
Methyl Alcohol - 100% Methyl Alcohol
- Fixes & dehydrates at the
same time
- If lower conc., RBC will
hemolyze & WBC
Ethyl Alcohol - 70-100% Ethyl Alcohol,
inadequately preserved
- For Pap’s smear

ALCOHOL Carnoy’s Fluid - Fixing chromosomes, lymph

- Absolute Alcohol
FIXATIVES - Chloroform, Glacial Acetic Acid
glands
MOST RAPID - For urgent biopsies
FIXATIVE
Alcoholic Formalin/ - 95% ETOH with Picric acid
- Useful for sputum
Gendre’s Fixative - Formalin, Glacial Acetic Acid
- Isopropyl Alcohol, Propionic
- Recommended for
Acid
Newcomer’s Fluid mucopolysaccharides &
- Petroleum Ether, Acetone,
nuclear proteins
Dioxane
- Normally used in conjunction with - Solidifies at 17ºC
GLACIAL ACETIC ACID other fixatives to form a - Fixes nucleoprotein
compound solution - Causes tissue to swell
OSMIC ACID/ - Most common Chrome-
- 1% Aq Chromic Acid
Flemming’s Osmium Acetic Acid fixative
OSMIUM Solution
- 2% Aq Osmium Tetroxide
- Excellent fixative for nuclear
- Glacial Acetic Acid
TETROXIDE structures
(up to 6%)
Flemming’s
- Made up of only Chromic & - Removal of GAc improves
MUST BE KEPT IN Solution Without
osmic Acid cytoplasmic details of cell
DARK-COLORED Acetic Acid
CONTAINERS
TRICHLOROACETIC ACID - May be used as weak
- Sometimes incorporated into
decalcifying agent,
compound fixatives
(TCA) precipitates proteins
- Used at ice cold temperature
(-5º to 4ºC)
ACETONE - Pure Acetone
- Used in Freeze substitution
- For dx of rabies
Heat from fire (as in alcohol lamp)
HEAT FIXATION Microwave
- For rapid diagnosis

8
SECONDARY FIXATION
- Process of replacing an already fixed tissue in a second fixative order
- Usually with 10% formalin or 10% formol saline as primary fixative

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

WASHING-OUT
- process of removing excess fixative from the tissue after fixation in order to improve staining
and remove artefacts from the tissues

WASHING OUT
WASH PURPOSE
excess chromates
(e.g. Helly’s, Zenker’s and Flemming’s)
Tap Water excess osmic acid
excess formalin
excess picric acid
50-70% Alcohol (e.g. Bouin’s)
Alcoholic Iodine excess mercuric fixatives

REMOVAL OF FORMALIN PIGMENT

KARDASEWITCH’S LILLIE’S PICRIC ACID
Bring down to water Bring down to water Bring down to water
  
Place in mixture of:
Place in mixture of:
Acetone Place in Saturated
70% ETOH
H2O2 Picric acid
28% Ammonia water
28% Ammonia water
5mins-3hrs 1-5 mins 10-15 mins
  
Wash with 70% ETOH
Wash with water for 10-
Wash with water 
15mins
Wash with water

9
DECALCIFICATION
 IDEAL VOLUME: (20:1)
 More concentrated acid solutions decalcify bone marrow rapidly but are more harmful to tissue
 High concentrations and greater amount of fluid will increase the speed of the process
 Heat will serve to hasten decalcification BUT it also increases the damaging
effects on tissues.
 At 37°C = impaired nuclear staining of Van Gieson’s stain for collagen fibers
 At 55°C = tissue will undergo complete digestion within 24-48 hours
 OPTIMUM TEMPERATURE: Room temperature (18-30°C)
 Dense bone tissues usually require up to 14 days or longer to complete the
process

DECALCIFYING AGENTS
MAJOR COMPOSITION/
EXAMPLE/S
COMPONENT EQUIPMENT
- Conc. Nitric Acid
10% Aqueous Nitric Acid
- Distilled H2O
- Conc. Nitric Acid
Formol-Nitric Acid - 40% Formalin
- Distilled H2O
Nitric Acid - 10% Nitric Acid
Perenyi’s Fluid - 0.5% Chromic Acid
- Absolute Ethanol
- Conc. Nitric acid
Phloroglucin-Nitric Acid - Phloroglucin
- 10% Nitric acid
- Conc. Hydrochloric acid
Hydrochloric
Von Ebner’s Fluid - 36% Aquoues NaCl
ACIDS Acid - Distilled H2O
- Conc. Formic acid
10% Formic Acid
- 10% Formol-Saline
Formic Acid Formic Acid-Sodium Citrate - 45% Formic acid
Solution - 20% Aq. Na citrate
- Trichloroacetic acid
Trichloroacetic Acid (TCA) - 10% Formol-saline
Sulfurous Acid
- 1% Chromic acid
Chromic Acid Flemming’s Fluid - 2% Osmium tetroxide
- Glacial acetic acid
Citric Acid-Citrate Buffer Solution
- EDTA Disodium salt
CHELATING - 37-40% Formalin
Ethylene Diamine Tetraacetic Acid (EDTA)
AGENTS - Distilled H2O
- Ion exchange resin (Ammonium
form of polysterene resin)
ION EXCHANGE RESIN - Formic acid-containing
decalcifying solutions
- Electrophoresis Machine
ELECTROPHORESIS/ - 88% Formic acid
- Conc. Hydrochloric acid
ELECTRICAL IONIZATION - Distilled H2O
10
 by neutralizing with 5% Sodium sulphate and washing in

PREVENT
running water for at least 12 hours
NITRIC ACID imparts YELLOW
color on tissue!!!
by adding 0.1% Urea to Nitric acid

SET-UP OF DECALCIFICATION TECHNIQUES

ACID DECALCIFICATION ION-EXCHANGE RESIN ELECTROPHORESIS

Measurement of EXTENT OF DECALCIFICATION

1. Physical/Mechanical Test – least accurate
a. by touch
b. by pliability
c. by resistance to fingernail
d. by needling
2. X-ray/Radiological Method – very expensive
3. Chemical Method (Calcium Oxalate Test) – simple, reliable, recommended for routine purposes

CALCIUM OXALATE TEST

BLUE litmus paper + 5mL discarded decalcifying agent  RED due to acidity
Add Ammonia drop by drop to neutralize (litmus change to BLUE)

IF CLOUDY IF CLEAR
INCOMPLETE Add AMMONIUM OXALATE

30 mins 
IF CLOUDY IF CLEAR
INCOMPLETE COMPLETE

TISSUE SOFTENERS
- For unduly hard tissues that may damage the microtome knives
 4% Aqueous Phenol
 Molliflex
 2% Hydrochloric Acid
 1% HCl in 70% Alcohol

11
DEHYDRATION 
 AIM: to remove fixative and water from the tissue and replacing them with dehydrating fluid in
preparation for impregnation
 Dehydrating fluids are generally used in increasing strengths (ASCENDING GRADES)
 Increasing strengths = all the aqueous fluids are removed but with little disruption to the tissue
due to diffusion currents

DEHYDRATING AGENTS
DEHYDRATING AGENTS CHARACTERISTICS
- BEST DEHYDRATING AGENT
Ethyl Alcohol
- for routine dehydration
Methyl Alcohol - employed for blood & tissue films
ALCOHOL Butyl Alcohol - utilized in plant & animal microtechniques
Industrial Methylated Spirit - combination of ethanol + small amount of
(Denatured Alcohol) methanol
Isopropyl Alcohol - rarely used alcohol dehydrating agent
- used in four changes
ACETONE - BOTH FIXATIVE & DEHYDRATING AGENT
- BOTH DEHYDRATING & CLEARING AGENT
Two Methods:
- Graupner’s Method
DIOXANE 3 changes of Dioxane (1h, 1h, 2h)and 3 changes
of Paraffin Wax (15’,45’,2h)
(Diethylene glycol) - Weiseberger’s Method
Set-up similar to acid decalcification
Quicklime/Ca oxide added to Dioxane to absorb
water from tissue
CELLOSOLVE - Combustible at 110-120°F
(Ethylene glycol monoethyl ether) - Toxic by inhalation, skin contact & ingestion
- causes conjunctival irritation
THF (Tetrahydrofuran) - BOTH FIXATIVE & CLEARING AGENT
- good dehydrating agent, produces minimum
TRIETHYL PHOSPHATE shrinkage and soluble to almost all possible
reagents used in tissue processing

ADDITIVES TO DEHYDRATING AGENTS

1. 4% Aqueous Phenol + each 95% ETOH baths
2. Anhydrous copper sulphate – indicator of water content
- Original color: White
- If hydrated: Blue

INDICATORS OF INCOMPLETE DEHYDRATION

1. Anhydrous copper sulphate
2. Xylene – will turn milky

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CLEARING 
 DEALCOHOLIZATION
 Process of replacing the dehydrating fluid with a fluid that is miscible with BOTH the dehydrating
fluid and the impregnating/embedding medium

CLEARING AGENTS
Clearing Agent Group Characteristic
MOST COMMONLY USED
XYLENE/XYLOL
MOST RAPID
- substitute for xylene &
benzene
TOLUENE - toxic upon prolonged
exposure
- expensive
- toxic to liver after prolonged
Organic inhalation
- DOES NOT make the tissue
CHLOROFORM
Solvents transparent
- for clearing tough tissues
- doesn’t clear
CARBON - similar to chloroform but
cheaper
TETRACHLORIDE
- penetrates and clears tissue
rapidly
BENZENE - highly carcinogenic
- causes aplastic anemia
METHYL BENZOATE - slow-acting
- used when double
METHYL SALICYLATE embedding techniques are
(Oil of Wintergreen) required

CEDARWOOD OIL Essential - recommended for CNS tissues

& cytological studies
- extremely slow

CLOVE OIL
Oils - aniline dyes are removed
- celloidin is dissolved
- expensive
- for delicate tissues
ANILINE OILS e.g.insects, embryo

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IMPREGNATION  AND EMBEDDING 
IMPREGNATION – process whereby the clearing agent is completely removed from the tissue
EMBEDDING – process by which impregnated tissue is placed into a precisely arranged position in a
mold containing a medium which is then allowed to solidify

INFILTRATING/EMBEDDING MEDIUM
MEDIUM MP CHARACTERISTIC
PARAFFIN 55-60°C 56°C – temperature of wax normally used for routine work
- for hard and dense tissues (e.g. bones and teeth)
- for large tissue sections of the whole embryo
- supplied as 1:1 mixture of ethanol & ether
Parloidoin – hard, amber, platelet-chips
TYPES
Low Viscosity Nitrocellulose (LVN) “nitrated variety”
- used in higher concentrations (to penetrate tissue rapidly)
- usually marketed while wet with alcohol
WET CELLOIDIN METHOD
CELLOIDIN - for bones, teeth, large brain sections and whole organs
Process:
- thin 2%
- medium 4%
- 1. Fixation and Dehydration
2. Equal parts Ether and alcohol - 12-24 hours
METHODS

- thick 8% 3. Infiltration:  Thin or Medium celloidin - 5-7 days &

 Thick celloidin - 3-5 days
4. Embed in dessicator (open from time to time)
Stored in 70% alcohol
DRY CELLOIDIN METHOD
- for processing the whole eye sections
Process: (same with Wet Method)
Stored in GILSON’S MIXTURE (1:1 chloroform & cedarwood oil)
- best stored in air-tight jar
for delicate specimens and frozen tissue sections because it prevents
fragmentation of tough and friable tissues
Process:
GELATIN 1. Fixation (No Dehydration and Clearing)
(water - 2. 10% Gelatin with 1% Phenol - 24 hours
soluble) 3. 20% Gelatin with 1 % Phenol - 12 hours
4. 20% Gelatin with 1 % Phenol - cooled to refrigerator
5. 10% Formalin - 12-24 hours to harden the tissue
VOLUME: should be at least 25 times the volume of the tissue
- superior results for light microscopic studies
- for renal biopsies, bone marrow biopses
PLASTIC/ EPOXY (Araldite, Epon, Spurr) - made up of epoxy plastics, catalysts
-
TYPES

RESIN POLYESTER PLASTICS - for electron microscopy (now seldom used)

ACRYLIC PLASTICS (Polyglycol methacrylate, Methyl methacrylate) -
made up of acrylic acid or methacrylic acid ; for light microscopy

14
TYPE OF PROCESSING
Fixation: (10% buffered formalin) – 24 hours
Dehydration: 70% Alcohol – 6 hours
95% Alcohol – 12 hours
100% Alcohol – 2 hours
100% Alcohol – 1 hour
MANUAL 4 changes of wax at 15 mins
interval to ensure complete
100% Alcohol – 1 hour
Clearing: Xylene 1 – 1 hour
PROCESSING removal of clearing agent Xylene 2 – 1 hour
Impregnation: Paraffin Wax 1 – 15 minutes
Paraffin Wax 2 – 15 minutes
Paraffin Wax 3 – 15 minutes
Paraffin Wax 4 – 15 minutes
Embedding: Paraffin Wax – 3 hours
Use of automatic processing Agitation & Heat is used to hasten automatic
AUTOMATIC machine to perform tissue process
processing
PROCESSING Machine: Elliot Bench-Type Processor
Air bubbles and clearing Negative Atmospheric Pressure is used to
agent from tissue block are hasten the process
VACUUM remove to promote rapid
wax penetration
EMBEDDING
GIVES THE FASTEST RESULT Machine: Vacuum Embedding Bath

PARAFFIN WAX SUBSTITUTES

MELTING CHARACTERISTIC
INFILTRATING MEDIUM
POINT
Paraffin + Plastic
Mixture of highly
Paraplast 56-57°C purified paraffin
polymers
Synthetic Wax
Embeddol 56-58°C similar to paraplast
Bioloid 50-52°C for embedding eyes
Paraffin + Rubber;
Tissue Mat 56-58°C excellent cutting
properties
not soluble in water;
Ester Wax 46-48°C soluble in alcohol
(no clearing required)
soluble & miscible w/
Water Soluble Waxes 38-42°C water
Carbowax 45-56°C (no dehydration &
clearing required)

15
BLOCKING-OUT MODELS
L-shaped strips of heavy brass
arranged in a flat metal plate
Leukhart’s
and which can be moved to
Embedding Mold adjust the size of mold to the
size of the specimen

Compound interlocking plates resting on a

Embedding Unit flat metal base

special stainless steel base mold

Plastic Embedding
fitted with plastic embedding
Rings and Base Mold ring
Peel-Away
- disposable thin plastic
embedding molds available
in sizes

Plastic Ice Tray

Disposable - recommended for busy
Embedding Molds routine laboratory

Paper Boats
- used for embedding
celloidin blocks but are
equally useful for paraffin
blocks

TRIMMING 
- process of removing the excess wax from the block to form a
four-sided prism or a truncated pyramid; opposite sides being parallel
- excess wax is cut-off in thin slices to prevent cutting
- at least 2mm of wax should surround the tissue block
- The small paper tag bearing the tissue number (label) which may be removed after removal of tissue
block from embedding mold or during trimming may be affixed by using a hot spatula

REMEMBER:
Paraffin blocks –REQUIRE chilling in ice cold water or refrigerator
Celloidin blocks – DO NOT REQUIRE chilling

16
SECTIONING 
- CUTTING OR MICROTOMY
- The process by which a processed tissue is cut into uniformly thin slices (sections) to facilitate
studies under the microscope

SECTION SIZES
Paraffin Section 4-6 micra Rotary/Rocking
Celloidin Section 10-15 micra Sliding
Frozen Section 10 micra Freezing/ Cryostat
50-120 millimicra
Ultrathin Section 500-1200 angstrom
Ultrathin

MICROTOMES MAJOR PARTS OF MICROTOME

- equipment used to perform sectioning 1. BLOCK HOLDER – holds the tissue in place
- machine specifically designed to cut 2. KNIFE CARRIER and KNIFE
very thin sections of tissue 3. ADJUSTMENT SCREW and RACHET –
device that line up tissue in proper
relation to the knife & feed the proper
thickness of tissue for successive sections
TYPES OF MICROTOMES
Microtome Inventor Description Purpose
William Hay Caldwell and Serial sections of large
Rocking Richard Threlfall (1881)
Simplest
paraffin embedded sections

Sliding George Adams (1789) Most Dangerous Celloidin embedded sections

Charles Sedgwick Minot

Rotary (1885)
Most common Paraffin embedded sections

Freezing John Queckett (1881) for Frozen Section Unembedded frozen section

uses glass
Ultrathin Marfred von Ardenne
diamond knives
Sections for EM

*Paldwell Trefall – a non-existent person credited by authors such as Gregorios for inventing the Rocking Microtome

MICROTOME KNIVES
BICONCAVE PLANE-CONCAVE PLANE WEDGE
-cut paraffin section -cut paraffin & celloidin section -cut frozen section

120mm 25 mm 100 mm

17
SHARPENING OF KNIVES
HONING STROPPING
-removal of “burrs” formed during honing and to
-removal of gross nicks and blemishes
polish the cutting edge
HEEL to TOE TOE to HEEL
20-30 strokes 40-120 strokes
Types of Hones Leather Strops
 Fine Carborundum – gives the best result (natural skin of horses/pigs/calf)
 Arkansas
 Belgium Yellow – fine
Lubricant: Soapy water, Oil Lubricant: Castor Oil (NEVER USE MINERAL OIL)

Section cut is pricked by:

 Index finger
 Camel’s hairbrush
 Spatula
 Flat-bladed forceps

0-15° (Gregorios) -inclination/tilt angle

CLEARANCE ANGLE 5-10° (Lynch) -angle between the block & cutting edge
BEVEL ANGLE 27-32° -angle between the cutting edges of knife
WEDGE ANGLE 14-15° -angle of the sides of wedge knives
-angle between the inclination/tilt and the side of
RAKE ANGLE 90°
the block

FISHING-OUT
- After cutting, tissue sections are floated out on a water bath
- When the sections are flattened out, slide is immersed in water bath and tissue is fished out
- Adhesives are used to promote adhesion of sections

CHARACTERISTIC OF A GOOD RIBBON

 Thin
 Transparent
 Without irregularities
 Not wrinkled
 Uniform in thickness
 Can easily be separated from each other
 Continuous

METHODS TO FLATTEN RIBBON METHODS OF DRYING SLIDES

 50% Alcohol  Using Paraffin oven – 56-60°C for 2 hours
 Float out bath – 45-50°C  Using Incubator – 37°C overnight
 Distilled water in hotplate  Using hotplate – 45-55°C for 45 minutes
 Pearse  Blower type slide dryer – 50-55°C for 20-30
 Blank & McCarthy for Carbowax minutes
 Bunsen burner/Alcohol lamp – pass over
flame until wax melts
18
ADHESIVES
- used to promote adhesion of tissues to slides

Adhesives Component
Egg white
Mayer’s Egg Albumin Glycerin
Thymol
Dried Albumin
Dried Albumin Sodium Chloride
Thymol
Distilled Water
should be placed
1% Gelatin Glycerol
in float-out bath
Phenol
1% Gelatin brushed in slides and
Gelatin-formaldehyde 2% Formaldehyde use after 24 hours
Powdered Starch
Distilled Water
Starch Paste Hydrochloric Acid
Thymol
Plasma Pooled outdated plasma
Poly-L-Lysine
APES (3-
aminopropylthriethoxysilane)

DEPARAFFINIZATION
- performed to remove surrounding paraffin wax from tissue sections in slides to prevent over-
staining of tissue

METHODS OF DEPARAFFINIZATION
 Alcohol lamp
 Xylene bath
 Hot oven – 55-60°C
 Cotton swab (soaked in Xylene)

19
TROUBLESHOOTING

FAULTS DURING TISSUE PROCESSING

FAULTS REASON REMEDY
Prolonged fixation
Prolonged dehydration
Tissue may be softened by
Prolonged clearing
soaking in a small dish or bowl
Brittle or Hard tissue Prolonged paraffin infiltration
containing detergent, phenol or
Overheated paraffin oven
Molliflex
Drying out of tissue before actual
fixation
Clearing agent turns milky
Water not completely removed due to Repeat dehydration with
as soon as tissue is placed
incomplete dehydration absolute alcohol then clear again
in it
Block is trimmed down nearest
to the tissue, remaining wax is
Clearing agent not completely
On trimming, tissue smells melted on embedding oven and
removed due to insufficient
of clearing agent paraffin impregnation is
impregnation
repeated, changing paraffin at
least once
Repeat clearing; if object has
Tissue is opaque, section
already been embedded prolong
cutting is difficult due to Insufficient clearing
clearing up to 12 hours then re-
the presence of alcohol
embed
Tissue shrinks away from Insufficient dehydration; therefore
Repeat the whole procedure
the wax when trimmed incomplete clearing and impregnation
Tissue is soft when block is
Incomplete fixation Repeat fixation
trimmed
Air holes found on tissue
Incomplete impregnation Repeat impregnation
during trimming
On trimming, wax appears Contaminated wax
Re-embed in freshly filtered wax
crystalline Block not cooled rapidly enough
Paraffin block after cooling Repeat paraffin impregnation,
Insufficient paraffin impregnation
is moist and crumbles then re-embed

FAULTS OBSERVED DURING FROZEN SECTION-CUTTING

FAULTS REASON REMEDY
Frozen tissue crumbles and
comes off the block holder Freezing is not adequate Refreeze the tissue block
when cut
Frozen tissue chips into
Tissue is frozen too hard Warm the tissue with the fingers
fragments when cut

20
FAULTS OBSERVED DURING SECTION-CUTTING
FAULTS REASON REMEDY
Surfaces and edges of the block are
Re trim the block
not parallel
Horizontal surface of the block is not
Re-adjust and re-orient the block
parallel to the knife
Sections fail to form Coat horizontal edge of the block
Paraffin wax is too hard
ribbons with wax of lower melting point
Knife tilted too much Reduce the tilt
Re-adjust the thickness of the
Sections are too thick
sections
Knife is dull Hone and strop
Sections roll-up on cutting Knife is blunt Sharpen the knife
so that they adhere and Tilt of the knife is too great Reduce the tilt
get broken against the
Knife edge is dirty Clean the knife edge
knife edge
Adjust the knife so that knife
Blunt or dull spot on the knife edge will present a uniformly
producing an irregular knife edge sharp edge to the block; or
Ribbon is curved, crooked sharpen
or uneven instead of Edges of the block are not parallel but
Re-trim the block
straight round or wedge-shaped
Knife is not parallel to the block Readjust the knife and the block
Repeat impregnation using pure
Paraffin is impure
wax
Knife is blunt or dull Re-sharpen the knife
Cool the block on ice water until
Paraffin block is warm and soft
firm
Sections are compressed,
Knife edge is coated with paraffin Clean the knife edge
wrinkled or jammed
Sections are too thin Readjust the thickness of section
Microtome set screw is loose Tighten the screw
Tilt of the knife is too vertical Reduce the tilt
Sections are
squashed(width of each Bevel of the knife is lost due to Re-sharpen, using a knife back or
section is less than that of incomplete sharpening automatic knife sharpener
the block
Bubble or dirt formed in the Re-embed in freshly filtered wax
embedding medium if necessary
A hole is formed in the Once embedded in paraffin wax,
section Hard spot in tissue possibly due to decalcification is impractical; use
calcium a base sledge microtome with
wedge knife

21
 FAULTS REASON REMEDY
Tilt of the knife is too great or bevel is
not cleared, hence object is Reduce the tilt
compressed against the knife edge
Sections of unequal Clamp set screw on knife or block
Tighten the screw
thickness are produced holder is loose
Blocks are too large Cut blocks into smaller fragment
Soften the blocks in detergent or
Blocks are too hard
phenol
Breath out or blow gently on the
Sections adhere to the Static electricity due to low block and knife to break the
knife or other parts of the atmospheric humidity static electricity, or boil water in
machine the room to increase humidity
Knife edge is dirty Clean the knife edge
Ribbon is split or Nicks or damage on the knife edge Sharpen knife
lengthwise vertical Dirty embedding medium Re-embed in filtered wax
scratches are seen on Knife edge is dirty Clean the knife edge with Xylene
sections Tilt of the knife is too great Reduce tilt
Knife tilt is too great Reduce the tilt
Sections are lifted from the Knife is dull Sharpen the knife
knife on upstrokes Paraffin is too soft or room temp is
Cool paraffin wax in ice water
warm
Tilt of the knife is insufficient; paraffin
Resistance is felt on the
block is therefore compressed against
lower part of the section Increase the tilt
the base of the knife towards the end
during cutting
of the stroke
Horizontal or parallel lines Knife edge vibrates due to hardness Treat with phenol during
or furrows across the of the tissue processing; or collodionize
section (CHATTERS) are
seen, forming thin and Tilt of the knife is too great Reduce the tilt
thick zones
Knife is blunt Sharpen the knife
Knife is not clamped properly Adjust the knife
Tilt of the knife is too great Reduce the tilt
Section cut is sometimes Tighten adjusting and locking the
Knife or block holder is loose
thin, sometimes thick screws
Knife tilt is too small that block is
compressed by bevel end section is Increase the tilt
not cut
Tilt of the knife is too slanted or too Readjust the angulation of the
Knife makes a hard
big knife
metallic scraping or ringing
Take fresh block treated with
sound on backstroke, Tissue is too hard
phenol during processing
when section is cut
Knife blade is too thin Change the knife

22
STAINING 
- The process of applying dyes on the sections to study architectural pattern of the tissue and
physical characteristics of the cells
- Different tissues and cells have varying affinities for most dyes and stains

METHODS OF STAINING
- tissue elements are stained in definite sequence
PROGRESSIVE - staining with specific periods of time or until desired color is
STAINING attained
- not washed or decolorized
REGRESSIVE - first over-stain the tissues to obliterate cellular details
- excess stain is removed or decolorized from unwanted parts of the
STAINING tissue and until the desired color is obtained
- selective removal of excess stain from the tissue during regressive
DIFFERENTIATION/ staining so that a specific substance may stain distinctly from the
surrounding tissue
DECOLORIZATION - Usually done by washing the section in simple solution (e.g. water or
alcohol) or use of acids and oxidizing agents
- Makes use of specific dyes which differentiate particular substances
by staining it with a color that is different from that of the stain itself
- Water is necessary for most metachromatic staining techniques
METACHROMATIC - Metachromasia is usually lost if section is dehydrated in alcohol
after staining
STAINING Methyl violet Basic fuchsin
Crystal volet Methylene blue
Cresyl blue Thionine
Safranin Toluidine blue
Bismarck brown Azure A, B, C
- Application of a different color or stain to provide contrast and
background to the staining of the structural components to be
demonstrated
Cytoplasmic stains
Red Yellow Green
Eosin Y Picric acid
Light green SF
Eosin B Orange G
COUNTERSTAINING Phloxine B Rose Bengal
Lissamine green
Nuclear Stains
Red Blue
Neutral red
Methylene bue
Safranin O
Toluidine bue
Carmine
Celestine blue
Hematoxylin
- process where specific tissue elements are demonstrated not by
METALLIC stains but by colorless solutions of metallic salts which are deposited
on the surface of the tissue
IMPREGNATION - it is not absorbed by the tissues, could be a precipitate or a
reduction product on certain tissues
- e.g. Gold chloride, Silver nitrate

23
 - The selective staining of living cell constituents
- Demonstrates cytoplasmic structures
 By engulfment of the dye particle
 By staining of pre-existing cellular components
- Nucleus is resistant to vital stains
VITAL STAINING Intravital staining Supravital staining
- by injecting the dye into any - used immediately after removal
part of the animal body of cells from the living body
e.g. Lithium, Carmine, India ink e.g. Neutral red (best), Janus green
(mitochondria), Trypan blue, Nile
blue, Thionine and Toluidine blue
- uses aqueous or alcoholic dye solutions (e.g. methylene blue, eosin)
DIRECT STAINING to produce a color
- uses a mordant or accentuator to intensify the action of the dye
MORDANT ACCENTUATOR
- serves as link or bridge  not essential and does not
INDIRECT STAINING between the tissue and dye participate to the chemical
reaction of the tissue and dye
 accelerates the speed of the
staining reaction by increasing
the staining power and selectivity
of the dye

H&E (Hematoxylin & Eosin) PROCEDURE

XYLENE (2 CHANGES)  Deparaffinization


DESCENDING GRADES OF ALCOHOL  Rehydration or Hydration

HEMATOXYLIN  Primary Stain

ACID ALCOHOL  Differentiation or Decolorization

AMMONIA WATER  Blueing

EOSIN Y  Secondary Stain or Counterstain

ASCENDING GRADES OF ALCOHOL  Dehydration

XYLENE (2 CHANGES)  Clearing

H& E RESULT
 NUCLEI – blue to blue black
 CYTOPLASM – pale pink

24
CATEGORIES OF STAINS
NATURAL SYNTHETIC (Artificial)
 Also known as “coal tar dyes”
 Derived from plants and animals
 Derived from hydrocarbon benzene
 Collectively known as “aniline dye”

NATURAL DYES
DYE CHARACTERISTIC
- not a stain
- active coloring agent – HEMATIN
- used in combination with a mordant such as alum, iron,
Hematoxylin chromium and copper salts
Hematoxylin campechianum - oxidized through the process of “RIPENING”
Natural Ripening
 Expose the substance to air and sunlight
 A slow process, 3-4 months
Artificial Ripening
 Chemical oxidation
 uses Hydrogen peroxide, Mercuric oxide, Potassium
permanganate, Sodium perborate, Sodium iodate
Over-Ripening
 excessive oxidation
- with alum
Cochineal dyes - Carmine dye
Cochineal bug (Coccus cacti) - Chromatin and nuclear stain for fresh and smear preparation
- if with picric acid – Picrocarmine
- if with aluminum chloride – Best’s Carmine
- vegetable dye
Orcein - colorless, treated with ammonia, exposed to air to produce a
Extracted from lichens blue or violet color
- weak acid, soluble in alkali
- used for staining elastic fibers

SYNTHETIC DYES
- the coloring agent is found in the acid
Acid Dye component e.g. Picric acid, Eosin
- the base is usually Sodium
- the coloring substance is found in the basic
Basic Dye component
e.g. Methylene blue
- formed by combining aqueous solutions of
Neutral Dye basic and acid dyes
e.g. Giemsa, Leishman

25
COMPONENT OF SYNTHETIC DYES
Substances that are capable of producing visible color but is not
CHROMOPHORE permanent and can be easily removed
Substances that are added to a chromogen, which alters the property of
AUXOCHROME the chromogen by altering its shade, enabling it to form salts with another
compound and enables it to retain its color in the tissue

COMMON STAINING SOLUTIONS

HEMATOXYLIN
HEMATOXYLIN MORDANT RIPENING AGENT PURPOSE
Erhlich’s Sodium Iodate for regressive staining
Harris Potassium Alum Mercuric Oxide for Exfoliative cytology
Cole’s (Aluminum Potassium Alcohol Iodine Solution for Celestine Blue
Sulfate)
for Celestine Blue
Mayer’s Sodium Iodate
Hemalum Method
for demonstration of
Weigert’s Ferric Chloride
muscle fibers and CT
Iron Alum Ferric Ammonium for regressive staining
Heidenhain’s (Ferric Ammonium Sulfate of thin sections
Chloride) for demonstrating
PTAH Light/Potassium structures in celloidin &
frozen sections

EOSIN
- A red acid dye
- Routinely used as a counterstain after hematoxylin and before methylene blue
- Stains connective tissues and cytoplasm differentially

EOSIN
EOSIN SHADE COLOR CHARACTERISTIC
green yellow
Eosin Y Yellowish
fluorescence
most commonly used

Eosin B Bluish deeper red color

26
OTHER STAINS
Acid Fuchsin-Picric Acid
 for demonstration of connective tissues
(Van Gieson’s Stain)
 for DNA – green fluorescence
Acridine Orange
 for RNA – red fluorescence
Acridine Red 3B  demonstration of calcium salt deposits and phosphatase activities
Alcian Blue  for connective tissue and epithelial mucin
Aniline Blue  for counterstaining of epithelial sections
 for staining acid-fast organisms, for mitochondria, for differentiation
of smooth muscles (with the use of picric acid)
Kinds:
Basic Fuchsin A. Carbol-Fuchsin
B. Coleman’s Feulgen Reagent
C. Schiff’s Reagent
D. Mallory’s Fuchsin Stain
E. Aldehyde Fuchsin (Gomori’s stain)
Benzidine  for staining hemoglobin
 used as counterstain for Gram’s technique, for acid fast, for
Bismarck Brown
Papanicolau method
 used for staining diphtheria organisms
Carmine  used as chromatin stain for fresh materials in smear preparations
 combined with aluminum chloride to stain glycogen (Best Carmine)
Celestine Blue  used for routine staining of fixed sections
 best known as an indicator
Congo Red
 stains elastic tissues, amyloid, myelin
 stains amyloid in frozen sections, platelets in blood
Crystal Violet
 Gentian violet (Crystal violet, Methyl violet, Dexterin)
Giemsa Stain  used for staining blood to differentiate WBCs
 stain used for metallic impregnation
Gold Sublimate
 made up of gold chloride and mercuric chloride
 stains amyloid, cellulose, starch, carotenes, glycogen
Iodine  Gram’s iodine -stains microorganisms and fibrin in tissue sections
 Lugol’s iodine -used as test for glycogen, amyloid
Janus Green B  for demonstrating mitochondria during intravital staining
 counterstain for Ascaris eggs, erythrocytes, bacterial spore stain
Malachite Green
 used as a decolorizer and counterstain
 common basic nuclear stain used with eosin
 stains plasma cells, cytological examination of sputum for malignant
Methylene Blue
cells, evaluation and differentiation of bacteria, diagnosis of
diphtheria, vital staining of nervous tissues
Neutral Red  for demonstration of cell granules and vacuoles of phagocytic cells
 stains elastic fibers
Orcein
 recommended for dermatological studies
Osmium Tetroxide  used to stain fat – black
 counterstain for acid fuchsin, connective tissues (in Van Gieson’s
Picric Acid stain), cytoplasmic stain in contrast to basic dyes, counterstain for
crystal violet

27
  colored salt of ferric ferrocyanide
Prussian Blue  used for the manufacture of paints
 used as contrast stain, intravital staining of the circulatory system
Rhodamine B  used with osmic acid to fix and stain blood and glandular tissues
Silver Nitrate  used for identification of spirochetes, reticulum, fiber stains
 used as nuclear stain in fixed tissues, stains Nissl granules or
Toluidine Blue chromophilic bodies

OIL SOLUBLE DYES (LYSOCHROMES)

- Not real dyes, lack auxochrome
- Gives color to lipids because they are more soluble in lipid medium of the tissues than in 70%
alcohol

OIL SOLUBLE DYES

OIL SOLUBLE DYE COLOR CHARACTERISTIC PURPOSE
MOST SENSITIVE;
Sudan Black B Blue-Black has 2 secondary amino stains phospholipids
groups per molecule
Sudan IV has no secondary amino
Deep & Intense Red stains neutral fats
(Scharlach IV) group
first Sudan dye to be
Sudan III Light Orange introduced in stain for CNS
histochemistry

CHIEF SOLVENTS USED FOR STAINS

WATER should be distilled unless otherwise stated
Ethyl Alcohol – used in varying concentrations
ALCOHOL Methyl Alcohol – used as absolute and acetone free
ANILINE WATER Aniline added to water
PHENOL used in 0.5-5% aqueous solution

28
SPECIAL STAINS
FOR CARBOHYDRATES
Special Stain Reaction
PAS positive red/magenta red
Periodic Acid Schiff
Nuclei blue
PAS with Diastase Glycogen red
( for glycogen) Nuclei blue-black
Glycogen bright red granules
Best Carmine Nuclei blue or grayish blue
Mucin and Fibrin weak red
Langhan’s Iodine Glycogen mahogany brown
(for glycogen) Tissue constituents yellow
Azure A Glycoaminoglycans red-purple
(for glycoaminoglycans) Tissue background blue
Acid mucin blue
Alcian Blue
Nuclei red
Glycoaminoglycans red-purple
Toluidine Blue
Tissue background blue
Sulfated mucin purple
Aldehyde Fuchsin-Alcian Blue
Carboxylated mucin blue
Mucin red
Mucicarmine
Nuclei blue
Acid mucin dark blue
Hale’s Dialyzed Iron Technique
Nuclei red
Acid mucopolysaccharides black
Fluorescent Acridine Orange Fungi greenish red fluorescence
Background reddish orange fluorescence

FOR FATS/LIPIDS
Special Stain Reaction
Lipids (triglycerides red
Sudan IV / Scharlach R
Nuclei blue/black
Fat brilliant red
Oil Red O
Nuclei blue
Osmic Acid Nuclei yellow-orange
(not a stain) Fats black
Neutral fat pinkish red
Cholesterin esters & fatty acids light red
Nile Blue Sulfate
Cerebrosides light blue
Fatty acids and soaps deep blue to violet
Toluidine Blue (Acetone Method)
Sulfatide deposits metachromatic red
(for sulfatide)
Borohydride-Periodic-Schiff (BHPS) Gangliosides red
(for glycosides) Nuclei blue

29
FOR PROTEINS
Special Stain Reaction
Alkaline Fast Green Histones and protamines
green
(for basic proteins) (found in nuclei)
Peracetic Acid-Alcian Blue
Cystine and Cysteine blue-green
(for cystine and cysteine)
Sakagushi’s Test
Arginine orange-red
(for arginine)

FOR ENZYMES
Special Stain Reaction
Gomori Calcium Method ALP activity brownish-black
(for ALP) Nuclei green
Gomori Lead Method ACP activity black
(for ACP) Nuclei green
Wachstein & Meisel Lead Method
5’nucleotidase blackish-brown
(for 5-nucleotidase)
α-Naphthyl Acetate Method Esterase reddish-brown
(for non-specific esterase) Nuclei green
Holt & Withers Indoxyl Acetate Esterase activity blue
(for non-specific esterase) Nuclei red
Tetrazolium Method
Monoamine oxidase activity bluish black
(for monoamine oxidase)

FOR NUCLEIC ACIDS

Special Stain Reaction
Feulgen Technique DNA red-purple
(for nuclear DNA) Cytoplasm green
Methyl Green-Pyronin Method DNA (chromatin) green or blue green
(for RNA and DNA) RNA (nucleoli) rose-red
Acridine Orange Fluorescent Staining DNA yellow-green fluorescence
(for RNA and DNA) RNA brick to orange red

30
FOR CONNECTIVE TISSUES
Special Stain Reaction
Gomori’s Silver Impregnation
Reticulin fibers black
(for reticulin)
Collagen pink or deep red
Van Gieson’s Stain
Muscle, cytoplasm, RBC, fibrin yellow
(for collagen)
Nuclei blue-black
Muscle, RBC, Keratin red
Masson’s Trichrome Stain Nuclei blue-black
Collagen, mucus blue
Weigert’s Stain dark blue or blue-black on
Elastic fibers
(for elastic tissue) clear background
Elastic fibers black
Verhoeff’s Stain/ Nuclei gray to black
Verhoeff’s Van Gieson’s Stain Collagen red
Cytoplasm, muscle yellow
Orcein/ Taenzer-Unna Orcein Elastic fibers dark brown
MOST EXCELLENT for elastic fibers Nuclei blue
Fibrin, muscle striation,
dark blue
neuroglia, amoeba
Nuclei, cilia, RBC blue
Mallory’s Phosphotungstic acid
Myelin lighter blue
Hematoxylin (PTAH)
Collagen, osteoid, cartilage,
deep-brownish red
elastic fibers
Cytoplasm pale-pinkish brown
Highman’s Congo Red Method Amyloid deep pink to red
(for amyloid) Nuclei blue
Amyloid, elastic fibers,
Alkaline Congo Red Technique red
eosinophil granules
Krajian’s Amyloid Stain Amyloid red on a clear background
Amyloid purplish red
Methyl Violet-Crystal Violet Method
Nuclei, cytoplasm, CT shades of violet

FOR BONE MARROW AND BLOOD ELEMENTS

Special Stain Reaction
Eosinophil granules red
Rapid Toluidine-Eosin Stain Basophils, mast cell granules blue
(for glycolmethacrylate sections) Nuclei blue
Vytoplasm blue/pink
Nuclei purple/blue
Wright-Giemsa or Jenner-Giemsa Cytoplasm pink/blue
Eosinophils pink/red
Peroxidase Reaction
Myeloid cells (except basophils) green to dark blue granules
(for myeloid cells)

31
FOR MUSCLES AND BONES
Special Stain Reaction
Muscle fibers red
Modified Gomori’s Trichrome Stain Collagen green
Nuclei blue to black
Mallory’s PTAH -same as above-
Muscle striations, mitochondria, gray-black
Heidenhain’s Iron Hematoxylin
myelin, chromatin
Muscles, RBC red
Lissamine Fast Red – Tartrazine
Collagen yellow
(for muscles and bones)
Nuclei black
Lacunae, canaliculi dark brown-black
Schmorl’s Picro-Thionin Method Bone matrix yellow or brownish yellow
Cells red

FOR CENTRAL NERVOUS TISSUE

Special Stain Reaction
Bielchowsky’s Technique
Neurofibrils, axons, dendrites black on grayish background
(for neurons, axons & neurofibrils)
Large & small peripheral neuritis black
Sevier-Munger Technique
Axons black
(for neural tissues)
Myelin sheath light brown
Cresyl Fast Violet Stain
Nissl substance purple-dark blue
(for Nissl)
Weigert-Pal Technique Myelin sheath blue-black
(for normal myelin sheath) Cells brown
Kluver & Barrers Luxol Fast Blue Myelin blue-green
(for myelin with Nissl counterstain) Cells violet
Myelin blue-green
Luxol Fast Blue – H&E
Nuclei dark blue
(for myelin)
Cytoplasm shades of pink
Myelin blue-green
Fungi & PAS (+) elements rose to red
Luxol Fast Blue – PAS
Nuclei dark blue
(for myelin)
Cytoplasmic nucleoproteins bluish purple
Capillaries red
Weil’s Method Myelin black
(for myelin sheath) Background yellow
black on a light brown
Astrocytes
Cajal’s Gold Sublimate background
(for astrocytes) Nerve cells red
Nerve fibers unstained
Glial fibril blue
Modified Holzer’s Method
Nuclei pale blue
(for astrocytic process)
Background colorless

32
FOR TISSUE PIGMENTS AND DEPOSITS
Special Stain Reaction
Hemosiderin, ferric salts deep blue
Perl’s Prussian Blue
red (according to
(for ferrous iron/hemosiderin) Tissue, nuclei
counterstain)
Iron pigments bright blue
Gomori’s Prussian Blue Stain
Nuclei red
(for iron)
Cytoplasm pink to rose
Turnbull’s Blue Reaction Hemosiderin blue
(for ferrous iron/hemosiderin) Nuclei red
Hemoglobin, oxidase
dark blue
Benzidine granules
(for hemoglobin) Nuclei red
Other tissues faint pink
Bile pigments emerald to blue green
Modified Fouchet’s Technique
Muscle yellow
(for bile liver pigments)
Collagen red
Bile, lipofucsin, melanin dark blue
Schmorl’s Ferric Cyanide Method Argentaffin cells, chromaffin dark blue
(for reducing substances) Thyroid colloid dark blue
Nuclei red
Gomori’s Aldehyde Fuchsin Lipofucsin purple
(for lipofucsin) Background yellow
Hemofucsin red
Mallory’s Fuchsin Stain
Hemosiderin unstained
(for hemofucsin)
Nuclei blue
Masson-Fontana Technique Melanin black
(for melanin, argentaffin cell) Argentaffin cell granules black
Von Kossa’s Silver Nitrate Method Calcium salts black
(for calcium) Nuclei red
Copper,
red to orange-red
Linquist’s Modified Rhodamine Technique copper-associated protein
(for copper) Nuclei blue
Bile green

33
FOR MICROORGANISMS
Special Stain Reaction
Gram positive blue-black
Gram negative pink-red
Gram-Twort Stain
Nuclei red
(for bacteria)
Cytoplasm, RBC green
Elastic fibers black
Gram positive blue
Brown and Brenn (B&B) Gram negative red
(for bacteria, Nocardia, Actinomyces) Nuclei red
Other tissue elements yellow
AFB red
Ziehl-Neelsen Method
Cells, nuclei blue
(for AFB)
RBC pink
M. leprae, other Mycobacteria,
red
Nocardia
Wade-Fite
Background blue
(for leprosy bacilli, Nocardia)
blue-black
Nuclei
(if Hematoxylin is used)
Auramine-Rhodamine Stain Mycobacteria golden yellow
(Fluorescent Method)
Background dark green
(for Mycobacteria)
Toluidine Blue
Helicobacter dark blue
(for Helicobacter)
Dieterle Method L. pneumophila, spirocetes black
(for Legionella pneumophila) Background yellow
Levaditi’s Method Spirochetes black
(for spirochetes) Background yellowish brown
Warthin-Starry Method Spirochetes black
(for spirochetes) Background golden yellow
Spirochetes, Donovan bodies,
Modified Steiner & Steiner black
fungi, bacteria
(for spirochetes)
Background yellow to brown
Fungi black
Mucin, glycogen gray-black
Grocott Methenamine Silver Stain
Mycelia, hyphae old rose
(for fungi)
RBC yellow
Background pale green
Viral inclusions bright red
Lendrum’s Phloxine Tartrazine Method RBC yellow to orange-red
(for viral inclusions) Nuclei blue-gray
Background yellow to pink
Orcein Method HBsAg, some mucins brown-black
(for HBsAg) Background yellow
Bacteria blue
Mast cell granules deep blue
Rapid Giemsa Stain
Nucleus blue
Cytoplasm pink
34
MOUNTING 
- process that involves the use of a mounting medium and a coverslip to facilitate the ease of
handling and storage of the slide and to prevent damage to the section

CHARACTERISTICS OF A GOOD MOUNTING MEDIUM

 A refractive index which is near to that  Does not cause shrinkage and distortion
of the glass (1.518) of tissues
 Miscible with xylene and toluene  Does not leach out any stain or affect
 Does not dry quickly staining
 Does not produce artifacts on the slides  Does not change in color or pH
 Does not dissolve out or fade tissue  Sets hard and produces permanent
sections mounting of sections

MOUNTING MEDIA
AQUEOUS RESINOUS
“temporary” mounting “semi-permanent” mounting
to mount water-miscible preparations directly from used for preparations that have been dehydrated
water in cases where the stain is removed or and cleared in xylene or toluene, and are
decolorized with alcohol or xylene recommended for majority of staining methods
Example Refractive Index Example Refractive Index
Glycerin 1.47 Canada Balsam 1.524
Farrant’s Medium (Gum Arabic) 1.43 Entellan 1.500
Apathy’s 1.52 DPX 1.532
Water 1.455 XAM 1.52
Fructose 1.47 Clarite 1.544
Highman’s 1.52 Coverbond 1.53
Gurr’s 1.51
Histoclad (Clay Adams) 1.54
Pro-Texx 1.495
Technicon Resin 1.62
UV Inert 1.57
Eukitt 1.510

RINGING
- process of sealing the margins of the coverslip 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

RINGING MEDIA
1. Kronig cement
2. Durofix
3. Nail polish
4. Varnish

LABELING 
- process of indicating the year and specimen number on one end of the prepared slide for proper
identification

35
QUALITY ASSURANCE AND DOCUMENTATION
A. Histopathology Reports
1. Surgical Pathology 1 Patient ORIGINAL COPY
2. Cytopathology Report 3 copies 1 FILE
3. Autopsy Report 1 Physician
B. Signatories
1. Request Forms – Patient’s Doctor
2. Result Forms – Pathologist
C. Specimen Handling
1. FIX FIRST
2. Label
D. Routine Turn-over of Results
1. Surgical pathology and Cytology – 24 hours
2. Frozen Section – 5 to 15 minutes
3. Autopsy Report – 1 week
E. Storage of Specimen, Tissue Blocks, Slides
1. Specimen – 1 month to 1 year
2. Tissue Blocks – 3 to 10 years
3. Slides – Indefinite

SUGGESTED GUIDELINES FOR RECORDS AND SPECIMEN RETENTION (Henry)

RECORD/SPECIMEN TYPE RETENTION
RECORDS
Requisitions 2 years
Quality Control 2 years
Instrument Maintenance 2 years
REPORTS
Clinical Pathology Laboratory Reports 2 years
Autopsy Forensic Reports Indefinitely
Surgical Pathology (and Bone Marrow) 10 years
Reports
SPECIMEN
Pathology/Bone Marrow slides 10 years
Pathology Blocks 10 years

36
EXFOLIATIVE CYTOLOGY
– branch of science dealing with the study of cells that are scrapped off or removed off or coming
from lining epithelium and mucosa of different organs of the body.

APPLICATIONS:
 assessing malignant or cancerous condition
 detection of asymptomatic cancer in women (Vaginal Cytology)
 assessment of female hormonal activity in case of sterility and endocrine disorders
 determination of genetic sex
 determination of presence of possible infection

SPECIMENS:
1. Cervicovaginal smear (Pap smear) 5. Pleural and peritoneal fluids
2. Nipple discharge 6. Sputum
3. Gastric secretions 7. Urine sediments
4. Bronchial secretions 8. CSF

GYNELOGICAL SPECIMEN
- sampling of T-zone (Transformation zone) for detection of dysplasias and carcinoma of the
cervix
COMMON
SAMPLING TECHNIQUES MATERIALS
Endocervical brush samples of endocervical canal USED:
Vaginal scrape for patients with hysterectomy
Lateral vaginal hormonal evaluation
scrape
Four quadrant localization of vaginal adenosis
vaginal scrape
Vulvar scrape for detection of herpetic lesions or
carcinoma SPECULUM

ADHESIVES:
1. Pooled human serum or plasma – PRACTICAL
2. Celloidin ether-alcohol
3. Leuconostoc culture AYRES SPATULA
PREPARATION OF SMEARS:
1. Streaking
2. Spreading – recommended for fresh sputum and bronchial aspirates
3. Pull-apart – for thick secretions such as serous fluids, concentrated sputum, blood smears
4. Touch Impression

50% Ethanol for all effusions

If Smears cannot be
made immediately!!! Saccomano’s Fluid
for sputum
(50% Ethanol and Carbowax)

37
COMMON FIXATIVES
1. Equal parts of 95% Ethyl Alcohol and Ether
2. 95% Ethyl Alcohol
3. Carnoy’s Fixative – rapid fixative
4. Spray-Cyte or Cytospray (Unscented) – has alcohol
5. 3 parts of tertiary butyl alcohol and 1 part 95% ETOH
6. Schaudinn’s Fluid – sat. aq. Hg2Cl, absolute HAc
7. MTOH–for dried films

METHODS OF STAINING
1. Pap’s or Papanicolau staining - named by Dr. George Papanicolaou in 1943
2. H & E
3. Modified staining (H & E)

PAP’S STAINING REAGENTS

1. Harris Hematoxylin – Nuclear stain (Basophilic stain) – Blue color to the nucleus
2. OG 6 (Orange-Green 6) – made up of 0.5-1% solution of OG in 95% alcohol and phosphotungstic
acid. This acts as the intensifier or differentiator for different structures of the cells.
3. EA 36 or EA 50 – made up of the following:
a. Bismarck brown Y dye – 0.5% in 95% alcohol
b. Eosin-Y
c. Phosphotungstic acid
d. Light-green SF yellow solution – 0.5% in 95% alcohol
e. Lithium carbonate

PAP’S STAINING PROCEDURES

FIXATIVE

Descending Grades of Alcohol 80%, 70%, 50%  Rehydration or Hydration

Harris Hematoxylin  Initial Stain or Basophilic Stain

Acid Alcohol  Differentiation or Decolorization

Ascending Grades of Alcohol  Dehydration

OG6  Second Stain or Intensification

95% Ethyl Alcohol (2 changes)

EA36 or EA50  Counterstain or Acidophilic Stain

95% Ethyl Alcohol (2 changes)

Xylene (3 changes)  Clearing

RESULTS:
Nucleus - blue
Cytoplasm - shades from pink, blue, green and yellow-orange.
38
CELLS FOUND IN CERVICO-VAGINAL SMEARS
CELLS SIZE SHAPE NUCLEUS CYTOPLASM
Presence of small
may be
Large dark pyknotic
SUPERFICIAL (30-60u)
Polyhedral flat cells
nuclei
acidophilic or
basophilic
(less than 6u)
Polyhedral or
elongated
Medium Navicular cells: Boat-
vesicular nuclei basophilic with
INTERMEDIATE large shaped
(6-9u) vacuoles
(20-30u) Pregnancy Cells:
Round or Oval boat
shaped
Thick;
Round to oval cells
PARABASAL smaller than vesicular nuclei strong basophilc
“sunny-side up” like
intermediate (6-9 u ) cytoplasm
cells
(15-25 u)
Relatively large
BASAL Small Round, slightly oval nucleus occupying strongly
(13-20u) cells half or more of the basophilic
cell volume
deeply basophilic
ENDOCERVICAL Slightly cylindrical
than that of the
appearance
parabasal cells
Endometrial stromal
cells: tight clusters of basophilic and
ENDOMETRIAL small and
small, oval dark cells maybe
moderately dark
Glandular cells: vacuolated
slightly larger

QUANTITATION OF VAGINAL CYTOLOGY

percentage of cells staining pink-orange to red with Pap’s smear
Acidophilic Index (A.I.) not a reliable index due to possible pseudoacidophilia
Percentage of cells with shrunken, dark, small structureless
Pyknotic Index (P.I.) nucleus
reliable criterion for identification of superficial cells
percentage of cells from the main layers of the vagina (Superficial,
Maturation Index (M.I.) Intermediate, Parabasal)

39
INTERPRETATION OR READING OF RESULTS OF PAP’S SMEAR:
I. Classify the cells counted according to location especially in vaginal smears as to the following:
A. Mature Superficial cells
B. Intermediate or Parabasal cells
C. Basal cells
Get the percentage (%) of cells of each according to location in order to determine the site and
extent of the injury, infection or cancer.
% = CC (according to location) x 100
TC
II. Report the cytologic smears into Classes or Grades:
Class I – Normal cytology 0-25% cells atypical
Class II – Infection of Inflammatory Cytology 26-49% cells atypical
Class III – Doubtful cytology at least 50 % cells undifferentiated
Class IV – Frankly Malignant Cytology 51-75% cells undifferentiated
Class V – Conclusively Malignant Cytology 76-100% cells undifferentiated
with metastasis

BETHESDA SYSTEM
- name came from the location of the conference that established the system (Bethesda,
Maryland)
 BETHESDA SYSTEM 1988
 BETHESDA SYSTEM 1991
 BETHESDA SYSTEM 2001

CELL BLOCK
– used for the diagnosis of cancer in the body fluids and other tissue juices. This is a method
wherein fluids undergo preparation before actual embedding in paraffin.
PROCESSING METHODS:
1. Agitate the fluid into a test tube and centrifuge for 15-30 minutes.
2. Pour off the supernatant fluid leaving the cell sediments at the bottom of the tube.
3. Pour a small amount of fixative directly to the test tube and let stand for 15-30 minutes or
longer. The fixative will cause the specimen to coagulate into a soft mass.
4. Wrap the recovered sediment in fine gauze. Place in tissue container.
5. The material must be fixed to 6-24 hours, depending on its size.
6. After complete fixation, the material is ready for the routine processing.
7. Proceed as in paraffin embedding.

40
FROZEN SECTION
 technique in which tissue is frozen rapidly at -20°C and sections are cut and stained
 In this way, tissue can be examined microscopically within 5-10 minutes of its
removal from the body
 reduces the time of processing from 18 hours  5 minutes
 Disadvantage: only 8-16 micron section can be cut and finer details of tissue
can’t be examined
 Performed on a machine called CRYOSTAT – a refrigerated cabinet in which a modified
microtome is housed

Situations Where Frozen Sections are Helpful:

1. When rapid diagnosis regarding benign or malignant nature of lesion is required to decide the
extent of surgery while the patient is still on the operation table
2. When study of fat, proteins or antigenic markers is required, as they are destroyed by routine
processing of tissue.

PRECAUTIONS:
1. Frozen section is an emergency.
2. Laboratory workers should always be informed about frozen section before hand.
3. All preparations are completed before arrival of tissue.
4. Cryostat should preferably remain “ON” all the time to maintain its temperature at -20°C.
5. Deal with tissue urgently on arrival in the laboratory.

METHODS OF PREPARING FROZEN SECTIONS

- almost any microtome can be used
- uses Carbon dioxide
Optimum temperature for sectioning:
Cold Knife Procedure Knife: -40 to -60°C
Tissue: -5 50 -10°C
Environment: 0 to -10°C
Cryostat Procedure - uses rotary microtome
(Cold Microtome) Optimum working temperature: -18 to -20°C

COMMONLY USED METHODS OF FREEZING MOUNTING MEDIA FOR CRYOSTAT

1. Liquid Nitrogen SECTIONS
2. Isopentane cooled by liquid nitrogen 1. Water
3. Carbon dioxide gas 2. 20-30% Bovine albumin
4. Aerosol sprays 3. Apathy’s
4. O.C.T. – BEST
STAINING METHODS FOR FROZEN
SECTIONS
1. Hematoxylin and Eosin
2. Thionine
3. Polychrome Methylene Blue
4. Alcoholoic pinacyanol Method

41
AUTOPSY
– process of taking pieces of tissue (representative out sections) from a dead person (Cadaver) for
the purpose of examination or investigation, in order to determine the cause of death or extent of
injury leading to the death of the person.

PRE-REQUISITE OF AUTOPSY PROCEDURES:

1. Written consent or permission from the nearest of kin or relative.
2. Type of autopsy must be specified according to purpose and completeness.
3. Common apparatuses or instruments needed during autopsy procedures as:
a. Bone marrow borer j. Spatula
b. Kwaksaw or electric saw for bones k. Specimen bottles with fixative
c. Forceps – different sizes and types l. Weighing scale
d. Knives m. Surgical needles and sutures
e. Scissors n. Sterile cotton
f. Sterile syringes and needles o. Surgical gloves
g. Culture medium p. Surgical mask (secondary)
h. Clean dry slide q. Pails – for organs or specimens
i. Alcohol lamp
4. Pathologist on duty – performer of the procedure

TYPES OF AUTOPSY DONE IN THE PHILIPPINES

According to Purpose
done in private hospitals for the cause of death of the person especially
Routine Hospital if the cause cannot be determined clinically or the cause of death is
problematic to the clinician
done at NBI or other government institution for the purpose of
Medico-Legal persecution (criminal case)
According to Completeness of the Procedure or Technique
autopsy request involved only the examination of a region or regions of
Partial the body as head only, thorax only or abdomen only.
autopsy request involved the examination of the whole body from head
Complete to foot for complete diagnosis and investigation.
According to the Manner of Incision or Opening the Cadaver
the cadaver is open from both shoulder regions down to the xiphoid
Y-Shaped Incision area, then incised down to the pubis; commonly done in adults
the cadaver is open from the middle of the body from the suprasternal
Straight Cut Incision notch down to the pubis; commonly done in children and infants

PRINCIPAL TECHNIQUES
Rudolf Virchow
VIRCHOW “FATHER OF MODERN PATHOLOGY”
organs are removed one by one

ROKITANSKY Carl Rokitansky in situ dissection

“en bloc”
GHON Anton Ghon altogether
“in toto”
“en masse”
LETULLE Maurice Lettule
in one group or body
42
43