HISTOPATHOLOGIC TECHNIQUES Main Factors Involved in Fixation

METHODS OF FRESH TISSUE EXAMINATION 1. Hydrogen ion concentration

• pH 6-8
1. Teasing or Dissociation
• Tissue in watch glass with isotonic salt 2. Temperature
solution and carefully dissected or separated • Room temperature – routine surgical
specimen
2. Squash Preparation or Crushing • 0-4°C – for electron microscopy and
• Tissue in between two glass slides and histochemistry
forcibly compressed
3. Thickness of section
3. Smear Preparation • 1-2 mm2 for electron microscopy
• Streaking • 2 cm2 for light microscopy
• Spreading
• Pull-apart 4. Osmolality
• Touch preparation or Impression smear • Isotonic solution

4. Frozen section 5. Concentration

• Utilized for rapid diagnosis • Formaldehyde normally 10%
• Recommended for lipids and nervous tissue
• Thickness: 10-15 micra 6. Duration of fixation
• Temperature: -10 to -20°C
Types of Fixative:
Freezing Agents
1. Liquid nitrogen – most common and most According to Composition
rapid 1. Simple Fixative
2. Isopentane 2. Compound Fixative
3. Carbon dioxide gas
4. Aerosol spray According to Action
1. Microanatomical
PROCESSING OF TISSUES • General microscopic study of the tissue
F – ixation structures
D – ehydration 2. Cytological
C – learing • Preserve specific parts and particular microscopic
I – mpregnation elements
E – mbedding a. Nuclear
T – rimming • Contain glacial acetic acid (affinity to
S – ectiong nuclear chromatin
S – taining • pH 4.6 or less
M – ounting • Temperature usually 20-22°C
L - abeling • Ethanol, Methanol, Carnoy’s – common
for nuclei acid
FIXATION AND FIXATIVES b. Cytoplasmic
• First and most critical step • Does not contain glacial acetic acid
• The quality of the section in the slide is as good as the • pH 4.6 and above
quality of the fixed tissue specimen c. Histochemcal
• Primary purpose: preserve the morphological and • Preserve the chemical constituents of the
chemical integrity of the cell cells
• Secondary purpose: harden and protect the tissue ,
thus easier to cut Remember!!
• It prevents DEGENERATION, DECOMPOSITION, • Lipid – Frozen section
PUTREFACTION, DISTORTION of tissue after • Carbohydrates – Alcoholic fixative
removal from body • Protein – Formaldehyde or Neutral buffered formol-
saline

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I. Aldehyde Fixatives a. Zenker’s Fluid
• With glacial acetic acid added before
FORMALDEHYDE use
• Most common, widely used • Recommended for fixing liver,
• 10 % rexommended spleen, connective tissue fibers and
• Produced by the oxidation of methyl alcohol nuclei
• 24 hours usual fixation time • Recommended for Trichrome
• Buffered to 7.0 Staining
• Prolonged storage may induce precipitation – filter b. Zenker-Formol (Helly’s)
or add 10% methanol • Excellent microanatomic fixative for
pituitary glands and bone marrow
10% FORMOL-SALINE • Brown pigments maybe produced if
• Recommended for CNS tissues and post-mortem fixed for more than 24 hrours
tissues for histochemical examination o Reason: Lysis of RBC
o Solution: Immerse in
10% NEUTRAL BUFFERED FORMALIN (PHOSPHATE Saturared Alcoholic Picric
BUFFERED) Acid or NaOH
• Recommended for preservation and storage of c. Heidenhain’s Susa
surgical, post-mortem and research specimen • Recommended for tumor biopsies
• Best fixative for tissues containing iron pigments especially of skin
• Excellent cytological fixative
FORMOL-CORROSIVE (FORMOL-SUBLIMATE) • Mercuric Chloride deposits (black
• Recommended for routine post-mortem tissues ppt.) may be produced – immerse in
alcoholic iodine
ALCOHOLIC FORMALIN (GENDRE’S)
• Coagulates mucus – can be used to fix sputum CHROMATE FIXATIVE
a. Chromic Acid
GLUTARALDEHYDE b. Potassium Dichromate
• 2.5% for small tissue for 2-4 hours at RT c. Regard’s (Muller’s)
• 4% for larger tissue for6-24 hours at RT • Recommended for demonstration of
chromatin, mitochondria, Golgi
Methods of Removing Pigments Left by Formalin d. Orth’s Fluid
1. Kardesewitch’s Method • Recommended for early degenerative
 Specimen is placed in mixture of 70% ethanol process and tissue necrosis
and 30% ammonia-water then wash with
water LEAD FIXATIVE
2. Lillies Method • Recommended for acid mucopolysaccharides
 Specimen is placed in a mixture of acetone,
hydrogen peroxide and ammonia-water then III. Picric Acid Fixatives
wash in 70% alcohol and water • Excellent for glycogen demonstration
3. Picric Acid Method • May impart yellow color to tissue
 Specimen is placed in saturated picric acid o Solution: 70% ethanol followed by 5% sodium
then wash in running water thiosulfate then running water
a. Bouin’s SOlution
II. Metallic Fixatives • Recommended for embryo and pituitary biopsies
b. Brasil’s Alcoholic Picroformol
MERCURIC CHLORIDE
• Most common metallic fixative IV. Glacial Acetic Acid
• Recommended for renal tissue, fibrin, connective • Normally used in conjunction with another fixative
tissues and muscles • Solidifies at 17°C
• Produces black precipitates of mercury – remove by
0.5% iodine solution in 70% ethanol then decolorize
iodine using absolute alcohol

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V. Alcohol 2. Freeze substitution
a. Methyl Alcohol • The frozen tissue is fixed in Rossman’s fluid
• Excellent for dry and wet smears, blood or Osmium tetroxide in 1% acetone for 1-6
smears and bone marrow tissues days at temperature of -60°c to -70°c and
b. Isopropyl Alcohol dehydrated in 70% absolute alcohol
• For touch preparation 3. Fresh frozen tissue sectioning
c. Ethyl Alcohol
d. Carnoy’s Fluid DECALCIFICATION
• Recommended for fixing chromosomes, • Removal of calcium or lime salts from the tissue to
lymph glands and urgent biopsies facilitate and ensure normal cutting of sections
• MOST RAPID FIXATIVE • Calcium maybe removed by:
e. Newcomer’s Fluid a. Acids
b. Chelating agents
VI. Osmium Tetroxide c. Ion exchange resin
a. Flemming’s Solution d. electrophoresis
• Nuclear stain
b. Flemming’s without acetic acid a. Acid Decalcifying Agents
• Cytoplasmic fixative • Most widely used agent

VII. TrichloroaceticAcid 1. Nitric acid – most common and fastest, 5-10%

a. 10% Aqueous Nitric Acid
VIII. Acetone • Recommended for urgent biopsy, needle
• For enzyme studies biopsy
b. Formol-Nitric Acid
IX. Heat Fixation • Yellow color will be imparted – neutralize in
• Usually employed for frozen tissue sections and 5% sodium sulfate
bacteriologic smears c. Perenyi’s fluid
• Recommended for routine purposes
SECONDARY FIXATION d. Phloroglucin-Nitric acid
• Placing and already fixed tissue in a second • Most rapid
fixative
POST-CHROMATIZATION 2. Hydrochloric acid
• Form of secondary fixation which utilizes 2.5- a. Von Ebner’s
3% potassium dichromate (act as mordant) • Recommended for teeth and small pieces of
WASHING-OUT bone
• Removing excess fixative
• Tap water, 50-70% alcohol, Alcoholic iodine 3. Formic acid
• Recommended for autopsy materials, bone
Factors That Affect Fixation Time marrow, cartilage and tissues studied for
1. Size and thickness of tissue research purposes upon the addition of
2. Presence of mucus sodium citrate
3. Presence of fats
4. Presence of blood 4. Trichloroacetic acid
5. Temperature 5. Sulfurous acid
6. Agitation 6. Chromic acid
• Fixative and decalcifying agent
Methods resorted to if chemical fixation is to be avoided
b. Chelating Agents
1. Freeze-drying • Combine with calcium ions to form weakly dissociated
• Preserving tissue by rapid freezing complex to facilitate removal
(quenching) and removing water (dessication) • EDTA (versene) – most common
by a physical process from the still frozen o Will not bind calcium at pH 3.0 below
tissue block without the use of any chemical o EDTA inactivates alkaline phosphatase
fixative activity – add magnesium chloride

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c. Ion Exchange Resin CLEARING
• Hastens decalcification by removing calcium ions from • Removal of alcohol (de-alcoholization) and replaced
formic acid-containing decalcifying solutions with a substance the will dissolve the wax with which
the tissue is to be impregnated
d. Electrophoresis • Must be miscible with paraffin
• Positively charged ions are attracted to negatively • It will make tissue transparent due to their high index of
electrode refraction
• Satisfactory for small bone fragments • Viscosity, temperature will affect the procedure
1. Xylene
Factors Influencing Rate of Decalcification • Most common, most rapid
1. Concentration and volume of decalcifying agent • It turns milky when dehydration is incomplete
2. Temperature 2. Toluene
3. Mechanical Agitation • Substitute to xylene
4. Size of the tissue 3. Benzene
• Recommended for urgent biopsies
Test to Measure Completeness of Decalcification • Carcinogenic or may damage bone marrow resulting to
1. Physical or Mechanical aplastic anemia
2. X-ray 4. Chloroform
3. Chemical Method (Calcium Oxalate Test) • Recommended for tough tissues (skin, decalcified
tissues), nervous tissues, lymph nodes, embryos
DEHYDRATION • Toxic to liver after prolonged inhalation
• Removal of intercellular and extracellular water from 5. Cedarwood oil
the tissue • Use for both paraffin and celloidin sections
• Increasing concentration of alcohol • Recommended for CNS tissue and cytological studies
o Routine – starts with 70% usually ethyl 6. Aniline oil
alcohol • Recommended for clearing embryo and very delicate
o Embryonic tissues – starts with 30% of ethyl specimen
alcohol
o 10:1 ratio of dehydrating agent and tissue IMPREGNATION AND EMBEDDING
Impregnation (infiltration)
1. Alcohol • Removal of clearing agent and replaced by a medium
• Ethyl alcohol – most common, best dehydrating agent that will completely fill all the tissue cavities
• Methyl alcohol – toxic, employed for blood and tissue Embedding (casting or blocking)
films • Process by which the impregnated tissue is placed into
• Butyl alcohol – utilized for plants and micro-techniques a precisely arranged position in a mold containing a
• 37°C – hasten dehydration medium which is then allowed to solidify
Paraffin Wax Impregnation
2. Acetone • Simplest, most common, best embedding medium
• Utilized for urgent biopsies • Melting point: 54-58°C at 20-24°C, 50-54°C at 15-
18°C
3. Diethylene dioxide
• Dehydrating and clearing agent
• Manual Processing
4. Cellosolve (Ethylene glycol monoethyl ether) o At least 4 changes of wax at 15 minutes
interval each
5. Triethyl phosphate • Automatic Processing
o 2-3 changes of wax, decreased processing
6. Tetrahydrofuran time because of constant agitation
o E.g. Autotechnicon
* 4% phenol – softener for hard tissues • Vacuum Embedding
o Negative atmospheric pressure inside the
embedding oven
o Recommended for urgent biopsies – gives the
fastest result

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Factors Affecting Paraffin Wax Impregnation Types of Microtome
1. Nature and size of tissue 1. Rocking Microtome
2. Type of clearing agent used • Cambridge
• Benzene and xylene – easily • Invented by Paldwell Trefall
removed • Simplest
• Chloroform and cedarwood oil – • For serial sections of large blocks of paraffin
difficult to remove embedded tissues
• 10-12 u
Substitute for Paraffin Wax 2. Rotary Microtome
1. Paraplast – melting point of 56-57°C, more elastic and • Minot
resilient • For paraffin embedded tissues
2. Embeddol – melting point of 56-58°C • Most common type
3. Bioloid – recommended for embedding eyes 3. Sliding Microtome
4. Tissue Mat – contains rubber • Adams
5. Ester Wax – melting point of 46-48°C, can be used for • For celloidin embedded tissues
impregnation without prior clearing • 2 types: base-sledge, standard sliding
6. Water Soluble Wax – does not require dehydration and • Most dangerous – because of the exposed
clearing movable knife
4. Freezing Microtome
Celloidin Impregnation • Queckett
• Suitable for specimen with large hollow cavities which • For unembedded frozen section
tends to collapse • E.g. Cryostat – maintained at temperature -5
a. Wet Celloidin Method to -30°C
• Recommended for bones, teeth, large brain • 4u
sections and whole organs • Most commonly used for rapid preparation
b. Dry Celloidin Method 5. Ultrathin Microtome
• Preferred for whole eye section • For cutting sections for electron microscopy
• 0.5 u
Gelatin Impregnation • Fixative – osmium tetroxide
• Rarely used except when dehydration is to be avoided • Plastic embedding medium
• For tissues subjected to histochemical and enzyme
studies Microtome Knives
• Embedding medium for delicate specimen and frozen 1. Plane-Concave Knife
sections • One side is flat, the other is concave
• Less concave – celloidin embedded tissue
TRIMMING AND CUTTING/SECTIONING using sliding microtome
• More concave – paraffin embedded tissue
Types of Blocking –out Molds using rotary and rocking microtome
1. Leuckhart’s Embedding Mold 2. Biconcave Knife
• 2 L-shaped trip of heavy brass or metal • Both sides concave
2. Compound Embedding Mold • For paraffin embedded sections using rotary
• Series of interlocking plates resting on a microtome
metal base 3. Plane-Wedge Knife
3. Plastic Embedding Rings and Base Mold • Both sides straight
4. Disposable Embedding Mold • For frozen sections, extremely hard and tough
specimen in paraffin blocks using base-
ORIENTATION – tissue is arranged in precise position in the sledge microtome
mold during embedding, on the microtome before cutting, and
on the slide before staining BEVEL ANGLE – angle formed between the cutting edge,
normally 27°to 32°
MICROTOMY WEDGE ANGLE – angle formed by the sides of the wedge
Three essential parts knives, normally 14° to 15°
1. Block holder CLEARANCE ANGLE – angle formed between the cutting facet
2. Knife carrier and knife presenting to the block and the surface of the block, normally 5°
3. Pawl, ratchet feed wheel and adjust to 15°

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HONING 4. Regressive Staining
• Removal of gross nicks on knife edge to remove • The tissue is first overstained and decolorized
blemishes and grinding the cutting edge of the knife on until the desired intensity of color is obtained
a stone 5. Metachromatic Staining
• HEEL TO TOE (Edge first) • Entails the use of dyes which differentiate
• Purpose: to remove irregularities from the knife particular substances by staining them with
the color that is different from that of the stain
Hones itself
a. Belgium Yellow – gives the best result 6. Counterstaining
b. Arkansas • Application of a different color or stain to
c. Fine Carborundum provide contrast and background
d. Plate-glass hone
e. Machine hone 7. Vital Staining
• Selective staining of living cell constituents
STROPPING
• The burr formed during honing is removed and a. Intravital Stain
thecutting edge of the knife is polished • Staining of living cells is done by
• TOE TO HEEL (Edge last) injecting the dye into any parts of the
• Purpose: polish and sharpen the cutting edge animalbody
• Common dyes: lithium, carmine,
Common Lubricant Used for Honing india ink
1. Mineral oil b. Supravital Stain
2. Clove oil • Used to stain living cells immediately
3. Xylene after removal from the living body
4. Liquid paraffin
5. Soapy water Neutral Red – best vital dye
Janus Green – for mitochondria
STAINING
1. Histological Staining Hematoxylin and Eosin Technique
• Tissue constituents are demonstrated in • Most common method for tissue examination
sections by direct interaction with dye • Fixative – except Osmic Acid because it inhibit
• Also called as Micro-anatomical staining hematoxylin
2. Histochemical Staining • Harris Hematoxylin – primary stain
• Various constituents of the tissues are studied • Acid alcohol – differentiator
through chemical reactions • Ammonia water – blueing agent
3. Immunohistochemical Staining • Eosin - counterstain
• Combination of immunologic and
histochemical techniques STAINS AND STAINING SOLUTIONS

Methods of Staining: 1. Natural Dyes

1. Direct Staining A. Hematoxylin

• Giving color to the sections using aqueous or
alcoholic dye solution
2. Indirect Staining
• The action of the dye is intensified by adding
another agent
• MORDANT – link or bridge between tissue
and dye
• ACCENTUATOR – accelerates/hastens the
speed of the staining reaction
3. Progressive Staining
• Tissue elements are stained in definite
sequence, stain is applied until the desired
intensity of color is attained
• Derived from Mexican tree Hematoxylin
campechianum
• Hematin – active coloring agent formed by the
oxidation of hematoxylin (ripening)
• Natural ripening – exposing substance to air
or sunlight
• Artificial ripening – uses substance that will
accelerate the process
o Hydrogen peroxide, mercuric oxide,
potassium permanganate, sodium
perborate, sodium iodate
• Ripened hematoxylin + alum, iron, chromium
or copper salts

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B. Cochineal Dye a.4. Mayer’s
• Dye extracted from the female cochineal bug o Ripening agent – sodium iodate
(Coccus cati) o Regressive and progressive staining
• Treated with alum to produce dye – o Cytoplasmic glycogen
CARMINE
• Carmine + picric acid = picrocarmine b. Iron Hematoxylin
o For neuropathological studies
• Carmine + aluminum chloride = Best Carmine b.1. Weigert’s
o Glycogen demonstration o Mordant – ferric ammonium chloride
C. Orcein o Standard iron hematoxylin in the
• Vegetable dye extracted from lichens which laboratory especially when
are normally colorless – treated with ammonia demonstrating muscle fibers and
and exposed to air to produce blue or violet connective tissue
color b.2. Heidenhain’s
o Mordant – ferric ammonium sulfate
2. Synthetic Dyes
• Known as Coal Tar Dyes EOSIN
• Chromophores – substance capable of • Routinely used as counterstain
producing color (chromogen)
• Chromogen + auxochrome = dye a. Yellowish (Eosin Y)
o Most commonly used
A. Acid Dyes b. Bluish (Eosin B, Erythrosin B)
• Active coloring substance is found in the acid o Deeper red color
component and the inactive base c. Ethyl eosin (Eosin S, eosin-alcohol soluble)
• E.g. picric acid
B. Basic Dyes ADHESIVE AND MOUNTING MEDIA
• Active coloring substance is found in the
basic component ADHESIVE
• E.g. methylene blue • Essential for methods that require exposure of sections
C. Neutral Dyes to acids and alkalis
• Capable of staining cytoplasm and nucleus
• E.g. romanowsky, giemsa, irishman 1. Mayer’s Egg Albumin
o Most common
Common Staining Solutions: o Glycerin – clearing agent
o Thymol crystal – preservative
HEMATOXYLIN 2. Dried Albumin
• Most common for routine histology 3. 1% Gelatin
• Mordant – alum,iron 4. Gelatin-Formaldehyde mixture
5. Starch Paste
a. Aluminum Hematoxylin 6. Plasma
• Recommended for progressive 7. Poly-L-Lysine
staining
a.1. Ehrlich’s MOUNTING MEDIUM
o Ripening agent – sodium iodate • Added to slide before the application of coverslip for
o Stabilizer – glycerine protection
a.2. Harris
o Ripening agent – mercuric chloride A. Aqueous Mounting Media
o Stabilizer – 4% glacial acetic acid 1. Water – low RI, evaporates quickly, temporary
o Widely used in routine nuclear mounting
staining, exfoliative cytology 2. Glycerine – high RI 1.46
a.3. Cole’s 3. Farrant’s Medium - RI 1.4, gum arabic dissolved in
o Ripening agent – alcoholic iodine water
o Used in sequence with celestine blue 4. Apathy’s Medium – RI 1.52
5. Brun’s Fluid – frozen sections

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B. Resinous Mounting Media STAINS OF FATS
1. Canada Balsam – RI 1.524, extracted from tree, Abus
balmasea, common Sudan Black B – most sensitive
2. DPX – RI 1.532, recommended for small tissue Result: Lipids – blue black
sections Nuclei – red
3. XAM – RI 1.52 Sudan IV (Scharlach R)
4. Clarite – RI 1.544 Result: Lipids (mainly triglycerides) – red
Nuclei – blue or black
STAINS Oil Red O in Dextrin
Result: Fats – brilliant red
STAINS OF CARBOHYDRATES Nuclei – blue
Osmic Acid
Periodic Acid Schiff (PAS) Result: Fats – black
Result: PAS (+) – red or magenta Nuclei – yellow orange
Nuclei – blue
PAS with Diastase – method of choice for glycogen STAINS OF PROTEIN, ENZYMES AND NUCLEIC ACID
demonstration
Result: Nuclei – blue-black Alkaline Fast-Green
Glycogen – red Result: Histones and Protamines – green
Control – only the nuclei are stained Gomori calcium
Best Carmine – for glycogen demonstration Result: Alkaline phosphatase activity – brownish black
Result: Nuclei – blue or grayish-blue (Erlich’s Nuclei – green
hematoxylin as counterstain) Feulgen – for nuclear DNA
Glycogen – bright red granules Result: DNA – red-purple
Mucin, fibrin – weak red Cytoplasm – green
Langhan’s Iodine – oldest stain (obsolete) Methyl Green-Pyronin – for RNA and DNA
Result: Glycogen – mahogany brown Result: DNA – green or blue-green
Tissue constituents - yellow RNA – rose red
Fresh frozen Azure A -Metachromatic staining for Plasma cell cytoplasm – purple
glycosaminoglycans
Result: Glycosaminoglycans – red-purple STAINS OF CONNECTIVE TISSUE
Tissue background - blue
Metachromatic Toluidine Blue staining For Collagen:
Result: Glycosaminoglycans – red-purple
Tissue background – blue Gomori’s Silver Impregnation Stain – for reticulin
Alcian Blue technique Result: Reticulin fibers – black
Result: Acid mucin – blue Van Gieson’s Stain – for collagen
Nuclei – red Result: Collagen (fibrous connective tissue) – pink,
Combined Alcian Blue-PAS technique for acid and neutral deep red
mucin Nuclei – brownish black
Result: Acid mucin – blue Muscle, cytoplasm, RBC and fibrin – yellow
Neutral mucin – magenta Masson’s Trichrome Stain- for collagen
Nuclei – pale blue Result: Collagen and mucus – blue
Mucicarmine stain Muscle, RBC and Keratin – red
Result: Mucins – red Nuclei – blue-black
Nuclei – blue
Background – unstained Other stains for collagen: Mallory’s Aniline Blue
Hale’s Dialyzed Iron Technique Azocarmine
Result: Acid mucin – dark blue Krajian’s Aniline Blue
Nuclei – red
Fluorescent Acridine Orange For Amyloid :
Result: Acid mucopolysaccharides – black
Fungi – greenish red fluorescent o Gram’s iodine
Background – reddish orange fluorescent o Congo Red
o Methy violet-Crystal violet

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For Elastic Fibers : For Astrocytes :
• Cajal’s Gold Sublimate
o Weigert’s Elastic Tissue Stain • Modified PTAH
Result: Elastic fiber – dark blue, blue black • Modified Holzer’s Method
o Verhoeff’s
Result: Elastic fibers – black STAINS OF TISSUE PIGMENTS AND DEPOSITS
Nuclei – gray to black For Hemosiderin
Collagen – red • Perl’s Prussian Blue
Cytoplasm – yellow • Gomori’s Prussian Blue
o Taenzer-Unna Orcein • Turnbull’s Blue Reaction
o Gomori’s Aldehyde-Fuschin
o Krajian’s For Hemoglobin
• Benzidine-Nitroprusside Stain
For Fibrin:
Mallory’s PTAH For Bile Pigments and Hematoidin
• Modified Fouchet’s Technique
• Gmelin’s Technique
STAINS OF MUSCLES AND BONES • Stein’s Iodine

o Modified Gomori’s Trichrome Stain For Lipofuschin

Result: Muscle fiber – red • Gomori’s Aldehyde Fucshin Technique
Collagen – green • Mallory’s Fuschin Stain
Nuclei – blue to black
o Mallory’s PTAH For Melanin
o Heidenhain’s Iron Hematoxylin • Masson Fontana Technique – also for Argentaffin
o Lissamine Fast Red – tartrazine method for muscles granules
and bones
For Calcium
For Bones: • Von Kossa’s Silver Nitrate Method
Schmorl’s Picro-Thionin Method
For Copper
STAINS OF BONE MARROW AND BLOOD ELEMENTS • Lindquist Modified Rhodanine Technique

o Rapid Toluidine-Eosin Stain STAINS OF MICROORGANISMS

o Romanowsky For Bacteria
o Wright’s stain • Gram’s Method
o Giemsa stain • Brown and Brenn – for Nocardia and
o Peroxidase Reaction – for myeloid cells Actinomyces
• Ziehl Neelsen – for Mycobacterium
STAINS OF CNS • Wade-Fite Technique – M. leprae, Nocardia
o Bielschowsky’s Technique • Auramine-Rhodamine – Mycobacteria
• For neurons, axons and neurofibrils • Toluidine Blue and Cresyl Violet Acetate – H.
o Bodian’s Stain pylori
• Nerve fibers and nerve endings • Dieterle Method – L. pneumophilia
o Siever-Munger Technique • Levaditi’s – Spirochete
• Neural tissues • Modified Steiner and Steiner – Spirochete
o Cresyl Fast Violet • Warthin-Starry – Spirochete
• Nissl bodies For Fungi
Grocott Methamine Silver
For Myelin Sheath: For Virus
• Weigert-Pal Technique • Lendrum’s Phloxine-Tartrazine Method – viral
• Kluver and Barrera Luxol Fast Blue Stain inclusion
• Weil’s Method • Orcein Method – HBsAg
For Protozoa
Giemsa

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