Routine and Special Tissue

Processing in Histopathology
MODULE 19
 HISTOPATHOLOGIC TECHNIQUES
I. Quality Assurance and Documentation
II. Fresh tissue Examination
III. Preserve Tissue examination
 I. Quality Assurance and
Documentation
A. Histopath Repots
1. Surgical pathology
2. Cytopathology report
3. Autopsy report
Questions How many copies prepared per report?
Doctor
Patient
File
Note: Orfiginal results- will be given to the patient
B. Signatories
1. Request forms= patient’ doctor
2. Result forms= pathologist
C. Specimen handling
1. FIX FIRST- because we want to see the cell in
its live form
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
24 hours= an autopsy to be most informative
and helpful.
E. Storage of specimen, tissue blocks and slides
Specimen= 1 month to 1 year
Tissue block- 1 month to 1 year
Slides- indefinite
Suggested Guidelines for records and
specimen retention
Record retention

requisitions 2 years

Quality control 2 years

Instrumental maintenance 2 years

Blood bank/recipient indefinitely

records

Blood bank employee 10 years

signatures/initials

Blood bank quality control 5 years

Reports Retention

Clinical pathology 2 years

laboratory reports

Autopsy forensic reports indefinitely

Surgical pathology (and 10 years

bone marrow) reports

Cytogenetic reports 20 years

Specimens Retention
Serum/other body fluids 48 hours
Blood smears- routine 7 days
Pathology/bone marrow 10 years
slides
Pathology blocks 10 years
Microbiology smears 7 days
Blood bank 7 days post-transfusion
donor/recipient specimens
Cytogenetics slides 3 years
Cytogenetics diagnostic 20 years
images
 II. Fresh tissue Examination
Methods:
1. Teasing/Dissociation tissue spx-> watch glass(
isotonic solution)
Microscope: phase contrast/Bright field
2. Crushing/ squash preparation
>1 mm-> sandwiched between 2 slides-> Vital
stain (living state)
3. Smear preparation= Cellular materials are spread
lightly over a slide by means of a wire
loop/applicator stick/another slide
Techniques:
Streaking- applicator stick, platinum loop,
Spreading- sputum, aspirates, mucoid secretion,
bronchial
Pull-apart- blood/sputum
Touch prep/Impression
4. Frozen section= Normally used when a rapid diagnosis of
tissue is required

Applications:
1. Rapid pathologic diagnosis during surgery
2. Enzyme histochemistry
3. Demonstration of soluble substances such as lipids and
carbohydrates
4. Immunofluorescent and immunocytochemical staining
5. Some specialized silver stains, particularly neuropathology
• Freezing of fresh unfixed tissue
- Best frozen section (if tissue frozen rapidly)

• Freezing of fixed tissue

- To localize hydrolytic enzymes and other antigen

Fixative: Formal (formol) calcium (4C for 18 hours)

 2 methods of preparing Frozen
sections:
1. Cold Knife procedure- almost any microtome
can be used , uses carbon dioxide
Optimum condition for sectioning
Knife= -40 to – 60 C
Tissue= 5 to -10 C
Environment= 0 to -10 C
2. Cryostat procedure (Cold Microtome)
-18 to -20 C
CRYOSTAT- a refrigerated cabinet in which a
modified microtome is housed
§ All the controls to the microtome are operated
from the outside the cabinet
§ Presently, the rotary microtome is the type of
choice
• Mounting media for cryostat sections:
Water
20-30% bovine albumin
Von Apathy’s gum syrup
O.C.T=best (synthetic water-soluble glycols and
resins
• Commonly used method of freezing
Liquid nitrogen, isopentane cooled by N, CO2 gas,
Aerosol sprays

Stains
H and E – used for progressive and regressive
Thionine
Polychrome methylene blue
Alcohol pinacyanol method
• Progressive- frozen tissue
• Regressive- routine paraffin section
 III Preserved Tissue examination
Fixation
Dehydration
Clearing/Dealcoholization
Impregnation/Infiltration
Embedding/casting/Blocking
Trimming
Sectioning/Cutting/Microtomy
Staining
Mounting
Labeling- used Diamond pencil
 Fixation
• Preserving fresh tissue for examination
• First and most critical step in histotechnology
• Primary aim: to preserve the morphologic and
chemical integrity of the cell in as life-like manner
as possible
• Secondary aim: to harden and protect the tissue
from the trauma of further handling
• Fixatives have the property of forming cross-links
between proteins
• Stabilization of proteins
Practical consideration of Fixation:
1. Speed
2. Penetration
3. Volume
4. Duration of fixation
Two mechanism involved in fixation:
1. Additive fixation- whereby chemical constituent
of the fixative is taken in and becomes part of
the tissue
2. Non-additive fixation- whereby the fixing agent
is NOT taken in, but changes in tissue
composition and stabilizes the tissue by
removing the bound water attached to hydrogen
bonds of certain groups within the protein
molecule.
Main factors involved in fixation:
1. Hydrogen ion concentration (pH)
Satisfactory ph= pH 6.8
2. Temperature
Surgical spx.- rm temp.
Electron microscopy and histochem- 0-4 C
3. Thickness of the section
4. Osmolality
5. Concentration
6. Duration of fixation
 Types of fixatives:
• According to Composition
a. Simple fixative- made up of only 1 component
b. Compound fixative- 2 or more fixatives
• According to Action
A. Microanatomical- for general microscopic study
of tissue structure
B. Cytologic fixatives- specific parts and particular
microscopic elements of the cell itself
 A. Microanatomical
• Ex. 10% formol solution
• 10% BNF
• Heidenhain’s SuSa
• Bouin’s
• Formol sublimate
• Zenker’s solution
• Zenker- formol
• Brasil’s
 B. Cytological Fixatives
1. Nuclear fixatives- with Glacial acetic acid (pH 4.6
or less)
Fleming’s
Carnoy’s
Bouin’s
New Comer’s
Heidenhain Susa
*GAC- fixes the nucleoproteins and destroys
mitochondria and Golgi bodies
2. Cytoplasmic Fixatives- no Glacial acetic acid
Flemming’s fluid w/o GAA
Helly’s fluid
Formalin w/o post chroming
Regaud’s (moller’s)
Orth’s
3. Histochemical fixatives
10% formol saline- new comer’s fluid
ABS. ETOH
Acetone
 A. Aldehyde Fixatives
1. Formaldehye- conc. Solutions of Formaldehdye must never be
neutralized- “VIOLENT EXPLOSIONS”
2. 2. 10% Formol-Saline= CNS tissues
3. 10% Buffered Neutral Formalin (BNF)- best fixative for tissue
containing iron pigments
Best general tissue fixative pH=7
4. Formol-Corrosive(formol-Sublimate)= w/mercuric chloride
5. Glutaraldehyde- preserve plasma protein better- for electron
microscopy
6. Formol-calcium- for preservation of lipids
7. karnovsky’s paraformaldehyde glutaraldehyde solution= for electron
cytochem
8. acrolein- for electron cytochemistry
Formaldehyde
10% working solution
37 to 40 %- stock solution
 B. Metallic Fixatives
1. Mercuric chloride- most common metallic
fixative
- May produce black granular deposits on
tissues (except SuSa) Sublimate-HgCl2 Saure-
acid
2. Chromate- preserve chromatin tissue
3. Lead fixatives- generally for acid
mucopolysaccharides
 1. Mercuric chloride
1. Zenker’s fluid (with glacial acetic acid)
2. Zenker-Formol (Helly’s solution)
3. Heidenhains SuSa- for tumor biopsies of the skin.
4. Schaudinn’s fluid-smears of alcohol stool
5. Ohlmacher’s fluid
6. Carnoy-Lebrun fluid
7. B-5 fixative-Bone marrow biopsies/commercial
solution
* Solution of mecrcuric chloride corrode all metals except
the nickel alloy called MONEL.
 2. Chromate
1. Chromic acid
2. Potassium dicromate
3. Regaud’s (moller’s)- Molliflex (24-48 hours)
long fixation
4. Orth’s fluid- for Ricketssia and other bacteria
- For study of early degenerative process
 3. Lead fixatives
1. Alcian Blue
For Umbilical cors/wharton’s jelly
Produce white precipitate
Removed by alcohol drop by drop
 C. Picrate Fixatives
• Highly explosive when dry
• Will produce excessive yellow staining in tissue
• Picrates are formed upon protein: precipitates
are soluble in water; hence tissues must be first
rendered insoluble by direct immersion in 70 %
ETOH
Tissue-> 70% ETOH->5% sodium thiosulfate->wash
in running water
• Picrates fixative MUST NEVER be washed in water
before dehydration
a. Bouin’s solution- for fixation of embryos-
contains picric acid
b. Brasil’s alcoholic picroformol- less messy than
Bouin’s
c. Gendre’s- alcoholic-formalin- sputum
 D. Glacial Acetic acid
• Fixes nucleoprotein
• Destroy mitochondria and Golgi bodies
• Causes tissue to swell (used to counteract the
shrinkage produced by some fixatives)
• Never used in cytoplasmic specimen
 E. Alcoholic Fixatives
Disadvantage: Polarization- glycoprotein granules
move towards the poles or ends of cells
a. Methanol- blood smears and BM tissues
b. Ethanol
c. Carnoy’s fluid- MOST RAPID FIXATIVE (fixation
time: 1 to 3 hours) alcoholic chloroform acetic
acid- chromosomes
d. Alcoholic formalin (Gendre’s fixative)- useful in
preserving sputum
e. Newcomer’s fluid
 F. Osmium Tetroxide fixatives
• For electron microscopy
• Should be kept in a dark colored, chemically clean bottle to
prevent evaporation and reduction by sunlight or organic
matter.
• Inhibits hematoxylin and makes counterstaining dificulty
Produces black precipitate (osmic oxide)
Prevention: add saturated aqueous HgCl2
Remedy: dissolve in cold water
Precaution: may cause conjuctivitis / blindness
a. Flemming’s solution
b. Flemming’s without acetic acid to improve cytoplasmic
details
G. Tricholoracetic acid
H. Acetone- used for Dx. Of rabies
I. Heat fixation
- Direct flaming fixation
- Microwave fixation (opt. tem. 45-55C)
Under heating –poor sectioning
Over heating- above 65C- vacuolation,
ovestained cytoplasm-pyknotic nuclei
 Fixatives for EM
• Glutaraldehyde- 4 to 6 %
• Platinic chloride (PtCl3)
• Platinic chloride-formalin (Zamboni’s fixative)
• Gold chloride (AuCl)
• Osmium tetroxide
• 10% BNF
Phosphotungstic acid- first general stain used for
EM
Uranyl acetate- Best stain for EM
Lead
 Factors that affect fixation
Retarded by:
1. Size and thickness of tissue
2. Mucus
3. Fat
4. Blood
5. Cold temperature
Enhance by:
1. Size and thickness of tissue
2. Agitation
3. Moderate heat (37-56C)
 Decalcification
• More concentrated acid solutions decalcify bone
more rapidly but more harmful to the tissue
• High concentrations and greater amount of fluid
will increase the speed of the process
• The recommended ratio of fluid to tissue for
decalcification is 20 to 1. osmium tetroxide= 5-
10:1
• Heat will serve to hasten decalcification BUT it
also increases the damaging effects on tissues.
• At 37C=impaired nuclear staining of Van Gieson’s
stain for collagen fibers
• At 55C= tissue will undergo complete digestion
within 24-48 hours
• Optimum temperature- rm temperature (18-30 C)
• The ideal time required for decalcifying tissue is
24-48 hours
• Dense bone tissues usually require up to 14 days
or longer to complete the process
 Decalcifying agents
• Acids
• Chelating agents
• Ion exchange resins
• Electronic ionization
** chelating agents- most common is EDTA salts-
Versene- commercial name)
 Some acid decalcifying agents
• Nitric acid- most common
Examples: Perenyi,s fluid
Both tissue softener and decalcifying agent
Phloroglucin-Nitric acid
Most rapid decal. Agent
• Formic acid- both fixative and decalcifying agent
5% formic acid is considered to be the BEST GENERAL DECALCIFYING
AGENT
Formic acid is recommended for small pieces of bones and teeth.
• HCL
• Von Ebner’s fluid- recommended for teeth and small pieces of
bones
• TRC
• Sulfurous acid
• Chromic acid
• Citric acid
 Extent of decalcification
3 ways to measure extent of decalcification
1. Physical/mechanical- innacurate
2. Xray or Radiologic method- very expensive-
most reliable, most ideal.
KODAK X-OMAT or F axitron- xray paper
3. Chemical method- (calcium oxalate test)-
simple, reliable, recommended for routine
purposes
 Tissue softener
• For unduly hard tissue that may damage the
microtome knives
1. 4% aq. Phenol
2. Molliflex
3. 2% HCK
4. 1% HCL in 70% alcohol
5. Perenyi’s fluid
 Dehydration
Aim: to remove fixative and water from the tissue and
replacing them with dehydrating fluid in preparation
for impregnation
Dehydrating fluid are generally used in increasing
strengths- ascending grade
Increasing strengths- all the aqueous tissue fluid are
removed but with little disruption to the tissue due to
diffusion currents
30% ETOH- delicate specimen
65%->70%->75%->85%->95%absolute alcohol
General Rule: amount in each stage; should not be less
than 10 x the volume of the tissue
 Commonly used dehydrating agents:
1. Alcohol
2. Acetone- BOTH fixative and dehydrating agent
3. Dioxane (diethylene dioxide)-BOTh dehydratinga nd
clearing agent
4. Cellosolve (ethylene glycol monoethyl ether)
5. THF (tetra hyrofura)- BOTH dehydrating and clearing agent
6. Triethyl phosphate
Additives to dehyrating agents
1. 4% phenol+95% ETOH baths- phenol acts as tissue
softener
2. Anhydrous copper sulfate (white)- BOTH dehyrating and
used as indicator
 Alcohol- most common
a. ethanol- for routine dehydration of tissues
BEST DEHYDRATING AGENT
b. methanol- for blood and tissue films
c. Butyl alcohol-for plants and animals
microtechniques
d. Industrial methylated spirit (denatured
alcohol)= ethanol+small amount of methanol
e. Isoprophyl alcohol
Indicators of complete dehydration
1. Anh. CuSo4( white-blue if presence of water)
2. Xylene (turned milky)
 Clearing
• Dealcoholization
• Process of replacing the dehydrated fluid with
a fluid that is miscible with BOTH the
dehydrating fluid and the
impregnating/embedding medium
Clearing agent suitable for routine use
1. xylene- most common clearing agent
2. Toluene- subst. of xylene
3. Chloroform- toxic to liver after prolonged inhalation and it does
not make the tissue transparent
4. Methyl benzoate and methyl salicylate
5. Cedarwood oil and clove oil- smooth muscles, CNS
6. Citrus fruits oils
7. Trichlorethane petrol
8. Benzene- carcinogenic, cause aplastic anemai
9. Aniline- partial dehydrant- embryo, insects, very delicate
specimen
10. Carbon tetrachloride
* Gum syrup and glycerin- makes tissue transparent but do not
dealcoholized
 Impregnation
• Also known as infiltration
• 25 times the tissue volume
• After clearing, tissue is submerged in 2 or more changes of
melted paraffin wax
• Process of replacing the clearing agent with infiltrating
medium
• The medium used to infiltrate the tissue is usually the same
medium used for embedding.
• Four types of tissue impregnation and embedding media
a. Paraffin wax c. Gelatin
b. Celloidin (Cellodion) d. Plastic
Filling in tissue cavities/holes/spave by using melted wax
 Paraffin
• Butschlii- first used the paraffin wax
• Simples/most common and BEST
infiltrating/embedding medium
• Is NOT recommended for fatty tissues- frozen
section
• Temperature of paraffin oven- 55 to 60C-paraffin
oven must be maintained at a temperature 2-5C
above the MP of the paraffin wax.
• To remove excess water- heat the wax to 100-
105C
 Substitute for paraffin wax- xylene
based
1. paraplast- MP= 56-57 C
- Mixture of highly purified paraffin and synthetic
plastic polymers
- More elastic and resilient than paraffin
- For large dense tissue blocks such as bones and brain
2. Embeddol- MP- 56-58C
- Less brittle and less compressible than paraplast
3. Bioloid- recommended for embedding eyes MP-56-58
C
4. Tissue mat- a product of paraffin, containing rubber,
with the same property as paraplast
5. Ester wax- MP-46-48 C
Harder than paraffin
Not soluble in water
Soluble in 95% EOH and other clearing agents
Can be used for impregnation without prior clearing of tissue
(can omit clearing)
6. Water soluble waxes- MP 38-42 C or 45-56C
Mostly polyethylene glycols
Most commonly used: Carbowax
Soluble and micible with water (hence does not require
dehydration and clearing of the tissue)
Suitable for many enzyme histochemical studies
 Celloidin
• Purified form of nitrocelluloase
- Suitable for specimens with large hollow cavities and dense
tissues (bone and teeth), large tissue section of the whole
embryo.
2 methods for celloidin impregnation
1. Wet- recommended for bones, teeth, large brains and
whole organ
2. Dry- preferred for processing whole eye sections.
L.V.N. (low viscosity Nitrocellulose) another form of celloidin
- Soluble in equal concentration of ether and alcohol with
lower viscosity, allowing it to be used in higher conc. And
still penetrate tissues rapidly.
 Gelatin
• Rarely used when dehydration is to be avoided
• Used when tissues are for histochem. and
enzyme studies
• Embedding medium for delicate specimens and
frozen sections because it prevents fragmentation
of tough and friable tissues when frozen sections
are cut
• Water soluble
• Does not require dehydration and clearing
 Plastic/Resin
• Classified into epoxy, polyester, acrylic
• Epoxy brand
1. Bisphenol A (araldite)
2. Glycerol (Epon)
3. Cyclohexene dioxide (spurr)
 Embedding
• 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.
ORIENTATION- process by which tissue is arranged in precise
positions in the mold during embedding, on the microtome
before cutting, and on the slide before staining.
Temperature of melted paraffin used for embedding = 5-10C
above its melting point.
To solidify embedded tissue- cooled rapidly in a ref (-5 C) or
immersed in cold water
The surface of the section to be cut should be placed parallel
to the bottom of the mold in which it is oriented
 Trimming
• process of removing excess wax after
embedding
• Excess wax is cut off from the block to expose
the tissue surface in preparation for actual
cutting
• Knife/blasé may be used
• Four sided prism/ truncated pyramid
• At least 2 mm of wax should surround the
tissue blocked.
 Sectioning
• CUTTING or MICROTOMY
• The process by which a processed tissue is cut
into uniformly thin slices (sections) to facilitate
studies under the microscope
• 4-5u- routine histologic procedure (rotary
microtome) Binconcave knife
• 10-15 u- frozen sections (cryostat)unfixed
• 0.5 u- electron microscopy
 Kinds of Microtomes
1. Rocking Microtome (Cambridge Rocking mucrotome)-
simplest among the microtomes
Disadvantage: difficult in re orienting the block
Inventor: Paldwell Trefall-1881
2. Rotary/Minot microtome- inventor- Minot 1885-1886
Most common type used today especially for paraffim-
embedded tissue
3. Sliding microtome- MOST DANGEROUS type due to
movable expose knife
Inventor: Adams 1789
 Types of sliding Microtome
a. Base-sledge
- For all forms of media
- Block holder: moving
- Knife: stationary
b. Standard Sliding microtome
Block: stationary
Knife: moving
4. Rotary rocking microtome
5. Vibrotome- used for unfixed, unfrozen specimen
sectioning for enzyme demonstrations
Disadvantage: sections are liable to disintegrate
6. Ultrathin Microtome- for cutting sections for Elec.
Microscopy
Uses DIAMOND knives or broken plate glass
Specimen is small, fixed in osmium tetroxide, embedded
in plastic
7. Freezing Microtome- invented by Queckett in 1848
• Clearance angle- 0-15 degrees
• Bevel angle- 27-32 degrees
• For routine work: 76x25mm slides that are
1.0-1.2 mm thick are usually preferred
because they do not break easily.
• Water bath 45-50C
• Approximately 6-10C lower than the MP
 STAINING
• Natural Dyes- obtain from plants and animals
1. Hematoxylin
2. Cochineal dyes- extracted from female cochineal
bug-coccus cati
3. Orcein- Hepatitis B surface Ag
4. Saffron
• Synthetic dyes- “Coal tar dyes”
• Derived from benzene and collectively known as
“aniline dye”
 Definition if terms
1. Chromophores: ( Gr. “color-bearers”)- group
of benzene ring with confers color
2. Auxochromes: (Gr.”increasers”) dyeing
properties
3. Dye modifiers=substances that affect either
the color or properties of a dye
4. Lake- the resultant complex of stain-
mordant-tissue
 Chromophores
• Quinoid ring – basic fuchsin
• Azo groups – congo red
• Xanthene-Eosin
• Quinone-imine group
Oxazin- cresyl fast violet
Thiazins- Toluidine blue
 Auxochromes
• Cationic auxochrome: amino group
• Anionic auxochrome: Hydroxyl and Carboxyl
groups
 Dye modifiers (attached on benzene
ring)
• Ethyl groups
• Methyl groups
• Suphonic Acid
 Dye-to Tissue-Mechanisms
• Tissue will bind dyes by one of the following mechanisms
1. Electrostatic- majority of tissue-dye reactions
Ex. Neutral red and Light Green
2. Hydrogen bonding- Congo red, Carmine, Weigert-type
resorcinol dye
3. Van der waals Forces- Alum Hematoxylin solutions
4. Physical Staining- sudan dyes
SUDANOPHILIA- property of tissue to be stained with fat or
oil-soluble dyes, regardless of the type of dye used, due to
the essential lipid nature.
5. Natural Affinity- Janus green
 Methods of staining
I. According to presence of mordant
1. Direct- w/o mordant, methylene blue and eosin.
A staining process by which sections are stained
with simple aqueous alcoholic solutions of dye
2. Indirect- w/mordant
II. According to the presence of Differentiator
1. Progressive staining- w/o decolorizer
2. Regressive- w/ decolorizer
III. Counterstaining- application of different color or stain
to prove counter stain or background
IV. According to resultant color
1. orthochromatic- color of dye =color of the same
tissue
2. Metachromatic- color of dye =/ color of the tissue
V. Metallic impregnation- specific tissue elements are
demonstrated by color less solutions of metallic salts
Metallic salts-> reduced by tissue->black deposit on the
surface.
VI.
1. Vital staining (live)
Injection of dye to the animal body
Ex. Lithium, carmine, india ink
2. Supravital staining
The cells is applied onto cells after removal form the animal body.
Neutral red- best vital dye
Janus green –mitochondria
Trypan blue
Nile blue
Thionine
Toluidine blue
 H and E staining
Hematoxylin
- A natural dye derived from extraction from
the heartwood of the mexican tree known as
“Hematoxylin Campechianum” or
“Hematoxylon Campechianum”- Jamaica
- Waldeyer- first person to use hematoxylin in
Histology
 Ripening/Oxidation
• May be done by exposing the substrate to air or
sunlight (SLOW)
• May be done by adding oxidizing agents such as:
- H2O2
- HgCl2- ripening agent of Harris hematoxylin
- KMNO4
- Sodium perborate
- Sodium iodate- ripening agent of Erlich’s
hematoxylin
 A. Alum Hematoxylins
• Mordant alum
• Mordant: Potash alum (potassium aluminum
sulfate or simply alum)
• Produce good nuclear stain (red)
• Examples
- Erlich’s- slowely Ripened/Sodium iodate
- Delafield’s-slowely ripened
- Harri’s- mercuric oxude
- Gill’s- sodium iodate
- Mayer’s – sodium iodate
 B. Iron Hematoxylins
• Iron salts are used as oxidizing agents and mordants
• Examples
1. Weigert’s-Ferric chloride
In combination with van Gieson’s stain, can demostrate
conenctive tissue elements and Entamoeba histolytica in
sections.
Standard iron hematoxylin
For nucleus/connective tissue fibers
Van Gieson’s stain- good for demonstrating collagen
2. Heidenhain’s-Ferric ammonium sulfate
- For mitochondria, muscle striations, chromatin and myelin
 C. Tungsten Hematoxylin
• Mordant: Tungsten
Mallory PTAH(Phosphotungstic acid
Hematoxylin)
To ripen: stand in the light for several weeks or
use potassium for immediate ripening
For staining muscle striations
 D. Copper Hematoxylins
• Used for study of spermatogenesis
• Spermatogonia-spermatid-sperm cell
 Eosin
• A red avid dye
• Routinely use din histopathology as a
counterstain after hematoxylin and before
methylene blue
3 forms
1. Eosin Y (yellowish)-MOST COMMONLY USED
2. Bluish-Eosin B
3. Ethyl eosin
 H and E staining Steps
1. Xylol ( 2 changes)- deparafinized
2. DESCENDING GRADE OF ALCOHOL
3. Water
*removal of pigments is done after rehdyration and right before
primary staining
*removal of mercuric pigments:
Place Weigert’s iodine
Wash in dist. Water
Remove iodine with 5 % sodium thiosulfate
Wash in running water
Proceed with stain
4. Stain with Harris/Erlich’s/delafields- red nucleus
5. Rinse slides in Tap water
6. Acid alcohol (differentiator) –red nucleus
7. Ammonia water(ammonium Hydroxide, lithium
carbonate, Scott’s tap water)- Blueing agent
8. Wash well in running water
9. Satin with Eosin Y
10. Ascending grade of alcohol- dehydration
11. Xylol/Xylene- Dealcoholization-clearing
12. Mount the label
 Results
Nuclei- blue to blue black
Karyosome- dark blue
Cytoplasm, proteins in edema fluid- pale pink
Calcium and calcified bone- purplish blue
Muscle fibers- deep pink
H- 1 stain(nuclear)
E- 2 countertain- cytoplasmic
Modified H and E staining- Progressive staining, no
differentiation phase, frozen section
• Blueing step- bridging mordant and dye
 PAP smear staining
• Uses 3 stains: Hematoxylin, OG-6, EA
• OG-6- cytoplasm of superficial/mature cell
• EA-cytoplasm of immature cells
(intermediate,parabasal)
1. Fix with 95% ETOJ
2. Stain wuth Harris Hematoxylin
3. Acid alcohol
4. Blueing step- ammonia water
5. Stain with OG-6
6. 70-95% ETOH- for washing
7. Stain with EA 36/50
8. Dehydrate
9. Xylol
10. Mount and label
EA 65- for body fluids
Modified Pap staining technique
- Ommitted the Bismark bron dye from EA formula
- Sharpness of color and brilliant staining are
improved

- Eozin Azur- Eosin Y

- Bismark brown
- Light green SF (,36,50,65)
- Lithium carbonate Phosphotungstic acid
 Other stains and their uses
1. Benzidine- used for staining hemoglobin
2. Acridine orange- DNA (green fluorescence) RNA (red
fluorescence)
3. Cystal violet- for amyloid in frozen sections and platelets
in blood
4. Gentian violet – formed by the mixture of crystal violet,
methyl violet, dexterin
5. Congo red- stain for axis cylinders in embryos, used as a
4% aqueous solution in krajan methods for staining,
elastic tissue, myelin
6. Iodine- probably the oldest of all stains, stains for amyloid,
cellulose, starch, carotenes and glycogen, widely used for
removal of mercuric fixative pigments
7. Malachite green- contrast stain for staining
Ascaris eggs and erytrocytes, bacterial spore
stain
8. Janus Green B- demonstrate mitochondria
during intravital stain
9. Night blue- substitute for carbol fuchsin in
acid fast staining
10. Victoria blue- demonstrate neuroglia in
frozen sections
11. Lysochromes (oil soluble dyes)- not real dyes, no auxochromes
groups, give color to lipids simply because they are more soluble in
lipid medium of the tissues than in their medium of 70% alcohol
Examples of oil soluble dyes used for demonstration of intracellular
fats
Sudan black B- black
Sudan III- orange
Sudan IV- (Scharlach R)- red
12. Periodic Acid-Schiff Reaction- for fungal wall
Red/magenta red
Mucoproteins are most common PAS positive substances
13. Toluidine blue- used to demonstrate mast cells in tissues
14. Gomori’s trichome- The best staining
procedure for demonstrating reticulin
 Mounting
• Refractive index- ratio of speed of light in air and speed of
light in a specific medium
• Refractive index of glass= 1.518
A. Resinous media (RI greater than or equal to 1.518)
1. Eukitt
2. Entallan
3. DPX (1.532)
4. Histomount
5. XAM (1.52)
6. Paramount
7. Canada balsam – Abus Balsamea (1.524)
8. Clarite (1.544)
B. Aqueous Media (usually for lipids because
resinous media contain xylene which dissolve
fats)
1. Glycerin (1.47)
2. Gum arabic ((Farrant’s medium) (1.43)
3. Karo Corn syrup
4. Apathy’s medium (1.52)
5. Brun’s Fluid- Recommended for mounting frozen
sections from water
6. Water- evaporates easily