HISTOPATHOLOGY W E E K｜08

MODULE WRITER: MR. RENE JESUS ALFREDO R. DINGLASAN MTAP 2

OUTLINE MAIN FACTORS INVOLVED IN FIXATION

I. Fixation
II. Decalcification 1. Hydrogen ion concentration (pH)
III. Dehydration ● Satisfactory fixation = pH 6-8
IV. Clearing 2. Temperature
V. Impregnation ● Surgical spx – room temperature
VI. Embedding ● Electron Microscopy and some histochem:
VII. Trimming 0-4ºC
VIII. Sectioning 3. Thickness of section
IX. H & E Staining ● Tissue blocks should be:
X. Pap Smear Staining SMALL
XI. Mounting ○ EM: 1 to 2 mm2
○ Light Microscopy: 2 cm2
THIN
FIXATION
○ (NO more than >0.4 cm for Light
microscopy), or as prescribed by
● First and most critical step in histotechnology tissue processor manufacturer
● Process of preserving fresh tissue for examination ● Brain: usually suspended whole in 10%
● Ideal time to perform fixation: 20-30 minutes after buffered formalin for 2 to 3 weeks :
interruption of blood supply 4. Osmolality
● Primary aim: To preserve the morphologic & chemical 5. Concentration
integrity of the cell in as life-like manner as possible 6. Duration of Fixation: The longer the organ stays in the
fixative, the harder it gets
● Secondary aim: To harden and protect the tissues
from the trauma of further handling TYPES OF FIXATIVES
● Fixatives have the property of forming cross-links
between proteins ● According to Composition
● Stabilization of proteins: Most important reaction for a. Simple Fixatives
maintaining tissue morphology - one component
● Correct fixative-to-tissue ratio: 20:1 ☆ - E.g., Glacial Acetic Acid - it solidifies at 17°C ☆
● Usual fixation temperature (surgical specimens): Room
b. Compound Fixatives
temperature - Made of 2 or more fixatives
- These are made to minimize the adverse
PRACTICAL CONSIDERATIONS OF FIXATION effects of the simple fixatives as they neutralize
each other’s effect.
1. Speed: specimen must be placed in the fixative as soon - E.g., Zenker’s solution
as it is removed from the body. - Major components:
2. Penetration: Formalin = 1mm. per hour (rate of 1. Mercuric Chloride - causes the
penetration) ☆ tissue to shrink
3. Volume: 20x the tissue volume 2. Glacial Acetic Acid - causes the
4. Duration of fixation: tissue to swell
(Done within 24 hours)
● According to Action
2 MECHANISMS INVOLVED IN FIXATION A. Microanatomical - for general microscopic study of
tissue structures, able to preserve almost all components
1. Additive fixation of tissues.
- The chemical constituent of the fixative is taken Examples:
in and becomes part of the tissue. ● 10% Formol Saline
- E.g., formalin, mercury (mercury chloride), and ● 10% BNF
osmium tetroxide ● Heidenhain’s SuSa
● Formol sublimate (Formol corrosive)
2. Non-additive fixation ● Zenker’s solution
- The fixing agent is NOT taken in, but changes ● Zenker-Formol (Helly’s solution)
the tissue composition and stabilizes the tissue ● Bouin’s
by removing the bound water attached to ● Brasil’s
hydrogen bonds of certain groups within the
protein molecule. B. Cytological Fixatives - able to preserve specific parts
- E.g., alcoholic fixatives and particular microscopic elements of the cell itself

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1. Nuclear Fixatives
- preserves the nuclear structure of the cell
- Usual component: Glacial acetic acid (preserves the
nucleoproteins)
- General pH: ≤4.6
- Acidic due to the presence of glacial acetic acid
- e.g.,
● Flemming’s
● Carnoy’s
● Bouin’s (Both microanatomical and nuclear)
● Newcomer’s
● Heidenhain’s SuSa (Both microanatomical and
nuclear)
○ Although this can preserve almost all
parts of the tissue, it preserves the
nucleus more effectively.
● Both microanatomical and nuclear fixatives:
○ Bouin’s
○ Heidenhain’s SuSa
2. Cytoplasmic Fixatives
- Preserves the cytoplasmic structure of the cell
- Must NEVER contain glacial acetic acid
- Glacial acetic acid: destroys the mitochondria
and Golgi bodies of the cells
- General pH: >4.6
- e.g.,
● Flemming’s fluid w/o acetic acid
● Helly’s fluid (Zenker’s formol)
● Formalin with post-chroming
● Regaud’s (Moller’s)
● Orth’s
☆ BOARD EXAM RECALLS ☆
Characteristics of Glacial Acetic Acid
1. It solidifies at 17°C
2. Causes the tissue to swell
3. Preserves the nucleoproteins
4. Destroys the mitochondria and Golgi bodies of the cells
5. Can act as a simple fixative
Sample board exam questions:
“All of the following contain glacial acetic acid except: ”
- Cytoplasmic fixatives
3. Histochemical Fixatives
- preserve the chemical constituents of the tissue
- e.g.,
● 10% Formol Saline
● Absolute Ethanol (Abs. ETOH)
● Acetone
● Newcomer’s Fluid- (both nuclear
histochemical)
A. ALDEHYDE FIXATIVES
1. Formaldehyde (a.k.a. Formalin)
● usual fixation time: 24 hours
● A gas produced by the oxidation of methanol
● Two concentrations:
A. 40% formaldehyde (“Stock solution”)
- Too strong
- Tends to overharden the outer surface
of the tissue
- Not used in routine fixation
B. 10% formaldehyde (“Working Solution”)
- Unstable solution (may produce
artefacts
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Artefacts:
1. Paraformaldehyde - white precipitates that may form
after prolonged standing of the solution esp at low temp
- may be removed by: Filtration or addition of
10% Methanol
2. Acid formaldehyde hematin - brown or black granules
that may obscure microscopic details of the cells
- May be removed by:
● Kardasewitsch method
● Lillie’s mtd
● Saturated alcoholic picric acid ☆
● Alcoholic KOH ☆
2. 10% Formol-Saline
- Recommended for CNS and general post-mortem
tissues
3. 10% BNF (Buffered Neutral Formalin)
- pH = 7.0
- BEST fixative for tissue containing iron pigments & for
elastic fibers ☆
- BEST general tissue fixative
4. Formol-Corrosive (Formol-Sublimate)
- Recommended for routine post-mortem tissues (same as
10% Formol Saline)
- It is corrosive as it contains mercuric chloride (derived
from its name sublimate)
5. Glutaraldehyde
- Enzyme histochemistry and electron microscopy
- 2 concentrations.:
a. 2.5% solution: small tissue fragments and needle biopsy
b. 4% solution: larger tissue spx
6. Formol-calcium
- For lipids preservation
7. Karnovsky’s paraformaldehyde-glutaraldehyde solution
(KPG)
- For electron cytochemistry
8. Acrolein
- For electron cytochemistry
B. METALLIC FIXATIVES (Mercuric Chloride, Chromate, Lead)
1. Mercuric Chloride (most common)
● Advantages:
- Routine fixative of choice for tissue
and photography ☆
- Permits brilliant metachromatic staining of cells
● Disadvantages:
- Causes tissue shrinkage
- Decreases amount of demonstrable glycogen
- Never use if glycogen determination
is desired.
- Corrodes all metals except for the nickel alloy
(Monel)
- Produce black granular deposits
- removed by de-zenkerization
technique - uses alcoholic iodine ☆
- e.g.,
a. Zenker’s Fluid (with glacial acetic acid)
- Small pieces of liver, spleen,
connective tissue fibers and nuclei
b. Zenker-Formol (Helly’s Sol’n)
2
 - Pituitary gland, Bone Marrow and - Used for cytological smears (e.g., Pap
blood containing organs (eg.: spleen smear - fixed using 95% ethanol)
and liver) c. Carnoy’s fluid
c. Heidenhain’s SuSa - Most rapid fixative of all (Fixation
- “SuSa” from the German words: time: 1-3 hours)
- Sublimat = mercuric - Uses:
chloride - Preservation of
- Saure = acid chromosomes ☆
- Tumor biopsies (esp. of the skin) - Preservation of lymph
d. Schaudinn’s fluid glands and urgent biopsies
e. Ohlmacher’s fluid - Preservation of brain tissue
f. Carnoy-Lebrun fluid for rabies diagnosis (same
g. B-5 fixative as acetone)
- Commonly used for Bone Marrow - Preservation of glycogen
biopsies d. Alcoholic Formalin (Gendre’s Fixative)
- useful in preserving sputum
2. Chromate e. Newcomer’s fluid
a. Chromic acid - Nuclear and histochemical fixative
b. Potassium dichromate - Used for the preservation of nuclear
c. Regaud’s (Moller’s) proteins and mucopolysaccharides
d. Orth’s fluid
- for Rickettsia and other bacteria Biopsies:
- for the study of early degenerative processes ● Heidenhain’s Susa: Tumor biopsy
● B-5 fixative: Bone marrow biopsy
3. Lead fixatives ● Bouin’s Solution: embryos and pituitary biopsies
- Generally for ACID Smears:
MUCOPOLYSACCHARIDES ● Methanol: Blood and Bone marrow smear
- E..g., Umbilical Cord/ Wharton’s jelly ● Ethanol: Cytological smears
Rabies dx:
C. PICRIC ACID FIXATIVES ● Carnoy’s fluid
- Used as: ● Acetone
● Fixative Post-mortem tissues fixatives:
● Decalcifying agent ● 10% Formol-Saline
● Stain ● Formol-Corrosive (Formol-Sublimate)
- Highly explosive when in dry powder form For electron cytochemistry:
- Produce excessive yellow staining of tissues ● KPG
- Picrates are formed upon protein; precipitates ● Acrolein
are soluble in water; hence tissue
- Must be first rendered insoluble by direct F. OSMIUM TETROXIDE FIXATIVES
immersion in 70% ETOH - should be kept in a dark-colored, chemically
- picrate fixatives MUST NEVER be washed in clean bottle to prevent evaporation and
water before dehydration. reduction by sunlight or organic matter.
a. Bouin’s Solution - inhibits hematoxylin and makes
- Recommended for embryos counterstaining difficult.
and pituitary biopsies - produces black precipitate (Osmic oxide)
b. Brasil’s Alcoholic Picroformol - Prevention: add saturated aqueous mercuric
chloride
D. GLACIAL ACETIC ACID - Remedy: Black osmic oxide crystals may be
- It solidifies at 17°C dissolved in cold water.
- Causes the tissue to swell - Precaution: may cause conjunctivitis or
- Preserves/fixes the nucleoproteins (nuclear blindness.
fixative) - e.g.,:
- Destroys the mitochondria and Golgi bodies of a. Flemming’s Solution (w/ acetic acid)
the cells - Nuclear fixative
- Can act as a simple fixative b. Flemming’s without acetic acid
- Cytoplasmic fixative
E. ALCOHOLIC FIXATIVES
- GENERALLY RECOMMENDED FOR: G. TRICHLOROACETIC ACID
Glycogen fixation - Both fixative and decalcifying agent
- One of the disadvantages of these fixatives is - Same as Newcomer’s fluid (dual functions - nuclear and
POLARIZATION - movement of glycogen histochemical fixative)
granules towards the ends or poles of the cells
- E.g.: H. ACETONE
a. Methanol - Used at ice cold temp. (-5 to 4 °C)
- Used for the fixation of blood smears - USES:
and bone marrow smears - Preservation of water-diffusible enzymes (such as
b. Ethanol lipases and phosphatases)

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 - Preservation of BRAIN tissue for RABIES dx (same as - Optimum temperature = Room Temperature (18-30°C)
Carnoy’s fluid) ☆
- The ideal time required for decalcifying tissue is 24-48
I. HEAT FIXATION hours
- direct flaming fixation- may be utilized for bacterial smear - Dense bone tissues usually require up to 14 days or
- microwave fixation (optimum temp. 45-55°C) longer in order to complete the process.
- a non chemical technique for the demonstration of
neurochemical subs. in the brain (such as DECALCIFYING AGENTS
ACETYLCHOLINE) 1. Acids
2. Chelating agents
FIXATIVES FOR ELECTRON MICROSCOPY 3. Ion exchange resins
4. Elec. ionization
● Glutaraldehyde ☆
● Platinic chloride (PtCl3) Some examples of acid decalcifying agents:
● Platinic Chloride-formalin (Zamboni’s fixative) ● Nitric acid
● Gold chloride (AuCl) - most commonly used decalcifying agent
● Osmium tetroxide ○ Perenyi’s fluid - functions as both a
● 10% BNF decalcifying agent and a tissue softener
○ Phloroglucin-Nitric Acid - most rapid
FACTORS THAT AFFECT FIXATION decalcifying agent
○ Carnoy’s fluid - most rapid fixative
Factors that affect fixation:
a. Fixation may be retarded (slow down) by: ● Formic acid
1. Size and thickness of the tissue - both fixative and decalcifying agent (same as
2. Presence of mucus TCA)
3. Presence of blood - recommended for small pieces of bones and
4. Presence of fats teeth.
5. Cold temperature ● 5% formic acid: BEST general decalcifying
agent
b. Fixation may be enhanced (speed up) by: ● 10% BNF: BEST general tissue fixative
1. Agitation
2. Size and thickness of the tissue ● Hydrochloric acid
3. Moderate heat (37-56°C) ○ Von Ebner’s Fluid – recommended for teeth
and small pieces of bones ☆
Mnemonics for Routine Tissue Processing Steps:
(F DCIETS SML) EXTENT OF DECALCIFICATION
1. Fixation ● 3 ways to measure extent of decalcification:
2. Dehydration 1. Physical/Mechanical Test
3. Clearing - Inaccurate
4. Impregnation - Done by touching or bending tissue
5. Embedding with the fingers
6. Trimming - Decalcified tissues: commonly have
7. Sectioning diminished consistency and are softer
8. Staining to touch
9. Mounting - Alternate method: pricking the tissue
10. Labeling with a fine needle or a probe or even
applicator stick
*Decalcification is done after fixation, before dehydration - only 2. X-ray or Radiological Method
performed on certain tissues such as nails and bones. - very expensive
- Most ideal, most sensitive, most
DECALCIFICATION reliable method
3. Chemical Method (Calcium Oxalate Test) -
- Process whereby the calcium and lime salts are removed simple, reliable, recommended for routine
from the tissues purposes
- GOAL: to soften the tissues
- After fixation and before impregnation TISSUE SOFTENERS
- More concentrated acid solutions decalcify bone more - For unduly hard tissues that may damage the microtome
rapidly but are more harmful to the tissue knives
- High concentrations and greater amounts of fluid will ● 4% aq. phenol.
increase the speed of the process. ● Molliflex
- The recommended ratio of fluid to tissue volume for ● 2% HCl
decalcification is 20:1 ☆ (same as fixation ratio) ● 1% HCl in 70% alcohol
- Heat will serve to hasten decalcification BUT it also
increases the damaging effects on tissues. DEHYDRATION
- At 37°C = impaired nuclear staining of Van Gieson’s stain
for collagen fibers - Aim: to remove fixative and water from the tissue and
- At 55°C = tissue will undergo complete digestion within replacing them with dehydrating fluid in preparation for
24-48 hours. ☆ impregnation.

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 - Dehydrating fluids are generally used in increasing 2. Toluene
strengths (all the aqueous tissue fluids are removed but 3. Chloroform
with little disruption to the tissue due to diffusion currents) - Toxic to the liver after prolonged inhalation and
it does not make the tissues transparent
COMMONLY USED DEHYDRATING AGENTS - Used for for tough tissue spx
1. Alcohol – MOST COMMON (used in ascending grades) 4. Methyl benzoate and methyl salicylate
a. Ethanol - Best dehydrating agent (for routine 5. Cedarwood oil and clove oil
dehydration of tissues) - Especially recommended for CNS tissues and
- 5% formic acid: BEST general decalcifying cytological studies, particularly of smooth
agent muscles and skin (cedarwood oil)
- 10% BNF: BEST general tissue fixative 6. Citrus fruits oils
b. Methyl alcohol - employed for blood and tissue films 7. Trichloroethane and petrol
c. Butyl alcohol - utilized in plant and animal 8. Benzene
microtechniques 9. Aniline oil - rather recommended for clearing embryos,
d. Industrial methylated spirit (denatured alcohol) - insects and very delicate specimens
ethanol + small amt. of methanol 10. Carbon tetrachloride
e. Isopropyl alcohol
2. Acetone: fixative & dehydrating agent IMPREGNATION
3. Dioxane (Diethylene dioxide)
4. THF (Tetrahydrofuran) - a.k.a. INFILTRATION
- 3 & 4 function as dehydrating & clearing agents - Process of replacing the clearing agent with the
5. Cellosolve (Ethylene glycol monoethyl ether) infiltrating medium
6. Triethyl phosphate - The medium used to infiltrate the tissue is usually the
same medium used for embedding.
Additives to dehydrating agents: - 25x the volume
1. 4% phenol - Most commonly used impregnating medium - paraffin
2. Anhydrous copper sulfate wax
- Both dehydrating agent & indicator of water
content (100% ETOH)
- W/o water: White in color 4 types of tissue impregnation and embedding media:
- Hydrated: blue in color 1. Paraffin wax: most commonly used impregnating
medium; not soluble to water & alcohol
2. Celloidin (Collodion)
3. Gelatin
4. Plastic

Paraffin
Hydrated: blue in color - the man who introduced paraffin wax embedding:
Butschlii
- simplest, most common and the BEST
infiltrating/embedding medium.
- NOT recommended for fatty tissues
- Temperature of paraffin oven = 55-60°C (Paraffin oven
must be maintained at a temperature 2-5°C above the
MP of the paraffin wax)
W/o water: White in color
SUBSTITUTES FOR PARAFFIN WAX
A typical dehydration sequence for specimens NOT more than 4
1. Paraplast
mm thick would be:
- Melting point: 56-57°C
1. 70% ethanol: 15 min
- Mixture of highly purified paraffin and synthetic
2. 90% ethanol: 15 min
plastic polymers
3. 100% ethanol: 15 min
- More elastic and resilient than paraffin
4. 100% ethanol: 15 min
- For large dense tissue blocks such as bones
5. 100% ethanol: 30 min
and brain
6. 100% ethanol: 45 min
2. Embeddol
- MP: 56-58°C
CLEARING - Less brittle and less compressible than
paraplast.
- a.k.a: DEALCOHOLIZATION 3. Bioloid
- Process of replacing the dehydrating fluid with a fluid that - Recommended for embedding eyes.
is miscible with BOTH the dehydrating fluid and the - Dry method celloidin can also be used
impregnating/embedding medium. for embedding the whole eyes
section.
Clearing agents suitable for routine use: 4. Tissue Mat
1. Xylene/xylol (most commonly used and most rapid) - A product of paraffin, containing rubber, with
- Milky - incomplete dehydration (water is the same property as paraplast.
detected)

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 5. Ester Wax - The surface of the section to be cut should be placed
- MP: 46-48°C parallel to the bottom of the mold in which it is oriented.
- Harder than paraffin
- Insoluble in water TRIMMING
- Soluble in 95% ETOH and other clearing
agents (alcohol soluble) - Correct shape of the block : Four-sided prism or
- When utilized, clearing step can be skipped truncated pyramid
as it is soluble in alcohol - Process of removing excess wax after embedding.
- Can be used for impregnation without prior - Excess wax is cut off from the block to expose the tissue
clearing of the tissue. surface in preparation for actual cutting.
- Sliding microtome or base-sledge type - Knife/blade may be used
microtome
6. Water-soluble waxes
SECTIONING
- Most commonly used: CARBOWAX
- MP: 38-42°C or 45-56°C
- a.k.a Cutting or Microtomy
- Mostly polyethylene glycols
- The process by which a processed tissue is cut into
- Carbowax – soluble and miscible with water
uniformly thin slices (sections/ ribbons) to facilitate
(hence does not require dehydration and
studies under the microscope.
clearing of the tissue).
- 4-6 u: routine tissue microtomy
- Suitable for many enzyme histochemical
- 10-15 u: frozen sections
studies
- 0.5 u: electron microscopy
Celloidin (Collodion)
KINDS OF MICROTOMES
- purified form of nitrocellulose
- suitable for specimens with large hollow cavities, hard
1. Rocking Microtome (Cambridge Rocking Microtome)
and dense tissues (bones and teeth), large tissue
- Simplest among the microtome
sections of the whole embryo.
- Disadvantage: difficulty in reorienting the block.
- Inventor: Paldwell Trefall in 1881
Two methods for celloidin impregnation:
1. Wet Celloidin – recommended for bones, teeth, large
brain sections and whole organs.
2. Dry Celloidin – preferred for processing of whole eye
sections.
- Bioloid can be used as well for processing eye
sections

Gelatin
- Water-soluble impregnating medium
- rarely used except when dehydration is to be avoided.
- used when tissues are for histochem and enzyme
studies.
- embedding medium for delicate specimens and frozen 2. Rotary/Minot Microtome
sections because it prevents fragmentation of tough and - MOST COMMON type used today especially for
friable tissues when frozen sections are cut. paraffin-embedded tissues
- Housed in cryostat
Plastic/Resin - Refrigerated cabinet
- Used for electron microscopy - Inventor: Minot in 1885-1886
- classified into: epoxy, polyester, acrylic
- 3 types of epoxy: 3. Sliding Microtome
1. Glycerol (EPON) - MOST DANGEROUS TYPE DUE TO MOVABLE
2. Bisphenol A (Araldite) EXPOSED KNIFE (STANDARD SLIDING)
3. Cyclohexene dioxide (Spurr) - Developed by Adams in 1789

EMBEDDING There are 2 types:

a. Base-Sledge
- a.k.a casting/blocking - For all forms of media
- Temp of melted paraffin (embedding): 5-10°C above its - Block holder: moving
Melting point - Knife: stationary
- Process by which the impregnated tissue from the plastic b. Standard Sliding Microtome
cassette is placed into a precisely arranged position in a - Block: stationary
mold containing a medium which is then allowed to - Knife: moving
solidify. - MOST dangerous
- ORIENTATION – the process by which a tissue is
arranged in precise positions in the mold during 4. Rotary Rocking Microtome
embedding, on the microtome before cutting, and on the
slide before staining. 5. Vibrotome
- To solidify embedded tissue = cooled rapidly in a ref (-5 - used for unfixed, unfrozen specimen sectioning for
°C) or immersed in cold water. enzyme demonstrations.

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 - disadvantage: sections are liable to disintegrate.
6. Ultrathin Microtome
- for cutting sections for Electron Microscopy
- uses DIAMOND KNIVES ☆ or broken plate glass
- specimen is small, fixed in osmium tetroxide, embedded
in plastic
- Very thin sections (0.5 u)
8. 3-APES (3-aminopropyltriethoxysilane)
- APES-coated slides: very useful in cytology, particularly
for cytospin preparations of proteinaceous of bloody
material
STAINING
A. NATURAL DYES: obtained from plants and animals

7. Freezing Microtome 1. Hematoxylin

2. Cochineal dyes
- extracted from the female cochineal bug
MICROTOME KNIVES (Coccus cacti)
Carmine
Microtome knives Usual Length Description - Cochineal dye + alum
- chromatin stain
I. Picrocarmine
Plane concave 25 mm One side: FLAT
- used for neuropathological studies
Other side:
- Carmine + Picric acid
CONCAVE
II. Mucicarmine
- Used for demonstration of mucins and
Biconcave 120 mm Both sides: Cryptococcus neoformans
CONCAVE - Carmine + Aluminum hydroxide
III. Best’s Carmine
Plane wedge 100 mm Both sides: - Demonstration of glycogen
STRAIGHT - Carmine + Aluminum chloride
3. Orcein
4. Saffron
● CLEARANCE ANGLE: 0-15° or 5-10°
B. SYNTHETIC DYES
● BEVEL ANGLE: 27-32°
- Aka “Coal Tar Dyes”
- Derived from benzene and collectively known as
STAGES OF KNIFE SHARPENING
“Aniline Dyes”
a. Honing
- Purpose: removal of gross nicks
Chromogen
- Knife direction: “HEEL to TOE”
- Imparts color temporarily
- Types of hones:
- Benzene + chromophore → Chromogen
1. Arkansas
Dye
2. Fine carborundum
- Imparts color almost permanently
3. Belgium yellow (gives the best honing result)
- Chromogen + auxochrome → DYE
b. Stropping
- Purpose: removal of burr
Chromophores:
- Knife direction: “TOE to HEEL”
● Part of the benzene ring which is responsible for
- Instrument used: Paddle strop
“coloring property”
- Made from horse leather
● Quinoid ring
● Azo groups
● 5% formic acid: BEST general decalcifying agent
● Xanthene
● 10% BNF: BEST general tissue fixative
● Quinone-imine group
● Paraffin wax: BEST embedding media
○ OXAZIN
● Belgium Yellow: BEST honing results
○ THIAZINS
● TEMPERATURE OF FLOTATION WATER BATH:
Auxochromes:
45-50°C (approximately 6-10 lower than the MP of wax)
● Particular part of benzene ring which is responsible for
● Microwave fixation (optimum temp. 45-55°C)
the “dyeing property”
● CATIONIC AUXOCHROME: Amino group
● ANIONIC AUXOCHROME: Hydroxyl and Carboxyl
ADHESIVE AGENTS
Groups
1. Mayer’s Egg Albumin
- Contains: Egg white (a.k.a Albumen) , glycerin, crystals
Dye Modifiers (attached on benzene ring) EMS
of Thymol (to prevent the growth of molds)
● Ethyl groups
- ***3-APES (3- aminopropyltriethoxysilane) : very useful in
● Methyl groups
cytology, particularly for cytospin preparations of
● Sulphonic Acid
proteinaceous of bloody material
2. Dried Albumin
Dye-to-Tissue Mechanisms - tissues will bind dyes by one of the
3. Gelatin
following mechanisms:
4. Gelatin-formaldehyde mixture
1. Electrostatic
5. Starch paste
- majority of tissue-dye reactions
6. Plasma
- e.g., Neutral Red and Light Green
7. Poly-L-Lysine
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 2. Hydrogen bonding - Neutral red (probably the BEST vital
- e.g., Congo Red, Carmine (natural dye & dye)
chromatin stain), Weigert-type resorcinol dye - Janus green (for mitochondria)
- Congo red is considered as the gold standard - Trypan blue
for amyloid demonstration ☆ - Nile blue
- Toluidine blue
3. Van der Waals Forces - Thionine
- e.g., Alum Hematoxylin Solutions
H & E STAINING
4. Physical Staining
- e.g.,: Sudan dyes H and E staining
- SUDANOPHILIA – property of tissues to be - Most widely used histological stain
stained with fat or oil-soluble dyes, regardless
of the type of dye used, due to their essential I. HEMATOXYLIN
lipid nature - Serves as:
5. Natural Affinity - Basic stain
- e.g., Janus Green - Primary stain
- Nuclear stain
METHODS OF STAINING - a natural dye derived from extraction from the
1. According to the presence of mordant heartwood of the Mexican tree (Hematoxylin
A. Direct staining campechianum)
- Gives color to the sections by using - Ripening/oxidation: needed to convert
simple aqueous or alcoholic dye hematoxylin to hamatei
solutions - First person to use hematoxylin in histology
- e.g., methylene blue (1862): Waldeyer
B. Indirect staining
- Action of dye is intensified by using Ripening/Oxidation
mordant - may be done by exposing the substance to air and
- e.g., hematoxylin sunlight (SLOW)
- Hematein (active coloring substance - Needs to convert hematoxylin to Hematein
of hematoxylin) - Hematein: Active coloring substance
- Still requires a mordant to attach to tissues
2. According to the presence of a differentiator - DYE LAKE: stain + mordant + tissue
(decolorizer) - may be done by adding oxidizing agents such as:
A. Progressive staining ● Hydrogen peroxide
- When dye is taken up by tissue, it is ● Mercuric oxide
NOT decolorized - ripening agent of Harris Hematoxylin
- e.g., H & E (for frozen sections) ☆
B. Regressive Staining ● Potassium permanganate
- Requires differentiator ● Sodium perborate
- Tissue is first overstained, then ● Sodium iodate - ripening agent of Ehrlich’s
excess stain is removed/decolorized Hematoxylin ☆
from unwanted parts of the tissue
- e.g., H & E (routine tissue staining) A. Alum Hematoxylins
Differentiator/Decolorizer - Used in routine H and E
- Primary stain is basic = differentiator - Mordant: Potash alum (potassium aluminum
should be acid solution sulfate or simply “alum”)
- Primary stain is acidic = differentiator - Produce good nuclear stain
should be an alkaline solution - examples:

3. According to the resultant color of the tissue

A. Orthochromatic staining Alum Hematoxylin Ripening Agent (Oxidant)
- Ortho = same
- Color of the dye = color of the tissue Harris Mercuric oxide
B. Metachromatic staining ***- useful general purpose
- Meta = after or change hematoxylin
- Color of the dye ≠ color of the tissue
Ehrlich’s Natural or sodium iodate
4. Vital Staining
- Selective staining of living cell constituents
Delafield’s Natural
A. Intravital staining
- Involves injection of the dye into any
part of the animal body Gill’s Sodium iodate
- e.g., lithium, carmine, india ink
B. Supravital staining Mayer’s Alcoholic iodine
- Staining of living cells immediately
after removal from the body Cole’s Potassium iodate
- e.g.,

aquino.bacani.pineda.policarpio.punsalan.rubiano.santos 8
 - Cytoplasm : colorless
Carazzi - Nucleus: red
7. Ammonia water (Ammonium hydroxide, lithium
carbonate, Scott’s tap water)
B. Iron Hematoxylins
- Blueing phase
- iron salts are used as oxidizing agents and
- Nucleus: blue
mordant
- Cytoplasm: colorless
- e.g.,
8. Wash well in running tap water
1. Weigert’s – ferric chloride
9. Stain with Eosin Y
- in combination with van Gieson’s stain, can
- Secondary staining, Acidic staining,
demonstrate connective tissue elements and
Cytoplasmic staining
Entamoeba histolytica in sections
- Nucleus: blue
- standard iron hematoxylin
- Cytoplasm: pale pink
- for muscle/connective tissue fibers
10. Ascending grades of alcohol
- Van Gieson’s stain: good for demonstrating
- Dehydration
collagen (can be used alone)
11. Xylol/xylene
2. Heidenhain’s- ferric ammonium sulfate
- clearing/dealcoholization
- for mitochondria, muscle striations, chromatin,
12. Mount then label
and myelin
3. Verhöeff – used for staining elastic fibers
Results:
4. Loyez - used for staining myelin
● Nuclei – blue to blue black
● Karyosome – dark blue
C. Tungsten Hematoxylin
● Cytoplasm, proteins in edema fluid – pale pink
● Mallory’s PTAH (Phosphotungstic Acid Hematoxylin)
● Calcium and calcified bone – purplish blue
- to ripen: stand in the light for several weeks or
● Muscle fibers – deep pink
use potassium for immediate ripening
- for staining muscle striations, fibrin and glial
fibers PAP SMEAR STAINING
- Staining method of choice for exfoliative cytology
D. Copper Hematoxylin - Uses 3 stains: Hematoxylin, OG-6, EA
- used for: study of spermatogenesis Steps:
- Seminiferous tubules: site of 1. Fix with 95% ETOH
spermatogenesis 2. Stain with Harris Hematoxylin - nuclear stain
3. Acid Alcohol
E. Molybdenum Hematoxylin 4. Blueing step
● Thomas Hematoxylin: used for collagen and endocrine 5. Stain with OG-6 (Orange green) = stains the cytoplasm
cell granules of mature
- For superficial cells
F. Lead Hematoxylin 6. 70-95% ETOH = for washing
● Solcia Hematoxylin – used for endocrine cell granules 7. Stain with EA 36 or 50 (Eosin Azure) = Stains the
cytoplasm of immature cells
II. EOSIN - Cytoplasmic stain
- a red acid dye - for intermediate and parabasal cells
- routinely used in histopathology as a counterstain after - A polychrome stain, composed of:
hematoxylin and before methylene blue - Light green SF (special formulation)
- In H and E, eosin serves as: - Eosin Y
- Acidic stain - Bismarck Brown
- Secondary stain 8. Dehydrate
- Counterstain 9. Xylol
- Cytoplasm stain 10. Mount and label
- Three forms:
1. Eosin Y (Yellowish): most commonly used OTHER STAINS AND THEIR USES
2. Eosin B (Bluish): deeper red color
3. Eosin S (Ethyl eosin) 1. Benzidine
- used for staining hemoglobin
Routine H and E Staining: 2. Acridine Orange
1. XYLOL ( 2 CHANGES) - DNA (green fluorescence)
- Deparaffinization - RNA (red fluorescence)
2. DESCENDING GRADE OF ALCOHOL 3. Crystal violet
- Rehydration - for staining amyloid in frozen sections and
3. WATER platelets in blood
- Removal of pigments is done after rehydration 4. Gentian violet
and right before primary staining - formed by the mixture of crystal violet, methyl
4. Stain with Harris/ Ehrlich’s/Delafield’s violet, and dexterin
- primary/basic/nuclear staining 5. Congo Red
- Cytoplasm: Colored - stain for axis cylinders in embryos
- Nucleus: Light transparent blue - used as a 4% aqueous solution in Krajian’s
5. Rinse slides in tap water method of staining elastic tissues, amyloid, and
6. Acid alcohol (Differentiator) myelin

aquino.bacani.pineda.policarpio.punsalan.rubiano.santos 9
 6. Iodine 5. Brun’s Fluid – recommended for mounting frozen
- probably the oldest of all stains sections from water
- stains amyloid, cellulose, starch, carotenes, 6. Water – evaporates easily
and glycogen
- widely used for removal of mercuric fixative
pigments VIDEO DISCUSSIONS:
7. Malachite Green
- contrast stain for staining Ascaris eggs and AM Session
erythrocytes
- used also as a bacterial spore stain
8. Janus Green B
- used for demonstrating mitochondria during
intravital staining (or supravital?)
9. Night Blue
- used as a substitute for carbol fuchsin in
acid-fast staining
10. Victoria Blu
- used for demonstration of neuroglia in frozen
sections
11. Lysochromes (Oil Soluble Dyes) PM Session
- not real dyes
- They do not have auxochrome groups.
- They 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
- PAS-positive substances = red/magenta red
- Mucoproteins are the most common
PAS-positive substances

ADDITIONAL STAINS:
1. Warthin-Starry method
- Used for the demonstration of spirochetes
2. Orcein method
- Used for the demonstration of HBsAg
3. Feulgen Technique
- Most reliable and specific histochemical
technique for DNA

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 (Refractive Index: 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 may dissolve fats)
1. Glycerin (1.47)
2. Gum Arabic (Farrant’s medium) (1.43)
3. Karo Corn Syrup
4. Apathy’s medium (1.52)

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