Objectives System of lenses used to increase or decrease

magnification
CHAPTER 1 : RISK MANAGEMENT AND SAFETY IN THE LABORATORY
Magnifies the object being viewed and
focuses a real image in the upper part of the
Risk Management Personal as well as environmental health and body tube
safety
Pathologist Views the slide to identify a disease process or
abnormality that will directly affect the Eyepiece/Ocular Lens system nearest to the eye (10x)
patient’s treatment Further magnifies the image formed by the
Histotechnologist Views the same slide microscopically for objective
quality control, to determine whether all Focal Length Distance between outer lens of objective and
technical processes are functioning properly the cover glass of the slide
and if a slide of diagnostic quality has been Magnification and its Calibration
achieved
Magnification Increases the size of the structure under
Stains and dyes Used to give contrast to the tissue by creating
examination
light absorption of varying degrees, uniquely
Total Magnification Objective x Eyepiece
taken up by each tissue element, and seen
microscopically as colors Illumination Systems and Their Parts
Microscope Instrument that enlarges images and allows Light Source Source of illumination located below the
the visualization of morphologic cellular stage and substage
details that are too small to be seen by the Iris Diaphragm For the passage of the light to the object
unaided eyes under examination
Condenser Receives light rays from the source of
Parts of the Microscope
illumination and forms a cone of light
Base Provides support for the microscope
Arm Supports and holds the magnifying and
Brings the parallel rays of light
adjustment system. Used as a handle for
Resolving Power Ability to allow the examiner to see two
carrying the microscope.
particular points that are close together, as
Stage Where the slide is placed for examination
separate and distinct
Substage Located under the stage and holds the
Numerical Aperture (NA) Light gathering capacity of of a lens governs
condenser and diaphragm
the resolution and the wavelength if light
Mechanical Stage Permits movement of the stage while holding
employed
the slide in the phase of focus
Nosepiece Used for holding the objectives
Health Hazards
Parfocal When one objective is focused on the turret Biohazards Cause disease in humans, regardless of its
and all lenses will also be in focus source

Includes infectious agents, contaminated

solutions, specimens or objectives
Irritants Cause reversible inflammatory effects at the
site of contact with living tssue
Corrosive Chemicals Cause destruction or irreversible alterations

Histopathology 1
 when exposed to living tissue, or destroy
certain inanimate surfaces
Sensitizers Cause allergic reactions in a substantial
proportion of exposed subjects because of
the high exposure level
Carcinogens Substances that induce tumors
Carcinogenic chemicals - chloroform,
chromic acid, formaldehyde, nickel chloride,
potassium dichromate
Carcinogenic dyes - auramine, basic fuschin,
any dye derived from benzidine including
Congo red and diaminobenzidine
Toxic Materials Capable of causing death by ingestion, skin
contact or inhalation
Includes methanol, chromic acid, osmium
tetroxide, uranyl nitrate
Physical Hazards
Combustibles Substances that ignite at or above a certain
temperature (or flash points)
USA, OSHA “flash point: as 100°F (38℃)
Department of Transportation 141°F (60.5℃)
Flammables Flash points below the temperature specified
above
Explosive Include picric acid
Silver solutions may explode upon aging. This
is why they should never be stored after use
Oxidizers Harmless by themselves, but may initiate or
promote combustion
Include sodium iodate, mercuric oxide,
chromic acid
Permissible Exposure Limits (PELs), Threshold Used todefine the maximum allowable
Limit Values (TLVs), or Occupational Exposure airborne concentration of a chemical (vapor,
Histopathology
Limitis (OELs) fume or dust) to which a worker may be
exposed
Storage of Hazardous Chemicals
Dangerous Liquids Below countertop height
Dangerous Reagents Stored in plastic or plastic-coated glass
bottles
Certain Flammable Liquids Must never be stored in a refrigerator or
freezer
Only small quantities must be made available
as needed and must be used up completely
First Aid
Emergency Eyewash Stations Have it no more than 100 feet from hazardous
work areas, and the water temperature
should be controlled to a range of 15-35℃
Accidental Eye Splashing Should be rinsed for 15-30 minutes, pulling the
lids away from the eyeball
Accidental Skin Contact Wash with water for 15-30 minutes
Handling of Potentially Infectious Specimen
- fresh tissue and body fluids must always be considered potentially infectious
Prions Infectious agents that cause spongiform
encephalopathies such as Creutzfeld-Jakob
disease (CJD), scrapie, and mad cow disease
Tissues from patients with suspected CJD Can be decontaminated by immersing the
specimen in formalin for 48 hours, followed by
treatment in concentrated formic acid for 1
hour, and additional formalin fixation for
another 48 hours
Hazards and Handling of Common Histological Chemicals
Acetic Acid Can irritate the skin, eyes, and respiratory
system
1-10% dilute solution is relatively safe
Concentrated solution should not be mixed
with chromic acid, nitric acid or
sodium/potassium hydroxide
Ammonium hydroxide Stored away from acids, and should not be
2
 mixed with formaldehyde
Irritating to the respiratory system
Aniline Toxic to the skin, can cause severe irritation of
the eyes, and is potential carcinogen
Can cause cyanosis (blue discoloration of the
skin)
Chloroform Toxic when inhaled or ingested
Carcinogenic and can affect the liver,
reproductive organs, central nervous system,
blood, and gastrointestinal tract
Chromic acid (Potassium dichromate) Toxic to the kidneys, is corrosive to skin and
mucous membranes, and can cause cancer
Environmental toxin
Ethanol Skin and eye irritant
Ether Mild to moderate irritation of skin and eyes
Should never be stored in a refrigerator or
freezer unless these appliances are certified
as suitable for an explosive atmosphere
Ethylene glycol Toxic to reproductive, urinary and blood
systems
Propylene-based glycol ether should be used
as substitute
Must be handled under a fume hood, with
butyl gloves
Formaldehyde and paraformaldehyde Can cause severe irritation of the skin and
eyes and are carcinogens
Formic acid Can irritate the skin and eyes and can
corrode metal
Should be handled under a chemical fume
hood
Glutaraldehyde Can cause severe irritation of the eyes and
skin and is toxic by ingestion
Hydrochloric Acid Can cause severe irritation of skin, eyes, and
Histopathology
respiratory system
Should be handled under a fume hood, using
goggles, apron and gloves
Hydrogen peroxide Harmless if used in concentration less than 5%
Hydroxide (sodium or potassium) Corrosive to eyes and skin
Isopentane Should be stored only in a refrigerator or
freezer that is especially suited for explosive
atmosphere
Chilled isopentane can cause frostbite
Excessive exposure can cause irritation of the
respiratory tract, cough, and irregular
breathing
Isopropanolol Can cause mild to moderate irritation of the
skin and eyes
Mercuric chloride and mercuric oxide Can cause severe irritation of the eyes and
skin and are corrosive to metal
Methanol Moderate skin and eye irritant
Nitric acid Corrosive to skin, mucous membranes and
most metals, and toxic by inhalation
Nitrogen (liquid) Can cost frost bite or thermal (cold) burns
Osmium tetroxide Vials must be scored, broken and opened
under a hood, not in open air
Oxalic aicd Relatively safe when used in dilutions
prescribed for histologic use
Periodic Acid Relatively safe when used in dilutions
prescribed for histologic use
Phenol Should be used with extreme caution, under
a hood, especially when mixing
concentrated formaldehyde and phenol
Picric acid Should not be disposed by pouring down the
drain
Jars and threads containing this chemical
should always be wiped with a damp towel
to prevent the substance from drying
Potassium ferricyanide and potassium Relatively safe when used in dilutions
3
ferrocyanide prescribed for histologic use
Potassium permanganate It should not be mixed with acetic acid,
ammonium hydroxide, ethanol, ethylene
glycol, formaldehyde, glycerol, hydrocholoric
acid, hydrogen peroxide or sulfuric acid
Propylene glycol Less toxic substitute for ethylene-based ethers

Silver salts Relatively safe when used as fresh solution,

but becomes explosive as it becomes old
Should not be discarded down the drain
since it is a serious environmental hazard
Sodium azide Very toxic and may be fatal

Can explode when placed in contact with

metals, and should not be discarded down
the drain
Sodium bisulfate Should be kept away from oxidants
Sodium hypochlorite (liquid chlorine bleach) Do not mix bleach with formaldehyde or
diaminobenzidene (DAB)
Sodium iodate Used to replace mercuric oxide when
reconstituting Harris hematoxylin
Sodium thiosulfate Contain significant amounts of mercury and
should not be discarded down the drain
Sulfuric acid Concentrated solutions require the use of
fume hood, apron, goggles, and gloves
Toluene Its use should be restricted or avoided if
possible, except as a diluent in mounting
media or for removing coverslips
Xylene Same risk as Toluene
Zinc chloride Should not be used in tissue processors as it is
a skin and eye irritant and can cause severe
gastrointestinal problems if ingested
General Rule:
Concentrated acids should be added to water, (never water to acid) in order to prevent
splashing, and should be done under a chemical fume hood

Histopathology 4
CHAPTER 2 : FRESH TISSUE EXAMINATION B. Spreading
- a selected portion of the material is gently spread into a moderately thick film by teasing the
mucous strands apart with an applicator stick
Methods of Fresh Tissue Examination
- Advantage: Maintains cellular interrelationships of the material to be examined
1. Teasing or Dissociation
- Recommended for: fresh sputum, bronchial aspirates, thick mucoid secretions
- selected tissue specimen is immersed in a watch glass containing isotonic salt solution
- Phase Contrast Microscopy: used for unstained specimen
- Differential dyes: used to stain specimen

2. Squash Preparation (Crushing)

- Tissue size (in diameter): note more than 1mm
- tissues are placed in microscopic slide and forcibly compressed with another slide or with a
cover glass

3. Smear Preparation
- cellular materials are spread lightly over a slide by means of a wire loop or applicator, or by
making an apposition smear with another slide
- especially useful in cytological examinations, particularly for cancer diagnosis C. Pull-Apart
- useful for preparing smears of thick secretions such as serous fluids, concentrated sputum,
A. Streaking enzymatic lavage samples from the gastrointestinal tract, and blood smears
- the material is rapidly and gently applied in a direct or zigzag line throughout the slide with
an applicator stick or a platinum loop

Histopathology 5
D. Touch Preparation (Impression Section) A. Liquid nitrogen
- the surface of a freshly cut piece of tissue is brought into contact and pressed on to the - used in histochemistry and during operative procedures
surface of a clean glass slide - most rapid of the commonly available freezing agents
- Phase Contrast Microscopy: for direct examination - Disadvantage:
- Light Microscopy: for stained smears ** Soft tissue is liable to crack due to the rapid expansion of the ice within the issue, producing
- Advantage: cells may be examined without destroying their actual intercellular relationship, ice crystals or freeze artifacts
and without separating them from their normal surroundings ** Overcools urgent biopsy blocks, causing damage to both block and blade if done at -70℃
or below
- majority of non-fatty unfixed tissues are sectioned well between -10° and -25℃
B. Isopentane cooled by liquid nitrogen
- Isopentane is liquid at room temperature
C. Carbon dioxied gas
D. Aerosol sprays
- is adequate for freezing small pieces of tissue except muscle

PROCESSING OF TISSUES
1. Fixation F
2. Dehydration D
3. Clearing
4. Infiltration (Impregnation)
5. Embedding
q
4. Frozen Section
- normally utilized when rapid diagnosis of the tissue in question is required 6. Trimming E
- recommended when lipids and nervous tissue elements are to be demonstrated 7. Section-Cutting T
- Tissue thickness: 10-15µ 8. Staining
- frozen in a microtome with CO2 or on a Cryostat (a cold chamber) kept at an atmospheric 9. Mounting SC
temperature of -10° to -20℃ 10. Labelling
- processed for light microscopic study
- Are commonly used for:
A. Rapid pathologic diagnosis during surgery
B. Diagnostic and research enzyme histochemistry
¥
C. Diagnostic and research demonstration of soluble substances such as lipids and
carbohydrates
D. Immunofluorescent and immunohistochemical staining
E. Some specialized silver stains, particularly in neuropathology

- Most commonly used methods of freezing:

Histopathology 6
CHAPTER 3 : FIXATION AND FIXATIVES B. Non-additive Fixation
- fixing agent is not incorporated into the tissue, but alters the tissue composition and stabilizes
Histotechnology
the tissue by removing the bound water attached to H-bonds of certain groups within the
- the art and science performed by the histotechnologist to produce a tissue section of good
protein molecule
quality that will enable the pathologist to diagnose the presence or absence of disease
- Examples: alcoholic fixatives

FIXATION
MAIN FACTORS INVOLVED IN FIXATION
- first and most critical step in histotechnology
1. Hydrogen Ion Concentration
- involves fixing or preserving fresh tissue for examination
- occurs between pH 6-8
- inadequate or poor fixation will result in a poorly processed tissue and will make it difficult for
the pathologist to render a proper diagnosis
2. Temperature
- Primary Aim : to preserve the morphologic and chemical integrity of the cell in as life-like a
Surgical specimens Room temperature
manner as possible
Electron microscopy 0-4℃, although some cells such as mast cells
- Secondary Aim : to harden and protect the tissue from the trauma of further handling, so
that it is easier to cut during gross examination are best fixed at room temperature
Nucleic acids Do not react with fixatives to any extent at
room temperatures, and chemical reactions
Purpose:
including those involved in fixation are more
1. To preserve the tissue.
rapid at higher temperatures
- by stopping all cellular activities so that the cells can be viewed under the microscope as if
they are still in their original living state.
Bacteriology & blood films Use of heat fixation
- the tissue must be fixed as soon as possible after removal from the body.
Very urgent biopsy specimen Formalin heated at 60℃ for rapid fixation
- Leaving a tissue specimen in air : will cause it to dry out and will result in distortion of its
morphologic appearance Tissues with tuberculosis Formalin heated at 100℃
- Leaving a tissue in a hypotonic solution : will cause the cell to swell
- Leaving the tissue in a hypertonic solution : will cause the cell to shrink 3. Thickness of Section
- measurements should not be compromised in order to obtain full penetration and
2. To prevent breakdown of cellular elements. satisfactory fixation
- each cell has a structure called lysosome (“suicide sac”) containing hydrolytic enzymes that Electron microscopy 1-2mm2
are released when the integrity of the cell is destroyed. Light microscopy no more than 0.4 cm to 2cm2
- Postmortem decomposition (“autolysis”) occurs due to the action of these hydrolytic Large solid tissue (uterus) opened or sliced thinly
enzymes Brain suspended whole in 10% buffered formalin for
- fixation prevents autolysis by inactivating the lysosomal enzymes, or by chemically altering, 2-3 weeks
stabilizing, and making the tissue components insoluble
- fixation protects the tissue from further decomposition (“putrefaction”) after death due to 4. Osmolality
bacterial or fungal colonization and overgrowth - hypertonic solution : cell shrinkage
- isotonic and hypotonic fixatives : cell swelling and poor fixation
3. To coagulate or precipitate protoplasmic substances. - best results are obtained at 400-450 mOsm (slightly hypertonic solutions); 340 mOsm (isotonic
- Two basic mechanisms involved in fixation: solution)
A. Additive Fixation - Sucrose : commonly added to osmium tetroxide fixatives for electron microscopy
- chemical constituent of the fixative is taken in and becomes part of the tissue by forming - the vehicle osmolality is generally more important than the total osmolality of the fixative,
cross-links or molecular complexes and giving stability to the protein and should be more or less isotonic with tissues in their normal living environment (for
- Examples: formalin, mercury, osmium tetroxide glutaraldehyde fixatives should be more or less 300 mOsm)

Histopathology 7
5. Concentration Types of Fixatives
- Formaldehyde (10%) I. According to COMPOSITION
- Glutaraldehyde (3%) A. Simple Fixatives - made up of only one component substance
- Buffer : causes polymerization of the aldehyde, with consequent decrease in its effective B. Compound Fixatives - made-up of two or more fixatives
concentration
- 0.25% glutaraldehyde : ideal for immuno-electron microscopy II. According to ACTION
A. Microanatomical Fixatives - permit the general microscopic study of tissue structures
6. Duration of Fixation * 10% Formol Saline
- Primary fixation in buffered formalin : carried out for 2-6 hours during the day the specimen is * 10% Neutral Buffered Formalin
obtained, but may remain in fixative over the weekend without much adverse effect * Heidenhain’s Susa
- Electron microscopy : diced tissues should be fixed for 3 hours and then placed in a holding * Formol Sublimate (Formol Corrosive)
buffer * Zenker’s Solution
- prolonged fixation may cause shrinkage and hardening if tissue, and may severely inhibit * Zenker-Formol (Helly’s Solution)
enzyme activity and immunological reactions * Bouin’s Soluton
* Brasil’s Solution
Practical Consideration of Fixation
1. Speed. Should be placed in fixative as soon as the specimen is removed from the body to B. Cytological Fixatives - preserve specific parts and particular microscopic elements
prevent autolysis and putrefaction of the cell itself (usually contain glacial acetic acid)
2. Penetration. Formalin diffuses into the tissue at the rate of approximately 1 mm per hour and 1. Nuclear Fixatives - preserve the nuclear structures
slows down as it goes deeper * Flemming’s Fluid
3. Volume. 20 times the tissue volume. * Carnoy’s Fluid
4. Duration of Fixation. Fibrous organs such as uterus or intestinal tract take longer that small or * Bouins’s Fluid
loosely textured tissues such as biopsies or scrapings. Fixation time can be cut down by using * Newcomer’s Fluid
heat, vacuum agitation, or microwave * Heidenhain’s Susa

2. Cytoplasmic Fixatives - preserve cytoplasmic structures (must never contain

Miscellaneous Consideration glacial acetic acid)
- Fixation is done slowly over one day or more * Flemming’s Fluid without acetic acid
- To maintain an adequate fixation time of 4-6 hours,the recommended size of the tissue is * Helly’s fluid
2cm2, and no more than 4mm thick * Formalin with “post-chroming”
- Brain : must be fixed before “grossing” or sectioning * Regaud’s Fluid (Muller’s fluid)
- Refrigeration : used to slow down decomposition * Orth’s Fluid
- Brain Cells : deteriorate very quickly
- Bone marrow : undergo mitosis (growth) up to 30 minutes after death when refrigerated 3. Histochemical Fixatives - preserve the chemical constituents of cells and
tissues
* Formol Saline (10%)
* Absolute Ethyl Alcohol
* Acetone
* Newcomer’s Fluid

Histopathology 8
Aldehydes
- satisfactory for routine paraffin sections, for electron microscopy, and when histochemical
and enzyme studies are indicated
A. Formaldehyde (10% Formalin)
A1. 10% Formol-Saline
A2. 10% Neutral Buffered Formalin or Recommended for preservation and storage
Phosphate-Buffered Formalin (pH 7) of surgical, post-mortem, and research
specimens
One of the most widely known fixatives
Best fixative for tissues containing iron
Formaldehyde is a gas produced by the pigments and for elastic fibers
oxidation of methyl alcohol, and is soluble in
water to the extent of 37-40% weight in Fixation time : 4-24 hours
volume
A3. Formol-Corrosive (Formol-Sublimate) Recommended for routine post-mortem
tissues
Usual fixation time : 24 hours
Fixation time : 3-24 hours
Buffered to pH 7 with phosphate buffer
A4. Alcoholic Formalin (Gendre’s Fixative) Fixes and dehydrates at the same time
Recommended for nervous tissue
Good for preservation of glycogen and for
preservation and for colored tissue
micro-incineration technique
photography
Used to fix sputum, since it coagulates mucus
Used for mailing specimen
B. Glutaraldehyde
- made up of two formaldehyde residues linked by three carbon chains
Does not require washing out
- recommended for histochemistry and electron microscopy
White paraformladehyde precipitates may
2.5% solution : used for small tissue fragments and needle biopsies fixed in 2-4 hours at room
be removed by filtration or by addition of 10%
temperature
methanol
Simple microanatomical fixative
4% solution : recommended for larger tissues less than 4 mm. thick, fixed in 6-8 hours up to 24
hours
Recommended for fixation of central nervous
tissues and general post-mortem tissues for
histochemical examination

Fixation time:
24 hours at 35℃(95ºF)
48 hours at 20-25℃ (65-77ºF)

Histopathology 9
Metallic Fixatives
A. Mercuric Chloride Most common metallic fixative, frequently
used in saturated aqueous solutions of 5-7%
Widely used as a secondary fixative
Routine fixative of choice for preservation of
cell detail in tissue photography
Recommended for renal tissues, fibrin,
connective tissues and muscles
De-zenkerization : process of removing
mercuric deposits by immersing tissues in
alcoholic iodine solution prior to staining
Mercury deposits → bring slides to water →
treating the section with 0.5% iodine solution
in 70% ethanol for 5-10 minutes → rinsed in
water for 5 minutes → decolorized for 5
minutes in 5% sodium thiosulfate → washed in
running water → proceed with required water
soluble stain
Add glacial acetic acid = Zenker’s solution
Add strong formaldehyde = Helly’s solution
A1. Zenker’s Fluid Glacial acetic acid has been added just
before its use to prevent turbidity and
formation of a dark precipitate
Recommended for trichrome staining
Fixation time : 12-24 hours
A2. Zenker-formol (Helly’s solution) Excellent microanatomic fixative for pituitary
gland, bone marrow and blood containing
organs such as spleen and liver
Fixation time : 12-24 hours
Histopathology
A3. Heidenhain’s Susa Solution
A4. B-5 Fixative
B. Chromate Fixatives
B1. Chromic Acid
B2. Potassium dichromate
B3. Regaud’s (Muller’s) Fluid
B4. Orth’s Fluid
Recommended mainly for tumor biopsies
especially of the skin
Excellent cytologic fixative
Fixation time : 3-12 hours
Commonly used as cytologic fixative for bone
marrow biopsies
Fixation time : 1 1/2 - 2 hours
Used in 1-2% aqueous solution
Strong oxidizing agent; hence a strong
reducing agent must be added
Used in 3% aqueous solution
Fixes but does not precipitate cytoplasmic
structures; preserves mitochondria
Preserves lipids
Recommended for demonstration of
chromatin, mitochondria, mitotic figures,
Golgi bodies, RBC and colloid-containing
tissues
Fixation time : 12-48 hours
Recommended for study of early
degenerative processes and tissue necroses
Demonstrates rickettsiae and other bacteria
Fixation time : 36-72 hours
10
C. Lead Fixatives Used in 4% aqueous solution Ethyl Alcohol Used at concentrations of 70-100%

Recommended for acid Used for histochemistry, especially enzyme

mucopolysaccharides studies
Picric Acid Fixatives
- normally used in strong saturated aqueous solution May be used as a simple fixative, however,
- yellow color may be removed by treatment with another acid dye or lithium carbonate more frequently incorporated into compound
- excellent fixative for glycogen demonstration fixatives for better results
- suitable for Aniline stains
Bouin’s Solution Recommended for fixation of embryos and Fixation time : 18-24 hours
pituitary biopsies
Carnoy’s Fluid Recommended for fixing chromosomes,
lymph glands and urgent biopsies
Does not need washing out
Used to fix brain tissue for the diagnosis of
Fixation Time : 6-24 hours
rabies

Most rapid fixative

Brasil’s Alcoholic Picroformol Fixative
Excellent fixative for glycogen
Preserves Nissl granules
Overnight tissue fixation by automatic
Fixation time : 1-3 hours
processing technique may utilize 3-4 changes
Newcomer’s Fluid Recommended for fixing
at 1/2 to 2 hours each, succeeded directly by
mucopolysaccharides and nuclear proteins
absolute alcohol
Fixation time : 12-18 hours @ 3℃

Glacial Acetic Acid Osmium Tetroxide (Osmic Acid)

- normally used in conjunction with other fixatives to form a compound solution - pale yellow powder which dissolves in water (up to 6% at 20℃) to form a strong oxidizing
- solidifies at 17℃ solution
- fixes and precipitates nucleoproteins - causes complete denaturation of protein
- very useful in the study of nuclear components of the cell Flemming’s Solution Most common chrome-osmium acetic acid
- it is an essential constituent of most compound nuclear fixatives fixative used
Alcohol Fixatives
Fixation time : 24-48 hours
- used both as a fixative and dehydrating agent
- excellent for glycogen preservation
Flemming’s Solution without acetic acid Recommended for cytoplasmic structures
- preserves nuclear stains
particularly the mitochondria
Methyl Alcohol Excellent for fixing dry and wet smears, blood
smears, and bone marrow tissues
Fixation time: 24-48 hours
Fixes and dehydrates at the same time Trichloroacetic Acid
Isopropyl Alcohol (95%) Used for fixing touch preparations - incorporated into compound fixatives

Histopathology 11
Acetone Washing out Process of removing excess fixative from the
- used at ice cold temperature ranging from -5℃ to 4℃ tissue after fixation
- recommended for the study of water diffusible enzymes especially phosphatases and lipases
Heat Fixation Tap water
- used to remove:
- involves thermal coagulation of tissue proteins for rapid diagnosis
a. Excess chromates from tissues fixed in
Helly’s, Zenker’s and Flemming’s solution
b. Excess formalin
c. Excess osmic acid
50-70% alcohol
- used to wash out excess amount of picric
Secondary Fixation Process of placing an already fixed tissue in a acid (Bouin’s solution)
second fixative
Alcoholic iodine
May be done before dehydration and on - to remove excessive mercuric fixatives
deparaffinized sections before staining Lipid Fixation
Cryostat or Frozen sections Used for demonstrating lipid in tissues
10% formalin or 10% formol saline is usually the Fixatives containing mercuric chloride and Can be effective for preservation of lipids in
primary fixative potassium dichromate cryostat sections
Post-chromatization Form of secondary fixation whereby a Aldehydes Used to fix phospholipids which contain
primarily fixed tissue is placed in aqueous amino groups
solution of 2.5-3% potassium dichromate for 24 Baker’s formol calcium May be used to preserve phospholipids
hours to act as mordant Imidazole osmium tetroxide Used for post-fixing to achieve improved
ultrastructural demonstration of lipids
Digitonin Fixative used for the ultrastructural
demonstration of cholesterol

Carbohydrate Fixation
Alcoholic Fixatives Recommended for glycogen fixation

Alcoholic formaldehyde Better fixative in human skin than neutral

buffered formaldehyde
Protein Fixation
Neutral buffered formol saline or Most commonly used fixatives for amino acid
Formaldehyde vapor histochemistry

Histopathology 12
Glycogen Fixation
Alcohol-based fixatives
Mixture of Fixatives
- two aldehyde fixative mixtures
- used for electron cytochemistry
Karnovsky’s paraformaldehyde-
glutaraldehyde
Acrolein
Microwave Technique Works as a physical agent similar in
Most useful fixatives for preserving glycogen mechanism to vacuum, oven (heat) and
agitation to increase the movement of
Rossman’s fluid molecules and accelerate fixation
Cold absolute alcohol
Used to accelerate staining, decalcification,
immunohistochemistry and electron
microscopy
Best known aldehyde fixative mixtures
Oscillation at a frequency of 2450 mHz
Aldehyde mixture with glutaraldehyde or
Allows light microscopic techniques used in
formaldehyde
routine histopathology to be performed
adequately
Penetrates tissues rapidly, preserves
morphology and enzyme activity at low
Non-chemical technique that is useful in
concentrations
preserving neurochemical substances in
brain, such as acetylcholine

Preparation of Tissue:
Tissue less than 1mm. thick
Ethanol (1 change, 5 minutes) → Isopropanol
(1 change, 3 minutes) → Paraffin (2 changes,
1 @ 67℃ for 2 minutes; 2 @ 82℃ for 5 minutes)

Tissue 1-2mm thick

Ethanol (15 minutes) → Isopropanol (15
minutes) → Paraffin (1 @ 67℃ for 10 minutes; 2
@ 82℃ for 12 minutes)

Tissue 2-5 mm thick

Immunofluorescence Techniques
Enzyme Histochemistry
Ethanol (60 minutes) → Isopropanol (45
minutes) → Paraffin (1 @ 67℃ for 30 minutes; 2
@ 82℃ for 60 minutes)
Commonly used in pathology for the
demonstration of various antibodies
To preserve the maximum enzyme activity at
its original localization

Histopathology 13
Electron Microscopy Most useful primary fixatives:
a. Osmium tetroxide
b. Glutaraldehyde
c. Paraformaldehyde

Difficulties Encountered Because of Improper Fixation

Difficulties Cause
Failure to arrest early autolysis of cells Failure to fix immediately (the tissue was
probably allowed to dry before fixing)

Insufficient fixative
Removal of substances soluble in fixing agent Wrong choice of fixative
Presence of artefact pigments on tissue Incomplete washing of fixative
sections
Tissues are soft and feather-like in consistency Incomplete fixation
Loss or inactivation of enzymes needed for Wrong choice of fixative
study

Shrinkage and swelling of cells and tissue Overfixation

structure
Tissue blocks are brittle and hard Prolonged fixation

Histopathology 14
CHAPTER 4 : DECALCIFICATION
Decalcification Procedure whereby calcium or lime salts are
removed from tissues following fixation
Carried out by acids to form soluble calcium
salts, or with chelating agents that bind to
calcium ions
Should be done after fixation and before
impregnation
Microcalcifications Appear as dark purple granular masses with
lighter purple halos after hematoxylin staining
Acid Decalcifying Agents
- most widely used agents for routine decalcification of bony tisses
A. Nitric Acid
- most common and the fastest decalcifying agent
Aqueous Nitric Acid Solution (10%) Recommended for urgent biopsy, and for
needle small biopsy
C. Formic Acid -
organic acid
- moderate acting decalcifying agent
- recommended for routine decalcification of postmortem research tissues
- used both as a fixative and decalcifying agent
- recommended for small pieces of bones and teeth
- suitable for most routine surgical specimens, particularly when immmunohistochemical
staining is needed
Decalcification Time : 2-7 days
Formic Acid-Sodium Citrate Solution Recommended for autopsy materials, bone
marrow, cartilage and tissues studied for
research purposes
Decalcification Time : 3-14 days
D. Trichloroacetic Acid Decalcification Time : 4-8 days
E. Sulfurous Acid Very weak decalcifying solution suitable only
for minute pieces of bone
F. Chromic Acid (Flemming’s Fluid) Used both as a fixative and decalcifying
agent

Decalcification Time : 12-24 hours For decalcifying minute bones

Formol Nitric Acid Recommended for urgent biopsies
Environmental toxin and carcinogenic
Decalcification Time : 1-3 days G. Citric Acid-Citrate Buffer Solution (pH 4.5) Decalcification Time : 6 days
Perenyi’s Fluid Recommended for routine purposes
Chelating Agents
- substances which combine with calcium ions and other salts to form weakly dissociated
Decalcifies and soften tissues at the same
complexes and facilitate removal of calcium salt
time
Ethylene diamine tetraacetic acid (EDTA) salt Most common chelating agent in the market
Commercial Name: Versene
Decalcification Time : 2-7 days
Recommended only for detailed microscopic
Phloroglucin-Nitric Acid Most rapid decalcifying agent
studies
recommended for urgent works
Very slow decalcifying agent
Decalfication Time : 12-24 hours
B. Hydrochloric Acid
Decalcification Time:
- produce good nuclear staining
1-3 weeks (small specimens)
- 1% solution with 70% alcohol : may be recommended for surface decalcification of tissue
6-8 weeks or longer (totally decalcify dense
blocks
cortical bone
Von Ebner’s Fluid Recommended for teeth and small pieces of
bone

Histopathology 15

Ion Exchange Resin
Electrophoresis (Electrical Ionization)
Factors Influencing the Rate of Decalcification
20 to 1
Room temperature range of 18℃ to 30℃
24-48 hours
14 days or longer
Measuring the Extent of Decalcification
Physical or Mechanical Test
Hastens decalcification by removing calcium
ions from formic acid-containing decalcifying
solutions
Not recommended for fluids containing
mineral acids such as nitric acid or
hydrochloric acid
Degree of decalcification may then be
measured by physical or X-ray method
Process whereby positively charged calcium
ions are attracted to a negative electrode
and subsequently removed from the
decalcifying solution
Utilizes electricity and is dependent upon a
supply of direct current to remove the
calcium deposits
Recommended ratio of fluid to tissue volume
for decalcification
Optimum temperature recommended
Ideal time required for decalcifying tissue
Required time for decalcifying dense bone
tissues in order to complete the process
Done by touching or bending the tissue with
the fingers to determine the consistency of
tissues
Chemical Method (Calcium Oxalate Test) Simple, reliable and convenient method
recommended for routine purposes, to
detect the presence of calcium in the
decalcifying solution
Involves the detection of calcium in acid
solutions by precipitation of insoluble calcium
hydroxide or calcium oxalate
Post-Decalcification After decalcification is complete, the acid
can be removed from tissues or neutralized
chemically yby immersing the decalcified
bone in either saturated lithium carbonate
solution or 5-10% aqueous sodium
bicarbonate solution for several hours
Tissue Softeners
Perenyi’s Fluid Act both as decalcifying agent and tissue
softener
4% Aqueous phenol solution For 1-3 days may also cause considerable
tissue softening
Others Moliflex
2% Hydrochloric acid
1% Hydrochloric acid in 70% alcohol

Mechanically done by pricking the tissue with

X-ray or Radiological Method
a fine needle or a probe
Very expensive although the most ideal, most
sensitive and most reliable method of
determining extent of decalcification due to
its ability to detect even the smallest focus of
calcium which appears opaque in an X-ray
plate

Not recommended for mercuric chloride-

fixed tissues

Histopathology 16
CHAPTER 5 : DEHYDRATION
Dehydration Process of removing intercellular and
extracellular water from the tissue following
fixation and prior to wax impregnation
Fixed specimen → 70% ethyl alcohol in water
→ 95% ethyl alcohol → 100% ethyl alcohol
Note: Amount in each stage should not be
less than 10x the volume of the tissue
Delicate tissues : dehydration starting 30%
ethanol is recommended
Dehydrating Agents
- solutions utilized to make dehydration possible
A. Alcohol
- most common dehydrating agent
Ethyl Alcohol Alcohol recommended for routine
dehydration of tissues
Best dehydrating agent
Methyl Alcohol Toxic dehydrating agent
Primarily employed for blood and tissue films
and for smear preparations
Butyl Alcohol Utilized in plant and animal micro-techniques
C. Dioxane (Diethylene Dioxide)
D. Cellosolve (Ethylege glycol monoethyl
ether)
E. Triethyl Phosphate
F. Tetrahydrofuran (THF)
Excellent dehydrating and clearing agent
readily miscible in water, melted paraffin,
alcohol, and xylol
Graupner’s Method
(1st) Pure dioxane solution - 1 hour
(2nd) Pure dioxane solution- 1 hour
(3rd) Pure dioxane solution - 2 hours
(1st) Paraffin wax - 15 minutes
(2nd) Paraffin wax - 45 minutes
(3rd) Paraffin wax - 2 hours
Embed in mold and cool in water
Dehydration Period : 3-24 hours
Combustible at 110-120ºF
Removes water very readily and produces
very little distortion and hardening of tissue
Used to dehydrate sections and smears
following certain stains
Reagent that both dehydrates and clears
tissues

Slow dehydrating agent

Recommended for tissues which do not

require rapid processing
B. Acetone Cheap, rapid-acting dehydrating agent
utilized for most urgent biopsies which it
dehydrates in 1/2 to 2 hours

Histopathology 17
CHAPTER 6 : CLEARING Aniline Oil
A
Recommended for clearing embryos, insects
and very delicate specimens
Clearing Process whereby alcohol or dehydrating
Clove Oil Causes minimum shrinkage of tissues,
agent is removed from the tissue and C
however, its quality is not guaranteed due to
replaced with a substance that will dissolve
its tendency to become adulterated
the wax with which the tissue is to be
Carbon Tetrachloride May be used in clearing tissues for
impregnated or the medium on which the
tissue is to be mounted C embedding
Glycerin and Gum Syrup Used when the tissue is to be cleared directly
Properties are very similar to chloroform
from water, as in a frozen section
although it is relatively cheaper
Xylene Most commonly used in histology laboratories
Methyl Benzoate and Methyl Salicylate M Used when double embedding techniques
✗ Most rapid clearing agent
are required

Clearing Time : 1/2 to 1 hour

Toluene Substitute for xylene or benzene for clearing
both during embedding and mounting
T processes

Clearing Time : 1-2 hours

Benzene Preferred by some as clearing agent in the

B embedding process of tissues because it

penetrates and clears tissues rapidly

Recommended for urgent biopsies (15-60

minutes)
Chloroform Recommended for routine work (6-24 hours)

:
Recommended for tough tissues, for nervous
tissues, lymph notes and embryos

Suitable for large tissue specimens

Cedarwood Oil Used to clear both paraffin and celloidin
sections during embedding process

Recommended for central nervous system

tissues and cytological studies

Clearing Time : 2-3 days

Clears celloidin in 5-6 days

Histopathology 18
CHAPTER 7 : IMPREGNATION AND EMBEDDING
Impregnation (Infiltration)
Embedding (Casting or Blocking)
Embedding Medium
Paraffin Wax Impregnation
Paraffin
Histopathology
Paraffin Oven/Incubator Regulated at 55-60℃
Common Waxes Melting Point 56℃ wax
Process whereby the clearing agent is
- normally used for routine work
completely removed from the tissue and
replaced by a medium that will completely fill
54-58℃ wax
all the tissue cavities
- indicated in a laboratory with temperature
20-24℃
Gives a firm consistency to the specimen
50-54℃ wax
Allows easier handling and cutting of suitably
- indicated if the laboratory temperature is
thin sections without any damage or
between 15-18℃
distortion to the tissue and its cellular
Precautions Paraffin oven must be maintained at a
components
temperature 2-5℃ above the melting point of
Process by which the impregnated tissue is
paraffin to be used for impregnation
placed into a precisely arranged position in a
mold containing a medium which is then
Paraffin wax must be pure (free from dust,
allowed to solidify
water droplets, and other foreign matter)
The medium used to infiltrate the tissue and
the same medium utilized for embedding
Fresh wax should be filtered before use in a
wax oven at a temperature 2℃ higher than its
Simplest, most common and best embedding melting point
medium used for routine tissue processing
Wax that has been trimmed away from the
Very rapid medium, allowing sections to be impregnated tissue may be melted and
prepared withing 24 hours filtered for future use, with coarse filter paper
(e.g. Green’s No. 904)
Tissue blocks and unstained mounted sections
may be stored in paraffin for indefinite period When was has been reused, some amount of
of time after impregnation without water inevitably is mixed with it. Water must
considerable tissue destruction therefore be removed by heating the wax at
100-105℃ thereby raising the melting point.
Not recommended for fatty tissues
Paraffin wax may be used only twice
For fixed knife microtomes, a relatively hard
wax with a higher melting point is
recommended
19
By Manual Processing At least four changes of wax is required at 15 Elliot Bench-Type Processor
minutes intervals in order to insure complete - Makes use of 12 individual processing steps, with ten 1-liter capacity glass beakers and two
removal of the clearing agent from the tissue thermostatically controlled wax baths with a safety device cut-out switch to protect the wax
Example Time Schedule (Manual Processing): against over-heating. A transfer arm moves the tissues from one processing reagent to
another. An electrical clock serves to control the time needed for each processing step.
Fixation :
10% Buffered Formalin - 24 hours Precautions:
Dehydrating fluids should be changed frequently since dehydration is the most critical stage
Dehydration (Increasing Grades of Alcohol) : of tissue processing and inadequate dehydration is difficult to correct once the tissue is in
70% Alcohol - 6 hours paraffin.
95% Alcohol - 12 hours
100% Alcohol - 2 hours The clearing agent and the dilute ethanols should be changed at least once a week.
100% Alcohol - 1 hour
100% Alcohol - 1 hour Wax bath thermostats should be set at least 3 degrees above the melting point of the wax.
Vacuum Embedding Wax impregnation under negative
Clearing atmospheric pressure inside an embedding
Xylene or Toluene - 1 hour oven to hasten removal of air bubbles and
Xylene or Toluene - 1 hour clearing agent from the tissue block thereby
promoting a more rapid wax penetration of
Impregnation tissue
Paraffin wax - 15 minutes
Paraffin wax - 15 minutes Recommended for urgent biopsies, for
Paraffin wax - 15 minutes delicate tissues such as lung, brain,
Paraffin wax - 15 minutes connective tissues, decalcified bones, eyes,
spleen and central nervous system
Embedding
Paraffin wax - 3 hours The time required for complete impregnation
is thereby reduced from 25-75% of the normal
time required for tissue processing

Gives the fastest result

By Automatic Processing Method which makes use of an automatic
tissue processing machine (e.g.
Autotechnicon) which fixes, dehydrates,
clears and infiltrates tissues

Only 2-3 changes of wax is required

Histopathology 20
Vacuum Embeding Oven
- Consists of flat-bottomed heavy brass chamber covered with a heavy glass lid resting on a
wide and thick rubber valve which produces an airtight seal when the chamber is being used.
The thermostatically controlled water jacket is usually maintained at a temperature of 2-4℃
above the melting point of the wax. There are two screw valves ; one valve allows the
readmission of air when the bath is under negative pressure and the other valve us connected
ti a tube which is in turn connected to a suction pump, to allow exhaustion of 400-500 mm
inside. A stopcock prevents water from being sucked back into the trap bottle and vacuum
chamber when the water or suction pump is closed.
Substitutes for Paraffin Wax
Paraplast Mixture of highly purified paraffin and
synthetic plastic polymers
Melting point : 56-57℃
Embeddol Synthetic wax substitute similar to paraplast
Melting point : 56-58℃
Bioloid Semi-synthetic wax recommended for
embedding eyes
Tissue Mat Product of paraffin, containing rubber, with
the same property as paraplast
Ester Wax Cellosolve (ethylene glycol monoethyl ether)
or Xylene : may be used as clearing agent
Sectioning must be done on a heavy duty
microtome (e.g. Sliding or Sledge type
microtome)
Melting point : 46-48℃
Water Soluble Waxes Mostly polyethylene glycol
Melting point : 45-46℃
Carbowax : most commonly used which
appears solid at RT
(Carbowax 70% for 30 minutes → Carbowax
90% for 45 minutes → Carbowax 100% for 1
hour → Carbowax 100% for 1 hour) all at a
temperature of 56℃
Histopathology
Celloidin Impregnation
Celloidin (Collodion) Purified form of nitrocellulose soluble in many
solvents, suitable for specimens with large
hollow cavities which tend to collapse
For hard and dense tissues such as bones and
teeth and for large tissue sections of the
whole embryo
Thin : 2%
Medium : 4%
Thick : 8%
(dissolved in equal parts of ether and alcohol)
Recommended for processing neurological
tissues
Recommended in cases when minimum
shrinkage is required and frozen section
techniques cannot be done
Wet Celloidin Method Recommended for bones, teeth, large brain
sections and whole organs
Placed in equal parts of ether and alcohol for
12-24 hours
Tissue → Thin celloidin (2-4%) for 5-7days →
Medium celloidin (4-6%) for another 5-7 days
→ Drained off → Thick celloidin (8-12%) until
the specimen has become impregnated,
usually between 3-5 days
When the ball of the finger leaves no mark on
the surface of the tissue block, evaporation
and embedding is considered to be
complete
Storage : 70-80% alcohol until ready for
cutting
21
Dry Celloidin Method
Nitrocellulose Method (Low Viscosity
Nitrocellulose LVN)
Gelatin Impregnation
Gelatin Impregnation
Histopathology
Preferred for processing of whole eye sections
Same principle with that of Wet Celloidin
Method, except that 70% alcohol is not used
for storage, instead Gilson’s mixture.
Gilson’s Mixture
- made up of equal parts of chloroform and
cedarwood oil, added to the celloidin block
before hardening, to make the tissue
transparent
Another form of celloidin soluble in equal
concentration of ether and alcohol, with a
lower viscosity, allowing it to be used in higher
concentrations and still penetrate tissues
rapidly
Rarely used except when dehydration is to be
avoided and when tissues are to be
subjected to histochemical and enzyme
studies
Used as an embedding medium for delicate
specimens and frozen tissue sections
10% gelatin with 1% phenol for 24 hours → 20%
gelatin with 1% phenol for the next 12 hours →
20% gelatin with 1% phenol → Tissue is
refrigerated until impregnation and
embedding are completed → 10% formalin
for 12-24 hours
Tissues should not be more than 2-3 mm thick
1% Phenol : serves to prevent the growth of
molds
The volume of the impregnating medium
should be at least 25 times the volume of the
tissue
Embedding
Embedding Process after impregnation, wherein the tissue
is placed into a mold containing the
embedding medium and this medium is
allowed to solidify
Paraffin embedded tissues are immersed in
melted paraffin at temperature between 5-
10℃ above its melting point → Cooled rapidly
in a refrigerator at -5℃ or immersed in cold
water to solidify
Orientation Process by which a tissue is arranged in
precise positions in the mold during
embedding, on the microtome before
cutting, and on the slide before staining
The surface of the section to be cut should be
placed parallel to the bottom of the mold in
which it is oriented
Types of Blocking-out Molds
Leuckhart’s Embedding Mold Consists of two L-shaped strips of heavy brass
or metal arranged on a flat metal plate and
which can be moved to adjust the size of the
mold to the size of the specimen
Blocks produced are even, with parallel sides,
and with a fairly shaped initial setting of the
wax
Recommended for routine use, although, too
slow and cumbersome for use in a busy
laboratory
22
Compound Embedding Unit Made up of a series of interlocking plates Disposable Embedding Mold A. Peel Away
resting on a flat metal base, forming several - available in 3 different sizes, simply peeled
compartments off one at a time, as as the was has solidified,
giving perfect even block without trimming

Plastic Embedding Rings and Base Mold
Tissue Tek
Consist of a special stainless steel base mold
B. Plastic Ice Trays
fitted with a plastic embedding ring, which
- such as those used in ordinary refrigerators
later serves as the block holder during cutting
may be recommended for busy routine
Warm Plate : used to manage the
laboratories
impregnated specimen

Cold Plate (-5℃) : for rapid solidification of the

block

White Plastic Cassette Mold (with detachable,

perforated stainless steel hinge and snap-on
lid) : used hold the tissue specimen through-
out fixation, dehydration, clearing, and wax
impregnation

C. Paper Boats
- normally utilized for embedding celloidin
blocks but are equally useful for paraffin wax
blocks
- provide easy and accurate identification of
specimens

Histopathology 23
Other Embedding Methods
Cellodin or Nitrocellulose Method
Double-Embedding Method
Plastic Resin Embedding
Plastic Resin Embedding Media
Epoxy Embedding Plastics
Polyester Plastics
Histopathology
Acrylic Plastics Made up of esters of acrylic and methacrylic
Recommended for embedding hard tissues acid
such as bones and teeth, and for large
sections of whole organs like the eye Used extensively for light microscopy when
Process in which tissues are first infiltrated with high resolution is required
celloidin and subsequently embedded in
paraffin mass Polyglycol methacrylate (GMA)
- proved to be popular embedding medium
Used to facilitate cutting of large blocks of for light microscopy
dense firm tissues like the brain - extremely hydrophilic
Recommended for making small sections of Methyl methacrylate (MMA)
celloidin blocks - widely used because of its hardness as the
ideal embedding medium for undecalcified
bone and other hard tissues
Provided superior results for light microscopic
- used for routine and immunohistochemical
studies, particularly in hard tissues such as
staining
undecalcified bone and for high resolution
light microscopy of tissue sections thinner than
the usual 4-6 μm, such renal biopsies and
bone marrow biopsies
Made up of a carefully balanced mixtures of
epoxy plastic, catalysts, and accelerators
Types:
Araldite
- based on bisphenol A
- infiltration is slow
Epon
- based on glycerol
- has lower viscosity but are often sold as
mixtures of isomers
Spurr
- based on cyclohexene dioxide
- can be obtained pure
- have very low viscosity
- infiltrate fastest
Originally introduced for electron microscopy
in the mid-1950s, but have been superseded
by more superior epoxides, and are now
seldom used
24
CHAPTER 8 : MICROTOMY
Microtomy Process by which processed tissue, most commonly Rocking (Cambridge) Microtome
a paraffin embedded tissue, is trimmed and cut into
uniformly thin slices or “sections” to facilitate studies
under the microscope
Microtome Basic instrument used that is capable of cutting a
section at a predetermined thickness by sliding the
block into a cutting tool
Principle:
Spring-balanced teeth or pawl is brought into
contact with, and turns a ratchet feed wheel
connected to a micrometer screw, which is in turn
rotated, moving the tissue block at a predetermined
distances towards the knife for cutting sections at
uniform thickness
Three Essential Parts Block Holder
- where the tissue is held in position
Knife Carrier and Knife
- for actual cutting of tissue section
Pawl, Ratchet Feed Wheel and Adjustment Screws Rotary (Minot) Microtome
- to line up the tissue block in proper position with
the knife, adjusting the proper thickness of the tissue
for successive sections
Types of Microtomes
For cutting serial sections of large blocks of paraffin
embedded tissues
Invented by Paldwell Trefall in 1881
Simplest among the different types of microtomes
Consists of:
Heavy Base
Two Arms
Lower Arm : Resting on pivots and a supporting
column, and attached to the micrometer screw, at
the base of which is found the ratchet wheel with
feed mechanism
Upper Arm : Carrying the block holder on one end
by means of a screw, is connected to a lever by a
piece of nylon thread
Knife : Slightly curved plane
Invented by Minot in 1885-1886
Most common type used for both routine and
research laboratories, especially for sectioning
paraffin-embedded tissues

Electrically Driven Rotary Microtome

- ideally used when to produce ribbons for serial
sections

Knife : Perfectly flat plane

Histopathology 25
Sliding Microtome Developed by Adams in 1789 The Cryostat or Cold Microtome Refrigerated apparatus used in fresh tissue
microtomy, for freezing the tissue into the block
Most dangerous type of microtome due to the holder to the correct degree of hardness to
movable exposed knife facilitate easier and faster sectioning

Base-Sledge Microtome Microtome : usually Rotary microtome

- favored in laboratories where very hard tissue or
large blocks are usually sectioned The microtome us kept inside a cold chamber
- suited for sectioning specimens embedded in all maintained at a temperature between -5 to
forms of media, especially for cutting sections from -30℃ (average is -20℃)
tough tissues blocks
- modern models are electrically driven and are Provides a means of preparing thin sections of fresh
ideal for resin-embedded decalcified bone frozen tissues especially for fluorescent antibody
staining techniques or histochemical enzyme studies
Standard Sliding Microtome
- developed mainly for cutting celloidin embedded Most commonly used for rapid preparation of urgent
tissue blocks tissue biopsies for intraoperative diagnosis

Knife :
Oblique - for celloidin sections
Straight - large refractory paraffin blocks

Ultrathin Microtome Primarily used for cutting tissue sections at 0.5 micra,
for electron microscopy
Freezing Movement Invented by Queckett in 1848
Care for the Microtome
Used to cut undehydrated tissues in a frozen state, 1. All the accumulated paraffin and small pieces of tissues must be brushed away with a soft
especially in instances when rapid diagnosis is brush and not allowed to stay in the microtome
required, when histological demonstration of fat is 2. Parts should be wiped with xylol
needed, when certain neurological structures are to 3. Movable portions should be oiled thoroughly to prevent rusting
be studied, and when sensitive tissue constituents to 4. Microtome must always be covered when not in use
be studied are damaged or destroyed by heat Microtome Knives
Plane-Concave Knife Biconcave Knife Plane-Wedge Knife

Size Usually 25 mm in length Usually 120 mm in length Usually 100 mm in length

Description One side of the knife is Both sides are concave Both sides are straight
flat while the other is
concave

Histopathology 26
 Function Flat Side Recommended for Recommended for Minot or Plane-Wedge Knife
- recommended for cutting paraffin frozen sections or for - the knife turned over so as to sharpen the
cutting Celloidin- embedded sections cutting extremely hard other surface every 10-20 strokes
embedded tissue blocks and tough specimens
embedded in paraffin
blocks
Concave side Plane-Concave Knife
- used to cut paraffin - only the concave surface should be rubbed
sections on the hone
Type of Flat side Rotary Microtome Base-sledge or Sliding Hone A natural sharpening stone or hard grinding
Microtome - Sliding Microtome Microtome surface (Carborundum), which serves to
remove nicks and irregularities on the knife
Concave side edges
- Base-sledge, Rotary, or Types of Hones
Rocking Microtome Belgium Yellow For manual sharpening when cutting edge
Bevel Angle Angle formed between the cutting edges has been rendered blunt or nicked

Normally about 27-32º
Slide-on Back Spring-loaded semi-circular metal sheet on to
the knife with one or more plane surfaces to
hold the cutting edge at a constant, correct
angle during the process of honing and
stropping
Honing and Stropping
Honing (Hard Sharpening) Purpose : To remove irregularities from the
knife
This type usually gives the best result
Arkansas Gives more polishing effect than the Belgium
Yellow
Fine Carborundum Much coarser
Used only for badly nicked knives followed by
either one of the first two knife sharpeners
Mechanical Honing with Machines Make use of a vibrating frosted glass plate or
a wheel driven by an electrical motor

1. Coarse Honing Approximately 30 double strokes are given

- involves the removal of gross nicks of the
knife edge to remove blemishes
2. Honing Proper
- grinding the cutting edge of the knife on a
stone to acquire an even edge
EDGE FIRST, with a HEEL TO TOE direction
each side of the knife
Precautions During Honing:
1. Hone should be long enough (about 8” x 3”) to allow the whole length of the knife edge to
be sharpened
2. Wide enough to sufficiently support and prevent the rocking of the knife
3. Hone should be lubricated with warm soapy water or fine oil before using
4. Hone should be cleaned before, during, and after use

Histopathology 27
Stropping Purpose : To polish and sharpen the cutting Forceps (fine pointed or curved) and squirrel Needed for handling sections during cutting,
edge brush and for removing folds and creases on the
sections during “floating out” in water bath
Process whereby the “burr” formed during Clean Slides For routine work : 76 x 25 mm slides that are
honing is removed and the cutting edge of 1.0-1.2 mm thick
the knife is polished
Frost-ended slides are generally used
EDGE LAST, in a TOE TO HEEL direction Ice Tray
Pencil
40-120 double strokes are usually required
Precautions Observed in Stropping
1. Knife should always be wiped clean with a soft cloth before and after a series of stropping
strokes
2. After stropping is completed, the knife edge is the oiled in a grease to prevent it from rusting
3. Knife is kept covered in a suspension box to prevent the settling of dust and grit on its
surface
4. Knife should not be allowed to rest on its sides
5. Leather strops are usually dry and require oiling before they are used
6. Strops are usually treated with vegetable oil (e.g castor oil) applied into the back of the
strop, NOT the surfaces
7. Wax must not be allowed to come in contact with the strop
Disposable Blades Cheaper to use than conventional steel
knives
Glass Knives Used for trimming and semi-thin sectioning of
tissue blocks for electron microscopy
Diamond Knives Used to cut any type of resin block for
electron microscopy
Other Equipment For Sectioning
Water bath Temperature of the water should be about
10℃ below the melting point of the paraffin
wax

Small amount of detergent may be added to

reduce the surface tension and allow the
section to flatten out
Drying Oven or Hot Plate Temperature setting at the melting point of
the wax to allow sections to dry

For delicate tissue (brain), a lower drying

temperature is used to avoid splitting and
cracking of the section due to excessive heat

Histopathology 28
CHAPTER 9 : PARAFFIN SECTIONS Faults Occuring During Tissue Processing
Sectioning Process whereby tissues are cut into uniformly Faults Reason Remedy
thin slices or “sections” with the aid of a Brittle or Hard Tissue Prolonged fixation May be softened by soaking
machine, to facilitate the studies under the Prolonged dehydration in a small dish or bowl
microscope Prolonged clearing containing water with
Microtome Machine or instrument used for cutting Prolonged paraffin infiltration detergent, phenol, or Molliflex
sections Overheated paraffin oven
General Types of Tissue Sections Drying out of tissue before
Paraffin Sections For paraffin embedded tissue blocks which actual fixation
may be cut by rocking and rotary microtome Clearing agent turns milky as Water not completely Repeat dehydration with
Celloidin Sections For celloidin embedded tissues which are soon as tissue is placed in it removed due to incomplete absolute alcohol then clear
usually cut by means of the sliding microtome dehydration again
Frozen Sections Cut from tissues that have been fixed and On trimming, tissue smells Clearing agent not Block is trimmed down
frozen with CO2 or for fresh or fixed tissues clearing agent completely removed due to nearest to the tissue.
frozen with the cryostat insufficient impregnation Remaining wax is melted on
embedding oven and
Paraffin Sections paraffin impregnation is
Trimming Process in which excess wax is cut off from the repeated, changing the
block to expose the tissue surface in paraffin at least once before
preparation for actual cutting blocking
Setting of a Microtome Knife Sections are usually cut between 4-6μ in Tissue is opaque, section Insufficient clearing Repeat clearing; if has
thickness for routine histologic procedures cutting is difficult due to already been embedded,
presence of alcohol prolong clearing up to 12
Knife is usually tilted at 0-15º hours, then re-embed
Tissue shrinks away from wax Insufficient dehydration, thus Repeat the whole procedure
Biconcave knives require smaller clearance
when trimmed incomplete clearing and
angles than wedge-shaped knives
impregnation
Removing Paraffin Ribbons from the Knife Sections are removed in ribbons of ten to
Tissue is soft when block is Incomplete fixation Repeat the whole procedure
allow easy location of serial sections
trimmed
Floating Out Sections are floated out on a water bath set
Air holes found on tissue Incomplete impregnation Repeat impregnation
at 45-50℃, approximately 6-10℃ lower than
during trimming
the melting point of the wax used for
On trimming, wax appears Contaminated wax Re-embed in freshly filtered
embedding tissue
crystalline Block not cooled rapidly wax
enough
Sections should not be left on the water bath
Paraffin block, after cooling, Insufficient paraffin Repeat paraffin
for a long time (30 seconds will be enough) to
is moist and crumbles impregnation impregnation, then re-embed
avoid undue expansion and distortion of
tissue
Celloidin Sections
- usually cut between 10-15μ in thickness

Histopathology 29
Faults Observed During Section-Cutting
Faults Reason
Sections fail to form ribbons Surfaces and edges of the
block not parallel
Horizontal surface of the
block is not parallel to the
knife
Paraffin wax is too hard
Knife is tilted too much
Sections are too thick
Knife is dull
Sections roll up on cutting so Knife is blunt
that they adhere and get Tilt of knife is too great
broken against the knife Knife edge is dirty
edge
Ribbon is curved, crooked or Blunt of dull spot on the knife
uneven instead of straight Edges of the block are not
parallel but round or wedge
shape
Knife is not parallel to the
block
Paraffin is impure
Sections are compressed, Knife is blunt or dull
wrinkled, or jammed Paraffin block is warm and
soft
Knife edge is coated with
paraffin
Sections are too thin
Microtome set screw is loose
Tilt of knife is too vertical
Sections are squashed (width Bevel of knife is lost due to
of each section is less than incorrect sharpening
that of block)
Histopathology
A hole is formed in the Bubble or dirt formed in the Re-embed in freshly filtered
Remedy section embedding medium wax
Re-trim block Hard spot in tissue due to Once embedded in paraffin
calcium wax, decalcification is
Re-adjust and re-orient the impractical; use a base-
block sledge microtome with a
wedge knife
Coat horizontal edges of the Sections of unequal thickness Tilt of knife is too great or Reduce the tilt
block with wax of lower are produced bevel is not cleared
melting point Clamp set screw on knife or Tighten the screw
Reduce the tilt blockholder is loose
Readjust the thickness Blocks are too large Cut blocks into smaller
Hone and strop fragments
Sharpen the knife Blocks are too hard Soften the blocks in
Reduce the tilt detergent or phenol
Clean the knife edge Sections adhere to the knife Static electricity due to low Breath out or blow gently on
or other parts of the atmospheric humidity the block and knife to
Adjust the knife, or sharpen microtome breakup static electricity, or
Re-trim the block boil water in the room to
increase the humidity
Knife edge is dirty Clean the knife edge
Readjust knife and block Knife edge is dull Sharpen the knife
Knife tilt is too great Reduce the tilt
Repeat impregnation using Ribbon is split or lengthwise Nicks or damage on the knife Sharpen the knife
pure wax vertical scratches are seen edge
Re-sharpen the knife on sections Dirty embedding Re-embed in filtered wax
Cool the block on ice water Knife edge is dirty Clean knife edge with xylene
until firm Tilt of knife is too great Reduce the tilt
Clean the knife edge Sections are lifted from the Knife tilt is too great Reduce the tilt
knife on upstrokes Knife is dull Sharpen the knife
Readjust thickness of section Paraffin is too soft or room Cool paraffin wax in ice
Tighten the screw temperature is warm water
Reduce the tilt Resistance is felt on the lower Tilt of knife is too small Increase the tilt
Re-sharpen part of the section during
cutting
Horizontal or parallel lines or Knife edge vibrates due to Treat with phenol during
furrows across the section hardness of tissue processing or collodionize
(“Chatters”) are seen, forming Tilt of knife is too great Reduce the tilt
thin and thick zone
30
Section cut is sometimes thin,
sometimes thick
Knife makes a hard metallic
scraping or ringing sound on
backstroke, when section is
cut
Frozen tissue crumbles and
comes off the block holder
when out
Frozen tissue chips into
fragments when cut
Histopathology
Knife is blunt
Knife is not clamped properly
Tilt of knife is too great
Knife or block holder is loose
Knife tilt is too small
Tilt of knife is too slanted or
too big
Tissue is too hard
Knife blade is too thin
Freezing is not adequate
Tissue is frozen too hard
Sharpen knife
Adjust the knife
Reduce the tilt
Tighten adjusting and locking
screws
Increase the tilt
Readjust the angle of the
knife
Take fresh block treated with
phenol
Change the knife
Refreeze the tissue block
Warm the tissue with the
fingers
31
CHAPTER 10 : FROZEN SECTIONS
Frozen Section Method
Frozen Section is Commonly Used For:
Methods of Preparing Frozen Sections
Cold Knife Procedure
Cryostat Procedure
Most Commonly Used Method of Freezing
Liquid Nitrogen
Isopentane (cooled by liquid nitrogen)
Carbon dioxide gas
Aerosol Sprays
Histopathology
Normally utilized when a rapid diagnosis of
the tissue in question is required
Especially recommended when lipids and
nervous tissue elements are to be
demonstrated
1. Rapid pathologic diagnosis during surgery
2. Diagnostic and research enzyme
histochemistry
3. Diagnostic and research demonstration of
soluble substances such as lipids and
carbohydrates
4. Immunofluorescent and
immunocytochemical staining
5. Silver stains - for neuropathology
Knife : 40 to -60℃
Tissue : 5 to -10℃
Environment : 0 to -10℃
Cryostat is an apparatus used in fresh tissue
microtomy
Refrigerated chamber
- maintained at temperatures near -20℃
where microtome, knife, specimen and
atmosphere are kept at the same
temperature
Optimum working temperature : -18 to -20℃
Generally used in histochemistry and during
intra-operative procedures
Most rapid of the commonly available
freezing agents
Isopentane is liquid at room temperature
Adequate for freezing small pieces of tissue
except muscle
Mounting of Tissue Block
Synthetic water-soluble glycols and resins Generally used as mounting media for tissue
blocks that need to be sectioned on a
cryostat
O.C.T. (Optimal Cutting Temperature Especially recommended for cryostat
compound, Lab-Tek Products, Division of
Miles Laboratories, Inc.) -5 to -15℃ : for brain, lymph nodes, liver,
spleen, uterine curetting, soft cellular tumors
-15 to -25℃ : for non-fatty breast tissue, ovary,
prostate, tongue, and GI tract
-35℃ : for fatty breast and omental tissue
Cryostat is usually set at -18 to -20℃
Freezing Previously Fixed Tissue Cryostat is also recommended for any
technique requiring cold sectioning of fixed
material and for some special methods for
the nervous system
Care for the Cryostat Microtome
1. Cryostat should be left on at all times
2. Spare knife should always be kept inside the cryostat cabinet
3. Knife as well as the undersurface and edge of the anti-roll plate must be kept scrupulously
clean and dry
4. Soft tissue paper, either dry or moistened with absolute alcohol, may be used for cleaning
the knife and anti-roll plate
5. Cryostat should be defrosted during the weekend
Special Processing Techniques
Frozen Section Deemed to be the most ideal and preferred
means of preserving tissues in order to avoid
complete or partial loss of enzymes
consequent to chemical fixation
Quenching Principle of rapidly preserving the tissue block
by freezing
Liquid Nitrogen The freezing agent commonly employed
32
Freeze Drying Special way of preserving tissues by rapid
freezing of fresh tissue at -160℃ and
subsequently removing ice water molecules
(dessication) by a physical process of
transferring the still frozen tissue block in a
vacuum at a higher temperature
(sublimation) without the use of any chemical
fixative

This technique is restricted to specialized or

research laboratories

May be used for:

1. Immunocytochemistry
2. Fluorescent antibody studies of polypeptide
and polypeptide hormones
3. Autoradiography
4. Microspectrofluorimetry of autofluorescent
substances
5. Formaldehyde-induced fluorescence of
biogenic amines (to demonstrate 5-
hydroxytryptamine, adrenaline, noradrenaline
and other catecholamines)
6. Scanning electron microscopy
Freeze-Substitution Frozen tissue is fixed in Rossman’s formula or in
1% Acetone and dehydrated in absolute
alcohol

Histopathology 33
CHAPTER 11 : PRINCIPLES OF STAINING
Staining
Silver nitrate
Three Major Groups of Staining
Histological Staining
Histochemical Staining (Histochemistry)
Immunohistochemical Staining
Histopathology
Process of applying dyes on the sections to
see and study the architectural pattern of the
tissue and physical characteristics of the cells
Basic dyes
- for certain parts of cells and tissues that are
acidic in character
- Hematoxylin : Nucleus
Acid dyes
- for basic constituents of cells
- Eosin : Cytoplasm
Most commonly used agent for impregnation,
can also function as a staining agent
Process whereby the tissue constituents are
demonstrated in sections by direct interaction
with a dye or staining solution, producing
coloration of the active tissue components
Used to demonstrate the general relationship
of tissues and cells with differentiation of
nucleus and cytoplasm
Process whereby various constituents of
tissues are studied thru chemical reactions
that will permit microscopic localization of a
specific tissue substance
Perl’s Prussian Blue
- reaction for hemoglobin
Periodic Acid Schiff
- staining for carbohydrates
Combination of immunologic and
histochemical techniques that allows
phenotypic markers to be detected and
demonstrated under the microscope
Methods of Staining
Direct Staining Process of giving color to the sections by
using aqueous or alcoholic dye solutions
Indirect Staining Process whereby the action of the dye is
intensified by adding another agent or
mordant
Mordant
- serves as a link or bridge between the tissue
and the dye, to make the staining reaction
possible
- Examples:
Potassium alum with hematoxylin in Ehrlich’s
hematoxylin
Iron in Weigert’s hematoxylin
Tissue-mordant-dye Complex
- mordant combines with a dye to for a
colored “lake”, which in turn combines with
the tissue
Accentuator
- accelerates or hastens the speed of the
staining reaction by increasing the staining
power and selectivity of the dye
- Examples:
Potassium hydroxide in Loeffler’s methylene
blue
Phenol in carbol thionine and carbol fuschin
Progressive Staining Process whereby tissue elements are stained
in a definite sequence, and the staining
solution is applied for specific periods of time
or until the desired intensity of coloring of the
different tissue elements is attained
34
Regressive Staining The tissue is first overstained to obliterate the Cytoplasmic Stains
cellular details, and the excess stain is Red Yellow Green
removed or decolorized from unwanted parts Eosin Y Picric acid Light green SF
of the tissue, until the desired intensity of color Eosin B Orange G Lissamine green
is obtained Phloxine B Rose Bengal
Nuclear Stains
Red Blue
Neutral red Methylene blue
Differentiation (Decolorization) Safranin O Toluidine blue
- selective removal of excess stain from the Carmine Celestine blue
tissue during regressive staining in order that Hematoxyline
specific substance may be stained distinctly Metallic Impregnation Process where specific tissue elements are
from the surrounding tissues demonstrated, not by stains, but by colorless
solutions of metallic salts which are thereby
Alcohol reduced by the tissue, producing an opaque,
- acts as a differentiator for both basic and usually black deposits on the surface of the
acidic dyes tissue or bacteria
Metachromatic Staining Entails the use of specific dyes which
differentiate particular substances by staining Amoniacal silver
them with a color that is different from that of - reduced by argentaffin cells (in melanin and
the stain itself (metachromasia) intestinal glands), forming black deposits seen
under the microscope
Employed for staining cartilage, connective
tissues, epithelial mucins, mast cell granules, Gold (gold chloride) and Silver (silver nitrate)
and amyloid - most valuable metals for this purpose
Vital Staining Selective staining of living cell constituents,
These are dyes belonging to the thizine and demonstrating cytoplasmic structures by
triphenylmethane groups: phagocytosis of the dye particle
1. Methyl violet or crystal violet (cytoplasmic phagocytosis)
2. Cresyl blue (for reticulocytes)
3. Safranin Trypan blue : for reticulo-endothelial system
4. Bismarck Brown
5. Basic fuschin True vital staining : staining of pre-existing
6. Methylene blue cellular components
7. Thionine
8. Toluidine blue Janus green : staining of mitochondria
9. Azure A, B, C
Counterstaining Application of a different color or stain to Death of the cell : demonstration of nuclear
provide contrast and background to the structures during vital staining suggests
staining of the structural components to be permeability of the membrane of the dye
demonstrated

Histopathology 35
Intravital Staining Done by injecting the dye into any part of the H & E Staining Technique
animal body (intravenous, intraperitoneal, or Routine Hematoxylin and Eosin (H&E) Staining Most common method utilized for
subcutaneous), producing specific coloration microanatomical studies of tissues, using the
of certain cells, particularly those of the regressive staining
reticulo-endothelial system H & E Staining of Frozen Sections for Rapid Harris hematoxylin (30-45 seconds)
Diagnosis (Progressive Staining) ↓
Common dyes: Lithium, carmine, India ink Tap water
Supravital Staining Stain living cells immediately after removal ↓
from the living body Blue in ammonia water (5 seconds)
↓
Neutral red : best vital dye Tap water
↓
Janus green : recommended for Counterstain with 5% aqueous eosin or 1%
mitochondria alcohol eosin (1 minute)
↓
Trypan blue : one gram of dye is dissolved in Tap water
100 ml of sterile distilled water to be used ↓
immediately Dehydrate in increasing grades of alcohol
↓
Nile blue, Thionine, Toluidine blue Clear with xylene
↓
Staining of Paraffin Sections
Mount
“Sections to Water” Paraffin wax should be removed from the
H & E Staining in Paraffin Embedded Section Harris alum hematoxylin (5 minutes)
section prior to staining
(Regressive Staining) or
Ehrlich’s hematoxylin (15-30 minutes)
Paraffin section → Xylene for 1-2 minutes →
↓
Xylene for another 1-2 minutes → Absolute
Tap water
alcohol → Descending grades of alcohol → ↓
Water → Actual staining of section Differentiate in 1% acid-alcohol (10-30
“Sections to Alcohol If an alcoholic stain is to be used, there is no seconds)
more need to replace the alcohol with water. ↓
After deparaffinization with xylene, the Blue in ammonia water (5 minutes)
section is subjected to decreasing grades of or
alcohol 1% aqueous lithium carbonate (30 seconds)
Materials for Staining ↓
Coplin Jar Holding from 5-9 slides Running water (5 minutes)
Slotted Staining Dishes Holding from 5-19 slides ↓
Metal or Glass Staining Racks or Carriers Holding from 10-30 slides upright Counterstain with 5% aqueous eosin (5
minutes)
or
Alcoholic eosin (30 seconds or 1 minute)
↓
Differentiate
Histopathology 36
 Aqueous eosin (tap water) Celestine Blue-Haemalum Sequence Staining
Alcoholic solution (70% alcohol) Celestine Blue An oxazine dye used as an alternative to iron
↓ hematoxylin nuclear stain, producing a strong
Dehydrate, clear, mount and precise nuclear stain that is resistant to
Results decolorization by succeeding acid stains and
Nucleus Blue to blue black solutions
Karyosome Dark blue Iron Alum Acting as a mordant to bind hematoxylin
Cytoplasm, proteins in edema fluid Pale pink Water
RBCs, eosinophilic granules, keratin Bright orange-red ↓
Basophil cytoplasm, plasma cells & osteoblast Purplish pink Stain in solution 1 (Celestine blue) (10-20 minutes)
Cartilage Pink or light blue to dark blue (darkest with ↓
Ehrlich’s hematoxylin) Water
Calcium and calcified bones Purplish blue ↓
Decalcified bone matrix, collagen and Pink Stain in Mayer’s acid alum hematoxylin (5-10 minutes)
osteoid ↓
Muscle fibers Deep pink Water
↓
Heidenhain’s Iron Hematoxylin Method Blue in running tap water
Water ↓
↓ Counterstain
Mordant in solution 1 (3 hours or longer) ↓
↓ Dehydrate, clear, and mount
Distilled water
Results
↓
Cell nuclei Blue
Stain in solution 2 (3 hours or longer)
↓ Other constituents Colored according to the counterstain used
Water Mallory’s Phloxine Methylene Blue Stain Originally known as Eosin-Methylene Blue
↓ (EMB) method
Differentiate in solution 1 controlling the degree microscopically
↓ Produces a sharp nuclear stain and reveals
Running water to remove the Iron alum (5-10 minutes) with marked differentiation the various
↓ structures in the tissues
Counterstain
↓ Fixed in Zenker’s fluid
Dehydrate, clear, mount Collodionization of Sections
Results Collodionization Process whereby paraffin ribbons containing
Cells nuclei, cytoplasmic inclusions, muscle Black air bubbles, torn or inadequately infiltrated
striations sections are more firmly attached by coating
Other constituents Colored according to counterstain the slide with dilute (thin) celloidin solutions

Recommended for sections that will be

subjected to strong alkaline or acid solutions
and for tissues that contain glycogen
Histopathology 37
Histopathology 38