HISTOPATHOLOGY

By: Dess Dagalea

STEPS IN TISSUE PROCESSING - formalin

“ FDCIETS SMoL” -osmium tetroxide

1. FIXATION 2. Non-additive fixative

2. DEHYDRATION  It is not incorporated in the tissue and it

stabilizing the tissue by removing the
3. CLEARING bond of the water.
4. IMPREGMENTATION Example:
5. EMBEDDING - Alcohol fixatives
6. TRIMMING FACTORS INVOLVE IN FIXATION
7. SECTIONAING / CUTTING  VOLUME
8. STAINING  pH
 TEMPERATURE
9. MOUNTING  THICKNESS OF SECTION
 OSMOLALITY
10. LABELING
 CONCENTRATION
FIXATION  DURATION OF FIXATION

 Is a process adopted to kill, harden and Difficulties caused by Causes:

improper fixation:
preserve materials for microscopic
1. failure to arrest early 1. Failure to fix
study by means of a substance known autolysis of cells immediately, insufficient
as FIXATIVE. 2. Removal of substance- fixation
 Fixation or fixative is the first or most soluble fixing agent 2. Wrong choice of fixative
critical part of tissue processing. 3. Presence of artifact 3. Incomplete washing of
pigments on tissue fixation
 Main goal: preserve cells and tissues for sections 4. Incomplete fixation
examination and important for 4. Tissues are soft and 5. Wrong choice of fixative
stabilization of proteins. feather-like in consistency 6. Over fixation
 Aims: to preserve the morphological 5. Loss or inactivation of 7. Prolonged fixation
enzymes needed for study
and chemical integrity of the cell and to 6. Shrinkage and swelling
harden and protect the tissue from of cells and tissue
further handling. structure
 Preferred volume: 10-20 times greater 7. Tissue blocks are brittle
and hard
or 15-20 times greater volume of
fixative in specimen.
 Uses 10% NBF
 pH: 8 or >7 (phosphate means alkaline). In fixation we have;
MECHANISMS INVOLE IN FIXATION PHYSICAL METHOD OF FIXATION
1. Additive fixative TYPES OF FIXATIVES:
 The fixative becomes part of the tissue 1. Heat fixation
were it stabilize the tissue proteins.
 Thermal coagulation of tissue proteins
Examples: for rapid diagnosis is usually employed
HISTOPATHOLOGY
By: Dess Dagalea
for frozen tissue sections and
preparation for bacteriologic smears.
2. Microwave fixation
FIXATIVE ACCORDING TO ACTION
 Usually it works as a physical agent
similar to mechanism of vacuum or MICROANATOMICAL -10% Formol saline
oven or heat fixation and it use -10% NBF
agitation. It use agitation to increase -Heidenhain’s Susa
-Formol Sublimate
the movement of the molecules and
(formol corrosive)
accelerating the fixation.
-Zenker’s solution
 It is useful in presering neuro chemical
-Zenker-formol
substancances like brain and fix usually
(Helly’s)
acetylcholine.
-Bouin;s Solution
3. Freeze drying -Brasil’s Solution

 Preserve the tissue by rapid freezing of CYTOLOGICAL -Nuclear Fixatives

fresh tissue and subsequently removing -Cytoplasmic
ice water molecules by a physical Fixatives
process of transfering the frozen tissue -Histochemical
block in a vacuum. Fixatives
4. Freeze substitute
 Those frozen tissue fixing in Roseman’s CYTOLOGICAL FIXATIVES
formula or 1% acetone and dehydrated Nuclear Cytoplasmic Histochemical
in absolute alcohol. Fixatives Fixatives Fixatives
Heidenhain’s Helly’s 10% Formalin
FIXATIVES ACCORDING TO COMPOSITION Susa Orth’s Acetone
Newcomer’s Regaud’s Newcomer’s
A. Simple Fixatives Bouin’s Flemming Absolute ethyl
1. Aldehyde a. Formaldehyde Flemming’s with without Acetic alcohol
b.Glutaraldehyde Acetic Acid Acid
Carnoy’s Formalin with
post chroming
2. Metalic Fixatives 1.. Mercuric Fixatives
2. Chromic Fixatives (Heide’s New (HORFF)
a. Potassium BoyFriend with (FANA)
Car)
Dichromate
b. Chromic Acid
3. Lead Fixatives ALDEHYDE FIXATIVES
a. Picric acid
b. Acetic Acid 1. FORMALDEHYDE (FORMALIN)
c. Acetone - Concentrated solutions should not be
d. Alcohol neutralized (explosion)
e. Osmium - STOCK SOLUTION: 37-40%
Tetroxide - WORKING SOLUTION: 10% Formalin
4. Heat
Formalin pigments:
B. Compound
Fixatives  Paraformaldehyde
HISTOPATHOLOGY
By: Dess Dagalea
- White crystalline precipitates due to
prolonged standing
- Removed by: 10% methanol
-
 Acid Formaldehyde hematin
- Brown/black granular deposits that may
obscure microscopic details
2. 10% FORMOL SALINE
- Used for fixation of CNS tissues and
general post-mortem tissues for
histochemical examination.
3. 10% NEUTRAL BUFFERED FORMALIN OR PO4
BUFFERED DORMALIN
- Recommended for preservation and storage of
surgical, post-mortem and research specimens.
- 1mm/hr- rate of tissue penetration
4. FORMOL-CORROSIVE (FORMOL SUBLIMATE)
-Excellent for stains like Silver Reticulin methods
-No washing out and fixes lipids especially
neutral fats and phospholipids
5. GENRE’S (ALCOHOL FORMALIN)
- has 95% ethanol picric acid and glacial acetic
acid
-Good for glycogen and microincineration
techniques
- can use for sputum
6. GLUTARALDEHYDE
- is made up of two formaldehyde residues,
linked by three carbon chains for routine light
microscope
2. CHROMATE FIXATIVES
-satisfactory for EM (electron microscopy)
-Less irritating but expensive
Percentage of glutaraldehyde:
 25% solution- for small tissue
fragments and needle biopsies (fixed in
2-4 hours at room temp)
 4% solution- recommended for larger
tissues less than 4mm thick fixed in 6-8
hours up to 24hours.
METALLIC FIXATIVE
1. MERCURIC CHLORIDE
- MOST COMMON FIXATIVE
-Frequently used in saturated Aqueous
solution of 5-7%
- Produce black granular deposits except
for Heidenhain’s Susa
Have 3 solutions:
A. ZENKER’S FLUID
 Made up of Mercuric chloride stock
solution to which Acetic acid has
been added just before its use.
 Recommended for fixing small
pieces of spleen, liver, CT fibers
and nuclei
 Recommended for trichrome stain.
B. ZENKER-FORMOL ( Helly’s Solution)
 Excellent microanatomical fixative for
pituitary gland, bone marrow and blood
containing organs
 Produce brown pigments
C. HEIDENHAIN SUSA
 Recommended mainly for tumor
biopsies especially of the skin
 Excellent cytologic fixative
D. B5 FIXATIVE
 For bone marrow biopsies
A. CHROMIC ACID
 Fixative, strong oxidizing agent
 Used concentration in 1-2% Acqueous
solution
 Precipitates all proteins and adequately
preserves carbohydrates
HISTOPATHOLOGY
By: Dess Dagalea
B. POTASSIUM DICRHOMATE - It fixes and precipitates nucleoproteins,
chromosomes and chromatin materials.
 Preserves lipids
 Preserves mitochondria (pH 4.5-5.2) 6. ALCOHOL FIXATIVES

C. REGAUD’S (MOLLER’S FLUID) - Act as fixative at the same time dehydrating

agent.
 Recommended for the demonstration
of chromatin, mitochondria, mitotic A. METHYL ALCOHOL
figures, golgi bodies, RBC and colloid-
 Excellent for fixing dry and wet smears,
containing tissues.
blood smears and bone marrow tissues
D. ORTH’S FLUID
B. ETHYL ALCOHOL
 Recommended study for early
 Concentration of 70-100%
degenerative processes and tissue
 If lower concentrations are used, RBC’s
necrosis
become hemolyzed and WBC’s
 Demonstrates Rickettsiae and other
inadequately preserved
bacteria
 Disadvantage: polarization
3. LEAD FIXATIVE
C. CARNOY’S FLUID
- are used in 4% aqueous solution of basic lead
acetate  recommended for fixing chromosomes,
lymph glands and URGENT BIOPSIES
-recommended for acid mucopolysaccharides  MOST RAPID ALCOHOL FIXATIVE
- fixes connective tissue mucin D. ALCOHOLIC FORMALIN (GENRE’S) FIXATIVE
4. PICRIC ACID FIXATIVES  -useful for sputum since it coagulates
mucus
-excellent fixatie for glycogen demonstration
E. NEWOMER’S FLUID
-suitable for Aniline stains (Mallory’s
Heidenhain’s or Masson’s methods)  recommended for fixing
mucopolysaccharides and Nuclear
A. BOUIN’S SOLUTION
proteins
 Recommended for fixation of embryos  Produces better reaction in Fuelgen
stain than Carnoy’s fluid
B. BRASIL’S ALCOHOLIC PICOFORMOL FIXATIVE
 Better and less messy than boiun’s
solution 7. OSMIUM TETROXIDE (OSMIC ACID)
 Excellent for glycogen
- color: pale yellow powder which dissolves in
5. GLACIAL ACETIC ACID water (up to 6% at 20 degree celcius) to form a
strong oxidizing solution
- Acetic acid is normally used in conjunction
with other fixatives to form a compound -fixes conjugated fats and lipids
solution
-may cause conjunctivitis or blindness
- It solidifies at 17 degree celcius
HISTOPATHOLOGY
By: Dess Dagalea
mordant for better staining effects and
to aid in cytologic preservation
A. FLEMMING’S SOLUTION -
 Most common Chrome-Osmium Acetic WASHING-OUT
Acid fixative
 Recommended for nuclear preparations - It is a process by removing excess
 Permanently fixes fats fixative from the tissue after fixation.
Why? In order to improve staining and
B. FLEMMING’S SOLUTION WITHOUT ACETIC remove artefacts from the tissue.
ACID Usually used:
 Made up only of chromic and osmic acid - TAP WATER
 Recommended for cytoplasmic - REMOVE EXCESS chromates from
structures particularly mitochondria tissues 50-70% alcohol
- Used to wash out excess amount of
8. TRICHLOROACETIC ACID picric acid Alcohol iodine
- Used to remove excessive Mercuric
- precipitates proteins and WEAK Fixatives
DECALCIFYING AGENT
FACTORS THAT AFFECTS FIXATION
9. ACETONE
RETARDED BY ENHANCED BY
- used at ice cold temperature ranging from -5
-SIZE AND THICKNESS -Thinner and small
degree celcius to 4 degree celcius
-Presence of Mucus size of tissues
-recommended for the study of water diffusible -Presence of Fat -Agitation
enzymes expecially phosphates and lipases -Presence of blood -Heat (37-56
Accelerates fixation)
-used in fixing brain tissues for the diagnosis of
RABIES
DECALCIFICATION
10. HEAT FIXATION
- Is the process that comes after fixation
- direct ( flaining) using alcohol lamp like bony substances
- Involve thermal coagulation of tissue proteins - Selected pieces of tissues are taken
for rapid diagnosis, usually employed for frozen from the specimen, properly labelled
sections and preparations of bacteriologic and identified and then subjected to the
smears subsequent steps of processing.
- This procedure wherby Calcium or Lime
SECONDARY FIXATION salts are fixation is known as
Decalcification
- Process of placing an already fixed
- Ratio: 20:1
tissue in a second fixative in order to
ensure further and complete hardening ACID DECALCIFYING AGENTS
and preservation of the tissues.
 NITRIC ACIDS (5-10%)
Note: heat fix is the first fixation  Imparts yellow color due to
formation of nitrous acid
POST CHORMATIZATION
 Most common acid decalcifying
- A primarily fixed tissue is placed in agent and rapid decalcifying
Aqueous solution of 2.5-3% Potassium agent
Dichromate for 24 hours, to act as
HISTOPATHOLOGY
By: Dess Dagalea
1. FORMOL NITRIC ACID- rapid; for urgent
biopsies
2. PERENYI’S FLUID- tissue softener and
decalcifying agents
3. PHLOROGLUCIN NITRIC ACID- for urgent
works (MOST RAPID DECALCIFYING AGENT)
Types of decalcifying agent:
 Acids
 Chelating agents (EDTA)
 Ion exchange resins
 Electrophoresis
 HYDROCHLORIC ACID
 Slower reaction
 Inferior compared to Nitric acid
in its role as decalcifying agent
because of its slower action and
greater distortion of the tissue
produced on the section
decalcified
A. VON EBNER’S
 For small pieces of bones and teeth
 For surface decalcification
FORMIC ACID
- Both fixative and dehydrating agent
TRICHOLOROACETIC ACID
- It is weak decalcifying agent used for
dense tissues and is suitable only for
small spicules of bones
- Permits good nuclear staining
CHELATING AGENTS
- Are substances that are combined with
Calcium ions and other salts to form
weakly dissociated complexes and
facility ate removal of Calcium salts
- Most common chelating agent is EDTA
with the commercial name VERSENE
- Tissue is placed in the agent form 1-3
weeks, the solution being changed
every 3 days and in the final stage,
every day to facilitate decalcification
ION EXCHANGE RESINS
-Ammonium form of Polystrene Resin
-Hastens the decalcification by removing Ca
ions from Formic-Acid containing
decalcifying solutions, thereby increasing
solubility from the tissue.
ELECTROPHORESIS
- Is a process whereby positively charged
Calcium ions are attracted to a negative
electrode and subsequently removed
from the decalcifying solution.
- The process of decalcification is
shortened due to the heat and
electrolytic reaction produced in the
process.
MEADURING THE EXTENT OF
DECALCIFICATION
A. PHYSICAL METHOD
 Done by touching or bending the
tissue with the fingers to determine
the consistency of tissues
 Decalcified tissue have diminished
consistency and are softer to touch
 Vague and inaccurate
B. X-RAY OR RADIOLOGICAL METHOD
 Very expensive although the most ideal
and most reliable method of
fetermining extent of decalcification
due to its ability to detect even the
smallest focus of Ca which appears in an
X-RAY plate
C. CHEMICAL METHOD
 A simple, reliable and convenient
method recommended for routine
purposes to detect the presence of
Calcium in the decalcifying solution
HISTOPATHOLOGY
By: Dess Dagalea
 Detection of Ca in acid solutions by - Excellent dehydrating and clearing
precipitation of Ca Hydroxide of Ca agent
oxalate - Readily miscible in water, melted
 Performed on discarded fluid paraffin, alcohol and xylol
TWO METHODS:
DEHYDRATION a. Graupner’s- pure dioxane + paraffin
- Intracellular and extracellular water b. Weiseberger’s- tissue is wrapped in
from the tissue are removed after gauze and suspended in a bottle
fixation and prior to wax infiltration containing dioxane and a little of
- Uses ascending grades of alcohol Anhydrous Ca oxide.
- Rate of dehydrating agent to tissue: CELLOSOLVE (Ethylene Glycol Monoethylether)
10:1
- Combustible and toxic
DEHYDRATING AGENTS
TRIETHYL PHOSPHATE
A. ALCOHOL
- Removes water very readily and
1. ETHYL ALCOHOL produces very little distortion and
 Recommended for routine dehydration hardening of tissue.
of tissues - Soluble in Alcohol, water, Ether,
 BEST DEHYDRATING AGENT –fast- Benzene, Chloroform, Acetone and
acting and it mixes with water and Xylene
penetrates tissue easily. - Minimizes shrinkage of tissue.
TETRAHYDROFURAN
2. METHYL ALCOHOL - both dehrydrating and clearing agent
- can cause conjuctival irritation
 Toxic dehydrating agent - has offension odor and toxic if ingested or
 For blood and tissue films and for smear inhale
preparations
CLEARING
3. BUTYL ALCOHOL
- DE- ALCOHOLIZATION
 Utilized in plants and animals - Process whereby alcohol is removed
microtechniques from the tissue
 SLOW dehydrating agent - Over clearing- tissue brittleness
4. DENATURED ALCOHOL - Most are flammable liquids
- Ehanol + methanol
5. ISOPROPHYL COMMONLY USED CLEARING AGENTS
- excellent substitute for ethanol
A. XYLENE
ACETONE
 Colorless, most common and most
- Is a cheap, rapid- acting dehydrating rapid clearing agent
agent utilized for most urgent biopsies  For tissue sections with a thickness of
which it dehydrates in ½ to 2 hours <5mm
- Used for small pieces of tissues only due  Time: ½ - 1 hours
to its extreme volatility and  MILKY- incomplete dehydration
inflammability
B.TOLUENE
DIOXANE (Diethylene Dioxide)
- substitute for Xylene or benzene
HISTOPATHOLOGY
By: Dess Dagalea
- tissues don’t become excessively hard and
brittle even if left in tuolene for 1 day
- clearing time: 1-2 hours

C. BENEZENE TYPES OF INFILTRATING MEDIUM

- recommended for urgent biopsies (15-60
minutes)
-carcinogenic
-excessive exposure to benzene may be
extremely toxic to man and may damage the
bone marrow resulting in plastic anemia
D. CHLOROFORM
- recommneded for tough tss (e.g. skin, fibroid
and delcified tss), for nervous tss, lymph nodes
and embryos-minimum shrinkage
-toxic to liver
-can be used for tissue blocks up to 1cm
A. PARAFFIN WAX
- simplest, most common and best embedding
medium used for routine tissue processing
- common waxes have melting point of 45
degree celcius, 52 degree celcius, 56 degree
celcius and 58 degree celcius.
- if the lab is at room temp (54-58 degree
celcius)
- if the lab is at 15-18 degree celcius (50 and 54
degree celcius)
Common waxes have 45, 52, 56, 58 degree
melting point of: celcius

E. CEDARWOOD OIL MP for routine works: 56 degree celcius

- recommended for CNS, and cytological Lab temp is between 54-58 degree celcius
studies, particularly of smooth muscles and skin 20-24 degree celcius:
- very slow (2-3 days)
Lab temp between 15- 50-54 degree celcius
F. ANILINE OIL 18 degree celcius:
- not for routine purposes
-recommended for embryos, insects and very
3 ways by which paraffin wax
delicate specimens due to its ability to clear
impregmentation and embedding tissues can
70% alcohol without excessive tss shrinkage and
be performed
hardening
A. MANUAL PROCESSING
G. CLOVES OIL
- at least 4 changes of wax are required at 15
- causes minimum shrinkage
minutes interval
-Not guaranteed quality- due to its tendency to
-makes use of a paraffin oven (2-5 degree
become adultered
celcius higher than the melting point) or 55-50
degree celcius temperature
H. CARBON-TETRACHLORIDE
- highly toxic upon prolonged exposure
B. AUTOMATIC
- makes use of automatic tissue processing
IMPREGMENTATION/INFILTRATION
machines wich fixes, dehydrates, clears and
infiltrates tissue.
- impregmentation (infiltration) is the process
- Usually 3(2-3) changes of wax are required to
whereby the clearing agent is completely
remove clearing agent and properly impregnate
removed from the tissue and replaced by a
the specimen
medium that will completely fill all the tissue
cavities
C. VACUUM
- ratio of infiltrating medium to tissue: 25:1
HISTOPATHOLOGY
By: Dess Dagalea
- wax impregmentation under negative - soluble in equal conc. Of ether and alcohol
atmospheric pressure
- for urgent bipsies, delicate tissues
- gives the fastest result

SUBSTITUTE FOR PARAFFIN WAX GELATIN IMPREGMENTATION

PARAPLAST 56-57 degree celsius - rarely used except when dehydration is to be
MELTING POINT avoided
- more elastic and -Volume: at least 25 times the volume of the
resilient tissue.
- tissue should not be more than 2mm-3mm
EMBEDDOL 56-58 degree celsius
thick 1% phenol prevents molds
MP
-less brittle and less EMBEDDING
compressible than
paraplast - after impregmentation, the tissue is placed
into a mold containing the embedding medium
BIOLOID - Recommended for and this medium is allowed to solidify.
embedding eyes

TISSUE MAT -contains rubber and ORIENTATION - process by which a tissue is

has the same arranged in precise positions in the mold during
properties as paraplast embedding, on the microtome before cutting,
and on the slide before staining.
ESTER WAX - 46-48 degree celsius
MP BLOCKING-OUT MOLDS
-soluble in 95% ETOH
and clearing agent 1. Leuckhart’s Embedding Mold
WATER SOLUBLE WAX -38-42 degree celsius/ - consist of 2 L-shaped strips of heavy brass or
45-56 degree celsius mental arranged on a flat metal plate.
MP
-mostly polythylene 2. Compound Embedding Unit
glycons - series of interlocking plates resting on a flat
metal base, forming several compartsments

CELLOIDIN 3. Plastic Embedding Rings and Base Mold

- Sustable for specimens with large hollow
cavities, hard and dense tissues, large tissue
sections of whole embryo
METHODS OF IMPREGMENTATION:
1. WET CELLOIDIN
- Recommended for bones, teeth, large brain
sections and whole
2. DRY CELLOIDIN
- preferred for processing of whole eyes
sections
3. LOW VISCOSITY NITROCELLULOSE
- consist of a special stainless steel base mold
fitted with a plastic embedding ring
Tisse Tek- equipped with a warm plate to
manage the impregnated specimen, and a cold
plate at -5 degree celsius for rapid solidification
of the block.
4. Disposable Embedding Molds
a. Peel-away - disposable thin plastic
embedding molds, peeled off one at a
time, as soon as the wax has solidified.
HISTOPATHOLOGY
By: Dess Dagalea
OTHER EMBEDDING METHODS:
2. GELATIN
1. Celloidin or Nitrocellulose Method - firmer attachments to the tissue
- embedding hard tissues - has to be gently heated before use to melt the
gelatin
2. Double Embedding Method -added to the floatation water bath
- infiltrate eith celloidin, embed with paraffin
3. CELLULOSE
TEMPERATURE - does not stain into appreciate extent
- in the form of 1% methyl cellulose
Manual 2-5 degree ABOVE the
Impregmentation melting point of the
4. SODIUM SILICATE
wax
- has strong adhesive properties
Embedding 5-10 degree celsius - little tendency of staining with most dyes
ABOVE the melting - not affected by the use of mild alkaline
point of the wax solutions
- DISADVANTAGE: blackening of the tissue black
Floatation Water Bath 6-10 degree celsius ( silver impregnation technique and reticulin
BELOW the melting
method )
point of the wax
5. RESIN
TRIMMING - greatest adhesion
-dilluted 1:10 acetone
- remove excess wax
- at least 2mm wax should surround the tissue METHODS OF DRING SLIDES
block 56-57 degree celsius Wax oven for 2 hours
- only thin slices are taken out at a time to
37 degree celsius Incubation for 24 hours
prevent the block from cracking
45-55 degree celsius Hot plate drying for 45
SECTIONING mins

- embedded tissue is trimmed and cut into 50-55 degree celsius Blower type slide dryer
for 20-30 mins
uniformly thin slice using a microtome
Urgent drying BUNSEN burner
TYPES OF SECTIONS USUAL THICKNESS
PARAFFIN 4-6 um SUMMARY OF FAULTS DURING TISSUE
CELLOIDIN 10-15 um PROCESSING

FROZEN 10 um Brittle/hard tissue Fixation, dehydration,

clearing, paraffin
ULTRATHIN 0.5-1 um
infiltration, Overheated
oven, Drying before
TYPES OF ADHESIVES fixation

On trimming, tissue Insufficient clearing

1. MAYER’S EGG ALBUMIN smells of clearing agent
- most common type of adhesive
HISTOPATHOLOGY
By: Dess Dagalea
Clearing agent turns Incomplete Hard spot in the tissue
MILKY dehydration
Sections of unequal Knife tilt
Tissue shrinks from wax Insufficient dehydration thickness Screw/holder is loose
Incomplete clearing Large and hard blocks
and impregmentation

Sections adhere to Static electricity

Tissue is opaque Insufficient clearing knife or other parts of Dirty knife edge
the microtome Dull knife edge
Tissue is soft when Incomplete fixation Tilt
block is trimmed
Ribbon slit Nicks/ damge on knife
Air holes on tissue Incomplete Dirty embedding
impregmentation Dirty knife
Wax appears crystalline Contaminated wax Tilt
Block not cooled rapidly Chatters are seen Knife vibrates (hard
enough tissues)
Paraffin block is moist Insufficient paraffin Tilt
and crumbles impregmentation Section: sometimes Blunt knife
Sections fail to form Surface and edges of thin and thick Tilt
ribbons block not parallel Knife/ block hiolder is
Wax too hard loose
Knofe titled too much Frozen tissue crumbles Inadequate freezing
Thick sections and comes off when
Dull knife the block holder when
Sections roll up on Blunt knife cut
cutting adhere and get Tilt knife Frozen tissue chips into Tissue is frozen too
broken against the Dirty knife edge fragments hard
knife edge

Ribbon is curved, Irregular knife edge STAINING

crooked or uneven Edges of block are not
parallel
- Process of applying dyes on the sections to see
Knife not parallel to the
and study the architectural pattern of the tissue
block
and physical characteristics of the cells.
Impure paraffin
- Acidic parts of cells/tissues have greater
Sections are Blunt/dull knife affinity for BASIC dyes while basic constituents
compressed, wrinkled Block is warm and soft take more of the ACID stains.
or jammed Knife edge coated with
paraffin Staining of tissues can be classified into 3
Thin sections major groups:
Microtome screw loose

Sections are squashed Bevel of knife is lost A. Histological Staining

Incorrect sharpening - process whereby the tissue constituents are
demonstrated in sections by direct interaction
Hole in sections Bubble/dirt with a dye or staining solution, producing
coloration of the active tissue component.
HISTOPATHOLOGY
By: Dess Dagalea
B. Histochemical Staining (Histochemistry)
- process whereby various constituents of
tissues are studied thru chemical reactions that
will permit micrroscopic localization of a specific
tissue substance.
C. Immunohistochemical Staining
- allow phenotypic markers to be detected by
using a wide range of antibodies
-combination of immunologic and histochemical
techniques
METHODS OF STAINING
A.DIRECT STAINING
- the process of giving colo to the sections by
using aqueous or alcoholic dye solutions (e.g.
methylene blue, eosin)
B. INDIRECT STAINING
_ process whereby the action of the dye is
intensified by adding another agents of
MORDANT which serves as a link or bridge
between the tissue and the dye to make
staining reaction possible
_ mordant combined with dye to form a colored
“ LAKE”
- lake combines with the tissue to form a
“tissue-mordant-dye-complex”
ACCENTUATOR- accelerates or hastens the
speed of the staining reaction
C.PROGRESSIVE STAINING
-tissue elements are stained in a definite
sequence
- No diffrentiation or decolorization is involved
D. REGRESSIVE STAINING
- tissue is first overstained to obliterate the
cellular details and the excess stain is removed
or decolorized.
E. METACHROMATIC STAINING
- the use of specific dyes which differentiate
particular substances by staining them with a
color that is different from that of the stain
itself.
F. COUNTERSTAINING
- application of different color or stain to
provide contrast and background to the stain of
the structural components to be demonstrated.
G. METALLIC IMPREGMENTATION
- specific tissue elements are demonstrated, not
by stains, but by colorless solutions of metallic
salts which are thereby reduced by the tissue,
producing am opaque, usually black deposit on
the surface of the tissue or bacteria.
H. VITAL STAING
- selective staining of living cell constituents,
demonstrating cytoplasmic structures by
phagocytosis of the dye particle (cytoplasmic
phagocytosis)
- Nucleus of a living cell is resistant to vital
stains.
2 types:
1. Intravital staining- staining of living
cells is done by injecting the dye into
any part of the animal body.
2. Supravital staining - used to stain
living cells immediately after removal
from the living body.
Biological stains or coloring sunstances are
prepared from dyes which may generally be
divided into two categories:
1. Natural dyes
2. Synthetic dyes
1. NATURAL DYES
- Those obtained from plants and animals
A. HEMATOXYLINE
- derived from the core or heartwood of a
Mexican tree known as “hEMATOXYLINE
cAMPECHIANUM”.
- hematoxyline itself is not a stain. The active
coloring agent is hetain, which is formed by
HISTOPATHOLOGY
By: Dess Dagalea
the oxidation of hematoxyline, aprocess MOUNTING
known as “ripening”. - use of medium and cover slip to facilitate the
handling storage and protection of the tissue
2 types: section
1. ALUM HEMATOXYLIN
- used in routine H and E KINDS OF MOUNTING MEDIUM
- Mordant: Potassium aluminum sulfate 1. AQUEOUS
- produce good nuclear stain (red) 2. RESINOUS

2. IRON HEMATOX
- WEIGERT’S AQUEOUS
mordant: Ferric chloride 1. Water based
-HEIDENHAIN’S 2. Glycerin
mordant: Ferric ammonium chloride 3. Farrant’s Medium (Gum Arabic Medium) RI:
For mitochondria, muscle striations, 1.43
chromatin and myelin 4. Apathy’s Medium= 1.52

B. COCHINEAL DYES ADDITIONAL NOTES

- old histologic dye extracted from the female
cochineal bug (Coccus cacti), which is treated
with alum to produce the dye, carmine.
C. ORCEIN
- vegetable dye extracted from certain lichens
which are normally colorless, but which is
treated with ammonia and exposed to air,
produce blue or violet colors.
MICROTOMES:
Different angles of cutting:
1. Bevel up
- formed between the cutting edges of the
knifes (27-32 degree celsius)
 Cutting angle- 15 degree celsius if incline
 Clearance angle - 5-10 degree celsius angle
from between the surface as block and the
cutting edges of the knifes.

SYNTHETIC DYES 2. Rake angle

- sometimes known as “Coal Tar Dyes” since
they were originally manufatured from
substance that have been taken from coal tar.
- They are derived from the hydrocarbon
benzene (C6H6) and are collectively known as
Aniline dyes.
- angle of the upper surface of the fucet and
surface of the tissue block
3. Wedge angle
- between the sides of the knifes
DECALCIFICATION

 Chromophore- responsible for the coloring Post decalcification

property
 Auxochrome- responsible for the dying
propety
 Lysochrome Dyes- oil soluble dyes
 Ampoteric Dyes- dyes with a neutral pH
- neutralize by rinsing the tissue in running tap
water to remove the chemicals excess fluid on
nitric acid.
WHEN IS DECALCIFICATION?
- done after following 2 fixation.