QUICK REVIEW NOTES in HISTOPATH –Fixation-Dehydration-Clearing-Infiltration-Embedding-Decalcification

FIXATION Osmium Tetroxide

Non-additive fixatives Glutaraldehyde
Fixation temp for EM & histochemistry paraformaldehyde
Fixation method that Involves thermal coagulation of proteins
Fixation temp using autotechnicon
Temperature for Manual Fixation Fixative & dehydrating agentfor
Penetration rate of formalin phosphatases & lipases
Presence of mucus, blood, fatty tissues, cold temperature
Fixative volume for maximu effective fixation
Temperature range that will accelerate fixation
Carnoy’s ; Bouin’s; Newcomer’s Heidenhain’s Susa;
Fixative volume for museum preparations
Flemming’s with HAc
Regaud’s / Moller’s; Orth’s; Formalin with post chroming Fixatives for Enzyme 4% formaldehyde & formol saline
Flemming’s without HAc histochemistry
100 ml of 37-40% formalin + 900 ml distilled water Smallest aldehyde
Bouin’s Brasil’s & Hollande’s
Added to prevent precipitation of formaldehyde to white
Can be used for myelin & peripheral nerves
paraformaldehyde
Proposed as mercuric chloride substitute
FORMALDEHYDE 10% formol saline
10% Nuetral Buffered formalin Used to wash out excess mercuric fixatives
/FORMALIN FIXATIVES
Formol corrosive/formol sublimate Used to wash out excess amount of picric acid fixatives
Alcoholic formalin/Gendres Can be used as fixative, decalcifying agent & stain
Mercuric chloride + formaldehyde Often used in conjunction with other fixatives to produce a compound
Glutaraldehdye – for EM for small tissue fragments/needle biopsies fixative
for larger tissues less than 4 mm thick DECALCIFICATION
Most common metallic fixative; Potent anticoagulant but weak decalcifying agent
Excellent for Trichromoe staining & Tissue photography Binds with calcium to form weakly dissociated complexes
MERCURIC CHLORIDE liver, spleen, CT & nuclei Decalcification method that involves attraction of positively charged
FIXATIVE bone marrow calcium ions with negatively charged electrodes
Heidenhain’s Susa Decalcifying acid used for surface decalcification of blocks
pituitary gland, BM, spleen, liver Both a decalcifying agent & a tissue softener
a.k.a helly’s fluid Most commonly used decalcifying agent especially recommended for
CHROMATE FIXATIVES for carbohydrates urgent biopsies, for needle & small biopsies
Potassium dichromate 3% - for lipids, mitochondria Temperature range for decalcification
mitochondria, RBC & colloid containing tissues Ratio of decalcifying agent to tissue volume
Rickettsiae & other bacteria, tissue necrosis Most reliable method of testing for the completeness of decalcification
Acid mucopolysaccharide Method of testing for the completeness of decalcification that uses
1. For brain tissues for the diagnosis of rabies calcium carbonate
2. Duration of decalcification thur ion exchange resin
Excellent for Glycogen demonstration Method of testing for the completeness of decalcification done by
For embryos; pituitary biopsies & endometrial curettings Not probing tissue with a needle
for kidney structures Most rapid nitric containing decalcifying agent
Not compatible with feulgen’s An excellent bone decalcifer for EM and enzyme histochemistry
For touch preparations Remedy for Inactivation ofalkaline phosphatase by EDTA
Solidifies at 17 degC
Must be used at ice cold temperature DEHYDRATION
Most rapid tissue fixative, for fixing chromosomes & lymph glands Important requirement when using alcohol as dehydrating agent
For mucopolysaccharides & nuclear protein For dehydrating sections and smears

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 QUICK REVIEW NOTES in HISTOPATH –Fixation-Dehydration-Clearing-Infiltration-Embedding-Decalcification

Toxic by ingestion, inhalation & by skin contact INFILTRATION / EMBEDDING

Highly corrosive on skin Celloidin infiltration method recommended for whole eye specimens
Toxic to reproductive, fetal, urinary system
Will require use of Gilson’s mixture to store blocks
Dehydrating agent whose main disadvantage is its being toxic Melting point of paraffin wax commonly used
Decomposes when expose to sunlight Substitute for paraffin wax recommended for bones and brain specimens
Temperature that will hasten dehydration time
Substitute for paraffin wax that is water soluble & is recommended for
Dehydrating agent that is both a skin & eye irritant enzyme histochemical studies
Has an offensive odor Temperature range of paraffin oven when in used
Rapid acting dehydrant suited for urgent biopsies but not for routine Approximate temperature of paraffin oven when in used
tissue processing Infiltration under negative atmospheric pressure inside the oven
Most commonly used alcohol for dehydration process Infiltration method not suited for fatty tissues
Alcohol for plant & animal microtechniques Substitute for paraffin wax used for eye specimens
Purpose of phenol in alcohol Substitute for paraffin wax that will require heavy duty type of microtome
bath
Melting point of paraplast
CLEARING/DEALCOHOLIZATION Melting point of ester wax
Clearing agent that can damage the bone marrow & may cause aplastic Infiltration method for enzyme & histochemical studies
anemia Celloidin infiltration method for bones, brain & teeth specimens
Toxic to liver; recommended for skin, fibroid & decalcified tissues Will require 70-80% alcohol to store blocks
For tough tissues , nervous tissues, lymph nodes, embryos Combination of Chloroform & cedarwood oil
Routinely used clearing agent but not for nervous tissues & lymph nodes Infiltration method recommended for specimens with large and hollow
Can become milky because of prolonged storage cavities that tends to collapse
Can become milky after placing an incompletely dehydrated tissue in it Plasticizers to prevent cracking tissues when infiltrated using LVN
For clearing insects, embryos & other delicate specimens Infiltration method that will require tissues not to be more than 2 -3 mm
When used, tissues become adulterated thick
Same property as chloroform A disposable Embedding mold that will require smearing of inner mold
Recommended for eye specimens with glycerine
Clearing agent obtain from citrus fruit An embedding mold consisting of a series of interlocking plates resting
With faint pleasant odor on a flat metal base
Clearing agents that can be used for dense tissues like uterus Disposable mold that gives perfect blocks without trimming
Clearing agent for CNS tissues & cytological studies , an extremely slow Other term for infiltration
clearing agent Other term for embedding 1.
can be used as xylene & benzene substitute 2.
Clearing agents for double 1. Process of infiltrating tissue with celloidin and embedding with paraffin
embedding which are slow 2. Embedding mold consisting of L shaped strips made of heavy brass
acting agents 3. Method of paraffin infiltration that will require 2-3 changes of wax
a.k.a. diethylene dioxide Use of phenol in gelatin
a.k.a. ethylene glycol monoethyl ether infiltration
Clearing agent that will not make tissues transparent Celloidin Infiltration Thin
Clearing agents difficult to remove from tissues Medium
Clearing agent easily removed from tissues Thick
Clearing agent that can attack rubber seal used in vacuum impregnation Spurr Fastest Epoxy resin
Steps in tissue processing 1. Polyester Plastic resin originally introduced for EM
that will require complete 2. Polyglycl methacrylate/GMA Extremely hydrophilic
clearing of tissues Methylmethacrylate/MMA Plastic resin considered ideal for undecalcified bones & hard tissues

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 QUICK REVIEW NOTES in HISTOPATH – Sectioning-Staining- Mounting

SECTIONING
Type of Microtome used to prepare serial sections SECTIONING
Microtome type used to cut tissues to
demonstrateneurological Type of microtome knife used to cut any
structures type ofresin block for EM
Thickness of sections produced using It is defined as the angle formed by the
RotaryMicrotome (range in micra) sides ofthe knife
Process of sharpening & polishing the Type of microtome developed by Adams
cuttingedge Thickness of sections produced using
Type of Microtome knife used to cut RockingMicrotome
paraffinembedded tissues Celloidin sections are usually cut
Defined as the angle formed between between
cuttingedges thickness
Actual angulation in degrees of clearance angle Type of hone used for badly nicked
toprevent uneven sections knives
Mayer’s egg albumin is prepared by adding In Honing, when using Plane wedge
equalamounts of egg white and knife, theknife is turned over as to
Block holder is also called sharpen the other surface every
Types of microtome which can be used to stroke
preparefrozen sections Type of microtome used to cut tissues
Type of microtome knife used for trimming with heatsensitive
andsemi-thin sectioning of tissue blocks for structures
EM Thickness of sections produced using
Type of Microtome invented by Trefall ultrathinmicrotome
Type of hone that gives the best result Type of microtome that has restriction as
Number of strokes required when doing Stropping tothe size of block that can be cut
The type of microtome that when used, will
makeblock re-orientation difficult Purpose of adding Thymol crystals in Mayer’s
Specific type of Sliding microtome in which egg albumin
themovable part is the block holder Type of microtome knife used to cut tough
A refrigerated apparatus used in fluorescent specimens embedded in paraffin blocks
antibody staining techniques or histochemical Type of microtome required for tissues fixed
enzyme studies using osmium tetroxide
Tissues embedded using Plastic resins i.e., Bevel angle is set at what angulation
epoxymust be cut using what type of
Microtome must be done to leather strop prior to use
Type of Microtome invented by Queckette Actual temperature of float out bath
Type of microtome operated by the rotation of Specific type of Sliding microtome
flywheel, causing reciprocal motion of knife regarded
overthe block as the most dangerous

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 QUICK REVIEW NOTES in HISTOPATH – Sectioning-Staining- Mounting

Mineral oil ; clove oil, xylene, liquid paraffin &

soapy water

To remove burrs and other irregularities

Cold knife procedure & Cold Microtome
procedure/Cryostat procedure
Liquid nitrogen, isopentane cooled by liquid
nitrogen, CO2 gas & aerosol sprays
Special way of preserving tissues by rapid
PERIODIC ACID SCHIFF For staining of carbohydrates
freezing (quenching) of fresh tissues at – 160 IMMUNOHISTOCHEMICAL • Staining technique which is a
degC combination of immunologic &
STAINING histochemical techniques
STAINING Application of dyes on tissue sections • For the detection of phenotypic
NUCLEUS Part of the cell with greater affinity for basic markers
dyes • Uses fluorescent labeled or
CYTOPLASM Part of the cell with greater affinity for acid enzyme labeled antibodies
dyes either polyclonal or monoclonal
DIRECT STAINING Staining technique that using aqueous
Staining Categories 1-HISTOCHEMICAL or
2-HISTOLOGICAL alcoholic solutions of dyes
3-IMMUNOHISTOCHEMICAL Examples of Direct stains 1-METHYLENE BLUE
HISTOLOGICAL STAINING Tissue components are demonstrated thru 2- EOSIN
direct interaction with a dye or staining INDIRECT STAINING Staining technique that employs use
solution of
To demonstrate the general relationship of mordant & accentuator
tissues & cells with differentiation of nucleus MORDANT Substance that serves as link or
& cytoplasm bridge
Histological stains are also called MICROANATOMICAL STAINING between dye & tissues
ACCENTUATOR Substance that accelerates or hastens
speedof staining reaction by increasing
HISTOCHEMICAL Tissue components are demonstrated thru the staining
chemical reactions power & selectivity of the dye
Examples of Mordants 1-POTASSIUM ALUM
Examples of Histochemical stains 1-PERIODIC ACID SCHIFF 2-IRON
2-PERL’S PRUSSIAN BLUE

PERL’S PRUSSIAN BLUE For Hemoglobin demonstration

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 QUICK REVIEW NOTES in HISTOPATH – Sectioning-Staining- Mounting
Example of Hematoxylin with potassium alum
as mordant
Examples of Hematoxylin with Iron as
mordants
Examples of Accentuators
Accentuator in Leoffler’s methylene blue
Accentuator in the stains: carbol fuchsin &
carbol thionine
PROGRESSIVE STAINING
Disadvantages of Progressive staining
REGRESSIVE STAINING
DECOLORIZATION
a.k.a DIFFERENTIATION
Commonly used decolorizer
Decolorization/differentiation
METACHROMATIC STAINING
Metachromatic stains are mainly used for
staining
Prepared by: Dr. Ma. Cristina Liwanag
1-HARRIS HEMATOXYLIN METACHROMATIC STAINS Methyl violet/ crystal violet/ Safranin
2-MAYER’S HEMATOXYLIN Bismarck Brown/ Basic Fuchsin
3-COLE’S HEMATOXYLIN Methylene blue/ Thionine/ Toluidine Blue
1-WEIGERT’S HEMATOXYLIN Azure A, B, C/ Cresyl Blue
2-HEIDENHAIN’S HEMATOXYLIN Metachromatic stain for Reticulocytes CRESYL BLUE
1-KOH ORTHOCHROMATIC STAINING Staining technique in which tissue
2-PHENOL components are stained with the same
KOH SHADE/HUE as that of the dye
PHENOL COUNTERSTAINING Staining technique that involves application
of different color to provide contrast and
• Staining technique that follows a background
definite sequence Most commonly used Counterstain EOSIN
• Staining solution is applied for METALLIC IMPREGNATION •
Tissue elements are demonstrated
specific periods NOT by stains but by colorless
• Stain is applied UNTIL the desired solutions of metallic salts that
color is achieved produces BLACK DEPOSITS on
• The dye/stain is applied gradually the surface of tissues/bacteria
to tissues • Especially used for silver staining
1-DIFFUSED COLORS of the nervous system and
2-OBSCURED DETAILS demonstration of reticulin
Staining technique in which the tissue is Examples of agents used in Metallic 1-GOLD CHLORIDE
OVERSTAINED and the excess dye is impregnation 2-SILVER NITRATE
removed selectively 3-AMMONIACAL SILVER
Process of removing excess dye Most commonly used agent for impregnation SILVER NITRATE
that can also function as a staining agent
ACID ALCOHOL VITAL STAINING staining of LIVING CELL
DONE in REGRESSIVE STAINING CONSTITUENTSI
NOT DONE in PROGRESSIVE involves cytoplasmic phagocytosis
STAINING Methods of VITAL staining 1-INTRAVITAL STAINING
Staining technique that involves use of 2-SUPRAVITAL STAINING
dye/stain that gives a different color , different INTRAVITAL STAINING Staining of Living cells done by injecting the
from the color of the dye itself dye into any part of LIVING body
1-MAST CELL GRANULES Examples of Intravital stains 1-LITHIUM
2-AMYLOID 2-CARMINE
3-INDIA INK
3-CONNECTIVE TISSUES SUPRAVITAL STAINING Staining of LIVING CELLS immediately done
4-CARTILAGE after removal from the LIVING BODY
5- EPITHELIAL MUCINS
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 QUICK REVIEW NOTES in HISTOPATH – Sectioning-Staining- Mounting

SUPRAVITAL STAINS Neutral red ; Janus Green CLEARING PRIOR TO LAST 2 changes of Xylene
Trypan Blue; Nile Blue MOUNTING
Thionine; Toluidine Blue
HEMATEIN Active coloring agent of hematoxylin
The best vital dye NEUTRAL RED COCHINEAL DYE Natural dye extracted from bug, producing the dye carmine
Supravital stain recommended for JANUS GREEN
PICROCARMINE Natural dye used extensively in neuropathological studies
Mitochondria ORCEIN Vegetable dye extracted from lichens, used for staining elastic
Both a Metachromatic & supravital stains 1-THIONINE fibers
2-TOLUIDINE BLUE LITMUS Obtained from lichens treated with lime & soda,
COPLIN JAR Can hold 5-9 slides Not used as a cytological stain but mainly as pH indicator
Slotted staining dishes Can hold10-19 slides COAL TAR DYES a.k.a. aniline dyes
Metal/Glass staining racks/carriers Can hold10-30 slides Basic cell structures have ACID DYES
affinity for
STAINING OF FROZEN SECTIONS 1- H&E
2- Thionine method Acidic cell structures have BASIC DYES

3- Polychrome Methylene Blue affinity for

4- Alcoholic Pinacyanol
METHYLENE BLUE Basic dye which may be used both as an indicator & dye,
widely used in bacterial staining
ALCOHOLIC PINACYANOL • Used for supravital staining of
Mitochondria
NEUTRAL DYES Giemsa, Irishman’s (for WBC differentiation)
Also known as AMPHOTERIC dye
• For color sensitization in
photography
ALUM HEMATOXYLIN Mayer’s, Gill’s, Ehrlich’s, Cole’s , Harris, Delafield’s
H& E staining • Commonly used stain in routine
IRON HEMATOXYLIN Weigert’s & Heidenhain’s Hematoxylin
tissue processing
SODIUM IODATE Ripening agent in Mayer’s, Ehrlich’s & Gill’s
H & E is classified as Stain Category
Histological
HARRIS HEMATOXYLIN Form of Hematoxylin used in exfoliative cytology & for staining
Staining Technique REGRESSIVE of sex chromosomes
Hematoxylin 1-basic dye MAYER’S HEMATOXYLIN Form of hematoxylin for Immunohistochemistry
2-nuclear stain Ferric ammonium chloride WEIGERT’S HEMATOXYLIN

3- will impart blue color Ferric ammonium sulfate HEIDENHAIN’S HEMATOXYLIN

Eosin 1-acid dye IRON HEMATOXYLIN Form of hematoxylin useful for photomicrography
2-cytoplasmic dye HEIDENHAIN’S Form of iron hematoxylin recommended for voluntary muscle
HEMATOXYLIN striations & myelin
3-will make cytoplasm pink COPPER HEMATOXYLIN Form of hematoxylin used to demonstrate spermatogenesis
STAINING SOLVENTS FOR STAINS Water, alcohol, aniline water & phenol
H& E staining 1st 2 changes of Xylene further deparaffinization CHROMOPHORE Component of the dye responsible for coloring property
Descending grades of alcohol HYDRATION AUXOCHROME Component of dye responsible for dyeing property
Hematoxylin - Nuclear stain/Primary dye LYSOCHROME Dyes without auxochrome component
Acid alcohol - DECOLORIZER Lysochrome dyes a.kA. a OIL DOLUBLE DYES
Ammonia water BLUEING AGENT used as fat stains
Eosin – counterstain; Cytoplasmic stain SUDAN BLACK Most sensitive of oil soluble dyes
Ascending grades of alcohol DEHYDRATION SUDAN III Sudan dye first introduced into histochemistry
SUDAN III Best fat stain for CNS tissues

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 QUICK REVIEW NOTES in HISTOPATH – Sectioning-Staining- Mounting

OTHER SPECIAL STAINS MOUNTING

Van Gieson stain Demonstration of CT Canada balsam Routinely used mountant
Mixture of picric acid & acid fuchsin BRUN’S FLUID Recommended for mounting frozen sections directly from water
Acridine orange Allows discrimination between dead & living cells 1,524 Refractive index of Canada balsam
Green fluorescens for DNA APATHY’S Used for methylene Blue stained nerve preparations
Red fluorescens for RNA Remedy for bleeding of 1. POTASSIUM ACETATE
Acridine red 3B Demonstration deposit s of calcium salts & possible sites of metachromatic stain for
phosphates activities amyloid due to the use of 2. SODIUM CHLORIDE
Aniline Blue Cytoplasmic stain for counerstaining of Epithelial sections Apathy’s
Basic Fuchsin For deep staining of Acid Fast organisms Refractive indices Glycerin jelly/ Kaiser’s 1.47
Main constituent of Feulgen’s & Schiff’s for aldehyde detection Farrant’s/Gum Arabic 1.43
Bismarck Brown For staining diphtheria organism Apathy’s 1.52
Mayer’s Carmalum Act as a basic dye & staining acidic substances DPX 1.532
solution XAM 1.52
Celestine Blue Recommended for routine staining of fixed sections Clarite 1.544
Congo Red Best known as indicator, stain for axis cylinders in embryos RINGING Sealing margins of coverslip
Crystal violet Nuclear stain for staining amyloid in frozen sections and Examples of Ringing 1.KRONIG CEMENT
platelets in blood Media 2. DUROFIX
Gentian violet Staining solution formed by mixture of crystal violet, methyl
violet & dextrin
Giemsa Staining blood to differentiate WBCs
Gold sub,imate Stain for metallic impregnation,
Made up of gold chloride & mercuric chloride Success comes easy to those
Iodine For microscopic study of starch granules, amyloid, cellulose,
starch & carotenes who believe & who worked
Malachite green Contrast stain for Ascaris eggs, RBCs hard to achieve it.
Bacterial spore stain
Used as decolorizer & as a counterstain Always give your best in
Methylene Violet Metachromatic dye formed whenever methylene blue is heated whatever you do.
Neutral red For observing cell granu;es and vacuoles of phagocytic cells
Night Blue Used as substitute for carbol fuchsin in acid fast staining
Osmium Tetrooxide Used to stain fats Always Pray
Picric acid Used as contrast stain to acid fuchsin For God sees everything in you!
Prussian Blue Demonstration of circulatory system trhu intravital staining
Rhodamine B Used with osmic acid to fix & stain blood and glandular tissues
Silver nitrate For identification of spirochetes, reticulum & other fiber stain
Toluidine Blue Used as substitute for thionine in fresh frozen tissues
For staining of nissle granules or chromophilic bodies
Victoria blue Demonstration of neuroglia in frozen sections
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