Examination of Fresh Tissues

 Teasing or Dissociation
 Squash Preparation (Crushing)
 Smear Preparation (Streaking, Spreading,
Pull – Apart, Touch or Impression Smear
 Frozen Section
FS indications
- rapid diagnosis (guide for intra-operative
patient management)
- to optimally process tissues for special
studies for diagnosis, treatment, or
research
- to confirm that lesional tissue is present for
diagnosis on permanent sections (sample
adequacy)
FS limitations
 Limited section sampling
 Ice crystal or freezing artifact
 Inferior quality compared to paraffin
sections
 Lack of special studies (time constraint)
 Special stains, immunohistochemistry, culture
 Lack of consultation for difficult cases
Consider these during RFS:
 Relevant clinical information / history
 Type of tissue or location of biopsy
 To determine beforehand what information
the surgeon requires from the FS and how
the information will be used.
 Optimal turn-around time is </= 15 mins
Consider these during RFS:
 Coordination between lab and OR
(personnel involved)
 Check cryostat (-17C)
 No fixative used
 Protection of laboratory personnel
 Selecting part of the tissue for FS
Examination of Fixed Tissues -
Histopathologic Techniques /
Steps:
 Numbering  Blocking
 Fixation  Trimming
 Dehydration  Sectioning
 Clearing  Staining
 Impregnation  Mounting
 Embedding  Labelling
Fixation
 Kills, hardens, preserves tissues for the next
histopath steps
 “life like” appearance – prevention of
degeneration, putrefaction, decomposition,
distortion – protein stabilization (cross links
formed between fixative and proteins)
 Reduce risk of infection
 Promotes staining
 Inhibit bacterial decomposition
Fixation
 To preserve the tissue
 Stop all cellular activities
 To prevent breakdown of cellular elements
 Inactivation of lysosomal hydrolytic enzymes
– post mortem decomposition (autolysis); or
by chemically altering, stabilizing, and making
tissue components insoluble
 Prevention of putrefaction after death
(bacterial / fungal colonization & overgrowth)
Fixation
 To coagulate or precipitate protoplasmic
substances
 Additive fixation – chemical constituent of
fixative is taken in & becomes part of the
tissue by cross – links or molecular
complexes  stable protein (formalin,
mercury, osmium tetroxide)
Fixation
 To coagulate or precipitate protoplasmic
substances
 Non – additive fixation – removes bound
water by attaching to H bonds of certain
groups within the protein molecule  new
cross links are established (alcoholic
fixatives)
Microwave Technique
 Physical agent like vacuum, oven (heat)
and agitation to increase movement of
molecules and accelerate fixation
 Accelerates staining, decalcification,
immunohistochemistry and electron
microscopy
 Oscillation frequency 2450 mHz
Microwave – advantages:
 Tissue is heated right through the block in
a very short time (main advantage)
 Non chemical technique (less
interference)
 Rapid
 Lesser time for immunohistochemistry and
in-situ hybridization
Microwave – disadvantages:
 Penetrates 10-15 mm only
 No significant cross linking of protein
molecules; subsequent chemical fixation
may be needed
 Viable spores/pathogens (alcohol based
fixatives or microwaving alone)
Fixative
 Cheap  Hardens tissues for
 Stable easier cutting
 Safe to handle  Isotonic
 Kills quickly
 Minimum tissue
shrinkage
 Rapid & even
penetration
Types of Fixative
 According to composition
- Simple – Aldehydes, metallic fixatives
- Compound
 According to action
- Microanatomical
- Cytological – Nuclear & Cytoplasmic
- Histochemical
Simple Fixatives
 Aldehydes  Picric Acid
 Formaldehyde  Acetic Acid
 Glutaraldehyde  Acetone
 Metallic Fixatives  Alcohol
 Mercuric Chloride  Osmium Tetroxide /
 Chromate Fixatives
Osmic Acid
 Lead Fixatives
 Heat
Microanatomical Fixatives
 10 % Formol Saline  Zenker’s
 10 % Neutral Buffered  Zenker – Formol
Formalin (Helly’s)
 Heidenhain’s Susa  Bouin’s
 Formol Sublimate  Brasil’s
(Formol Corrosive)
Cytological Fixatives
 Nuclear:  Cytoplasmic
 Flemming’s  Flemming’s w/o acetic
 Carnoy’s acid
 Bouin’s  Helly’s
 Newcomer’s  Formalin w/ post
chroming
 Heidenhain’s
 Regaud’s (Moller’s)
 Orth’s
Histochemical Fixatives
 Formol Saline 10%
 Absolute Ethyl Alcohol
 Acetone
 Newcomer’s Fluid
Formaldehyde
 Methanol  oxidized
 Cheap, readily available, easy to prepare,
stable, compatible w/ stains, penetrates
tissues well, preserves fat, mucin,
glycogen, for tissue photography
 Irritating fumes, prolonged fixation may
bleach tissues
Formaldehyde – precautions:
 Paraformaldehyde formation
 Well ventilated room
 Not neutralized if concentrated – explosion
 Buffered or neutralized by adding
magnesium carbonate/CaCO3 – wide
mouth bottle
 Bleaching prevented by changing formalin
10 % Formol Saline
 Penetrates and fixes tissues well,
minimum shrinkage & distortion, does not
overharden tissues
 Slow (>24 h)
10% Neutral Buffered Formalin
 Na dihydrogen PO4, Disodium H PO4
 For preservation and storage of surgical,
post mortem and research specimens
 Best fixative for Fe pigments, elastic fibers
 Longer to prepare – time consuming, inert
towards lipids
Formol corrosive/formol sublimate
 Formol mercuric chloride
 Minimum shrinkage and hardening
 No need for wash out from fixative to ROH
 Slow
 Forms mercuric chloride deposits
Glutaraldehyde
 For LM, EM
 Adv vs. HCHO: more stable effect, less
tissue shrinkage, less irritating
 Disadv: more expensive, slow penetration
Mercuric Chloride
 Most common metallic fixative; 5-7 %
 For tissue photography, recommended for
renal tissues, fibrin, CT, muscles
 Disadv: hardens outer layers only, black
granular deposits formed (removed by
adding iodine), corrosive to metals
Mercuric Chloride
 Zenker’s (HgCl2 + Glacial HAc) – liver,
spleen, CT fibers, nuclei; poor penetration,
wash thoroughly in running H20
 Zenker-Formol (Helly’s)–HgCl2 , K2Cr2O7
for pituitary, BM, spleen, liver; brown
pigment produced–remove by picric/NaOH
 Heidenhain’s Susa – HgCl2, NaCl, TCA
for skin biopsies; place in high grade ROH
Chromate Fixatives
 Chromic Acid – preserves CHO
 K2Cr2O7 – preserves lipids, mitochondria
 Regaud’s (Moller’s) – 3% K2Cr2O7 – for
chromatin, mitochondri, Golgi, RBC,
colloid, mitotic figures; slow, not for fats
 Orth’s – 2.5% K2Cr2O7 – for Rickettsia,
bacteria, myelin
Lead Fixatives
 For acid MPS
 Fixes connective tissue mucin
 Forms insoluble lead carbonate – remove
by filtering or adding HAc
Picric Acid fixatives (yellow)
 Bouin’s (picric, HCHO, glacial) – for embyros,
glycogen, does not need washing out; poor
penetration, not good for kidneys, mitochondria,
hemolyzes RBC
 Brasil’s alcoholic picroformol (w/TCA) – good for
glycogen; better & less messy than Bouin’s
 Remove yellow color by 70% ethanol followed
by 5% sodium thiosulfate & running water
 Highly explosive when dry
Glacial Acetic Acid
 Solidifies at 17 degrees C  glacial
 For nucleoproteins, chromosomes
 Contraindicated in cytoplasmic fixatives 
destroys mitochondria & golgi
Alcohol Fixatives (fixative/dehyd)
 Polarization – glycogen
 Methanol – BM / bld smears, slow
 Ethanol – strong reducing agent
 Carnoy’s-absolute ROH, CHCl3, glacial
HAc (most rapid); RBC hemolysis
 Alcoholic Formalin (Gendre’s) - sputum
 Newcommer’s – isopropyl ROH, propionic
acid, petroleum ether, acetone, dioxane –
for MPS
Osmium Tetroxide (Osmic Acid)
 Fixes fats, for EM
 Expensive, poor penetration, reduced w/
sunlight  black deposit; dark bottle
 Acid vapor  conjunctivitis, osmic oxide in
cornea  blindness
 Inhibits hematoxylin
 Extremely volatile
 Flemming’s
TCA
 Weak decalcifying agent
 Poor penetration
Acetone
 Use at ice cold temp (-5C to 4C)
 Fixes brain – for rabies
 Dissolves fat, evaporates rapidly,
preserves glycogen poorly
 Heat Fixation
 Secondary Fixation – post chromatization
 Washing Out
Fixation
 Retarded by:  Enhanced by:
 Large size  Small / thin tissue
 Mucus  Agitation
 Fat  Moderate heat (37 to
 Blood 56 degrees C)
 Cold
Decalcification
 Bones, teeth, calcified tissues –
tuberculous lungs, arteriosclerotic vessels
 Poor cutting of hard tissues / knife
damage
 Know patient’s case - if too large – use
saw
 Change decalcifying agent regularly
Decalcification*
 “grating” sensation during cutting = place
block in 10 % HCl for 1 hour
 Rapid decalcification – produces effect on
nuclear staining – (failure of nuclear
chromatin to take up hematoxylin)
Decalcification
 Acids
 Chelating Agents
 Ion Exchange Resins (Ammonium form of
polystrene resin)
 Electrical Ionization (Electrophoresis)
Decalcification
 Acids – HNO3, HCl, formic, TCA,
sulfurous, chromic, citric
 Chelating Agents – EDTA - slow
 Ion Exchange Resins (Ammonium form of
polystrene resin) – 1 – 14 days – spread
on bottom of container
 Electrical Ionization (Electrophoresis) –
attraction of Ca to negative electrode
Acids
 Most common
 Stable
 Easily available
 Cheap
 Nitric, hydrochloric, formic, TCA,
sulfurous, chromic, citric acid
Nitric Acid (5-10%)
 Most common
 Fastest
 Disadvantage: inhibits nuclear stain –
combine with formaldehyde or alcohol
 Aqueous nitric acid 10%, formol nitric acid,
Perenyi’s, Phloroglucin – nitric acid
Nitric Acid
 Aqueous nitric acid 10% = 12-24 hours
 Conentrated nitric acid w/ distilled water
 Rapid, with minimal tissue distortion (if
prolonged)
 Yellow color imparted
Nitric Acid
 Formol – Nitric Acid = 1 – 3 days
 Rapid acting
 Good nuclear staining
 Less tissue destruction than 10% aqeuous
nitric acid
 Use fume hood
 Lessen yellow tissue discoloration by 5%
sodium sulfate or 0.1 % urea
Nitric Acid
 Perenyi’s = 2-7 days
 10% nitric acid, 0.5% chromic acid, absolute
ethyl alcohol
 Decalcifies and softens
 Good nuclear and cytoplasmic staining
 Maceration avoided by chromic/ethyl
 Disadv: slow, difficult to assess complete
decalcification by chemical means
Nitric Acid
 Phloroglucin – Nitric Acid = 12 –24 hours
 Conc nitric + phloroglucin = dense white
fumes, then add 10% nitric acid
 Most rapid
 Disadv: poor nuclear staining
 * when decalcification is complete, acid must
be removed by 3 changes of 70 to 90%
ethanol
HCl
 Slower action, greater tissue distortion
 Good nuclear staining
 * rapid proprietary solutions- w/ HCl
 * slow proprietary solutions - w/ buffered
formalin/formic acid
 Von Ebner’s fluid – NaCl, HCl, H20
 Good cytologic staining
Formic Acid
 Better nuclear staining with less tissue
distortion & * safer to handle than nitric
and HCl
 2-7 days - slow
 Fixative & decalcifying agent
 Excellent nuclear & cytoplasmic staining
 Formic acid – sodium citrate solution (better
nuclear staining than nitric acid)
TCA
 4-8 days – very slow acting
 Good nuclear staining
 Weak
Sulfurous
 Weak
 For minute pieces of bone
Chromic Acid (Flemming’s fluid)
 Chromic acid, osmium tetroxide, glacial
HAc
 Fixative and decalcifier
 Nuclear staining with hematoxylin is
inhibited
 Forms precipitate at the bottom
 *carcinogenic, corrosive to skin
Citric Acid – citrate buffer pH4.5
 With zinc sulfate and chloroform
 6 days – slow
 Good nuclear and cytoplasmic staining
HNO3 - summary
 Most common, rapid (1-3 days)
 Yellow color imparted – prevented by 5%
sodium sulfate, wash in running water x 12
hours or + 0.1% urea
 Formol – Nitric (1-3 days)
 Perenyi’s – w/ chromic acid + abs. EtOH
 Decalcifies & softens; slow (2-7 days)
 Phloroglucin – Nitric – most rapid (12-24h); poor
nuclear staining
 HCl – slower than HNO3
 Formic – fixative/decalcifier, slow
 Formic Acid-Sodium Citrate solution - slow
 TCA – good nuclear staining, no washing
out; weak & slow acting
 Sulfurous – very weak
 Chromic (Flemming’s) – fixative/decalcifier
 Citric Acid – Citrate buffer pH4.5 - slow
 Von Ebner’s – NaCl, HCl
Chelating Agents
 EDTA (versene)
 Excellent staining
 Very slow
 Slight tissue hardening produced
Ion Exchange Resins
 * artifacts produced, usually caused by
CO2 bubbles
 slow
Electrophoresis
(Electrical ionization)
 Formic acid, conc HCl, distilled water
Tests for Completeness of Decal:
 Physical / Mechanical
 X – ray / Radiological
 Chemical – litmus paper  red if due to
acidity, add NH3 drop by drop  blue
litumus; if cloudy – still w/ calcium; if clear:
+ammonium oxalate, 30 mins  cloudy if
incomplete
Tissue softeners
 Perenyi’s – 12 –24 hours
 4% aqueous phenol – 1 – 3 days
 Molliflex (swollen & soapy appearance)
 2 % HCl
 1 % HCl in 70 % alcohol
*Post decalcification
 Remove acid by saturated lithium
carbonate solution or 5-10 % aqueous
NaHCO3 for several hours
 Running tap water
 If EDTA is used – use 70 % alcohol
*Rate of decalcification
 More concentrated acid solutions – more
rapid but more harmful to tissue
 Heat hastens decalcification, but
increases damaging effect of acids to
tissues
 Mechanical agitation, sonication
 Ideal time – 24 to 48 hours
Dehydration – most critical stage
of processing – difficult to correct
 Ideal Dehydrating Solution:
 Rapid acting w/ no tissue shrinkage or
distortion
 Should not evaporate fast
 Can dehydrate even fatty tissues
 Non toxic
 Not fire hazard
 Should not harden tissues excessively
Dehydrating Agents:
 Alcohol – most common
 Acetone
 Dioxane
 Cellosolve
 Triethyl phosphate
 THF
Alcohol – progressively
increasing
 Ethyl – best, fast, mixes with water and
penetrates easily, not poisonous, cheap
 Methyl – toxic, for blood films
 Butyl – slow, for Plant and Animal
microtechnique
* Alcohol
 Prolonged storage in 70 % alcohol –
macerates tissues
 Directly placed in high grade alcohol –
shrinkage & hardening of tissues
Acetone
 Cheap
 Rapid acting
 Volatile
 Tissue shrinkage
Dioxane (Diethylene Dioxide)
 Excellent dehydrating & clearing agent
 Less tissue shrinkage
 Tissues can be left here for a long time
 Ribbons poorly
 Expensive
 Dangerous – vapor – cumulative
 Graupner’s method (dioxane, paraffin wax)
 Weiseberger’s method (gauze, Ca oxide)
Cellosolve
 Ethylene Glycol Monoethyl Ether
 Rapid
 Storage without producing hardening or
distortion
Triethyl Phosphate
 Minimum shrinkage
 Minimum distortion and hardening of
tissue
Tetrahydrofuran - THF
 Dehydrates and clears
 Less shrinkage
 Easier cutting w/ fewer artifacts
 Non toxic; 6 months exposure –
conjunctival irritation
 Offensive odor (use well ventilated room)
*additives to dehydrating agents
 4 % phenol added to 95 % ethanol –
softens hard tissues
 Hard tissues – immerse in glycerol/alcohol
mixture or in Molliflex
Clearing / Dealcoholization
 Substance will dissolve wax with which the
tissue is to be impregnated
 After staining – transparent tissues
 High refractive index
Clearing Agents:
 Miscible with alcohol and paraffin &/or
mounting medium
 Should not produce excessive tissue
shrinkage & hardening
 Should not evaporate quickly in a
waterbath
 Should make tissues transparent
Clearing Agents:
 Xylene
 Toluene
 Benzene
 Chloroform
 Cedarwood oil
 Aniline oil
 Clove oil
 CCl4
* Clearing Agents (cont)
 * methyl benzoate and methyl salicylate
 slow
 `THF
 Dehydrates and clears
 Non toxic
 Offensive odor
Xylene (Xylol)
 Most common, colorless
 Most rapid
 Cheap
 Milky if incomplete dehydration
 Hardens/shrinks tissues – not for nervous
system & lymph nodes
 Hard/brittle tissues if > 3 hours
Toluene
 Not carcinogenic; but will emit fumes that
are toxic upon prolonged exposure
 Rapid acting
 Tissues do not become excessively hard
and brittle even if left here for 24 hours
 Slower than xylene & benzene
 expensive
Benzene
 Rapid acting
 Volatilizes rapidly in paraffin oven
 Tissue shrinkage if left for a long time
 Carcinogenic; aplastic anemia
Chloroform
 For tough tissues – decalcified, nervous,
lymph nodes, embyros – minimum
shrinkage and hardening
 For large specimens
 Toxic to liver (prolonged inhalation)
 Does not make tissues transparent
 Tissues tend to float
Cedarwood oil
 Very penetrating
 Extremely slow
 For CNS, smooth muscles, skin, cytology
 Milky on prolonged storage (filter)
 May produce crystals – heat to 200 C
 Very expensive
Aniline Oil
 For clearing embryos, insects and delicate
specimens
Clove Oil
 Wax impregnation here – slow / difficult
 Tissues become brittle
 expensive
CCl4
 Like chloroform
 Hepatotoxic
THF
 Dehydrates and clears
 Non toxic but with offensive odor
Methyl benzoate/Methyl salicylate

 Slow acting – for double embedding

Impregnation / Infiltration
 Types of Tissue Impregnation/Embedding:
 Paraffin Wax
 Manual – 4 changes of wax 15 mins interval
 Automatic – Autotechnicon, Elliott Bench type

 Vacuum – fastest, negative atmospheric pressure

 Celloidin (Collodion)
 Wet – bones, teeth, large brain sections
 Dry – whole eye sections – 70% ROH not used

 Gelatin
 *Plastic
Paraffin Wax Impregnation
 Simplest, most common, best, 56 C
 Ease in cutting
 Permanent paraffin blocks
 Good staining results
 Not recommended for fatty tissues
 Overheated paraffin  brittle specimen
 Inadequate impregnation  clearing agent
retained
Substitute for paraffin wax:
 Paraplast – more elastic / resilient
 Embeddol – less brittle
 Bioloid – for eyes
 Tissue Mat – with rubber
 Ester Wax – low melting point (46-48C)
 Harder than paraffin; water insoluble
 Water Soluble Wax
 Carbowax – water soluble (no dehydration/clearing);
for enzyme histochemistry; hygroscopic
Celloidin
 Permits cutting of thicker tissue sections
 Crumbling of tissues avoided
 Very slow
 Thin sections difficult to cut
 Serial sections difficult to prepare
 Photomicrographs difficult to obtain
 Very volatile
Nitrocellulose method
 LVN – low viscosity nitrocellulose
 A form of celloidin but more expensive
 Forms harder tissue block; makes thinner
sections
 When dry, striking or dropping the
container will cause explosion.
Gelatin Impregnation
 Rarely used – when dehydration is
avoided; for histochemical and enzyme
studies
 Does not require dehydration & clearing
 Low melting point
Embedding
 Casting / Blocking
 Place tissue in a precisely arranged
position in a mold with a medium which is
then allowed to solidify (orientation)
 Orientation – on the microtome before
cutting, on the slide before staining
Double Embedding
 2 % Celloidin for 3 days and subsequent
paraffin
 For large blocks of dense firm tissues –
brain
 Obsolete – due to paraffin waxes with
different types of resins
*Plastic (Resin) Embedding
 Epoxy, polyester, acrylic
 For hard tissues, renal and bone marrow
biopsies
 With VCD – vinylcyclohexane dioxide-
carcinogenic
Blocking Out Molds
 Leuckhart’s Embedding Mold
 Compound Embedding Unit
 Plastic Embedding Rings & Base Mold
 Tissue Tek – machine w/ warm plate
 Disposable
 Peel Away
 Plastic Ice Trays
 Paper boats
Trimming
 Sides, top and bottom of tissue block are
trimmed
 Celloidin blocks – do not require chilling or
refrigeration
Sectioning
 Microtome
 Block holder, knife carrier, knife, pawl, ratchet
feed wheel and adjustment screws
 Knife
 Plane Concave-celloidin embedded, sliding
 Biconcave – paraffin embedded, rotary microtome
 Plane wedge – frozen sections or hard specimens
in paraffin blocks with base sledge/sliding
Microtomes
 Rocking – serial sections of large paraffin embedded ;
simplest – Paldwell Trefall 1881
 Rotary – paraffin embedded tissues; most common –
Minot 1885-86
 Sliding – celloidin embedded; most dangerous – Adams
1789
 Base Sledge
 Standard Sliding
 Freezing – unembedded frozen sections; Queckett
1881; bursts of CO2 gas vs. cryostat
 Ultrathin – 0.5 u EM
Sliding Microtome
 Base Sledge
 Standard Sliding – block is stationary;
knife is moving
 Most dangerous type of microtome – movable
exposed knife
Honing – heel to toe (sharpening)
 Coarse Honing – gross nicks
 Honing Proper – even edge acquired
 Hone – carborundum – 8”x3”
 Belgium Yellow – best result
 Arkansas – more polishing effect
 Fine Carborundum – much coarser – for badly
nicked
Stropping – toe to heel (polishing)
 Burrs formed during honing is removed
 Cutting edge of knife is polished /
sharpened
Staining
 Natural Dyes
 Hematoxylin
 Cochineal Dyes & its derivatives
 Orcein
 Saffron
 Synthetic (Artificial) Dyes / Coal Tar Dyes /
Aniline Dyes
 Chromophore Dyes: Acid, Basic, Neutral
Methods of Staining
 Direct vs. Indirect (mordant, accentuator)
 Progressive vs. Regressive
 Differentiation (decolorizaton)
 Metachromatic – cations
 Counterstaining
 Microanatomical staining
 Cytoplasmic staining
 Negative staining
Methods of Staining
 Metallic Impregnation
 Vital – Intravital vs. Supravital
 Neutral Red (probably the best vital dye)
 Janus Green (mitochondria)
 Trypan blue
 Nile blue
 Thionine
 Toluidine blue
Hematoxylin
 Alum - blue salt lakes
 Ehrlich’s (Na iodate) – regressive staining
 Harris’ (Mercuric chloride) – for routine nuclear
staining, cytology, sex chromosome; large beaker
used – explosion (O2 liberation)
 Cole’s (w/ Celestine blue)
 Mayer’s – more vigorous than Ehrlich’s
 Fe – blue black salt lakes
 Weigert’s – w/ Ferric Ammonium Chloride
 Heidenhain’s solution – w/ Ferric Am. SO4 (Fe alum)
 Chromium
 Copper
Cochineal Dyes
 From female cochineal bug (Coccus cacti)
+ alum  carmine
 Powerful chromatin and nuclear stain
 With picric acid – picrocarmine –
neuropath
 With AlCl3 (Best’s Carmine) - glycogen
Orcein
 Vegetable dye from Lichens
 For elastic fibers
 Litmus – used as indicator
Aluminum Hematoxylin
 Blueing – with alkaline soln (1% OH) – to
neutralize acid and free the OH group 
insoluble blue Aluminum Hematin-Tissue
lake
Eosin
 Counterstain after hematoxylin and before
methylene blue
 2 shades – Bluish (Eosin B)
Yellowish (Eosin Y) – most
commonly used; in both aqueous and
alocholic solutions (yellow fluorescence)
Other stains:
 Methylene Blue – basic nuclear stain with
eosin; for plasma cells; sputum, bacterial
stain, vital staining of NS, indicator
 Methylene Violet – metachromatic dye
 Toluidine blue – nuclear stain – Nissl
granules, chromophilic bodies
 Crystal violet – if w/ methyl violet &
dexterin – Gentian violet
Other stains:
 Aniline blue – cytoplasmic counterstain
 Basic Fuchsin – acid fast, mitochondria,
smooth muscles
 Carbol Fuchsin
 Coleman’s Feulgen Reagent
 Schiff’s Rgt
 Mallory’s fuchsin
 Aldehyde fuchsin (Gomori’s stain)
Other stains:
 Van Gieson’s – picric acid, acid fuchsin –
(Masson stain) – for CT
 Giemsa – blood
 Celestine Blue – good nuclear stain w/
Alum hematoxylin
 Malachite green – Ascaris ova, RBC,
spore, decolorizer, counter stain
Other Stains:
 Methyl green – chromatin
 Bismarck brown – contrast, diphtheria
 Prussian blue
 Orcein – elastic fibers – Taenzer Unna;
dermatologic studies – fibers
 Picric acid – contrast stain to acid fuchsin
– demonstration of CT; counterstain to
crystal violet, tissue fixative, decalcifier
Other stains:
 Carmine +AlCl3 – glycogen- Best Carmine
and Fucin (Mucicarmine)
 Mayer’s Carmalum solution – basic dye
and stains acidic structures
 Iodine – oldest, starch, amyloid, cellulose,
starch, carotenes, glycogen
 Gram’s Iodine
 Lugol’s Iodine – glycogen, amyloid, corpora
amylacea
Other stains:
 Alcian blue – phthalocyanin dye similar to
chlorophyll – stains acid MPS – more
specific for CT and epithelial mucin
 Neutral red - basic dye – cell granules
 Congo red – indicator – stain for axis
cylinders in embryos – Krajan’s method –
elastic tissues, amyloid, myelin
Other stains:
 Janus Green B – mitochondria (intravital)
 Victoria blue – neuroglia (FS)
 Night blue – Carobl Fuchsin – Acid fast
 Acridine Red 3B – Calcium deposits
 Acridine Orange – green fluorescence for DNA
and red if RNA
 Rhodamine B – w/ osmic acid-bld/glands
 Benzidine - Hb
Oil soluble dyes - lysochromes
 No auxochrome
 Gives color to lipids because they are
more soluble in lipid medium of the
tissues, than in their medium of 70 %
alcohol
 Sudan Black, Sudan IV, III
Gold sublimate
 Metallic impregnation – gold chloride and
mercuric chloride
Osmium tetroxide
 Fixative, stains fat
Silver Nitrate
 Stains spirochete, reticulum, other fiber
stains
Chief Solvents used for Stains
 Water
 Alcohol
 Aniline water
 phenol
Mounting
 Near refractive index of glass 1.518
 Should not dry quickly
 Should not fade
 No shrinkage or tissue distortion
 permanent
Aqueous Media
 Water – low refractive index, temporary
 Glycerin – high refractive index
 Glycerin jelly – 1.47 (gelatin, glycerol, distilled water,
phenol crystals)
 Gum arabic (Farrant’s medium) 1.43
 Apathy’s medium 1.52 (pure gum arabic, pure
cane sugar or sucrose, dist water, thymol)
 Brun’s fluid (glucose, glycerin, spirits of camphor,
distilled water)
 Karo corn syrup
Resinous Media
 Canada Balsam
 DPX
 Xam
 Permount
 Clarite
Ringing
 Sealing margins of the coverslip
 Kronig cement (2 parts paraffin wax w/ 4-9
parts powdered colophonium resin)
 Cellulose Adhesives - Durofix
General Pathology
 Cell Adaptation, Cell Injury, Cell Death
(necrosis)
 Inflammation
 Cell Cycle
 Neoplasia – causes, benign vs. malignant,
grading vs. staging, parts of a tumor,
chemotherapy
Somatic Death
 Primary Changes – circulatory,
respiratory, nervous failure
 Secondary Changes – algor mortis (cold),
rigor mortis (rigidity), livor mortis (purplish
discoloration), post mortem clotting,
dessication, putrefaction, autolysis