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IMPREGNATION AND EMBEDDING o Hasten removal of air bubbles and

clearing agent
Impregnation (infiltration) o Recommended for urgent biopsies
Removal of clearing agent and replaced by a o Gives the fastest result
medium that will completely fill all the tissue o
cavities Factors Affecting Paraffin Wax Impregnation
1. Nature and size of tissue
Embedding (casting or blocking) 2. Type of clearing agent used
Process by which the impregnated tissue is placed Benzene and xylene easily
into a precisely arranged position in a mold removed
containing a medium which is then allowed to Chloroform and cedarwood oil
solidify difficult to remove
Substitute for Paraffin Wax
Embedding media: 1. Paraplast melting point of 56-57C, more elastic
1. Paraffin wax and resilient
2. Celloidin 2. Embeddol melting point of 56-58C, less brittle
3. Gelatin and less compressible
3. Bioloid recommended for embedding eyes
PARAFFIN WAX IMPREGNATION 4. Tissue Mat contains rubber
Paraffin 5. Ester Wax melting point of 46-48C, can be used
Simplest, most common, best embedding medium for impregnation without prior clearing
Melting point: 54-58C at 20-24C, 50-54C at 15- 6. Water Soluble Wax does not require
18C dehydration and clearing
Advantages: melting points 38-420C or 45-560C
1. Thin sections cut easily without distortion
3. Tissue blocks can be stored in paraffin for an Celloidin
indefinite period of time without tissue distortion - purified form of nitrocellulose soluble in many
4. Good result staining solvents
Disadvantages: - suitable for large hollow cavities
1. Overheated makes tissue brittle Advantages:
2. Prolonged cause excessive tissue shrinkage 1. Permits cutting of thicker tissues
3. Inadequate promote retention of clearing agent 2. Rubbery consistency to be cut without undue
tissue soft, shrunken distortion
4. Difficult to in filtrate 3. Helps to soften brittle layers of dense tissues
5. Paraffin not for fatty tissues 4. Does not require heat when processed
560C = normally used for routine work 1. Very slow
2. Very thin sections are difficult to cut
Three ways of Paraffin Wax Impregnation 3. Serial sections difficult prepare
Manual Processing 4. Vapor of ether very in flammable
o At least 4 changes of wax at 15 minutes 5. Photomicrographs difficult to obtain
interval each 6. Very volatile
o 3 hours impregnation time
Automatic Processing Two Methods for Celloidin Impregnation of tissue
o 2-3 changes of wax, decreased processing 1. Wet Celloidin Method - recommended for bones,
time because of constant agitation teeth, large brain sections & whole organs
o Makes use of an automatic tissue Process:
processing machine Fixation -> (12-24hours)
o Fixes, dehydrates, clears and infiltrates Thin, medium celloidin -> (5-7 days)
tissue Thick celloidin -> (3-5 days) stored in 70% alcohol
o E.g. Autotechnicon, Elliot Bench Type
Processor 2. Dry celloidin Method whole eye sections
Vacuum Embedding - same Principle except that of 70% Alcohol, it is
o Negative atmospheric pressure inside the GILSONS MIXTURE (equal parts of chloroform
embedding oven and cedarwood oil)
- Best stored in air-tight jar o Used in hard tissues such as undecalcified bone
and for high resolution light microscopy of
NITROCELLULOSE METHOD tissue sections thinner than usual 4- 6 um.
Low Viscosity Nitrocellulose o Plastic are classified as: epoxy, polyester and
- Equal concentration of ether and alcohol, with acrylic
lower viscosity o EPOXY: are made up of a carefully balanced
- Used in higher concentrations and still penetrate mixture of epoxy plastic, catalysts and
tissues rapidly accelerators.
3 types:
- More explosive handle with care
Bisphenol A (araldite)
Glycerol (Epon)
Cyclohexene dioxide (spurr)
Used as an embedding medium for delicate Disadvantages:
specimens and frozen tissue sections because it Hydrophobic
prevents fragmentation of tough and friable Reduce antigenicity
tissues Compromise the result of
Fixation -> 10% gelatin with 1% phenol (24H) immunohistochemistry staining
transferred to 20% gelatin with 1 % phenol (12H) Vinylcyclohexane dioxide carcinogenic
20% gelatin with 1 % phenol
o POLYESTER: were originally introduced
cooled to refrigerator; transferred to 10%
for electron microscopy.
formalin (12-24H) o ACRYLIC PLASTICS: are made up of esters
Volume of impregnating medium should be at of acrylic of methacrylic acid.
least 25 times the volume of the tissue Used extensively for light
EMBEDDING microscopy
After impregnation, the tissue is placed into a mold Polyglycol methacrylate (GMA) *hydrophilic* and Methyl
containing the embedding medium and this medium Methacrylate widely used because of its hardness as the
is allowed to solidify ideal embedding medium for undecalcified bone

ORIENTATION tissue is arranged in precise position in

1. Leuckharts Embedding Mold- consists of L
the mold during embedding, on the microtome before
shaped strips of heavy brass or metal arranged on
flat metal plate and which can be moved to adjust cutting, and on the slide before staining
the size of the mold to the size of specimen.
2. Compound Embedding unit is made up of a MICROTOMY
series of interlocking plates resting on a flat metal - Processed by which processed tissue is trimmed
base and cut into uniformly thin slices or sections
3. Plastic Embedding Rings and Base Mold MICROTOME
consists of a special stainless steel base mold - Capable of cutting a section at a predetermines
fitted with a plastic embedding ring. thickness by sliding the block into a cutting tool
4. Disposable Embedding Mold Three essential parts:
a. Peel Away- disposable thin plastic 1. Block holder
embedding molds available in 3 different 2. Knife carrier and knife
sizes. Giving perfect block even without
3. Pawl, ratchet feed wheel and adjust
b. Plastic Ice Trays
c. Paper boats are normally utilized for Kinds of microtome:
embedding celloidin blocks but are 1. Rocking microtome - for cutting serial sections of large
equally for paraffin wax blocks. blocks of paraffin
Other embedding methods: 2. Rotary microtome - for cutting paraffin embedded sxn
1. Celloidin or Nitrocellulose Method 4. Sliding microtome - for cutting celloidin embedded sxn
o Used to be recommended for embedding hard 5. Freezing microtome - for unembedded frozen sections
tissues such as bones, teeth and for large 6. Ultrathin microtome - for cutting sections for EM
sections of whole organs.
2. Double Embedding Method
Types of Microtome
o Is the process in which tissues are first
infiltrated with celloidin and subsequently 1. Rocking Microtome
embedded in paraffin mass. Cambridge
3. Plastic or Resin Embedding Invented by Paldwell Trefall
For serial sections of large blocks of paraffin Types of Hones
embedded tissues a. Belgium Yellow gives the best result
10-12 u b. Arkansas
2. Rotary Microtome c. Fine Carborundum
Minot d. Plate-glass hone
For paraffin embedded tissues e. Machine hone
Most common type
3. Sliding Microtome STROPPING
Adams The burr formed during honing is removed and
For celloidin embedded tissues thecutting edge of the knife is polished
2 types: base-sledge, standard sliding TOE TO HEEL (Edge last)
Most dangerous because of the exposed movable Purpose: polish and sharpen the cutting edge
knife Common Lubricant Used for Honing
4. Freezing Microtome 1. Mineral oil
Queckett 2. Clove oil
For unembedded frozen section 3. Xylene
E.g. Cryostat maintained at temperature -5 to - 4. Liquid paraffin
30C 5. Soapy water
Most commonly used for rapid preparation STAINING
5. Ultrathin Microtome - process of applying dyes on the sections to see and study
For cutting sections for electron microscopy the pattern of the tissue and physical characteristics of the
0.5 u cells
Fixative osmium tetroxide - Nucleus is acidic so it has greater affinity for basic dyes;
Plastic embedding medium Cytoplasm is basic so it takes more of the acidic stain,
Microtome Knives Combination of these two principles yields to the EXTRA
1. Plane-Concave Knife (25mm)
One side is flat, the other is concave

Less concave celloidin embedded tissue using
Three Major Groups of Tissue Staining
sliding microtome
More concave paraffin embedded tissue using 1. Histological Staining
rotary and rocking microtome Tissue constituents are demonstrated in sections by
2. Biconcave Knife (120mm) direct interaction with dye
Both sides concave Also called as Micro-anatomical staining
For paraffin embedded sections using rotary Micro-anatomic stains
Bacterial Stains
3. Plane-Wedge Knife (100mm)
Specific Tissue Stains (Muscles, Connective Tissue and
Both sides straight
Neurologic Stains)
For frozen sections, extremely hard and tough
specimen in paraffin blocks using base-sledge 2. Histochemical Staining
microtome Various constituents of the tissues are studied
BEVEL ANGLE angle formed between the cutting edge, through chemical reactions
normally 27to 32 = 15 best The final opacity or coloration produced from the
WEDGE ANGLE angle formed by the sides of the wedge substrate rather than the tissue
knives, normally 14 to 15 Perls Prussian Blue (Hemoglobin)
CLEARANCE ANGLE angle formed between the cutting Periodic Acid Schiff (Carbohydrates)
facet presenting to the block and the surface of the block,
normally 5 to 15 3. Immunohistochemical Staining
Combination of immunologic and histochemical
HONING techniques
Removal of gross nicks on knife edge to remove allows the phenotypic markers to be detected
blemishes and grinding the cutting edge of the knife uses a wide range of monoclonal and polyclonal,
on a stone fluorescent or enzyme labeled antibodies.
HEEL TO TOE (Edge first)
Purpose: to remove irregularities from the knife
Methods of Staining Neutral Red best vital dye
Janus Green for mitochondria
1. Direct Staining
3. Progressive Staining After the section is cut and mounted on the slide, it must be
4. Regressive Staining drained and dried thoroughly to ensure that all moisture has
5. Differentiation (Decolorization) evaporated, so section is firmly attached to the slide
6. Metachromatic Staining
8. Metallic Impregnation Coplin Jar (slotted jar that can hold 5 to 9 slides)
Slotted Staining Dishes (can hold 5 to 9 slides)
1. Direct Staining Metal or glass staining racks or carriers (can hold 10 to
Giving color to the sections using aqueous or 30 slides upright)
alcoholic dye solution
Methylene Blue and Eosin
The action of the dye is intensified by adding Most common method for tissue examination
another agent Fixative except Osmic Acid because it inhibit
MORDANT link or bridge between tissue and dye hematoxylin
ACCENTUATOR accelerates/hastens the speed of Harris Hematoxylin primary stain
the staining reaction Acid alcohol differentiator
Potassium Alum with Hematoxylin in Ehrlichs Reagent and Ammonia water blueing agent
Iron in Weigerts Hematoxylin Eosin counterstain
It is the most common method utilized for
3. Progressive Staining microanatomical studies of tissues
Tissue elements are stained in definite sequence, Four Staining Methods
stain is applied until the desired intensity of color is 1. Hematoxylin Eosin Method
2. Thionine Method
4. Regressive Staining
3. Polychrome Methylene Blue Method
The tissue is first overstained and decolorized until
4. Alcoholic Pinacyanol Method
the desired intensity of color is obtained
5. Metachromatic Staining PROGRESSIVE STAINING
Entails the use of dyes which differentiate particular H and E stains are generally arranged in
substances by staining them with the color that is sequence using a series of coplin jars. This
different from that of the stain itself method takes only 5 10 minutes and produces
for staining cartilage connective tissues, epithelial mucins, well differentiated sections that are semi-
mast cell granules and amyloid permanent and can be stored
Metachromatic dyes are basic dyes belonging to thizine RAPID METACHROMATIC STAINING
and triphenylmethane groups such as Methyl Violet or EYE DROPPER METHOD : It may be stained as in
Crystal Violet, Cresyl Blue (for Retics), Safranin, Bismarck paraffin sections although the duration of
Brown, Basic Fuchsin, Methylene Blue, Thionine, Toluidine staining is usually shorter
6. Counterstaining REGRESSIVE STAINING : Fixation most fixatives
Application of a different color or stain to provide can be used except osmic acid solutions which
contrast and background inhibit hematoxylin
Selective staining of living cell constituents Cells nuclei, cytolasmic inclusions and muscle
endothelial cell system (Trypan Blue), Mitochondria striations stain black-- other constituents are
(Janus green) colored according to counterstain
Staining of living cells is done by injecting the dye Cell Nuclei is BLUE
into any parts of the animalbody oxazine dye used as an alternative to iron
Common dyes: lithium, carmine, india ink hematoxylin nuclear stain= strong and precise
b. Supravital Stain nuclear stain resistant to decolorization by
Used to stain living cells immediately after removal succeeding acid stains and solutions
from the living body
Eosin Methylene Blue (EMB) method, produces Recommended for progressive staining
a sharp nuclear stain and reveals with marked a.1. Ehrlichs
differentiation the various structures in the o Ripening agent sodium iodate
tissues, which should be fixed in Zenkers Fluid o Stabilizer glycerine
STAINING OF CELLOIDIN SECTIONS-- cellulose nitrate a.2. Harris
o Ripening agent mercuric chloride
o Stabilizer 4% glacial acetic acid
STAINS AND STAINING SOLUTIONS o Widely used in routine nuclear staining,
1. Natural Dyes exfoliative cytology
A. Hematoxylin a.3. Coles
Derived from Mexican tree Hematoxylin o Ripening agent alcoholic iodine
campechianum o Used in sequence with celestine blue
Hematin active coloring agent formed by the a.4. Mayers
oxidation of hematoxylin (ripening) o Ripening agent sodium iodate
o Natural ripening exposing substance to air or o Regressive and progressive staining
sunlight o Cytoplasmic glycogen
o Artificial ripening uses substance that will b. Iron Hematoxylin
accelerate the process b.1. Weigerts
Hydrogen peroxide, mercuric oxide, potassium o Mordant ferric ammonium chloride
permanganate, sodium perborate, sodium iodate o Standard iron hematoxylin in the laboratory
Ripened hematoxylin + alum, iron, chromium or especially when demonstrating muscle fibers
copper salts and connective tissue
B. Cochineal Dye b.2. Heidenhains
Dye extracted from the female cochineal bug o Mordant ferric ammonium sulfate
(Coccus cati) EOSIN
Treated with alum to produce dye CARMINE Routinely used as counterstain
Carmine + picric acid = picrocarmine a. Yellowish (Eosin Y)
o For neuropathological studies o Most commonly used
Carmine + aluminum chloride = Best Carmine b. Bluish (Eosin B, Erythrosin B)
o Glycogen demonstration o Deeper red color
C. Orcein c. Ethyl eosin (Eosin S, eosin-alcohol soluble)
Vegetable dye extracted from lichens which are
normally colorless treated with ammonia and ADHESIVE AND MOUNTING MEDIA
exposed to air to produce blue or violet color ADHESIVE
2. Synthetic Dyes Essential for methods that require
Known as Coal Tar Dyes exposure of sections to acids and alkalis
Chromophores substance capable of producing 1. Mayers Egg Albumin
color (chromogen) o Most common
Chromogen + auxochrome = dye o Glycerin clearing agent
A. Acid Dyes o Thymol crystal preservative
Active coloring substance is found in the acid 2. Dried Albumin
component and the inactive base 3. 1% Gelatin
E.g. picric acid 4. Gelatin-Formaldehyde mixture
B. Basic Dyes 5. Starch Paste
Active coloring substance is found in the basic 6. Plasma
component 7. Poly-L-Lysine
E.g. methylene blue
Capable of staining cytoplasm and nucleus Added to slide before the application of coverslip
E.g. romanowsky, giemsa, irishman for protection
Common Staining Solutions 1. To avoid distortion of the image, the refractive index of
HEMATOXYLIN the mountant should be as near as possible to that of the
Most common for routine histology glass which is 1.518
Mordant alum,iron 2. It should be freely MISCIBLE with xylene and toluene
a. Aluminum Hematoxylin
3. It should NOT DRY QUICKLY Sections: paraffin, frozen
4. It should NOT CRACK or produce artefactual granularity PAS + subs = red or magenta
on the slide upon drying Nuclei = blue
5. It should NOT DISSOLVE OUT or fade tissue sections Mucoproteins are most common PAS + subs-- CHO,
6. It should NOT CAUSE SHRINKAGE and distortion of glycoproteins, phospholipids
tissues PAS rxn is a useful indicator for glycogen
7. It should NOT LEACH OUT any stain of affect stain
8. It should NOT CHANGE in color and pH STAINING OF GLYCOGEN
9. It should SET HARD, thereby producing permanent Main storage form of glucose, stored in liver
mounting of sections Fixatives Bouins, Brasils, Kellys, Gendre soln
Nuclei = blue black
A. Aqueous Mounting Media Glycogen = Red
1. Water low RI, evaporates quickly, temporary PAS Technique w/diastase = METHOD OF CHOICE
mounting Other methodsbest carmine & langhans iodine
2. Glycerine high RI 1.46
Best Carmine Method
3. Farrants Medium - RI 1.4, gum arabic dissolved in
Due to affinity of alkaline carminic acid for glycogen =
bright red color
4. Apathys Medium RI 1.52
5. Bruns Fluid frozen sections Ehrlichs rgtcounterstain = blue nuclei
B. Resinous Mounting Media FIXATION: neutral 10% formalin, formol-saline, carnoys
1. Canada Balsam RI 1.524, extracted from tree, SECTION: celloidin
Abus balmasea, common Nuclei = blue or grayish blue
2. DPX RI 1.532, recommended for small tissue Glycogen = bright red granules
sections Mucin, fibrin = weark red
3. XAM RI 1.52 Langhans Iodine Method
4. Clarite RI 1.544 Carletons Modification
OLDEST STAIN; not specific for glycogen
Colors amyloid and other CHON subs
RINGING-- Process of sealing of margins of the cover slip to
FIXATION: neutral 10% formol alcohol
prevent the escape of fluid
SECTIONS: paraffin sections
STAINING OF CARBOHYDRATES Glycogen: mahogany brown
CHO= main sources of energy in the body Tissue constituents: yellow


Demonstrates CHO and other subs Mucins are polysaccharides bound to other substances
Used to oxidized 1,2 glycol group of polysaccharides Light pink color w/eosin
and mucin red magenta or purplish-pink color Mucopolysaccharides Acid mucopolysaccharides
Intensity of PAS rxn is proportional to the content of Mucoproteins Neutral mucopolysaccharides
PAS rxn is specific for glycol or glycol amino grp Acid mucopolysaccharides
Polysaccharides w/hexuronic acid as seconday CHO
Schiff reagent const. bound to proteins
Basic fuchsin, mixtures of 3 dyesrosanilin, Hyaluronic acid(CT, fibroblasts), Heparan sulphate
pararosanilin and magenta (aorta, cardiac CT), chondroitin (cartilage)
Reoxidation by slow exposure to light and air will Metachromatic staining toluidine blue and azure a;
restore colorless leukofuchsin to magenta uranyl nitrate-azure method
Different ways : Alcian blue technique
1. The Bargery and de Lamater Method uses thionyl Colloidal iron technique
chloride to release sulfur dioxide Aldehyde fuchsin stain
2. The De Tomasi-Coleman Method addition of Na of Mucicarmine stain
potassium metabisulfate Fluorescent acridine orange technique
3. Oguru Method uses Sulfur dioxide gas
Metachromatic Staining
PAS REACTION AZURE A is the most useful
Fixation: most fixatives Mercurial fixatives are used
Uranyl nitrate = mordant-type; gives excellent results Neutral Mucopolysaccaharides
w/CT Contains hexoses as thei 2nd CHO component
Found in epithelial & intestinal glands
Fresh frozen Azure A Metachromatic Staining for PAS + : mucoproteins and glycolipids
Glycosaminoglycans = red purple Mucoproteinsepithelial, glandular, ductal mucins,
Tissue background = blue gonadotropins and TSH
Acian Blue Technique Glycoproteinsbound to lipid const; gangliosides
Forms electrostatic bonds w/certain tissue components
containing either carboxyl or sulphate groups
Nuclei = red Lipids are best demonstrated on cryostat sections
FORMALIN only preserves those lipids that are already
Combined Alcian Blue-PAS Techiques more or less firmly bound to proteins
Separating acid mucins and neutral mucins PHOSPHOLIPIDS and NEUTRAL FATS are only agents
Acid mucins = PAS + will not react in PAS rxn that truly fix lipids
Neutral mucins will be stained by PAS Formol calcium FIXATIVE OF CHOICE for lipid
Acid mucins = blue histochemistry; 2% Ca acetate to 10% formalin
Neutral mucins = magenta POLYETHYLENE GLYCOLS preserve lipids
Mixtures of above = color will range from blue-purple
thru purple-violet or mauve color SIMPLE LIPIDS: esters of FA w/alc;
Nuclei = pale blue COMPOUND: CNS; phospholipids= important in cell
Gomoris Aldehyde Fuchsin Stain mem, found in mitochondria; glycolipids= FA and
Employs basic fuchsin plus aldehyde to demonstrate
sulfur-containing compounds (strong mineral acid,
DERIVED LIPIDS: FA from hydrolysis of simple and
forms purplish dyes)
compound lipids
stain mast cells
Sulfated mucins = purple Sudanophilia is the properties of tissue to be stained
Carboxylated mucins = blue
with fat or oil-soluble dyes
Mucicarmine Stain
addition of Aluminum hydroxide improves ability of
I. Basic Aryl Amines
carmine to stain mucin
II. B-naphthols
southgates mucicarmine techniqueencapsulated fungi
(C. neoformans)
Basic Aryl Amines
Mucins = red a. Sudan Black B
Nuclei = blue
Most sensitive lipid stain known
BG = unstained
stains phospholipids and neutral fats
it does not stain crystalline cholesterol, and free fatty
Colloidal (dialyzed) Iron Technique
low pH, colloidal will be adsorbed onto tissues acids tend to be dissolved in alcoholic dye bath
visualized by conversion to ferric ferrocyanide (Prussian b. Sudan Red VII B
blue) by Perls technique
Acid mucin = dark blue
a. Sudan III (C.I. No. 26100)
Nuclei = red
b. Sudan IV (Scharlach B, C.I. No. 26105)
- staining fats with a more brilliant or deeper red color
Fluorescent Acridine Orange Technqiue
than Sudan III which stains lipid orange-red; RAPID
to demonstrate acid mucins
mucins stained w/iron hematoxylin and acridine orange
gives selective brilliant orange fluorescence
Sudan Black Method for Lipids
ONLY LAST FOR 2hrs Fixation : formaldehyde calcium with post-chroming
Acid mucopolysaccharides = black Section : Unfixed cryostat sections preferred
Fungi = greenish red fluorescence Lipids : Blue black
BG = reddish orange fluorescence Nuclei : Red
Sudan IV Stains for Lipids-- Scharlach R
Fixation : 10% Formalin
Sections : Frozen Sections
Lipids : Red
Nuclei : Blue/Black

Oil Red O
Fixation : Fresh frozen
Sections : 5um mount on Superfrost
Fat : Brilliant red
Nuclei : Blue

Osmic Acid Stain for Fats

Fixation : 10% formalin
Section : cryostat section
NOT A DYE but is an unstable oxide which is reduced to
a black substances by unsaturated fats and fatty acids

Nile Blue Sulfate Method-- for fats

preliminary indicator of the type of the lipid
Fixation : 10% formalin
Neutral fat : pinkish red
Cholesterin esters and fatty acids : light red
Cerebrosides : light blue
Fatty acids and Soap : deep blue / violet

- NBS is capable of differentiating two lipid classes by the

action of two components:
i. red oxazone = neutral lipids
ii. Blue oxazine = phospholipids & free fatty acids


1. Free Fatty Acids : binds heavy metal ions such as Cu to
form soaps which can be stained with Weigerts lithium
hematoxylin, dimethylaminobenzyldine rhodanine or
rubeanic acid
2. Toluidine-Blue Acetone : Std metachromatic dye for
acidic polymers and imparts a yellow brown or purple
color to sulfatide deposits
3. Cholesterol : Oxidized by cholesterol oxidase to release
hydrogen peroxide which reacts with diaminobenzidine
to produce an insoluble brown polymer
4. Cerebrosides : Stained by PAS designed to stain
mucopolysaccharides and can be distinguished from
glycogen by removal with diastase
5. Borohydride-Periodic-Acid-Schiff : Fixation : formol
calcium; Sections : cryostat sections > Gangliosides : red ;
Nuceli : blue
6. Gangliosides : distinguished by other glycolipids by their
constituents, neuramic acid and sialic acid; Tay-Sachs