CHAPTER 2

CHAPTER 3

FRESH TISSUES EXAMINATION

-vary according to structural and chemical components of
the cells to be studied
-may be done on fresh/preserved tissue

FIXATION AND FIXATIVES

Fixation fixing or preserving fresh tissue for examination

METHODS
1. Teasing/Dissociation
- selected tissue is immersed in watch glass containing
NSS then carefully dissected/separated and examined
under the microscope.
2. Squash Preparation/Crushing
- small pieces not more than 1 mm are placed in a slide
and forcibly compressed with another slide or with a cover
glass.
3. Smear Preparation
- examining sections of sediments, whereby cellular
materials are spread lightly over a slide by means of wire
loop or applicator.
Streaking zigzag line
Spreading teasing mucous strands
Pull-Apart two slides are used
Touch Preparation cells are transferred to the slide
4. Frozen Section Tissue is frozen with liquid nitrogen
and a section is examined under the microscope.
PROCESSING OF TISSUES
Fixation
Dehydration
Clearing
Infiltration (Impregnation)
Embedding
Trimming
Section-Cutting
Staining
Mounting
Labelling

- Quality of section on the slide is only as good as the

quality of the fixed specimen
Primary aim: preserve the morphological and chemical
integrity of the cell in as life-like manner.
- Shape, structure, intercellular relationship and
chemical constituents of tissues are preserved.
- Prevents degeneration, decomposition,
putrefaction, and distortion of tissues after
removal from the body.
Secondary goal: harden and protect the tissue from the
trauma of further handling
Stabilizing proteins: most important reactions for
maintaining morphology in the fixation of tissues for
routine histopathology
- They are fixed to structural proteins and thus
rendered insoluble
1. To preserve the tissue
2. To prevent breakdown of cellular elements
3. To coagulate or precipitate protoplasmic
substances
Additive fixation fixative becomes part of the tissue
Negative fixation fixative does not incorporate into the
tissue but alters the tissue composition and stabilizes the
tissue by removing the bound water attached.

MAIN FACTORS INVOLVED IN FIXATION:

1. Hydrogen Ion Concentration pH 6 and 8
2. Temperature Formalin heated at 60C
3. Thickness of section 2cm2 for light microscopy
4. Osmolality slightly hypertonic
5. Concentration low conc. of glutaraldehyde
6. Duration of fixation 2-6 h in buffered formalin
PRACTICAL CONSIDERATIONS OF FIXATION:
1. Speed
2. Penetration
3. Volume
4. Duration of Fixation

EFFECT OF FIXATIVES
- harden soft and friable tissues
- make the cells resistant to damage and distortion
- inhibit bacterial decomposition
- increase optical differentiation of cells and tissues
- act as mordants or accentuators
- reduce the risk of infection
CHARACTERISTICS OF A GOOD FIXATIVE
1. Cheap
2. Stable
3. Safe to handle
4. Kills the cell quickly producing minimum distortion
of cell constituents.
5. Inhibit bacterial decomposition
6. Produce minimum shrinkage of tissues
7. Harden tissues making cutting sections easier
8. Isotonic, causing minimal physical and chemical
alteration of the cells and their constituents.
9. Make cellular components insoluble to hypotonic
solutions

TYPES OF FIXATIVES
1. According to composition
a. Simple Fixative made up of only one
component substance.
i. Aldehydes
1. Formaldehyde
2. Glutaraldehyde
ii. Metallic Fixatives
1. Mercuric Chloride
2. Chromate Fixatives
a. Potassium
dichromate
b. Chromic acid
3. Lead Fixatives
a. Picric Acid
b. Acetic Acid
c. Acetone
d. Alcohol
e. Osmium
tetraoxide
4. Heat
b. Compound Fixative made up of two or
more fixatives

2. According to Action
a. Microanatomical Fixatives permits the general
microscopic study of tissue structures
1. 10% Formol Saline
2. 10% Neutral Bufered Formalin
3. Heidenhains Susa
4. Formol sublimate
5. Zenkers solution
6. Zenker formol
7. Ouins solution
8. Brasils solution
b. Cytological preserve the specific parts and
particular microscopic elements of the cell itself
1. Nuclear Fixative preserve nuclear structures
Flemmings fluid
Carnoys fluid
Bouins fluid
Newcomers fluid
Heidenhains Susa
2. Cytological Fixatives preserves cytoplasmic
structures
Flemmings fluid without acetic acid
Kellys fluid
Formalin with post-chroming
Regauds fluid (Mullers fluid)
Orths fluid
3. Histochemical Fixatives preserve chemical
contents of cells and tissues
Formol Saline 10%
Absolute Ethyl Alcohol
Acetone
Newcomers Fluid
LIPID FIXATION
Use mercuric chloride and potassium dichromate
- Bakers formol calcium for phospholipids
CARBOHYDRATE FIXATION Alcoholic formaldehyde
PROTEIN FIXATION Neutral buffered formol saline or
formaldehyde
GLYCOGEN FIXATION Rossmans fluid or absolute alcohol

ALDEHYDE FIXATIVES
Formaldehyde widely used (10% formalin)
- DA: fumes are irritating to the nose and eyes
- prolonged storage may induce precipitation of white
paraformaldehyde
- Removal of precipitate is addition of 10% methanol
10% Formol-Saline
- 40% Formaldehyde + NaCl + Distilled water
fixation of CNS Tissues and General post-mortem tissues
- preserves enzymes and proteins
10% Neutral Buffered Formalin/Phosphate-Buffered
Formalin
- Sodium dihydrogen phosphate + Disodium hydrogen
phosphate + 40%Formaldehyde + Distilled water
- preservation of surgical, post-mortem and research
specimens
- best fixative for iron-containing tissues
Formol-Corrosive (Formol Sublimate)
- Sat. Aq. Mercuric Chloride + 40% Formaldehyde
- routine post-mortem tissues
- excellent in silver reticulum methods
- fixes lipids, especially neutral fats and phospholipids
Alcoholic Formalin (Gendres Fixative)
- 95% Ethyl Alcohol saturated with picric acid + Strong
formaldehyde solution + glacial acetic acid
- immunoperoxidase studies on tissues
- used for rapid diagnosis
- good for preservation of glycogen and for microincineration
-used to fix sputum, since it coagulate mucus
Glutaraldehyde
-two formaldehyde residues linked by 3C chains
-used for enzyme histochemistry and electron microscopy
-preserves plasma proteins

METALLIC FIXATIVES
1. MERCURIC CHLORIDE
- Mercuric Chloride + Potassium Dichromate + Sodium
Sulfate + Distilled Water
- most common metallic fixative
- Tissues fixed with mixtures containing mercuric chloride
(except Susa) contain black precipitates of mercury.
-Routine fixative of choice for preservation of cell detail in
tissue photography.
- Renal tissues, Fibrin, Connective tissues and muscles
- Black deposits may be removed by adding saturated
iodine solution in 96% alcohol, the iodine being
decolorized with absolute alcohol in the subsequent
stages of dehydration.
Zenkers Fluid
- Mercuric Chloride + Glacial Acetic Acid
- fixing small pieces of liver, spleen, connective tissue and
nuclei
- may act as mordant
- Mercuric deposits may removed by immersing tissues in
alcoholic iodine solution. de-zenkerization
Zenker-formol (Hellys solution)
- Mercuric chloride + Potassium dichromate + Sodium
sulphate + Distilled water + Strong formaldehyde (40%)
- fixative for pituitary gland, bone marrow and blood
containing organs such as spleen and liver.
- preserves cytoplasmic granules
- Brown pigments are produced if tissues are allowed to
stay for more than 24 hours.
- Pigments can be removed by immersing the tissue in
saturated alcoholic pricric acid or sodium hydroxide
Heidenhains Susa Solution
- Mercuric chloride + Sodium chloride + Trichloroacetic
acid + Glacial Acetic Acid + Formaldehyde (40%) + Distilled
water
- tumor biopsies especially of the skin
- excellent cytologic fixative
-Mercuric chloride deposits may be removed by
immersion on alcoholic iodine solution
- the tissue should be transferred directly to a high-grade
alcohol, to avoid undue swelling of tissues caused by
treatment with low-grade alcohol or water.
B-5 Fixative
- Distilled water + Mercuric Chloride + Sodium acetate
- commonly used for bone marrow biopsies

CHROMATE FIXATIVES
Chromic Acid
- used in 1-2% aqueous solution
- precipitates all proteins and adequately preserves
carbohydrates.
- Strong oxidizing agent, strong reducing agent must be
added.
Potassium Dichromate
- used in 3% aqueous solution
- preserves lipids
- preserves mitochondria

GLACIAL ACETIC ACID

- Precipitates chromosomes and chromatin materials
- Essential constituent of most common compound
nuclear fixatives
ALCOHOLIC FIXATIVES
- Ideal for small tissue fragments
- Used as a fixative and dehydrating agent
Methyl Alcohol - fixing dry and wet smears, blood smears
and bone marrow tissues

Regards (Mullers) Fluid

- Potassium dichromate + Strong formaldehyde 40%
- Demonstration of chromatin, mitochondria, mitotic
figures, Golgi bodies, RBC and colloid-containing tissues
- Prolonged fixation gives out black deposits and can be
removed by running tap water.

Isopropyl Alcohol 95% - fixing touch preparations

Ethyl Alcohol blood, tissue films and smears
Carnoys Fluid
- Absolute alcohol + Chloroform + Glacial acetic acid
- fixing chromosomes, lymph node glands and urgent
biopsies
-fix brain tissue for diagnosis of rabies

Orths Fluid
- study of early degenerative proceses and tissue necrosis
- demonstrates rickettsiae and other bacteria
- preserves myelin

Newcomers Fluid
- Isopropyl alcohol + Propionic acid + Petroleum ether +
Acetone + Dioxane
- fixing mucopolysaccharides and other proteins

LEAD FIXATIVES
- used in 4% aqueous solution
- recommended for acid mucopolysaccharides
- fixes connective tissue mucin
- forms insoluble lead carbonate due to prolonged
standing which can be removed by filtration or adding
acetic acid drop by drop

OSMIUM TETRAOXIDE (OSMIC ACID)

- Pale yellow powder which dissolves in water to form
strong oxidizing solution.
-fixes conjugated-fats and lipids permanently by making
them insoluble during subsequent treatment with alcohol
and xylene
-helps preserve cytoplasmic structure
-fixes myelin and peripheral nerves well
-fixes materials for ultrathin sectioning in electron
microscopy, since it rapidly fixes small pieces of tissues
and aids in their staining
-black osmic oxide crystals may be dissolved in cold water
-Osmic acid-fixed tissues must be washed in running water
for at least 24 hours to prevent formation of artefacts

PICRIC ACID FIXATIVES

- Yellow color may be removed by treatment with another
acid dye or lithium carbonate
- excellent fixative for glycogen demonstration
- suitable for Aniline stains
Bouins Solution
- Sat. Solution of picric acid + Strong formaldehyde 40% +
Glacial acetic acid
- Fixation of embryos and pituitary biopsies
- Preserving soft and delicate structures (endometrial
curettings)
-yellow stain is useful for handling fragmentary biopsies.
- collagen, elastic connective tissues
Brasils Alcoholic Picroformal Fixative
- Formaldehyde + Picric Acid + Ethanol/Isopropyl Alcohol +
Tricbloroacetic acid
- Fixative for glycogen
- Less messy than Bouins solution

Flemmings Solution
- common chrome-osmium acetic acid fixative used
- Recommended for nuclear preparation of such sections
- Aqueous chromic acid 1% + Aqueous osmium tetraoxide
2% + Glacial acetic acid
-an excellent fixative for nuclear structures
-permanently fixes fat
Flemmings solution without acetic acid
- Made up only of chromic and osmic acid
- Removal of acetic acid from the formula serves to
improve the cytoplasmic detail of the cell

TRICHLOROACETIC ACID
- incorporated into compound fixatives
-precipitates proteins
-marked swelling effect on tissues serves to counteract
shrinkage produced by other fixatives
-may be used as a weak decalcifying agent
ACETONE
- Used at a cold temperature ranging from 5*C to 4*C
- Recommended for the study of water diffusible enzymes
especially phosphatases and lipases
- Used in fixing brain tissues for diagnosis of rabies
HEAT FIXATION
- Involves thermal coagulation of tissue protein for rapid
diagnosis
- Employed for frozen tissue sections and bacteriologic
smears
- Destroys RBC
- Dissolves starch and glycogen
SECONDARY FIXATION
- Process of replacing an already fixed tissue in a second
fixative order
- Usually with 10% formalin or 10% formol saline as
primary fixative
POST CHROMATIZATION
-form of secondary fixation whereby a primarily fixed
tissue is placed in aqueous solution of 2.5- 3 % potassium
dichromate for 24 hours to act as mordant for better
staining effects
WASHING-OUT
-process of removing excess fixative from the tissue after
fixation in order to improve staining and remove artefacts
from the tissues
1. Tap water- removes:-excess chromates from tissues
fixed in Kellys, Zenkers, and Flemmings solutions
-excess formalin -excess osmic acid
2. 50-70% alcohol used to wash out excess amount of
picric acid (Bouins solution)
3. Alcoholic iodine- used to remove excessive mercuric
fixatives

Factors that affect Fixation of the Tissues

A.RETARDED BY:
1. Size and thickness of the tissue specimen-largest tissues
require more fixatives and longer fixation time
2. Presence of Mucus-prevents complete penetration of
fixative; hence, tissues that contain mucus are fixed slowly
and poorly.Excess mucus may be washed away with
normal saline solution.
3. Presence of fat- fatty tissues should be cut in thin
sections and fixed longer.
4. Presence of blood- tissues containing large amount of
blood should be flushed out with saline before fixing
5. Cold temperature- inactivates enzymes
B.ENHANCED BY:
1. Size and thickness of tissues- smaller and thinner
tissues requires less fixative and shorter fixation time
2. Agitation- fixation is accelerated when automatic or
mechanical tissue processing is used.
CHAPTER 5
DEHYDRATION
- Process of removing intercellular and extracellular water
from the tissue following fixation and prior to wax
impregnation
Dehydration Agents - Solutions utilized
General Rule: Whatever the dehydrating agent is used, the
amount in each stage should not be less than 10 times the
volume of the tissue in order to ensure complete
penetration of the tissue by the dehydrating solution.
Characteristics of Ideal Dehydrating Agent
1. Should dehydrate rapidly
2. Should not evaporate very fast
3. It should be able to dehydrate even fatty tissues
4. It should not harden tissues excessively
5. It should not remove stains
6. It should not be toxic to the body
7. It should not be a fire hazard
Commonly used:
- Alcohol (most common)
- Acetone
- Dioxane 4 cellosolve
- Triethyl phosphate
- Tetrahydrofuran

ALCOHOL
- Routine dehydration of tissues
- Mixes with water and other organic solvents
Methyl Alcohol blood and tissue films; smear preparation
Butyl Alcohol plant and animal micro-techniques
37C temp hastens dehydration
Anhydrous copper sulphate
accelerates dehydration
- blue discoloration will indicate full saturation
ACETONE
- Urgent biopsies
- More miscible with epoxy and other resins & highly
flammable
- Small pieces of tissues due to extreme volatility and
inflammability
DIOXANE
- Less tissue shrinkage as compared to alcohol dehydration
- Its vapour produces a cumulative and highly toxic action
in man
I. Gaupners Method
1st Pure dioxane solution 1 hr
2nd Pure dioxane solution 1 hr
3rd Pure dioxane solution 2 hrs
1st Paraffin wax 15 minutes
2nd Paraffin wax 45 minutes
3rd Paraffin wax 2 hrs
II. Weisebergers Method
- the tissue is wrapped in a gauze bag and suspended in a
bottle containing dioxane and a little anhydrous calcium
oxide.
CELLOSOLVE
- can be stored in for months without distortion.
TRIETHYL PHOSPHATE
- removes water very readily and produces very light
distortion and hardening of tissues
TETRAHYDROFURAN
- dehydrates and clears tissue
- THF is toxic if ingested or inhaled. Vapors cause nausea,
dizziness, headache and anesthesia.
- May cause conjunctival irritation
Phenol 4% + 95% Ethanol acts as softener for hard
tissues such as tendons , nails or dense fibrous tissue.

CHAPTER 6
CLEARING de-alcoholization
- Alcohol or dehydrating agent is removed from the tissue
and replaced with a substance that will dissolve the wax
with which the tissue is to be impregnated.
CHARACTERISTICS OF A GOOD CLEARING AGENT
1. Should be miscible with alcohol.
2. Should be miscible with, and easily removed by melted
paraffin wax
3. Should not produce excessive shrinkage
4. Should not dissolve out aniline dyes
5. It should not evaporate quickly in a water bath.
6. Should make tissues transparent.
1.
2.
3.
4.
5.
6.
7.
8.

Xylene ( most common)

Toluene
Benzene
Chloroform
Cedarwood Oil
Aniline Oil
Clove Oil
Carbon tetrachloride

XYLENE (XYLOL)
- Routine histologic processing
- Urgent biopsies which it clears within 15-30 minutes
- Becomes milky when an incompletely dehydrated tissue
is immersed in it.
TOLUENE
- Substitute for xylene or benzene
- Miscible with both absolute alcohol and paraffin
- Much more expensive than xylene
BENZENE
- Urgent biopsies and routing purposes
- Excessive exposure to benzene may be extremely toxic to
man and may become carcinogenic or it may damage the
bone marrow resulting in aplastic anemia.
CHLOROFORM
- Slower action than xylene
- Recommended for tough tissues, for nervous tissues,
lymph nodes and embryos because it causes minimum
shrinkage and hardening of tissues
CEDARWOOD OIL
- Used to clear both paraffin and celloidin sections
- Recommended for CNS tissues and cytological studies,
particularly smooth muscles and skin.
- Becomes milky upon prolonged storage and should not
be filtered before use.

ANILINE OIL clearing embryos, insects and very delicate

specimens due to its ability to clear 70% alcohol without
excessive tissue shrinkage
CLOVE OIL Tissues become brittle, aniline dyes re not
removed and celloidin is dissolved.
CARBON TETRACHLORIDE Similar to chloroform except
its a lot cheaper.
METHYL BENZOATE AND METHYL SALICYLATE
- used in double embedding techniques

CHAPTER 7
IMPREGNATION AND EMBEDDING
IMPREGNATION - process whereby the clearing agent is
completely removed from the tissue
EMBEDDING process by which impregnated tissue is
placed into a precisely arranged position in a mold
containing a medium which is then allowed to solidify
1.
2.
3.
4.

Paraffin wax
Celloidin
Gelatin
Plastic

55-60C above the melting point of wax

56C temp of wax normally used for routine work

BY AUTOMATIC PROCESSING
- Use of automatic tissue processing machine which fixes,
dehydrates, clears and infiltrates tissues.
- Only 2-3 changes of wax are required to remove the
clearing agent
Agitation & Heat hastens automatic process
Ex: Elliott Bench-Type Processor
BY VACUUM EMBEDDING
- Principle is the negative atmospheric pressure hastens
the tissue processing
- Removal of air bubbles and clearing agent from the
tissue block thereby promoting a more rapid wax
penetration of tissue.
- gives the fastest result
SUBSTITUTES FOR PARAFFIN WAX
PARAPLAST
- Mixture of highly purified paraffin and synthetic plastic
polymers
- Melting point of 56-57c
- More elastic and resilient than paraffin wax
Embeddol similar to paraplast; melting point 56-58C
Bioloid recommended for embedding eyes
Tissue Mat Product of paraffin, containing rubber, with
the same property as paraplast.

1. By manual processing
2. By automatic processing
3. By vacuum embedding

ESTER WAX
- Melting point 46-48C
- Harder than paraffin wax
- 3-4 changes of wax are required to ensure complete
tissue impregnation

BY MANUAL PROCESSING
- 4 changes of wax are required at 15 minutes interval in
order to insure complete removal of the clearing agent.

WATER SOLUBLE WAXES

- Melting points 38-42C or 45-56C
Carbowax

Example of time schedule for manual processing:

FIXATION: 10% Buffered Formalin
24 hours
DEHYDRATION: 70% Alcohol
6H
95% Alcohol
12H
100% Alcohol
2H
100% Alcohol
1H
100% Alcohol
1H
CLEARING: Xylene or Toluene (2x)
1H
IMPREGNATION: Paraffin Wax (4x)
15minutes
EMBEDDING: Paraffin Wax
3H

CELLOIDIN IMPREGNATION
- purified form of nitrocellulose soluble in many solvents,
with large hollow cavities which tend to collapse, for hard
and dense tissues such as bones and teeth for large tissue
sections of the whole embryo
- supplied in thin 2%, medium 4%, or thick 8%
2 Types:
1. Parloidoin hard, amber, platelet-chips
2. Low Viscosity Nitrocellulose nitrated variety
- it can be used in higher concentrations and still
penetrate tissues rapidly.
- usually marketed while wet with alcohol.

2 Methods:
1. Wet Celloidin Method
- Recommended for bones, teeth, alrge brain
sections and whole organs
Process:
Fixation placed in equal parts of ether and alcohol (1224hours) thin, medium celloidin (5-7 days) thick
celloidin (3-5 days) stored in 70% alcohol
2. Dry Celloidin Method
- Recommended for processing the whole eye
sections
- Same Principle except that of 70% Alcohol, it is
GILSONS MIXTURE (equal parts of chloroform and
cedarwood oil)
- Best stored in air-tight jar
GELATIN IMPREGNATION
- Used as an embedding medium for delicate specimens
and frozen tissue sections because it prevents
fragmentation of tough and friable tissues
- Fixation 10% gelatin with 1% phenol (24H)
transferred to 20% gelatin with 1 % phenol (12H)
20% gelatin with 1 % phenol cooled to refrigerator
transferred to 10% formalin (12-24H)
-Volume of impregnating medium should be at least 25
times the volume of the tissue
EMBEDDING
- After impregnation, the tissue is placed into a mold
containing the embedding medium and this medium is
allowed to solidify.
Types of Blocking-Out Models
1. Leuckharts Embedding Model L-shaped strips of
heavy brass arranged in a flat metal plate and which can
be moved to adjust the size of mold to the size of the
specimen.
2. Compound Embedding Unit interlocking plates resting
on a flat metal base
3. Plastic Embedding Rings and Base Mold
- special stainless steel base mold fitted with plasticembedding ring
4. Disposable Embedding Models
-

PLASTIC (RESIN) EMBEDDING

- superior results for light microscopic studies
- high resolution light microscopy of tissue
- Epoxy, Polyester, Acrylic
EPOXY
- Benzoyl peroxide + epoxy (catalysts)
- epoxy plastics, catalysts and accelerators
- Cyclohexene dioxide-based plastics (Spurtt) can be
obtained pure, have very low viscosity and infiltrate
fastest.
Disadvantages:
1. Hydrophobic
2. Reduce antigenicity of embedded tissue and may
compromise the result of immunohistochemical staining
3. Cause sensitization if absorbed bt skin or inhalation
4. VCD is known to be carcinogenic
POLYESTER PLASTICS
- Introduced for electron microscopy
ACRYLIC PLASTICS
- made up of acrylic acid or methacrylic acid
- used for light microscopy
Ex.
1. Polyglycol methacrylate (GMA)
2. Methyl methacrylate (MMA) undecalcified bone

LABORATORY
1 FRESH TISSUE EXAMINATION
Fresh Tissue Examination has advantage of examining
the tissue in living state allowing protoplasmic activities to
be observed.
Methods
- Teasing (Dissociation)
- Squash Preparation (Crushing)
- Smear Preparation
-Streaking
-Spreading
-Pull-apart
-Touch Preparation
-Frozen Section
2- FIXATION
Fixation first and most critical step in histotechnology.
- 10% Neutral buffered formalin is mostly widely used
fixative because it is compatible with most stans.
- Physical characteristics to note: color, consistency,
texture and size of tissue to be processed
- Size: 1 x 1 x 0.5
3- DEHYDRATION
- Dehydration is the removal of intercellular and
extracellular water from the tissue following fixation.
- Alcohol is the most commonly used dehydrating agent
- Acetone provides a rapid method stat method
- Dioxane is a rapid dehydrating agent but its fumes are
highly toxic
-Cellosolve dehydrated rapidly and is not harmful to the
tissues
- Tetrahydrofuran possesses same properties as dioxane
-70%Ethyl alcohol 6H 90%Ethyl Alcohol 12H
Absolute Ethyl Alcohol 2H Absolute Ethyl Alcohol 1H
Absolute Ethyl Alcohol
4- CLEARING (DE-ALCOHOLIZATION)
- Clearing is the removal of dehydrating agent from the
tissue
-Xylene is the most rapid clearing agent, suitable for
urgent biopsies
- Xylene 1H Xylene 1 H

5- INFILTRATION/IMPREGNATION
- Impregnation is the complete removal of the clearing
agent by substitution as the medium (paraffin) penetrates
the tissue with the use of no less than two, and preferably
three baths of paraffin.
- Infiltrating Mediums are paraffin wax, celloidin, gelatin
and plastic
-Tissue into a beaker with melted paraffin wax 15minutes
Second beaker of paraffin wax 15 min Third beaker
of paraffin wax 15 min

6 EMBEDDING
- Embedding is the orientation of the tissue in melted
paraffin, which when solidified, provides firm medium for
keeping intact all the parts of the tissues when sections
are cut.
- Leuckhearts Embedding Model
- Compound Embedding Unit
- Plastic Embedding Unit
- Disposable Embedding Molds
-Paper boats, Peel-away, Plastic ice trays
-Arrange tissue in embedding mold Tissue is oriented at
the center of the mold Pour melted paraffin wax in a
mold and allow to solidify for 3 H
MANUAL TISSUE PROCESSING
FIXATION: 10% Buffered Formalin
DEHYDRATION: 70% Alcohol
95% Alcohol
100% Alcohol
100% Alcohol
100% Alcohol
CLEARING: Xylene or Toluene (2x)
IMPREGNATION: Paraffin Wax (4x)
EMBEDDING: Paraffin Wax

24 hours
6H
12H
2H
1H
1H
1H
15minutes
3H