CHAPTER 2 CHAPTER 3

FRESH TISSUES EXAMINATION FIXATION AND FIXATIVES

-vary according to structural and chemical components of Fixation – fixing or preserving fresh tissue for examination
the cells to be studied
-may be done on fresh/preserved tissue - Quality of section on the slide is only as good as the
quality of the fixed specimen
METHODS
1. Teasing/Dissociation Primary aim: preserve the morphological and chemical
- selected tissue is immersed in watch glass containing integrity of the cell in as life-like manner.
NSS then carefully dissected/separated and examined - Shape, structure, intercellular relationship and
under the microscope. chemical constituents of tissues are preserved.
- Prevents degeneration, decomposition,
2. Squash Preparation/Crushing putrefaction, and distortion of tissues after
- small pieces not more than 1 mm are placed in a slide removal from the body.
and forcibly compressed with another slide or with a cover
glass. Secondary goal: harden and protect the tissue from the
trauma of further handling
3. Smear Preparation
- examining sections of sediments, whereby cellular Stabilizing proteins: most important reactions for
materials are spread lightly over a slide by means of wire maintaining morphology in the fixation of tissues for
loop or applicator. routine histopathology
- They are fixed to structural proteins and thus
Streaking – zigzag line rendered insoluble
Spreading – teasing mucous strands
Pull-Apart – two slides are used 1. To preserve the tissue
Touch Preparation – cells are transferred to the slide 2. To prevent breakdown of cellular elements
3. To coagulate or precipitate protoplasmic
4. Frozen Section – Tissue is frozen with liquid nitrogen substances
and a section is examined under the microscope.
Additive fixation – fixative becomes part of the tissue
PROCESSING OF TISSUES Negative fixation – fixative does not incorporate into the
Fixation tissue but alters the tissue composition and stabilizes the
Dehydration tissue by removing the bound water attached.
Clearing
Infiltration (Impregnation)
Embedding MAIN FACTORS INVOLVED IN FIXATION:
Trimming 1. Hydrogen Ion Concentration – pH 6 and 8
Section-Cutting 2. Temperature – Formalin heated at 60C
Staining 3. Thickness of section – 2cm2 for light microscopy
Mounting 4. Osmolality – slightly hypertonic
Labelling 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 2. According to Action
- harden soft and friable tissues a. Microanatomical Fixatives – permits the general
- make the cells resistant to damage and distortion microscopic study of tissue structures
- inhibit bacterial decomposition 1. 10% Formol Saline
- increase optical differentiation of cells and tissues 2. 10% Neutral Bufered Formalin
- act as mordants or accentuators 3. Heidenhain’s Susa
- reduce the risk of infection 4. Formol sublimate
5. Zenker’s solution
CHARACTERISTICS OF A GOOD FIXATIVE 6. Zenker formol
1. Cheap 7. Ouin’s solution
2. Stable 8. Brasil’s solution
3. Safe to handle
4. Kills the cell quickly producing minimum distortion b. Cytological – preserve the specific parts and
of cell constituents. particular microscopic elements of the cell itself
5. Inhibit bacterial decomposition
6. Produce minimum shrinkage of tissues 1. Nuclear Fixative – preserve nuclear structures
7. Harden tissues making cutting sections easier Flemming’s fluid
8. Isotonic, causing minimal physical and chemical Carnoy’s fluid
alteration of the cells and their constituents. Bouin’s fluid
9. Make cellular components insoluble to hypotonic Newcomer’s fluid
solutions Heidenhain’s Susa

2. Cytological Fixatives – preserves cytoplasmic

TYPES OF FIXATIVES structures
1. According to composition Flemming’s fluid without acetic acid
a. Simple Fixative – made up of only one Kelly’s fluid
component substance. Formalin with “post-chroming”
i. Aldehydes Regaud’s fluid (Muller’s fluid)
1. Formaldehyde Orth’s fluid
2. Glutaraldehyde
ii. Metallic Fixatives 3. Histochemical Fixatives – preserve chemical
1. Mercuric Chloride contents of cells and tissues
2. Chromate Fixatives Formol Saline 10%
a. Potassium Absolute Ethyl Alcohol
dichromate Acetone
b. Chromic acid Newcomer’s Fluid
3. Lead Fixatives
a. Picric Acid LIPID FIXATION
b. Acetic Acid – Use mercuric chloride and potassium dichromate
c. Acetone - Baker’s formol calcium for phospholipids
d. Alcohol
e. Osmium CARBOHYDRATE FIXATION – Alcoholic formaldehyde
tetraoxide PROTEIN FIXATION – Neutral buffered formol saline or
4. Heat formaldehyde
GLYCOGEN FIXATION – Rossman’s fluid or absolute alcohol
b. Compound Fixative – made up of two or
more fixatives
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 (Gendre’s 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 micro-
incineration
-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.
Zenker’s 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 (Helly’s 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
Heidenhain’s 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 GLACIAL ACETIC ACID
Chromic Acid - Precipitates chromosomes and chromatin materials
- used in 1-2% aqueous solution - Essential constituent of most common compound
- precipitates all proteins and adequately preserves nuclear fixatives
carbohydrates.
- Strong oxidizing agent, strong reducing agent must be ALCOHOLIC FIXATIVES
added. - Ideal for small tissue fragments
- Used as a fixative and dehydrating agent
Potassium Dichromate
- used in 3% aqueous solution Methyl Alcohol - fixing dry and wet smears, blood smears
- preserves lipids and bone marrow tissues
- preserves mitochondria
Isopropyl Alcohol 95% - fixing touch preparations
Regard’s (Muller’s) Fluid Ethyl Alcohol – blood, tissue films and smears
- Potassium dichromate + Strong formaldehyde 40% Carnoy’s Fluid
- Demonstration of chromatin, mitochondria, mitotic - Absolute alcohol + Chloroform + Glacial acetic acid
figures, Golgi bodies, RBC and colloid-containing tissues - fixing chromosomes, lymph node glands and urgent
- Prolonged fixation gives out black deposits and can be biopsies
removed by running tap water. -fix brain tissue for diagnosis of rabies

Orth’s Fluid Newcomer’s Fluid

- study of early degenerative proceses and tissue necrosis - Isopropyl alcohol + Propionic acid + Petroleum ether +
- demonstrates rickettsiae and other bacteria Acetone + Dioxane
- preserves myelin - fixing mucopolysaccharides and other proteins

LEAD FIXATIVES OSMIUM TETRAOXIDE (OSMIC ACID)

- 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
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
Bouin’s 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
- 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
Flemming’s 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

Brasil’s Alcoholic Picroformal Fixative Flemming’s solution without acetic acid

- Formaldehyde + Picric Acid + Ethanol/Isopropyl Alcohol + - Made up only of chromic and osmic acid
Tricbloroacetic acid - Removal of acetic acid from the formula serves to
- Fixative for glycogen improve the cytoplasmic detail of the cell
- Less messy than Bouin’s solution
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 Kelly’s, Zenker’s, and Flemmings solutions
-excess formalin -excess osmic acid
2. 50-70% alcohol – used to wash out excess amount of
picric acid (Bouin’s 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 CHAPTER 6
- Routine dehydration of tissues CLEARING – de-alcoholization
- Mixes with water and other organic solvents - Alcohol or dehydrating agent is removed from the tissue
and replaced with a substance that will dissolve the wax
Methyl Alcohol –blood and tissue films; smear preparation with which the tissue is to be impregnated.
Butyl Alcohol – plant and animal micro-techniques
CHARACTERISTICS OF A GOOD CLEARING AGENT
37C temp – hastens dehydration 1. Should be miscible with alcohol.
Anhydrous copper sulphate 2. Should be miscible with, and easily removed by melted
– accelerates dehydration paraffin wax
- blue discoloration will indicate full saturation 3. Should not produce excessive shrinkage
4. Should not dissolve out aniline dyes
ACETONE 5. It should not evaporate quickly in a water bath.
- Urgent biopsies 6. Should make tissues transparent.
- More miscible with epoxy and other resins & highly
flammable 1. Xylene ( most common)
- Small pieces of tissues due to extreme volatility and 2. Toluene
inflammability 3. Benzene
4. Chloroform
DIOXANE 5. Cedarwood Oil
- Less tissue shrinkage as compared to alcohol dehydration 6. Aniline Oil
- Its vapour produces a cumulative and highly toxic action 7. Clove Oil
in man 8. Carbon tetrachloride

I. Gaupner’s Method XYLENE (XYLOL)

1st Pure dioxane solution 1 hr - Routine histologic processing
2nd Pure dioxane solution 1 hr - Urgent biopsies which it clears within 15-30 minutes
3rd Pure dioxane solution 2 hrs - Becomes milky when an incompletely dehydrated tissue
1st Paraffin wax 15 minutes is immersed in it.
2nd Paraffin wax 45 minutes
3rd Paraffin wax 2 hrs TOLUENE
- Substitute for xylene or benzene
II. Weiseberger’s Method - Miscible with both absolute alcohol and paraffin
- the tissue is wrapped in a gauze bag and suspended in a - Much more expensive than xylene
bottle containing dioxane and a little anhydrous calcium
oxide. BENZENE
- Urgent biopsies and routing purposes
CELLOSOLVE - Excessive exposure to benzene may be extremely toxic to
- can be stored in for months without distortion. man and may become carcinogenic or it may damage the
bone marrow resulting in aplastic anemia.
TRIETHYL PHOSPHATE
- removes water very readily and produces very light CHLOROFORM
distortion and hardening of tissues - Slower action than xylene
- Recommended for tough tissues, for nervous tissues,
TETRAHYDROFURAN lymph nodes and embryos because it causes minimum
- dehydrates and clears tissue shrinkage and hardening of tissues
- THF is toxic if ingested or inhaled. Vapors cause nausea,
dizziness, headache and anesthesia. CEDARWOOD OIL
- May cause conjunctival irritation - Used to clear both paraffin and celloidin sections
- Recommended for CNS tissues and cytological studies,
Phenol 4% + 95% Ethanol – acts as softener for hard particularly smooth muscles and skin.
tissues such as tendons , nails or dense fibrous tissue. - Becomes milky upon prolonged storage and should not
be filtered before use.
ANILINE OIL –clearing embryos, insects and very delicate BY AUTOMATIC PROCESSING
specimens due to its ability to clear 70% alcohol without - Use of automatic tissue processing machine which fixes,
excessive tissue shrinkage dehydrates, clears and infiltrates tissues.
- Only 2-3 changes of wax are required to remove the
CLOVE OIL – Tissues become brittle, aniline dyes re not clearing agent
removed and celloidin is dissolved. Agitation & Heat – hastens automatic process
Ex: Elliott Bench-Type Processor
CARBON TETRACHLORIDE – Similar to chloroform except
it’s a lot cheaper. BY VACUUM EMBEDDING
- Principle is the negative atmospheric pressure hastens
METHYL BENZOATE AND METHYL SALICYLATE the tissue processing
- used in double embedding techniques - Removal of air bubbles and clearing agent from the
tissue block thereby promoting a more rapid wax
penetration of tissue.
CHAPTER 7 - gives the fastest result
IMPREGNATION AND EMBEDDING
IMPREGNATION - process whereby the clearing agent is SUBSTITUTES FOR PARAFFIN WAX
completely removed from the tissue PARAPLAST
EMBEDDING – process by which impregnated tissue is - Mixture of highly purified paraffin and synthetic plastic
placed into a precisely arranged position in a mold polymers
containing a medium which is then allowed to solidify - Melting point of 56-57c
- More elastic and resilient than paraffin wax
1. Paraffin wax
2. Celloidin Embeddol – similar to paraplast; melting point 56-58C
3. Gelatin Bioloid – recommended for embedding eyes
4. Plastic Tissue Mat – Product of paraffin, containing rubber, with
the same property as paraplast.
55-60C – above the melting point of wax
56C – temp of wax normally used for routine work ESTER WAX
- Melting point 46-48C
1. By manual processing - Harder than paraffin wax
2. By automatic processing - 3-4 changes of wax are required to ensure complete
3. By vacuum embedding tissue impregnation

BY MANUAL PROCESSING WATER SOLUBLE WAXES

- 4 changes of wax are required at 15 minutes interval in - Melting points 38-42C or 45-56C
order to insure complete removal of the clearing agent. Carbowax

Example of time schedule for manual 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 CELLOIDIN IMPREGNATION
6H - purified form of nitrocellulose soluble in many solvents,
12H with large hollow cavities which tend to collapse, for hard
2H and dense tissues such as bones and teeth for large tissue
1H sections of the whole embryo
1H - supplied in thin 2%, medium 4%, or thick 8%
1H
15minutes 2 Types:
3H 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 (12-
24hours)  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
GILSON’S 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.
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

Types of Blocking-Out Models

1. Leuckhart’s 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 plastic-
embedding ring
4. Disposable Embedding Models
-
LABORATORY 5- INFILTRATION/IMPREGNATION
- Impregnation is the complete removal of the clearing
1 – FRESH TISSUE EXAMINATION agent by substitution as the medium (paraffin) penetrates
Fresh Tissue Examination – has advantage of examining the tissue with the use of no less than two, and preferably
the tissue in living state allowing protoplasmic activities to three baths of paraffin.
be observed. - Infiltrating Mediums are paraffin wax, celloidin, gelatin
and plastic
Methods -Tissue into a beaker with melted paraffin wax 15minutes
- Teasing (Dissociation)  Second beaker of paraffin wax 15 min  Third beaker
- Squash Preparation (Crushing) of paraffin wax 15 min
- Smear Preparation
-Streaking
-Spreading 6 – EMBEDDING
-Pull-apart - Embedding is the orientation of the tissue in melted
-Touch Preparation paraffin, which when solidified, provides firm medium for
-Frozen Section keeping intact all the parts of the tissues when sections
are cut.
2- FIXATION - Leuckheart’s Embedding Model
Fixation – first and most critical step in histotechnology. - Compound Embedding Unit
- 10% Neutral buffered formalin is mostly widely used - Plastic Embedding Unit
fixative because it is compatible with most stans. - Disposable Embedding Molds
- Physical characteristics to note: color, consistency, -Paper boats, Peel-away, Plastic ice trays
texture and size of tissue to be processed -Arrange tissue in embedding mold  Tissue is oriented at
- Size: 1 x 1 x 0.5 the center of the mold  Pour melted paraffin wax in a
mold and allow to solidify for 3 H
3- DEHYDRATION
- Dehydration is the removal of intercellular and MANUAL TISSUE PROCESSING
extracellular water from the tissue following fixation. FIXATION: 10% Buffered Formalin 24 hours
- Alcohol is the most commonly used dehydrating agent DEHYDRATION: 70% Alcohol 6H
- Acetone provides a rapid method “stat” method 95% Alcohol 12H
- Dioxane is a rapid dehydrating agent but its fumes are 100% Alcohol 2H
highly toxic 100% Alcohol 1H
-Cellosolve dehydrated rapidly and is not harmful to the 100% Alcohol 1H
tissues CLEARING: Xylene or Toluene (2x) 1H
- Tetrahydrofuran possesses same properties as dioxane IMPREGNATION: Paraffin Wax (4x) 15minutes
-70%Ethyl alcohol 6H  90%Ethyl Alcohol 12H  EMBEDDING: Paraffin Wax 3H
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