ROUTINE HISTOTECHNIQUES
> Overall size of the tumor
CHAPTER 1: SPECIMEN HANDLING AND IDENTIFICATION
Bar codes are frequently used by clinical laboratories.
The specimen container label and the accompanying request
form should include: Patients name, Age or birth date, A
medical record number
Label should be firmly attached to the body of the container --
- not to the lid of the container.
The request form should have a provisional diagnosis and
brief clinical details.
Any discrepancies in specimen identification or labeling
should be resolved prior to processing.
Incorrect identification of any specimen of any specimen
results in the wrong diagnoses and incorrect treatment.
GROSSING
Major components in grossing a specimen:
1) Reliable and rapid transfer of the specimen from surgery to
pathology
2) Accurate identification of the specimen
3) Description of additional specimens received from the same
patient
4) Gross description of the specimens normal and abnormal
features
5) Recording the sites from which blocks of tissue are taken
6) Recording markers that help with the correct orientation
7) Identifying special studies requested and/or needed.
SPECIMEN FROM DERMATOLOGY
1) CORE BIOPSIES
Larger core biopsies (4mm) --- should be bisected eccentrically
and embedded with cut surfaces down.
Small core biopsies (2mm) --- should be embedded totally
without cutting it.
2) SHAVE BIOPSIES OF SKIN
Depending upon the size of the biopsy, it may be bisected,
trisected, or cut into sections.
Most specimens of skin or other epithelial surfaces should be
cut --- all aliquots are embedded on edge.
Care should be taken with any pigmented lesions of the skin.
3) EXCISIONAL BIOPSY
Method of choice for surgical removal of melanomas but may
be sometimes removed by shaving
Biopsies of skin are examined to ensure that the lesion has
been completely removed and the original clinicians
diagnosis was correct.
Can be oriented using sutures or dyes.
4) RE-EXCISION SPECIMENS
Original site of a lesion may need to be re- excised if:
> The margins are invaded by tumor
> Too close to the tumor: melanoma / basal cell carcinoma
5) NON-SKIN SPECIMEN (Excisional biopsies)
Operative specimens --- tumors, unidentifiable inflammatory
masses, tissues removed prior to transplantation, traumatic,
congenital malformations, or cosmetic surgical specimens.
All specimens must be examined carefully --- may harbor
unsuspected malignant tumors
Important determinants of neoplastic specimens:
> Depth of invasion into or through the tissue walls
> Involvement of margins and lymph nodes
CHAPTER 2: METHODS OF FRESH TISSUE EXAMINATION
1. TEASING or DISSOCIATION
A process whereby a selected tissue specimen is immersed in
a watch glass containing isotonic salt solution, carefully
dissected or separated, and examined under the microscope.
2. SQUASH PREPARATION (Crushing)
A process whereby small pieces of tissue not more than 1 mm
in diameter are placed in a microscopic slide and forcibly
compressed with another slide or with a cover glass.
3. SMEAR PREPARATION
The process of examining sections or sediments, whereby
cellular materials are spread lightly over a slide by a wire loop
or applicator, or by making an apposition smear with another
slide.
Useful in cytological examinations --- particularly for cancer
diagnosis
a. STREAKING with an applicator stick or platinum loop, the
material is rapidly and gently applied in a direct or zigzag line
throughout the slide, attempting to obtain a relatively
uniform distribution of secretion.
b. SPREADING a selected portion of the material is transferred
to a clean slide and gently spread into a moderately thick film
by teasing the mucous strands apart with an applicator stick
o Little more tedious but with an advantage of maintaining
cellular interrelationships of the materials to be
examined.
o Recommended for smears preparations of fresh sputum
and bronchial aspirates and for thick mucoid secretions.
c. PULL-APART done by placing a drop of secretion or
sediment upon one slide and facing it to another clean slide.
o The material disperses evenly over the surface of two
slides.
o Slight movement of the two slides in opposite directions
may be necessary to initiate the flow of materials.
o The two slides are then pulled apart with a single
uninterrupted motion, and the specimen placed under
microscope for immediate examination, or applied with
vital stains.
o Useful for preparing smears of thick secretions such as
serous fluid, concentrated sputum, enzymatic lavage
samples from GIT, and blood smear
d. TOUCH PREPARATION (Impression smear) - a special method
of smear preparation whereby the surface of a freshly cut
piece of tissue is brought into contact and pressed on to the
surface of a clean glass slide, allowing the cells to be
transferred directly to the slide for examination.
o Advantage: Cells may be examined without destroying
their actual intercellular relationship
4. FROZEN SECTION
is normally utilized when a rapid diagnosis of the tissue in
question is required.
 Especially recommended when lipids and nervous tissue
elements are to be demonstrated
Applications:
1. Rapid pathologic diagnosis during surgery
2. Diagnostic and research enzyme histochemistry
3. Diagnostic and research demonstration of soluble
substances such as lipids and carbohydrates
4. Immunofluorescent and immunohistochemical staining
5. Some specialized silver stains --- particularly in
neuropathology
LIQUID NITROGEN generally used in histochemistry and during
operative procedures
> Most rapid of the commonly available freezing agents
> Disadvantage: Soft tissue is liable to crack due to rapid expansion
of the ice within the tissue --- producing ice crystals or freeze
artifacts
> PROBLEM: Use of liquid nitrogen can cause a vapor phase to form
around the tissue --- acting as an insulator that causes uneven
cooling of tissue (particularly in muscle biopsies) and making
diagnostic interpretation difficult.
> Can be overcome by freezing the tissue in Isopentane, OCT, or
Freon 2.2 --- has a high thermal conductivity.
CHAPTER 3: HISTOTECHNOLOGY
Is the art and science performed by the histotechnologist to
produce a tissue section of good quality that will enable the
pathologist to diagnose the presence or absence of disease.
FIXATION first and most critical step in histotechnology
> Fixing or preserving fresh tissue for examination.
> The quality of the section on the slide is only as good as the
quality of the fixed tissue specimen.
the process by which the constituents of the cells and tissues
are fixed in a physical, and partly in a chemical state, so that
they will withstand subsequent treatment with various
reagents with a minimum of loss, significant distortion, or
decomposition.
Inadequate or poor fixation will result in a poorly processed
tissue and will make it difficult for the pathologist to render a
proper diagnosis.
With fixation, the shape, structure, intercellular relationship
and chemical constituents of tissues are preserved.
Fixation prevents degeneration, decomposition, putrefaction,
and distortion of tissues after removal from the body.
To Preserve the tissue --- by stopping all cellular activities so
that cells can be viewed under the microscope as if they are
still in their original living state.
> Do not leave the tissue specimen in air for prolonged period
of time --- dry out and will result in distortion of its
morphologic appearance
> Leaving the tissue in water ---cause the cell to swell while a
strong salt --- cause the cell to shrink.
2 MECHANISM INVOLVED IN FIXATION:
1) ADDITIVE FIXATION
The chemical constituent of the fixative is taken in and
becomes part of the tissue by forming cross- links or
molecular complexes and giving stability to protein.
Example: Formalin, Osmium tetroxide, Mercury
2) NON-ADDITIVE FIXATION
The fixing agent is not incorporated into the tissue, but alters
the tissue composition and stabilizes the tissue by removing
the bound water attached to H- bonds.
Example: Alcoholic fixatives
MAIN FACTORS INVOLVED IN FIXATION:
1) Hydrogen Ion Concentration
2) Temperature
3) Thickness of section
4) Osmolality
5) Concentration - Low concentrations of glutaraldehyde have
been found to be an ideal concentration for immuno- electron
microscopy.
6) Duration of fixation - Buffered formalin is 2 to 6 hours during
the day the specimen is obtained
Prolonged fixation: May cause shrinkage and hardening of
tissue and may severely inhibit enzyme activity and
immunological reactions
For electron microscopy, tissues should be fixed for 3 hours
and then placed in holding buffer.
PRACTICAL CONSIDERATIONS OF FIXATION:
SPEED: Specimen should be placed in fixative as soon as it is
removed from the body.
* Done to prevent autolysis and putrefaction
PENETRATION: Time of fixation varies with different types of tissue
* Formalin diffuses into the tissue at the rate of 1mm per hour.
VOLUME: The amount of fixative used has been 10 25 times the
volume of tissue to be fixed
* Maximum effectiveness of fixation: 20x the tissue vol.
DURATION OF FIXATION: Some tissues take longer to fix than
others, depending on their structure.
* Fixation time can be cut down by using heat, vacuum,
agitation or microwave
MISCELLANEOUS FIXATION
Tissue selected for sectioning should be thin enough to allow
penetration by fixative within a reasonable amount of time.
To maintain an adequate fixation time of 4 6 hours, the
recommended size of the tissue is 2 square cm, and no more
than 4 mm thick.
Most tissue can be cut and trimmed without prior fixation,
EXCEPT for the brain.
FIXATIVES
EFFECTS OF FIXATIVE:
a. Harden soft and friable tissues and make the handling and
cutting of sections easier.
Usually accelerated by the action of alcohol during
dehydration process.
b. Make cells resistant to damage and distortion caused by
hypotonic and hypertonic solutions used during tissue
processing
c. Inhibit bacterial decomposition
d. Increase the optical differentiation of cells and tissue
components thereby rendering them more readily visible
during examination.
 e. Act as mordants or accentuators to promote and hasten
staining or inhibit certain dyes in favor of another.
f. Reduce the risk of infections during handling and actual
processing.
CHARACTERISTICS OF A GOOD FIXATIVE:
a. Must be cheap, stable and safe to handle.
b. Must kill the cell quickly thereby producing minimum
distortion of cell constituents
c. Must inhibit bacterial decomposition and autolysis
d. Must produce minimum shrinkage of tissue.
e. Must permit rapid and even penetration of tissues.
f. Must harden tissues thereby making the cutting of sections
easier.
g. Must be isotonic --- causing minimal physical and chemical
alteration of the cells and their constituents.
h. Must make cellular components insoluble to hypotonic
solutions and render them insensitive to subsequent
processing.
i. Must permit the subsequent application of many staining
procedures to facilitate easier and more profitable
examinations.
TYPES OF FIXATIVE:
A. ACCORDING TO COMPOSITION
SIMPLE FIXATIVES
are made up of only one component substance.
Aldehydes: Formaldehyde, Glutaraldehyde
Metallic Fixatives: Mercuric chloride, Chromate fixatives
(Potassium dichromate,Chromic acid), Lead Fixatives (Picric
acid, Acetic acid, Alcohol, Osmium tetroxide)
COMPOUND FIXATIVES
are those that are made up of two or more fixatives which
have been added together to obtain the optimal combined
effect of their individual actions upon the cells and tissue
constituents.
B. ACCORDING TO ACTION
MICROANATOMICAL FIXATIVES
those that permit the general microscopic study of tissue
structures without altering the structural pattern and normal
intercellular relationship of the tissues in question.
Includes: 10% formol saline, 10% Neutral buffered formalin,
Heidenhains Susa, Formol sublimate, Brasils solution,
Zenkers solution, Bouins solution,Zenker-formol
CYTOLOGICAL FIXATIVES
are those that preserve specific parts and particular
microscopic elements of the cell itself.
Types of Cytological Fixatives
1. NUCLEAR FIXATIVES those that preserve the nuclear structures
Usually contain GLACIAL ACETIC ACID as a primary
component due to its affinity for nuclear chromatin
Have a pH of 4.6 or less
Includes: Flemmings fluid, Bouins fluid, Heidenhains Susa,
Carnoys fluid, Newcomers fluid,
2. CYTOPLASMIC FIXATIVES are those that preserve cytoplasmic
structures.
Must never contain glacial acetic acid which destroys
mitochondria and Golgi bodies
Have a pH of more than 4.6
Includes: Flemmings fluid without acetic acid,Kellys fluid,
Formalin with post-chroming, Regauds fluid (Mullers fluid),
Orths fluid,
3. HISTOCHEMICAL FIXATIVE are those that preserve the chemical
constituents of cells and tissues
Includes: Formol Saline 10%, Absolute ethyl alcohol, Acetone,
Newcomers fluid
Fixation of tissues can be accomplished by:
A. PHYSICAL METHOD
1. HEAT FIXATION
Simplest form
Each component is less soluble in water after heat fixation
Primarily used to accelerate other forms of fixation as well as
the steps of tissue processing.
2. MICROWAVE HEATING
Speeds fixation and can reduce times for fixation of some
gross specimens and histological sections from more than 12
hours to less than 20 minutes.
Commercial glyoxal-based fixatives which do not form vapors
when heated at 55 degrees Celsius introduced as an
efficient method of microwave fixation.
3. FREEZE-DRYING
B. CHEMICAL METHODS
Used by most methods of fixation in processing of tissue for
histopathological diagnosis.
Utilizes organic or non-organic solutions to maintain adequate
morphological preservation.
1. COAGULANT FIXATIVES
Both organic and non-organic solutions may coagulate
proteins making them insoluble.
Result in cytoplasmic flocculation as well as poor preservation
of mitochondria and secretory granules
Not useful in ulltrastructural analysis.
Types of Coagulant fixatives:
DEHYDRANT COAGULANT FIXATIVES
most commonly used are ALCOHOL and ACETONE
Alcohol denatures protein differently depending on:
Choice and concentration of alcohol
Presence of organic and non-organic substances
pH, Temperature
o METHANOL has a closer structure with water
>Fixation begins at a concentration of 80% or more.
o ETHANOL competes more strongly in interaction with
hydrophobic areas of molecules
>Begins at a conc. Of 50-60%
ACIDIC COAGULANTS change the charges on the ionizable side chains
of proteins and disrupt electrostatic and hydrogen bonding
ACETIC ACID coagulates nucleic acids but does not fix or
precipitate proteins added to other fixatives to prevent the
loss of nucleic acids
2. CROSS-LINKING FIXATIVES (Covalent additive fixatives)
Has a potential actions forming cross-links between proteins
and nucleic acids or between nucleic acids and proteins.
Example: Formaldehyde, Glutaraldehyde
3. COMPOUND FIXATIVES
Useful for specific tissues
Example: Alcoholic formalin for fixation of fatty tissue
LIPID FIXATION
Lipids are largely removed during preparation of tissues
Cryostat or frozen sections should be used for demonstrating
lipid in tissues.
Fixatives containing mercuric chloride or potassium
dichromate can be effective for preservation of lipids in
cryostat.
Phospolipids are fixed by aldehydes Bakers formol-calcium
Ultrastructural demonstration of lipids is achieved by post-
fixing in imidazole osmium tetroxide.
Cholesterol may be fixed with digitonin for ultrastructural
demonstrations.
PROTEIN FIXATION
Neutral buffered formol saline or formaldehyde vapor most
commonly used fixative for amino acid histochemistry
CARBOHYDRATE AND GLYCOGEN FIXATION
Alcoholic fixatives are generally recommended for glycogen
fixation
Alcoholic formaldehyde is a better fixative in human skin
compared with neutral buffered formaldehyde.
Most useful fixatives for preserving glycogen - Rossmans fluid
or cold absolute alcohol
Better retention of glycogen if the section is coated with
celloidin
MIXTURE OF FIXATIVES
a. KARNOVSKYS PARAFORMALDEHYDE-GLUTARALDEHYDE
two aldehyde fixative mixtures useful for electron
cytochemistry.
b. ACROLEIN introduced as a mixture with glutaraldehyde or
formaldehyde
Penetrates tissue rapidly, preserves morphology and enzyme
activity at low concentrations
Useful for immersion fixation of surgical biopsies.
ALDEHYDE FIXATIVES
a. Formaldehyde
b. 10% Formol-Saline
c. 10% Neutral buffered formalin
d. Formal-corrosive
e. Alcoholic formalin
f. Glutaraldehyde
a. FORMALDEHYDE
Formalin (Most widely used fixative)
A gas produced by the oxidation of methyl alcohol
A staturated solution of formaldehyde gas in water
approximately 35% - 40% gas by weight
10% FORMALIN a mixture of 10 mL of formalin with 90mL of
water
FIXATION TIME: 24 hrs
Usually buffered to pH 7 with phosphate buffer
Advantages:
Cheap, readily available, easy to prepare, relatively stable, esp. if
stored in buffered solutions
Compatible with many stains therefore can be used with various
staining techniques depending upon the need.
Does not overharden tissues, even with prolonged periods of
fixation, as long as solutions are regularly changed.
Penetrates tissues well
Preserves fat and mucin making them resistant to subsequent
treatment with fat solvents, thereby allowing them to be stained
for demonstration.
Preserves glycogen
Preserves but not precipitate proteins, thereby allowing tissue
enzymes to be studied. It does not make tissue brittle, and is
therefore recommended for nervous tissue preservation.
Allows natural tissue colors to be restored after fixation by
immersing formalin fixed tissues in 70% alcohol for 1 hour and is
therefore recommended for tissue color photography
Allows frozen tissue sections to be prepared easily
Does not require washing out, unless tissue have stayed for
excessively long periods of time.
It is a tolerant fixative, used for mailng specimen because
specimen can be left in formalin indefinitely.
Disadvantages:
Fumes are irritating to the nose and eyes and may cause sinusitis,
allergic rhinitis, or excessive lacrimation.
Solution is irritating to the skin and may cause allergic dermatitis on
prolonged contact.
It may produce considerable shrinkage of tissues
It is a soft fixative and does not harden some cytoplasmic
structures adequately enough for paraffin embedding.
If buffered:
Reduces both basophilic and eosinophilic staining of cells
Forms abundant brown pigment granules on blood containing
tissues, ex: Spleen, due to blackening of hemoglobin.
Prolonged fixation may produce:
Bleaching of the specimen and loss of natural tissue colors
Dispersal of fat from the tissues into the fluid
Dissolution or loss of glycogen, biurate or sodium crystals and uric
acid
PRECAUTIONS
Prolonged storage of formalin at very low temperatures becomes
turbiddue to formation of paraformaldehyde resulting from
polymerization of formaldehyde (HCHO)
Turbidity may be removed by filtration or addition of 10%
methanol.
Concentrated solutions of formaldehyde must NEVER be
neutralizedmight precipitate violent explosion.
Room should be properly ventilated with adequate windows and
exhaust fan to prevent inhalation of fumes and consequent
injury to the eyes and nose
Dermatitis may be avoided by the use of rubber gloves.
Acid reaction due to formic acid formation can be buffered or
neutralized by adding magnesium carbonate or calcium carbonate
to 10-15% formalin.
In TISSUE CONTAINING MUCH BLOOD- Unbuffered formalin leads
to formation to dark brown pigment granules.
 REMOVAL OF FORMALIN PIGMENT is achieved by: Advantages: are similar to formol-saline with the ff. additions.
1. KARDASEWITSCHSMETHOD It prevents the precipitation of acid formalin pigments on post-
a mixture of 70% ethyl alcohol and 28% ammonia water mortem tissue.
for 5 minutes for 3 hours. It is the best fixative for tissues containing iron pigments and for
2. LILIESS METHOD elastic fibers which do not stain well after Susa, Zenker or
a mixture of acetone, 3 vol. hydrogen peroxide and 28% Chromate fixation.
ammonia water for 1 to 5 minutes followed by 70% and It requires no post-treatment after fixation goes directly into 80%
then running water. alc. For processing.
3. PICRIC ACID
for 5 minutes to 2 hours and then wash for 10 to 15 Disadvantages:
minutes in running tap water. It is longer to prepare; hence, is time consuming
Positivity of mucin to PAS is reduced
b. 10% FORMOL-SALINE It may produce gradual loss in basophilic staining of cells
A simple microanatomical fixative made up of saturated Reactivity of myelin to Weigerts iron hematoxylin stain is reduced
formaldehyde (40% by weight volume) diluted 10% with It is inert towards lipids, esp. neutral fast and phospholipids
sodium chloride.
Recommended for fixation of central nervous tissues and d. FORMOL CORROSIVE (Formol-sublimate)
general post mortem tissues for histochemical examination. Formol-mercuric chloride solution is recommended for
FORMULA: routine post- mortem tissues
Formaldehyde 40% = 100mL FORMULA:
NaCl = 9gm Sat. Aq. Mercuric chloride 90 mL
Distilled water = 900mL Formaldehyde 10 mL
Fixation Time: FIXATION TIME: 3 - 24 hours
24 hours at 35C(95F) penetrates small pieces of tissues rapidly - no need for
48 hours at 20-25C (65-77F) "washing- out" --- can be transferred directly from fixative to
alcohol
Advantages: forms mercuric chloride deposits
It penetrates and fixes tissues evenly inhibits the determination of the extent of tissue
Preserves microanatomic and cytologic details with minimum decalcification
shrinkage and distortion.
Large specimens may be fixed for a long time provided that the e. ALCOHOLIC FORMALIN (Gendre's fixative)
solution is changed every three months. can be used for rapid diagnosis --- it fixes and dehydrates at
It preserves enzymes and nucleoproteisns the same time
It demonstrates fats and mucin. good for preservation of glycogen and for microincineration
It does not overharden tissues, thereby facilitating dissection of the technique
specimen. FORMULA:
It is ideal for most staining techniques, including silver 95% ethyl alc. saturated w/ picric acid 80 mL
impreganation. strong formaldehyde soln. 15 mL
It allows natural tissue color to be restored upon immersion in 70% glacial acetic acid 5 mL
alcohol.
f. GLUTARALDEHYDE
Disadvantages: similar with formaldehyde with ff. additions. made up of 2 formaldehyde residues, linked by three carbon
It is a slow fixative. The period of fixation is required to be >24 hrs chains
Formol-saline fixed tissues tend to shrink during alcohol used for electron microscopy --- buffered glutaraldehyde then
dehydration; this may be reduced by secondary fixation. osmium tetroxide
Metachromatic rxn of amyloid is reduced. 2.5% soln. - used for small tissue fragments and needle
Acid dyes stains less brightly than when we fixed with mercuric biopsies fixed in 2-4 hours @ room temp.
chloride. 4% soln. - recommended for larger tissues less than 4 mm
thick fixed in 6-8 hours up to 24 hours
c. 10% NEUTRAL BUFFERED FORMALIN (pH 7) (Phosphate-buffered FIXATION TIME: 1/2 - 2 hours
formalin (pH 7)) preserves cellular structures better
recommended for preservation and storage of surgical, post- --- recommended for enzyme histochemistry and electron
mortem and research specimen microscopy
FORMULA: specimen vial must be kept refrigerated during fixation
Sodium dihydrogen phosphate (anhydrous) 3.5 gm process
Disodium hydrogen phosphate (anhydrous) 6.5 gm soln. may be changed several times during fixation by swirling
Formaldehyde 100 mL the vials
Distilled water 900 mL
FIXATION TIME: 4 - 24 hours
best fixative for tissues containing iron pigments and elastic
fibers which do not stain well after Susa,
Zenker or Chromate fixation
requires no post treatment after fixation
 METALLIC FIXATIVES * After using this fixative, the tissues should be transferred directly to a
high-grade alcohol --- 96% or absolute alcohol
a) Mercuric Chloride > To avoid undue swelling of tissues caused by treatment with low-grade
Zenkers fluid alcohol or water.
Zenker-formol
Heidenhains Susa FORMULA:
B-5 Fixative Mercuric chloride 45 gm
Sodium chloride 5 gm
b) Chromate Fixatives Trichloroacetic acid 20 gm
Chromic acid Glacial acetic acid 40 mL
Potassium dichromate Formaldehyde, 40% 200 mL
Regards fluid Distilled water 800 mL
Orths fluid
4) B-5 FIXATIVE
c) Lead Fixatives Commonly used for bone marrow biopsies.
Rapid fixation can be achieved in 1 1/2 2 hrs.
MERCURIC CHLORIDE Two working solutions are kept separate, since the mixture is
Most common metallic fixative --- frequently used in unstable.
saturated aqueous solutions of 5%-7%
Widely used as a secondary fixative FORMULA:
Penetrates poorly and produces shrinkage of tissue Distilled water 90 cc
Tissues fixed with mixtures containing mercuric chloride Mercuric chloride 6 gm
contain black precipitates of mercury Sodium acetate 1.25 gm
(Just prior to use, add 1 cc of formaldehyde 40% for 10 cc of B-5
Mercury deposits are removed by treating sections with 0.5%
fixative.)
iodine solutions in 70% ethanol for 5-10 minutes.
The use of metallic forceps and of metal caps to cover the
CHROMATE FIXATIVES
bottles containing the fixative should be avoided.

1) CHROMIC ACID
FORMULA:
Used in 1% - 2% aqueous solution --- usually a constituent of a
Mercuric chloride 5 gm
compound fixative.
Potassium dichromate 2.5 gm
Sodium sulfate (optional) 1 gm Precipitates all proteins and adequately preserves
Distilled water 100 mL carbohydrates.
A strong oxidizing agent --- in need of strong reducing agent
1) ZENKERS FLUID before use in order to prevent counteracting effects.
FIXATION TIME: 12-24 hrs.
2) POTASSIUM DICHROMATE
Made up of mercuric chloride stock solution plus GLACIAL
ACETIC ACID --- added before its use to prevent turbidity and Used in 3% aqueous solution
formation of dark precipitate. Preserves lipids
Good general fixative for adequate preservation of all kinds of Fixes but does not preserve cytoplasmic structures.
tissues and give excellent staining results Preserves mitochondria at a pH of 4.5 5.2
Solutions must always be freshly prepared
Do NOT let tissues stay in solution for more than 24 hrs.
Mercuric deposits may be removed by immersing tissues in
3) REGAUDS FLUID (Mullers fluid)
ALCOHOLIC IODINE prior to staining, through a process known
as DEZENKERIZATION. FIXATION TIME: 12-48 hrs.
Recommended for demonstration of chromatin,
2) ZENKER-FORMOL FLUID (Hellys solution) mitochondria, mitotic figures, golgi bodies, RBC, and colloid-
FIXATION TIME: 12-24 hrs. containing tissues.
An excellent microanatomic fixative for pituitary gland, bone
FORMULA:
marrow, and blood containing organs --- spleen and liver
Potassium dichromate 80 mL
Brown pigments are produced if tissues are allowed to stay in
Strong formaldehyde, 40% 20 mL
the fixative for >24 hrs. due to RBC lysis
(To be added just before use)
FORMULA:
4) ORTHS FLUID
Stock solution, Mercuric chloride 5gm
FIXATION TIME: 36-72 hrs.
Strong formaldehyde, 40% 5 mL
Recommended for study of early degenerative process and
3) HEIDENHAINS SUSA SOLUTION tissue necrosis.
FIXATION TIME: 3-12 hrs. Preserves myelin better and demonstrates rickettsiae.
Recommended mainly for tumor biopsies especially the skin.
An excellent cytologic fixative
FORMULA: METHYL ALCOHOL 100%
Potassium di chromate 2.5 % 100 mL Excellent for fixing dry and wet smears, blood smears and
Sodium sulfate (optional) 1 gm bone marrow tissues.
Strong formaldehyde 40% 10 mL Fixes and dehydrates at the same time.
If left in fixative for more than 48 hrs. --- tissues may be
LEAD FIXATIVES overharden and difficult to cut.
Used in 4% aqueous solution of basic lead acetate.
Recommended for acid mucopolysaccharides ISOPROPYL ALCOHOL 95%
Takes up CO2 to form insoluble lead carbonate on prolong Used for fixing touch preparations
standing --- removed by:
a) Filtration ETHYL ALCOHOL
b) Addition of acetic acid --- drop by drop to lower the pH FIXATION TIME: 18-24 hrs.
and dissolve the residue. Used at conc. of 70% - 100%
Lower concentrations --- RBCs become hemolyzed and WBCs
PICRIC ACID FIXATIVES are inadequately preserved.
Normally used in strong saturated aqueous solution. Used as a simple fixative
Tissue fixed with this fixative retain little affinity for basic
dyes. CARNOYS FLUID
Preserves glycogen well FIXATION TIME: 1-3 hrs.
To remove the yellow color --- tissue should be placed in 70% Recommended for fixing chromosomes, lymph glands and
ethyl alcohol followed by sodium thiosulfate. urgent biopsies.
Rapid in action
1) BOUINS SOLUTION Also used to fix brain tissue for diagnosis of rabies.
FIXATION TIME: 6-24 hrs.
Recommended for fixation of embryos and pituitary biopsies. FORMULA:
Absolute alcohol 60 mL
FORMULA: Chloroform 30 mL
Sat. solution of picric acid 75 mL Glacial acetic acid 10 mL
Strong formaldehyde 40% 25 mL
Glacial acetic acid 5 mL NEWCOMERS FLUID
FIXATION TIME: 12-18 hrs. at 3oC
2) BRASILS ALCOHOLIC PICROFORMOL FIXATIVE Recommended for fixing mucopolysaccarides and nuclear
An excellent fixative for glycogen proteins.
Overnight tissue fixation by automatic processing technique Produces better reaction in Feulgen stain.
may utilize 3-4 changes of Brasils fixative at to 2 hours Acts both as anuclear and histochemical fixative.
each.
FORMULA:
FORMULA: Isopropyl alcohol 60 mL
Formaldehyde 37% 2040 mL Propionic acid 30 mL
Picric acid 80 gm Petroleum ether 10 mL
Ethanol or Isopropyl alcohol 6000 mL Acetone 10 mL
Trichloroacetic acid 65 gm Dioxane 10 mL

GLACIAL ACETIC ACID OSMIUM TETROXIDE (Osmic acid)

Acetic acid is used in conjunction with other fixatives to form
a compound solution.
Solidifies at 17oC --- Glacial acetic acid
ALCOHOLIC FIXATIVES
Alcohol rapidly denatures and precipitates proteins by
destroying hydrogen and other bonds.
Used in concentrations ranging from 70% to 100%
Can be used to fix and preserve glycogen, pigments, blood,
tissue films and smears.
May be used both as a fixative and dehydrating agent.
Excellent for glycogen preservation
Tissue left in alcohol too long will shrink, making it difficult or
impossible to cut.
Alcohol-containing fixatives are contraindicated when lipids
are to be studied --- dissolves fats and lipids.
A pale yellow powder dissolves in water to form a strong
oxidizing solution.
Causes complete denaturation of protein
Should be kept in a dark-colored, chemically clean bottle to
prevent evaporation and reduction by sunlight or organic
matter.
Fixes conjugated-fats and lipids permanently.
A poor penetrating agent and very expensive
It should be kept in a cool place or refrigerated to prevent
deterioration.
Extremely volatile
1) FLEMMINGS SOLUTION
FIXATION TIME: 24-48 hrs.
Most common chrome-osmium acetic acid fixative used.
Recommended for nuclear preparation of each sections.
FORMULA: Mineral acids that may be used for decalcification are the
Aqueous chromic acid 1% 15 mL following:
Aqueous osmium tetroxide 2% 4 mL i. Nitric acid
Glacial acetic acid 1 mL ii. Hydrochloric Acid
iii. Formic Acid
2) FLEMMINGS SOLUTION w/o ACETIC ACID iv. Citric Acid
FIXATION TIME: 24-48 hrs. v. Tricholoroacetic Acid
Made up only of chromic and some osmic acid vi. Sulfurous Acid
Recommended for cytoplasmic structures particularly the vii. Chromic Acid
mitochondria
Removal of acetic acid from the formula serves to improve the
cytoplasmic detail of the cell. I. NITRIC ACID
Most common and very rapid decalcifying agent
TRICHLOROACTEIC ACID Can be used both as a simple aqueous solution (5% - 10%) or
Incorporated into compound fixatives combined with other reagents.
Precipitates proteins and may be used as a weak decalcifying Inhibit nuclear stains and destroys tissues.
agent. The endpoint of decalcification must be carefully watched for
With softening effect on dense fibrous tissues. --- to prevent progressive tissue damage and impaired
Poor penetrating agent staining.
> May be prevented by combining nitric acid with
ACETONE FORMALDEHYDE or ALCOHOL
Used at ice cold temperature ranging from -5 to 4oC
Recommended for the study of water diffusible enzymes --- A. AQUEOUS NITRIC ACID ADD SOLUTION 10%
phosphatases and lipases DECALCIFICATION TIME: 12 24 HOURS
Used in fixing brain tissues for diagnosis of rabies. Recommended for urgent biopsy, needle and small biopsy
Evaporates rapidly specimens to permit rapid diagnosis within 24 hours or less.

CHAPTER 4: DECALCIFICATION FORMULA:

The procedure whereby calcium or lime salts are removed Concentrated Nitric acid 10 mL
from tissues following fixation. Distilled water added up to 100 mL
Usually carried out by the use of chemical agents:
a. With acids to form to form soluble calcium salts B. FORMOL-NITRIC ACID
b. With chelating agents that bind to calcium ions DECALCIFICATION TIME: 1 3 days
Should be done after fixation and before impregnation --- Imparts a yellow color that will impair staining reaction of the
ensure and facilitate the normal cutting of sections and to cell.
prevent obscuring the microanatomic detail of sections by > Prevented by:
bone dust and other cellular debris. a. Neutralizing the tissue with 5% SODIUM SULFATE and washing
Inadequate decalcification may result in poor cutting of hard in running tap water for at least 12 hours.
tissues and damage to the knife edge during sectioning. b. Addition of 0.1% UREA to pure concentrated nitric acid.
Bones and calcified tissues are usually cut into small pieces
with a fine fret-saw and trimmed with hand razor --- permit FORMULA:
complete penetration of the decalcifying solution with Concentrated nitric acid 10mL
minimal surface damage and tissue distortion. Strong formaldehyde 5 mL
Distilled water 85 mL
If the tissue surface may reveal small foci of calcification ---
may cause resistance and a grating sensation when
C. PERENYIS FLUID
sectioned with a microtome knife.
> REMEDY: The block can be placed face down on a pad of DECALCIFICATION TIME: 2 - 7 days
cotton or gauze saturated with 10% HCl for 1 hour. Relatively slow decalcifying agent for dense bones
A good decalcifying agent must be capable of removing Complete decalcification cannot be determined by chemical
calcium salts from tissues completely without producing test --- a precipitate is formed upon the addition of ammonia
considerable destruction of cells and tissue components and REMEDY: Dissolved by adding glacial acetic acid drop by drop.
without adversely affecting the staining capacity of the cell. Reappearance of white precipitate within 30 minutes after
Calcium may be removed by any one of the following agents: addition of 0.5mL saturated aqueous AMMONIUM OXALATE
1. Acids will confirm the presence of calcium ---- signifying that
2. Chelating agents decalcification is still incomplete.
3. Ion exchange resins
4. Electrical ionization (electrophoresis) FORMULA:
Nitric acid 10% 40 mL
ACID DECALCIFYING AGENTS Chromic acid 0.5% 30 mL
Most widely used agents for routine decalcification of large Absolute ethyl alcohol 30 mL
amounts of bone tissues --- they are stable, easy available and
relatively inexpensive. Mix shortly before use. Chromic acid must be collected for proper
disposal
D. PHLOROGLUCIN-NITRIC ACID FORMULA:
DECALCIFICATION TIME: 12 24 HOURS Aqueous sodium citrate 20% 50 mL
Most rapid decalcifying agent Formic acid 45% 50 mL
Nuclear staining is poor
Yellow color must be neutralized with 5% SODIUM SULFATE IV. TRICHLOROACETIC ACID
and washed with running tap water for at least 24 hours. DECALCIFICATION TIME: 4 8 DAYS
When decalcification is complete, the acid must be removed Does not require washing out --- excess acid may be removed by
by three changes of 70% to 90% ethanol. several changes of 90% ALCOHOL
Very slow-acting and a weak decalcifying agent
FORMULA:
Concentrated nitric acid 10 mL FORMULA:
Phloroglucin 1 gm Trichloroacetic acid 5 gm
Nitric acid 10% 100 mL Formol Saline 95 mL
( To be added after diasappearance of dense white fumes formed by
combining the first two ingredients) V. CHROMIC ACID ( FLEMMINGS FLUID)
Used both as a fixative and decalcifying agent
II. HYDROCHLORIC ACID It tends to undergo reduction and forms precipitates at the
Inferior compared to nitric acid bottom of the container thus requiring frequent changes of
Slow decalcifying agent and produce greater distortion of solution
tissue produced on the section decalcified. An environmental toxin
If used in 1% solution with 70% alcohol --- Recommended Highly corrosive to skin and mucous membranes
for surface decalcification of the tissue blocks. Carcinogenic

A. VON EBNERS FLUID FORMULA:

Permits relatively good cytologic staining Chromic acid % 15 mL
Recommended for teeth and small pieces of bone Osmium tetroxide 2% 4 mL
Does not require washing out Glacial acetic acid 1 mL

FORMULA: VI. CITRIC ACID-CITRATE BUFFER SOLUTION

Saturated aqueous solution of NaCl 36% 50 mL DECALCIFICATION: 6 DAYS
Concentrated HCl 8 mL
Distilled water 50 mL FORMULA:
Citric acid (monohydrate) aqueous solution 7% 5.0 mL
III. FORMIC ACID Ammonium citrate (anhydrous) aqueous solution 7.4% 95.0 mL
A moderate-acting decalcifying agent Zinc sulfate aqueous solution 1% 0.2 mL
Produces better nuclear staining with less tissue distortion Chloroform a few drops
Safer to handle
Recommended for routine decalcification of postmortem CHELATING AGENTS
research tissues. Are substances which combine with calcium ions and other
The only weak acid used extensively as a primary decalcifying salts to form weakly dissociated complexes and facilitate
agent. removal of calcium salt.
Addition of CITRATE --- accelerates decalcification by chelating EDTA (Versene) most common chelating agent
the calcium liberated from the bone. > Recommended only for detailed microscopic studies
> Combines with calcium, forming an insoluble non-ionized
A. AQUEOUS FORMIC ACID complex --- used an anticoagulant and water softener
DECALCIFICATION TIME: 2 7 DAYS > Very slow decalcifying agent
Used both as fixative and decalcifying agent. > Tissue is placed in EDTA from 1 3 weeks for small
Suitable for most routine surgical specimen - -- specimen, but may take 6 8 weeks or longer to totally
immunohistochemical staining decalcifying dense cortical bone.
> The solution should be changed every 3 days
Relatively slow --- not suitable for urgent specimens
> EDTA will not bind to calcium below pH 3 and is faster at
Decalcification may be hastened by increasing the proportion
pH 7 7.4
of formic acid to 25 mL
An excellent bone decalcifier for immunohistochemical or
Requires neutralization
enzyme staining, and for electron micorscopy
EDTA inactivates alkaline phosphatase activity --- can be
FORMULA:
restored by addition of MAGNESIUM CHLORIDE.
Formic acid (Sp. Grav. 1.20) 10 mL
Formal 90 mL
FORMULA:
EDTA disodium salt 5.5 gm
B. FORMIC ACID-SODIUM CITRATE SOLUTION
Distilled water 90 mL
DECALCIFICATION TIME: 3 -14 DAYS
Formaldehyde 10 mL
Recommended for autopsy materials, bone marrow, cartilage
and tissues studied for research purposes.
ION EXCHANGE RESIN
Ammonium form of polystrene resin
Hastens decalcification by removing calcium ions from formic
acid-containing decalcifying solutions
Not recommended for fluids containing mineral acids ---
NITRIC ACID or HYDROCHLORIC ACID.
A layer of the ion exchange resin, about inch thick is spread
over the bottom of the container to be used --- specimen is
placed on top of it.
The decalcifying agent is then added, usually 20 30 times the
volume of the tissue.
Tissue may be allowed to stay in solution for 1 14 days.
The degree of decalcification may then be measured by
physical or X-ray method.
ELECTROPHORESIS
A process whereby positively charged calcium ions are
attracted to a negative electrode and subsequently removed
from decalcifying solution.
The time required for decalcification is thereby shortened due
to heat and electrolytic reaction produced in the process.
Satisfactory for small bone fragments
Prolonged decalcification of tissue is liable to prevent
hydrolysis and lead to maceration and destruction of tissue
components which are poorly stained.
Solution Used for Electrolytic Decalcification:
Formic acid 88% 100 mL
Concentrated hydrochloric acid 80 mL
Distilled water 1000 mL
Factors Influencing Rate of Decalcifcation
Concentration of decalcifying agent
- More concentrated acid solutions decalcify bone more
rapidly.
Volume of decalcifying agent
- Recommended ratio of fluid to tissue volume for
decalcification is 20 to 1.
- Greater amount of fluid will increase the speed of the
process.
Too rapid removal of calcium salts may produce complete
digestion of the tissue specimen, with marked swelling and
hydrolysis of the bony matrix and poor staining capacity of the
cell.
Temperature
- Heat will serve to hasten decalcification, but it also
increases the damaging effects of acids on tissue.
- 37C Will impaired nuclear staining of Van Giesons
stain for collagen fibers.
- 55C Tissue will undergo complete digestion within 24
48 hours
- The optimum temperature so far recommended is the
room temperature range of 18C - 30C
Increase in size and consistency of tissues will require longer
periods for complete decalcification.
The ideal time required for decalcifying tissue is 24 48 hours.
Dense bone tissues usually require up to 14 days or longer in
order to complete the process.
Solution should be changed daily to ensure better penetration
and to test for the degree of decalcification.
Measuring Extent of Decalcification
1. PHYSICAL or MECHANICAL TEST
Done by touching or bending the tissue with the fingers to
determine the consistency of tissues
By pricking the tissue with a fine needle or a probe --- to
produce needle tract artifacts and destroy important debris
2. X-RAY or RADIOLOGICAL METHOD
Most ideal, most sensitive and most reliable method of
determining extent of decalcification due to its ability to
detect even the smallest focus of calcium which appears
opaque in an X-ray plate.
3. CHEMICAL METHOD/CALCIUM OXALATE TEST
Simple, reliable and convenient method for routine purposes
to detect the presence of calcium in the decalcifying
solution.
Involves the detection of calcium in acid solutions by
precipitation of insoluble calcium hydroxide or calcium
oxalate
SOLUTIONS:
a. Ammonium hydroxide, concentrated
b. Saturated aqueous ammonium oxalate
Decalcifying fluid is usually changed every 24 48 hours and
the chemical test is performed on the discarded fluid.
If chemical method of determination is to be done, the
decalcifying agent should be prepared with distilled water.
POST- DECALCIFICATION
After decalcification is complete, the acid can be removed by
immersing the decalcified bone in:
a. Saturated lithium carbonate solution
b. 5% - 10% Aqueous sodium bicarbonate solution for
several hours
Adequate water rinsing can usually be accomplished in 30
minutes for small samples and 1 4 hours for larger
specimens.
Acid decalcified tissues for frozen sections must be:
a. Thoroughly washed in water
b. Stored in formol saline containing 15% sucrose
c. Stored in phosphate-buffered saline with 15 20%
sucrose at 4C before freezing.
Tissues decalcified in EDTA solutions should not be placed
directly into 70% alcohol --- formation of EDTA precipitate in
the alcohol and within the tissue.
Rinsing the decalcified tissue with water or storing overnight
in formol saline or phosphate buffered saline will prevent the
formation of crystalline precipitate.
TISSUE SOFTENERS
PERENYIS FLUID act as a decalcifying agent and tissue
softener
To soften hard tissues:
a. Selected portions are left in the fluid for 12 24 hours
and dehydrated in the usual manner
b. Cut the surface of the block may be submerged in the
fluid for 1- 2 hours before sectioning.
c. Washing out and immersion of fixed tissues in 4%
aqueous phenol solution for 1 3 days
Substances used as tissue softeners:
- Molliflex , 2% Hydrochloric acid and 1% Hydrochloric acid
in 70% alcohol
 CHAPTER 5: DEHYDRATION
Process of removing intercellular and extracellular water from
the tissue following fixation and prior to wax impregnation
Many of these dehydrating agents are ALCOHOLS of various
types that are generally used in increasing strengths to
remove aqueous tissue fluids with little disruption to the
tissue.
Starts by placing the fixed specimen in 70% ethyl alcohol in
water, progressing through 95% ethyl alcohol to 100% ethyl
alcohol
For delicate tissues --- starting with 30% ethanol is
recommended
GENERAL RULE: Whatever 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 dehydrating solution.
CHARACTERISTICS OF AN IDEAL DEHYDRATING SOLUTION:
1) Should dehydrate rapidly without producing considerable
shrinkage or distortion of tissues.
2) Should NOT evaporate very fast
3) Should be able to dehydrate even fatty tissue
4) Should NOT harden tissues excessively
5) Should NOT remove stain
6) Should NOT be toxic to the body
7) Should NOT be a fire hazard.
Commonly used Dehydrating agents are:
I. Alcohol ( most common)
II. Acetone
III. Dioxane 4 cellosolve
IV. Triethyl phosphate
V. Tetrahydrofuran
I. ALCOHOL
A. ETHYL ALCOHOL Ethanol
Alcohol recommended for routine dehydration of tissues.
A clear, colorless, flammable fluid
Considered to be the best dehydrating agent:
a. Fast -acting
b. Mixes with water and many organic solvents
c. Penetrates tissue easily
d. Not poisonous and not very expensive
B. METHYL ALCOHOL Methanol
A toxic dehydrating agent
Primarily employed for blood and tissue films and for smear
preparations
C. BUTYL ALCOHOL
A slow dehydrating agent
Producing less shrinkage and hardening
Recommended for tissues which do not require rapid
processing.
Concentrated alcohols (95% or absolute) tend to harden only
the surface of the tissue while the deeper parts are not
completely penetrated -- -- resulting in a relatively unequal
impregnation of tissue.
REMEDY: 70% or lower concentrations of alcohol, gradually
increased to 95% is used.
II. ACETONE
Cheap, rapid-acting dehydrating agent utilized for most urgent
biopsies.
Dehydration time: to 2 HOURS
A clear, colorless fluid that mixes with water, ethanol, and
most organic solvents.
Not recommended for routine dehydration purposes.
More miscible with epoxy resins than alcohol
Highly flammable and require considerable care in handling
Rapid in action but penetrates tissues poorly and causes
brittleness in tissues
Limited use only to small pieces of tissues due to extremely
volatility and inflammability
III. DIOXANE
An excellent dehydrating and clearing agent readily miscible in
water, melted paraffin, alcohol and xylol --- tissues may be
placed directly into the solution after washing out
Produces less tissue shrinkage
Expensive and extremely dangerous --- its vapor produces a
cumulative and highly toxic action in man.
TWO METHODS INVOLVED:
A. Graupners method
A combination of pure dioxane solution and paraffin wax embed
in mold and cool in water.
B. Weisebergers method
The tissue is wrapped in a gauze bag and suspended in a bottle
containing dioxane and a little anhydrous calcium oxide.
Dehydration period ranges: 3 24 hrs
IV. CELLOSOLVE
Ethylene glycol monethyl ether
Dehydrates rapidly
Tissues may be transferred from water or normal saline
directly to cellosolve and stored for months without
hardening or distortion
CAUTION: Combustible at 110 120F and toxic by inhalation,
skin contact and ingestion
> If cannot be avoided, PROPYLENE-BASED GLYCOL ETHERS
should be used.
V. TRIETHYL PHOSPHATE
Removes water very readily and produces very little distortion
and hardening of tissue.
Soluble in alcohol, water, ether, benzene, chloroform, acetone
and xylene.
VI. TETRAHYDROFURAN (THF)
A reagent that both dehydrates and clears tissues --- miscible
in both water and paraffin.
Can dissolve many substances --- fats
May be used for demixing, clearing and dehydrating paraffin
sections and before and after staining.
It does not dissolve out aniline dyes
Causes less shrinkage and easier cutting of sections with
fewer artifacts.
Toxic if ingested or inhaled
An eye and skin irritant and prolonged exposure may cause
conjunctival irritation
Vapor cause nausea, dizziness, headache and anesthesia.
 CHAPTER 6: CLEARING
DE-ALCOHOLIZATION
The process whereby alcohol or a 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 or the medium on which the tissue is to be
mounted.
When the dehydrating agent has been entirely replaced by
the solvent, the tissue has a translucent appearance ----
CLEARING AGENT
Clearing agents must be miscible with paraffin in order to
facilitate the penetration of this embedding medium.
When used after the tissue section has been stained, the
clearing agent will make microscopic tissue preparations
TRANSPARENT due to their high index of refraction.
Not all clearing agents exhibit the property of making tissues
transparent
A clearing agent must also be miscible with Canada balsam
and other resins that are used for mounting sections ---
XYLENE most commonly used
In frozen sections, GLYCERIN and GUM SYRUP are used when
tissue is to be cleared directly from water --- no de-
alcoholization process involved.
CHARACTERISTICS OF A GOOD CLEARING AGENT:
1) Should be miscible with alcohol to promote rapid removal of
the dehydrating agent from the tissue.
2) Should be miscible with, and easily removed by melted
paraffin wax and/or mounting medium to facilitate
impregnation and mounting of sections.
3) Should NOT produce excessive shrinkage, hardening or
damage of tissue
4) Should NOT dissolve out aniline dyes
5) Should NOT evaporate quickly in a water bath
6) Should make tissue transparent
- Most clearing agents are flammable liquids that warrant
considerable caution in their use.
- Clearing fluids with low boiling point are generally more readily
replaced by melted paraffin wax.
- Viscosity also affects the speed of penetration of the clearing
agent.
- Prolonged exposure to most clearing agents causes the tissue to
become brittle --- difficult to cut
Common Clearing Agents Used are:
A. Xylene ( most common)
B. Toluene
C. Benzene
D. Chloroform
E. Cedarwood oil
F. Aniline oil
G. Clove oil
H. Carbon tetrachloride
A. XYLENE ( XYLOL)
Most commonly used
CLEARING TIME: 30 minutes 1 hour
Used for clearing both for embedding and mounting
procedures
Suitable for most routine histologic processing schedules of
less than 24 hours and when tissue block size is less than 5
mm in thickness.
Most rapid clearing agent and cheap
Miscible with absolute alcohol and paraffin
For mounting procedures --- it does NOT dissolve celloidin
and can be used for celloidin sections
Highly inflammable and should be appropriately stored
If used longer than 3 hours - -- makes tissues excessively hard
and brittle
NOT suitable for nervous tissues and lymph nodes --- causes
considerable hardening and shrinkage of tissues
Becomes milky when an incompletely dehydrated tissue is
immersed in it.
B. TOLUENE
Used as a substitute for xylene or benzene for clearing both
during embedding and mounting processes.
CLEARING TIME: 1 2 hours
Acts fairly rapid and is recommended for routine purposes
Miscible with both absolute alcohol and paraffin.
If left for 24 hours --- tissues do NOT become excessively hard
and even brittle.
NOT carcinogenic
More expensive and relatively slower
Highly concentrated solutions will emit fumes that are toxic
upon prolonged exposure.
C. BENZENE
It penetrates and clears tissues rapidly
Rapid acting --- recommended for urgent biopsies (15 60
minutes) and routine purposes.
Volatilizes rapidly in paraffin oven --- easily eliminated from
the tissue
Miscible with absolute alcohol
Makes tissue transparent Does NOT make tissue hard and
brittle
Highly flammable
If a tissue section is left for a long time --- considerable
shrinkage may be observed.
Excessive exposure to benzene may be extremely toxic ---
carcinogenic or may damage the bone marrow ---APLASTIC
D. CHLOROFORM
Slower action and causes less brittleness
Thicker blocks (even up to 1 cm in thickness) can be
processed.
Tissue placed in chloroform do NOT become transparent.
Recommended for routine work (6 24 hours)
Miscible with absolute alcohol
Recommended for tough tissues, nervous tissues, lymph
nodes and embryos.
Suitable for large tissue specimens
Relatively toxic to the liver after prolonged inhalation
Does not make the tissues transparent
Not very volatile in paraffin oven --- difficult to remove from
paraffin sections
Tissues tend to float in chloroform
> May be avoided by wrapping the tissues with absorbent
cotton gauze --- to facilitate sinking of the section in solution.
E. CEDARWOOD OIL
Used to clear both paraffin and celloidin sections during the
embedding process
Especially recommended for central nervous system tissues
and cytological studies --- smooth muscles and skin.
Requires two changes in clearing solution
Clearing is usually complete in 2 3 days
Clears celloidin in 5 6 days
Tissues may be left in oil indefinitely without considerable
damage and distortion
Clearing with this agent often improves cutting of the
sections.
Hard to eliminate from tissues in paraffin bath making the
wax impregnation process very slow
> Remedy: Transfer the specimen from oil to benzene for
hour before finally placing the tissue in wax.
It becomes milky upon prolonged storage and should be
filtered before use.

F. ANILINE OIL
Recommended for clearing embryos, insects, and very
delicate specimens --- due to its ability to clear 70% alcohol
without excessive tissue shrinkage and hardening.

G. CARBON TETRACHLORIDE
Used in clearing tissues for embedding
Produces considerable tissue hardening
Dangerous to inhale on prolonged exposure due to its highly
toxic effects.

H. CLOVE OIL
Causes minimum shrinkage of tissues
Wax impregnation after clearing with clove oil is slow and
difficult.
Tissues become brittle, aniline dyes are removed, and
celloidin is dissolved.

PandaMT13