HISTOPATH

CLASS RULES:

•Be respectful to professors and classmates at all times.

•Arrive on time and stay for the full class.

•Avoid side conversations or disruptive behavior.

•Silence or turn off phones during class (unless allowed for class use).
Grading System: To pass this course, the student must accumulate 75% of all the
course requirements discussed above.

 Prelim Grade: Raw Grade

 Midterm Grade: 40% Prelim Grade + 60% Midterm Grade = Total Midterm Grade
 Pre final Grade: 40% Midterm Grade + 60% Pre Final Grade = Total Pre Final Grade
 Final Grade: 40% Pre Final Grade + 60% Final Grade = Total Final Grade

Grade Computation: Score/Total # of items x 60 + 40 = Equivalent Score

LECTURE LABORATORY

PARTICIPATION : 10% PARTICIPATION :

35%

PROJECT/ASS: 15% PROJECT/ASS/LAB

PERFORMANCE: 15%

RECITATION: 20% QUIZZES:

15%

QUIZZES: 20% EXAMINATION:

35%
OBJECTIVES
At the end of the topic the students will be able to do the
following:
 Name the different histotechnology technique
 Discuss the proper use and care of microscope
 Discuss the steps in tissue processing
 Identify types of fixative agents
 Discuss the importance of rapid tissue processing
 Determine methods of fresh tissue examination
HISTOPATHOLOGIC TECHNIQUES
It deals with the preparation for microscopic examination - It is accomplished by
submitting the total or a selected part of the tissue presented for examination to a
series of processes:
 Fixation - Preservation; 1st and most critical step
 Decalcification - Calcium or lime salts are removed from the tissues - Optional
process: for calcified tissues only such as bones and teeth
 Dehydration or Desiccation - Removing intracellular and extracellular
fluid/water
 Clearing or De-alcoholization - Removing the alcohol used in dehydration
 Infiltration or Impregnation
 Embedding - Casting or Blocking
 Trimming - Removing excess wax from the tissue block - Optional process:
not all tissue blocks have excess wax
 Sectioning - Cutting tissue blocks into uniformly thin size
 Staining - Dyeing
 Mounting
 Labelling - Proper labelling
Examination may be done on:

1. Fresh Tissues usually examined when there is an immediate need for

evaluation.
2. Preserved Tissues routinely done in the histopathology section.

1. FRESH TISSUE EXAMINATION

ADVANTAGE: Examined in the living state, thereby allowing protoplasmic

activities such as:

a. Mitosis (Cellular Reproduction)

b. Motion (Cellular Movement)
c. Phagocytosis (Cell-eating activity)
d. Pinocytosis (Cell-drinking activity)

DISADVANTAGE: Its use has been limited, however, because of the fact
that tissues examined in the fresh state are not permanent and therefore,
are liable to develop the changes that have usually been observed after
death.
PRIMARY & SECONDARY SIGNS OF DEATH

PRIMARY (Occur during somatic death)

1. CNS Failure
2. Respiratory failure
3. Cardiovascular failure
SECONDARY (Occur after somatic death)
1. Algor mortis - The cooling of the body (7°F/hr or 1-1.5
°C/hr) 1st demonstrable change
2. Rigor mortis - Stiffening of the skeletal muscles
3. Livor mortis - Post-mortem lividity or post-mortem
suggilation Purplish discoloration
4. Post-mortem clot
5. Autolysis
6. Putrefaction
 POST MORTEM CHANGES

1. Autolysis - The destruction of the tissues (breaking

down of the protein of the cell) by enzymes which are
produced by the tissues and eventually liquefy it. - It is
the first to occur among all post-mortem changes
2. Putrefaction or Decomposition - The decomposition
of organic matter under the influence of microorganisms
accompanied by the development of disagreeable
odors.
3. Degeneration - A retrogressive pathologic process in
cells in which the cytoplasm undergoes deterioration
while the nucleus is
 EXAMINATION OF FRESH TISSUE

Fine needle aspiration • Simplest, least invasive test and uses the smallest needle to remove cells from the
area of abnormality.

Core needle biopsy • Removes not only cells, but also a small amount of surrounding tissue.

Incisional biopsy • Takes out even more surrounding tissue

• Takes out some of the abnormality, but not all.

Excisional biopsy • Generally removes the entire area in question.

Punch biopsy • Primary technique for obtaining diagnostic full-thickness skin specimens.
• Use of circular blade that is rotated down through the epidermis and dermis and into
the subcutaneous fat yielding a 3- to 4-mm cylindrical core of tissue sample.

Shave biopsy • Small fragments of tissue are “shaved” from a surface usually (skin).

Curettings • Tissue is scooped or spooned to remove tissue or growths from body cavity such as
endometrium or cervical canal.
METHODS OF FRESH TISSUE EXAMINATION

Teasing or Dissociation  Process wherein selected tissue specimen is

immersed in a watch glass containing isotonic salt
solution (NSS or Ringer’s lactate), carefully dissected
or separated and examined under the microscope,
either unstained by Phase Contrast microscope or
Bright-field microscope, or stained with differential
dye.

Squash preparation/  Process where small pieces of tissue not more than
Crushing 1mm in diameter are placed in a microscopic slide
and forcibly compressed with another slide or with
coverglass.
 Vital dyes/stains are placed at the slide and coverslip
junction and absorbed through capillary action.
METHODS OF FRESH TISSUE EXAMINATION

Frozen Section  Normally utilized when a rapid diagnosis of

the tissue in question is required, and
especially recommended when lipids and
nervous tissue elements are to be
demonstrated.

Smearing  Useful in cytological examinations,

particularly for cancer diagnosis
SMEARING TECHNIQUE MATERIAL PROCESS/IMPORTANT NOTES

Streaking Applicator stick or platinum loop. Rapid and gentle direct or zigzag
application to obtain uniform
distribution. Too thick or too thin
smears are unsuitable for
examination.
Spreading technique Applicator stick to tease the mucous strands to make a Little more tedious than streaking,
moderately thick film. but has advantage in maintaining
the intracellular relationship. -
Especially recommended for fresh
sputum, bronchial aspirates and
thick mucoid secretions.

Pull-apart technique Slides facing each other as a drop of secretion is The material disperses evenly over
sandwiched in between. the surface of 2 slides. A single
uninterrupted motion of pulling
apart is applied. It is useful for
serous fluids, concentrated sputum,
enzymatic GIT lavage and blood
smears.

Touch preparation or Impression One slide. Special method where slide surface
smear is in contact and pressed on the
site. Cells may be examined without
destroying their actual intercellular
relationship and without separating
them from their normal
surroundings.
PRESERVED TISSUES

- A better and more effective means of studying tissues whether normal or abnormal is by
examination of their sections and smears which have been permanently preserved, stained
for the demonstration of specific structures and mounted on glass slides with coverslips for
permanent keeping.
- The aim of a good histopathological technique is to produce microscopic preparation of
tissue, usually stained, that represent as closely as possible their structure in life.
 STEPS IN PROCESSING PRESERVED TISSUES
FIXATION
AKA: Preservation
- The process by which the constituents of the cells, and therefore of the tissues are fixed in a physical, and partly also in a chemical
state so that they will withstand subsequent treatment with various reagents with minimum loss or significant distortion or
decomposition.
- The first and most critical (important) step in histotechnology .
- Why is it the most critical? Because it is the first procedure/step and is able to affect the subsequent procedures/steps
- Primary aim of fixation - Preserve the morphologic and chemical integrity of the cell in as life-like a manner as possible
- Secondary goal of fixation - To harden and protect the tissue from trauma of further handling
- For easy cutting during gross examination
- The most important reaction in fixation: Stabilization of proteins - This can be achieved by: Forming cross-links between proteins
- Leaving a tissue specimen in air cause it to: dry-out (distortion of morphologic appearance)
- Leaving the tissue in water (hypotonic solution) will cause the cell to: swell
- If the environment is hypotonic, more water is outside the cell and the solute concentration inside the cell is high, the water would
enter the cell and swells, bursts, or lyses (cytolysis)
- Leaving the tissue in strong salt (hypertonic solution) will cause the cell to: shrink
- More solutes are outside the cell (strong salt solution) and water is less, so water will move outside the cell and the cell shrinks.
Two Basic Mechanisms
in Fixation
Principle/ Notable Examples
Characteristics
Additives • The chemical constituent of the Formaldehyde
fixative is taken in and becomes Osmium tetroxide/ Osmic acid
part of the tissue through cross- fixative
link formation or molecular Mercuric chloride
complex.

Non-additives • The fixing agent is not Alcohol

incorporated into the tissue but
alters the tissue composition
and stabilizes it through water
removal.
• New cross-links are formed
preventing autolysis and
bacterial decomposition.
Main Factors Involved in Fixation
[Link] Ion Concentration  -pH: 6.0-8.0
 -Average: 7.0 (neutral pH)
[Link]  Traditional/usual: Room temperature (18-30°C)
 Tissue processors (Autotechnicon): 40-42°C
 Electron Microscopy and Histochemistry: 0-4°C
o Mast cells for EM: Room temperature
 Nucleic acids fixation: Rapid at higher temperature
 Formalin heated to 60°C → rapid fixation of very urgent
biopsy specimens –
 Formalin heated to 100°C → to fix tissues with
tuberculosis
[Link] of sections  Small
o Electron Microscopy: 1-2 mm2
o Light Microscopy: 2 cm2
 Thin
o Light Microscopy: ≤0.4 cm (4mm) or as prescribed by
tissue processor manufacturer
o Large solid tissue, such as uterus, should be opened
or sliced thinly
o Brain is usually suspended whole in 10% Neutral
Buffered Formalin for 2-3 weeks.
4. Osmolality  Best results are obtained using slightly hypertonic solutions (400-450
mOsm)
o Hypertonic solutions = Shrinkage
o Isotonic (340 mOsm)/Hypotonic solutions = swelling and poor fixation
 Added to Osmium tetroxide fixatives for EM: Sucrose

5. Concentration  Formaldehyde: 10%

 Glutaraldehyde: 3%
 Glutaraldehyde for Immunoelectron microscopy: 0.25%
6. Duration of fixation  Most formalin fixatives: 24 hours (washed out)
 Buffered formalin: 2-6 hours up to 1 week
 EM: 3 hours (New books: 0-4 hours; Average: 2 hours)
 Prolonged fixation may cause shrinkage and hardening of tissue
 PRACTICAL CONSIDERATIONS OF FIXATION
1. Speed - The specimen should be placed in fixative as soon as it is removed from the body.
 This is done to prevent autolysis and putrefaction/decomposition.
 If bacteriologic and toxicologic studies should be encouraged, fixation is not required.
o Fixation can kill microorganisms and prevent growth in culture
o Fixatives can neutralize drugs and toxins
2. Penetration
 Formalin diffuses into the tissue at the rate of approximately 1mm/hr
 Time of fixation varies with different types of tissue.

3. Volume/Amount of Fixative
 Traditionally, the amount of fixative used has been 10-25x the volume of the tissue to be fixed.
 Recently, 20x is known as the maximum effective concentration for fixation –
 Except when osmium tetroxide (5-10x) is used.
• Because Osmium tetroxide is very expensive
4. Duration - Some tissues take longer to fix than others, depending on their structures.
o Fibrous organs such as uterus and intestinal tract take longer.
o Fixation can be cut down by using Heat, Vacuum, Agitation, or Microwave.
 IDEAL SIZE OF TISSUE TO BE FIXED
 not more than 2cm2 in diameter 9. The optimum osmolality for a specific tissue and
 not more than 4mm thick fixative type

 Ideal number of hours for fixation: 4-6 hours (New 10. 10. Make cellular components insoluble
books: 6-18 hours) 11. Permit subsequent application of many staining
procedures
CHARACTERISTICS OF A GOOD FIXATIVE:
1. Cheap
2. Stable
“the fact that there many fixatives indicate that the ideal
3. Safe to handle
has not been found”
4. Kill the cells quickly
5. Produce minimum shrinkage
6. Harden the tissues properly
7. Inhibits bacterial decomposition and autolysis
8. Permit rapid and even tissue penetration
 TYPES/CLASSIFICATION OF FIXATIVES
I. According to Composition
A. SIMPLE FIXATIVES (made up of only 1 component substance)
1. Aldehydes (Formaldehyde, Glutaraldehyde)
2. Metallic Fixatives (Mercuric chloride, Chromic acid, Lead)
3. Picric acid
4. Acetic acid
5. Acetone
6. Alcohol
7. Osmium tetroxide
8. Heat

B. COMPOUND FIXATIVES (Made of up of 2 or more fixatives to obtain optimal results)

 II. ACCORDING TO ACTION
A. Microanatomical Fixatives: Permit the general
microscopy study of tissue structures a) Bouin’s fluid
b) Flemming’s fluid
1. 10% Formol saline c) Newcomer’s fluid
2. 10% Neutral Buffered Formalin (NBF) d) Carnoy’s fluid
3. Heidenhain’s Susa e) Heidenhain’s Susa
4. Formol sublimate (corrosive)
5. Zenker’s solution
6. Zenker-formol (Helly’s)
7. Bouin’s solution
8. Brasil’s solution

B. Cytological Fixatives: Preserve specific parts and

particular microscopic elements of the cell itself

9. Nuclear Fixatives - Preserve nuclear structures (ex.

chromosomes)
 They usually contain Glacial acetic acid as their primary
component due to its affinity for nuclear chromatin.
 The pH is <4.6
2. Cytoplasmic Fixatives - Preserve cytoplasmic structures.

 They must never contain glacial acetic acid because it destroys the mitochondria
and golgi bodies of the cytoplasm.
 The pH >4.6

a. Helly’s
b. Orth’s
c. Regaud’s
d. Flemming’s fluid w/o Acetic acid (HAc)
e. Formalin w/ post-chroming

3. Histochemical Fixatives - Preserve the chemical constituents of cells and tissues

a. 10% Formol saline

b. Absolute Ethyl alcohol
c. Newcomer’s fluid (Both Nuclear and Histochemical fixative)
d. Acetone
 PRINCIPLES AND PRECAUTIONS IN HANDLING AND FIXATION OF SPECIMENS IN GENERAL

1. Autopsy materials should be fixed as soon after death as possible to prevent decomposition and autolysis due to deprived
oxygen and metabolism. lf this is not possible, the body should be placed in mortuary refrigerator (4°C) or arterial embalming
should be carried out without delay, provided that toxicological or microbiological examinations are not indicated. Such
embalming, well done, is most advantageous and gives excellent preservation.
2. Surgical specimens should be fixed as soon as possible after removal, or refrigerated if fixation is to be delayed, to prevent
drying of surface layers and ultimate tissue distortion.
3. The amount of fixative must be adequate, approximately 20 times the volume of the tissue specimen except in Osmium
tetroxide which is very expensive, requiring only 5-10 times that of tissue volume for fixation.
4. For prolonged fixation (e.g. Museum preparation) volume of fixing fluid should not be less than 50-100 times that of the tissue.
5. Hollow organs (e.g. Stomach and intestines) should be packed with cotton soaked in fixative or completely opened before
being immersed in adequate fixing solution.
6. Air-filled lungs may float on fixative. To avoid this, the organ may be covered with several layers of gauze to maintain it under
surface. Note: normal lungs will float while diseased lungs will sink.
7. Eyes should not be dissected before they are fixed. Formol-alcohol must be injected before immersing the organ in the
fixative.
8. Frozen sections may lead to formation of ice crystal artifacts.
9. Water should not be used for glycogen-containing tissues because glycogen is soluble in water.
10. The choice of fixatives and mode of processing vary depending upon the following factors: a. Need for immediate examination
b. Tissue structure or component to be studied c. Type of tissue to be processed d. Staining technique to be applied e. Type of
section to be made, whether serial or individual
ROUTINE FIXATIVES
I. ALDEHYDE FIXATIVES

A. FORMALDEHYDE:FORMALIN

 Commercial formaldehyde/Formalin
 A saturated solution of formaldehyde gas in water, approximately 35-40% gas by weight (Gregorios: 37-40%
weight in volume).
 A mixture of 10 mL formalin with 90 mL of water/saline is known as 10% formalin (most widely used fixative of
all)
 It is usually buffered to pH 7 with a phosphate buffer.
o Calcium acetate can also be used as buffer but is prone to calcium deposit formation.
 Commercial formalin often becomes turbid, especially if stored in a very cold place, because of the formation
of paraformaldehyde (whitish precipitate) → may be removed by filtration, but almost all commercial
formaldehyde contains 10% methanol, which tends to inhibit the formation of paraformaldehyde.
 Formalin is the best fixative for the Nervous tissues.
 DISADVANTAGE:

 Troublesome dermatitis of the hands

 Its fumes are irritating to the nostrils
 It may also cause asthma in some allergic individuals
 In tissues containing much blood (spleen), unbuffered formalin leads to
the formation of dark brown artifact pigment granules → these granules
consist of acid formaldehyde hematin and are doubly refractile
o Treated with: Saturated alcoholic picric acid or alcoholic KOH (1% KOH
in 80% alcohol)
 Prolonged fixation may cause bleaching, fat dispersal (add cadmium or
cobalt salts) and dissolution or loss of glycogen, biurates of sodium
crystal and uric acid.
Reagent-grade formaldehyde contains 10% methanol as a preservative to retard decomposition
to formic acid. Prevents the formation of Paraformaldehyde.

• Why should formic acid formation be avoided?

o Acidity reduces the quality of routine cytologic staining
o It leaches out hemosiderin
o It can lead to formation of formalin pigments
o To prevent pigmentation, a buffer can be used, such as a handful of calcium carbonate or
magnesium carbonate added to 10-15% formalin
o Renders formalin unsuitable for the critical fixation required in electron microscopy and has
denaturing effects on proteins
 Formalin pigments – are also formed due to over-fixation.
 Fixation Time: 24 hours
REMOVAL OF FORMALIN PIGMENTS:
 Lillie’s Method
o Involves placing formaldehyde fixed specimens in acetone, 28% ammonia
water and hydrogen peroxide.
o It uses 70% alcohol as a rinsing agent.
 Kardasewitsch’s Method
o A method of formaldehyde clearance involving 70% ethanol and 28% ammonia
water.
 Saturated Alcoholic Picric Acid
 ROUTINE FORMALIN FIXATIVES

1. 10% Formol Saline - Traditionally, it is the most commonly used fixative in pathology
- Best fixative for central nervous tissues and general post-mortem tissues
for histochemical examination.
Fixation time: 24 hours at Advantages:
35ºC 48 hours at 20- 25ºc 1. Penetrates tissues evenly and preserves microanatomic and cytologic
details with minimum shrinkage and distortion
2. Should be changed every 3 months so large specimens can be fixed
3. Preserves enzymes and nucleoproteins, and demonstrates fats and lipids
4. Ideal for silver impregnation
Disadvantages:
*Similar to formaldehyde with the following additions
- Slow and requires 24 hours or longer
- It shrinks during alcohol dehydration Remedy: secondary fixation
- Amyloid metachromatic action is reduced
- Reduced staining when fixed with mercuric chloride
2. Calcium acetate formalin - It is used to preserve phospholipids
(Lillie’s Fixative) - Replaced formol-saline as the most commonly used fixative
in pathology because:
o Simple to prepare
o Buffered to pH 7 by acetate
3. 10% Neutral Buffered Formalin/ - Best general tissue fixative
Phosphate buffered Formalin (pH 7) - Best fixative for frozen sections
- Recommended for surgical, post mortem and research
Fixation time: 4-24 hours specimens.
- Prevents precipitation of acid formalin pigments.
- Best fixative for iron-containing pigments and elastic fibers.
Disadvantages:
1. It is longer to prepare; time-consuming
2. PAS positivity to mucin is reduced
3. Myelin reactivity to Weigert’s Iron hematoxylin is reduced
4. Inert to neutral fats and phospholipids.
4. Formol corrosive/ Formol-sublimate/ - Recommended for routine post-mortem tissues.
Formol-Mercuric Chloride - It is excellent for silver reticulum methods.
- It fixes lipids, especially neutral fats and phospholipids.

Fixation time: 3-24 hours Disadvantages:

1. Tissue sections should not be more than 1cm thick (low
penetration)
2. Forms mercuric/black deposits
3. No frozen sections are made
4. Inhibits determination of tissue decalcification

5. Alcoholic Formalin/ Gendre’s fixative - Formula: 95% ethyl alcohol saturated with picric acid, strong
formaldehyde solution, Glacial acetic acid
-For sputum cytology - It enhances immunoperoxidase studies for EM if posed-
fixed with phenol formalin for 6 hours or more.
- It fixes and dehydrates at the same time and fixes sputum
since it coagulates mucus

Disadvantages:
1. It produces gross tissue hardening and causes partial RBC
lysis
2. Preservation of iron pigments is poor
3. It causes little cross-linking of proteins
6. SPECIAL FORMALIN FIXATIVES: - Cajol’s formol ammonium bromide -
good fixative for nervous tissue
(astrocytes).
- Fixatives for acid mucopolysaccharides.
- Baker’s formol calcium - used for the
preservation of lipids since most formalin
fixatives are inert to lipids.
 B. GLUTARALDEHYDE
 It is made up of two formaldehyde residues,  ADVANTAGES:
linked by three carbon chains.  Has more stable effect in tissues giving firmer and
 It is utilized for Light microscopy. better tissue sections – CNS
 Buffered Formaldehyde with secondary fixation in  Preserves plasma proteins better
osmium tetroxide is satisfactory for Electron  Produces less tissue shrinkage
Microscopy.
 Recommended for Histochemistry and electron
o 2.5% - for small tissue fragments and needle
microscopy
biopsies for 2-4 hours at room temperature.
 More pleasant with less irritation to the nose
o 4% - large tissue fragments less than 4 mm thick
for 6-8 hours up to 24 hours.  It does not cause dermatitis
 Fixation time: ½-2 hours DISADVANTAGES:
o The most effective aldehyde for protein cross- • 1. It is more expensive.
linking • 2. It reduces PAS positivity of reactive mucin. *
How can this be prevented?
• → Immersion of Glutaraldehyde fixed tissues in a
mixture of Concentrated glacial acetic acid and
aniline oil.
 II. METALLIC FIXATIVES

A. MERCURIC CHLORIDE

 The most common metallic fixative

 Frequently used in aqueous saturated solutions of 5-7%.
 The routine fixative of choice for preservation of cell detail in tissue photography
 Tissues fixed with mercury chloride containing compounds produce black precipitates except
Heidenhain’s Susa
 Satisfactory for immunoperoxidase techniques but ultrastructural preservation is poor.
 Permits brilliant metachromatic staining
 Excellent for Trichrome staining - Recommended for renal tissues, fibrin, connective tissues, and
muscles

Disadvantages:
1. It causes marked shrinkage of cells.
o How can one counteract this?
 By addition of an acidic solution such as glacial acetic acid
 Through secondary fixation

2. Has black granular deposits and is extremely corrosive to metals

REMOVAL OF BLACK DEPOSITS CAUSED BY MERCURIC CHLORIDE:

a) 0.5% iodine in 70% ethanol (alcoholic iodine) for 5-10 minutes + water + 5% sodium thiosulfate
(decolorized-5mins)-wash in running water. o Alcoholic iodine is used to remove excess mercury o
5% sodium thiosulfate is used to remove excess iodine
b) Addition of saturated iodine solution in 96% alcohol (alcoholic iodine) and iodine decolorized with
absolute alcohol in the subsequent stages of dehydration.
c) Dezenkerization
o Bring slides to water
o Immerse in Lugol’s Iodine (5 minutes)
o Wash in running water (5 minutes)
o Immerse in 5% Na thiosulfate (5 minutes)
o Wash in running water (5 minutes)
o Proceed with required water soluble stain.
MERCURIC CHLORIDE FIXATIVES
1. Zenker’s fluid - Mercuric chloride + glacial acetic acid just before its use.
o To prevent turbidity and formation of dark precipitates
- Good general fixative for all kinds of tissue
Fixation time: 12-24 hours - Recommended for fixing small pieces of liver, spleen, connective tissue
fibers and nuclei
- May act as mordant to make certain special staining possible (ex. Brilliant
staining)
- Contains glacial acetic acid which makes the solution unstable
- Recommended for Trichrome staining

2. Zenker-formol/ Helly’s - Formula: Mercuric chloride, potassium dichromate, sodium sulfate, 40%
Solution formaldehyde, and distilled water
- Mercuric chloride + 40% formaldehyde just before its use.
- It is an excellent microanatomic fixative for pituitary gland, bone marrow
Fixation time: 12-24 hours and blood-containing organs such as the liver and spleen.
- Better nuclear fixation and staining than Zenker’s
“Bloody Helly” - It preserves cytoplasmic granules better than Zenker’s
Disadvantage: Brown pigments are produced if tissues are allowed to stay
in the fixative for more than 24 hours due to RBC lysis.
Remedy: immerse the tissue in alcoholic picric acid or sodium hydroxide
3. Heidenhain’s Susa - Recommended for tumor biopsies especially of the skin
solution - It is an excellent Cytologic fixative
- Produces minimum shrinkage and hardening of tissues due to the counter-
balance of the swelling effects of acid (trichloroacetic acid) and the shrinkage
Fixation time: 3-12 hours effect of a metal (mercury).
- Counterbalance effect
o Su (sublimat) = metal (mercury) → cell shrinkage
o Sa (saure) = acid (trichloroacetic acid) → cell swelling

4. Schaudinn’s Solution/ A solution of mercuric chloride, sodium chloride, alcohol, and glacial acetic acid
Sublimated alcohol - Used on wet smears for cytologic examinations.

5. B-5 Fixative - Composed of mercuric chloride and sodium acetate.

- Commonly used for bone marrow biopsies.
Fixation time: 1 ½ to 2 - Just prior to use, add 1 mL of 40% formaldehyde to 10 mL of B5
hours (Rapid fixation)

6. Carnoy-Lebrun
7. Ohlmacher
 B. CHROMATE

 A class of fixatives which are strong oxidizing agents used for precipitating proteins and
preserving carbohydrates.

 Recommended for Chromaffin tissues, Adrenal medulla, Mitochondria

CHROMATE FIXATIVES (“CROP”)

1. Chromic acid - Used in 1-2%, used as a constituent of a compound fixative.

- It precipitates all proteins and adequately preserves
carbohydrates.
- Formaldehyde must be added to chrome-containing tissues
before use to prevent counteracting effects and consequent
decomposition of solution upon prolonged standing

2. Regaud’s Fluid/ Moeller’s fluid - Recommended for demonstration of Chromatin, Mitochondria,

Mitotic figures, Golgi bodies, RBC’s and colloid-containing
tissues
- Penetrates the tissues well - It hardens tissue better and
more rapidly than Orth’s fluid.
- Low penetration and tends to produce precipitates of sub-
oxides
- Prolonged fixation blackens the tissue pigments.
- Remedy: wash in running tap water before dehydration
3. Orth’s Fluid - Recommended for study of early degenerative processes and tissue
necrosis
Fixation time: 36-72 hours - Demonstrates Rickettsia and other bacteria
- Preserves Myelin better than buffered formalin
Disadvantages: Same as that of Regaud’s

4. Potassium dichromate - Preserves mitochondria (pH 4.5-5.2)

- Fixes lipids
- Used in 3% aqueous solution
- It fixes but does not precipitate cytoplasmic structures
C. LEAD FIXATIVES

1. Lillie’s alcoholic lead nitrate formalin

2. Lead subacetate (4% aqueous solution)

Advantages:

1. It is recommended for acid mucopolysaccharides

2. It fixes connective tissue mucin

Disadvantage:

1. It takes up CO2 to form insoluble lead carbonate especially on prolonged standing.

o This may be removed by filtration or by adding acetic acid drop by drop to lower
the pH and dissolve the residue.
III. PICRIC ACID FIXATIVES (“PBB”)
1. Excellent for glycogen demonstration
2. Normally used in strong saturated aqueous solution
3. It dyes the tissues yellow, thus preventing the tissue fragments from being overlooked. On the other
hand, this hinders proper staining.
o Can be used for fragmentary biopsies
o What must be done to remove this yellow color?
• Add 50-70% ethanol
• Acid dye/lithium carbonate
• 70% ethanol → 5% sodium thiosulfate → Wash in running tap water
4. It is suitable for aniline stains and Trichrome method

- The chemical name for the general picric acid fixatives: 2,4, 6 - trinitrophenol
- Picric acid is highly explosive when dry, and therefore must be kept moist with distilled water or
saturated alcohol at 0.5 to 1% concentration during storage.
- Picrates are soluble in water and should be transferred directly from fixative to 70% alcohol
1. Bouin’s solution - Fixation of embryos and pituitary biopsies
- It is an excellent fixative for preserving soft and delicate structures
(endometrial curetting’s)
Fixation time: 6-24 hours - Produces minimal distortion (Picric - shrinking, Glacial HAc - swelling)
- Yellow stain is useful in fragmentary biopsies
- Preferred fixative for Masson’s trichrome staining for collagen, elastic
or connective tissue
- Also used for the preservation of glycogen
- Not suitable for fixing kidney structures, lipids, and mucus - It destroys
cytoplasmic structures (ex. Mitochondria)

2. Brasil’s Alcoholic Picroformol - Best routine fixative for glycogen

Fixative - It is better and less messy than Bouin’s solution
- Overnight tissue fixation by automatic processing technique may utilize
3-4 changes of Brasil’s fixative at ½ to 2 hours each, succeeded
directly by absolute alcohol.
IV. GLACIAL ACETIC ACID

1. It solidifies at 17ºC (New book: 16oC)

2. Fixes and precipitates nucleoproteins
3. It precipitates chromosomes and chromatin materials.
4. Causes tissues to swell specially those containing collagen.
Disadvantage: It is contraindicated for cytoplasmic fixation because it destroys mitochondria and
golgi elements.
 V. ALCOHOL FIXATIVES

- Denatures and precipitates proteins

- 70-100% concentrations are used; Less concentrations results to red blood cell lysis/hemolysis due
to hypotonicity
Advantages:
- 1. May be used both as a fixative and dehydrating agent
- 2. Excellent for glycogen preservation
Disadvantages:
- 1. Causes RBC hemolysis and dissolves fats and lipids
- 2. Tissue shrinks on long usage
- 3. Polarization (major disadvantage); glycogenic granules moves towards the ends or poles of cells
General Rule:
- Alcohol-containing fixatives are contraindicated when lipids are to be studied
1. Methyl Alcohol - Used in Wright’s stain as a diluent
100% (CH3OH/ Methanol) - Excellent for fixing wet and dry smears, blood smears and
bone marrow tissues

2. Isopropyl Alcohol - It is used for fixing touch preparations (impression smears),

95% (Isopropanol) although some are air dried and not fixed, for certain procedures
such as Wright-Giemsa staining.

3. Ethyl Alcohol - Is used in 70-100% concentrations.

(C2H5OH/ Ethanol) - Lower concentrations (70-80%) will cause RBC lysis and
inadequate WBC preservation
Fixation time: 18-24 hours - It fixes blood, tissue films and smears
- Used for histochemistry especially for enzyme studies
- Can be both used as a simple and compound fixative.
4. Carnoy’s Fluid - The most rapid tissue fixative
- Recommended for fixing chromosome, lymph glands and urgent biopsies
Fixation time: 1-3 hrs - It is used to fix brain tissue for rabies diagnosis
- It preserves Nissl granules (Tigroid bodies) and very suitable for curettings
(small tissue fragments).
- After fixation, tissues can be transferred directly to absolute alcohol, or an
alcoholchloroform mixture (1:1)
- It leads to polarization unless very cold temperatures (-70oC) are used

5. Alcoholic Formalin/ - Better preserves glycogen

Gendre’ - Also a formalin fixative
- Capable of coagulating mucus and is used as a fixative for sputum cytology

6. Newcomer’s Fluid - Formula: Isopropyl alcohol, propionic acid, petroleum ether, acetone, and
dioxane
Fixation time: 12-18 hours - For fixing mucopolysaccharides and nuclear proteins
@ 3ºC - It produces better reaction in Feulgen stain than Carnoy’s fluid
- It is both a nuclear and histochemical fixative.
 VI. OSMIUM TETROXIDE/OSMIC ACID
(“OFF”)

 It is a pale yellow powder which dissolves in water (up to 6% at 20°C) to form strong oxidizing
solution.
 Adequately fixes materials for ultrathin sectioning in electron microscopy, since it rapidly fixes small
pieces of tissues and aids in their staining

Disadvantage:
- Inhibits Hematoxylin and makes counterstaining difficult
- Very expensive (less amount is required for fixation; 5-10x volume of fixative used)
- Formation of artifact pigments/black precipitate
- Prolonged exposure to acid vapors causes eye irritation(conjunctivitis) or black osmic oxide
deposition in the cornea (blindness)
1. Flemming’s Solution - Formula: 1% Aqueous chromic acid, 2% aqueous osmium tetroxide,
glacial acetic acid
- The most commonly used Chrome-Osmium acetic acid, recommended
Fixation time: 24-48 hours for nuclear preparation of such sections.
- Excellent for nuclear structures such as chromosomes
- Permanently fixes fats/lipids

Disadvantages:
 Very expensive (less amount is required for fixation)
 Formation of artifact pigments/black precipitate
o Remedy: wash in running tap water for 24 hours before dehydration
 Prolonged exposure to acid vapors causes eye irritation (conjunctivitis)
or black osmic oxide deposition in cornea (blindness)
 Extremely volatile
 It depresses the staining power of Ehrlich hematoxylin

2. Flemming’s Solution - Recommended for cytoplasmic structures such as mitochondria

without Acetic Acid - The removal of acetic acid from the formula serves to improve the
cytoplasmic detail of the cell.
Fixation time: 24-48 hours
 VII. TRICHLOROACETIC ACID (TCA)

1. Sometimes incorporated into compound fixatives (Ex. Heidenhain’s Susa)

2. It precipitates proteins
3. Causes marked swelling effect on tissues
4. Can be used as a weak decalcifying agent.
5. Has softening effect on dense fibrous tissue
6. Suitable only for small pieces of tissues or bone because of its poor penetration
 VIII. ACETONE

1. It is used at ice cold temperature from -50C to 40C (New book: 0-4oC)
2. Recommended for the study of water diffusible enzymes especially lipases and phosphatases
3. Used in fixing brain tissues for diagnosis of rabies (Negri bodies).
4. Used in freeze substitution techniques as a solvent for certain metallic salts
5. Evaporates rapidly
 IX. HEAT FIXATION

- This procedure involves thermal coagulation of tissue proteins for rapid diagnosis, usually employed
for frozen tissue sections and preparations of bacteriologic smears.
Lipid fixation - Cryostat or frozen sections should be used for demonstrating lipids in tissues
- Mercuric chloride and potassium dichromate are used in cryostat sections
- Aldehydes
- Baker’s formol calcium
- Imidazole Osmium Tetroxide – improved ultrastructural demonstration of lipids
- Cholesterol may be fixed with Digitonin for ultrastructural demonstration

Carbohydrate - Alcoholic fixatives are generally recommended for glycogen demonstration

fixation - Alcoholic formaldehyde is a better fixative in human skin compared with BNF

Protein fixation/ - Neutral buffered formaldehyde or formaldehyde vapor are the most commonly used
Amino acid
histochemistry

Glycogen fixation - Most useful fixatives are alcohol based such as Rossman’s fluid or Cold Absolute
alcohol
- Section is coated with Celloidin for better retention of glycogen
• MIXTURE OF FIXATIVES
- Useful for Electron Microscopy (Mnemonic: KOGA)
o Karnovsky’s paraformaldehyde-glutaraldehyde solution
o Acrolein – mixture with glutaraldehyde or formaldehyde; simplest unsaturated aldehyde
o Other fixatives for Electron Microscopy:
• Glutaraldehyde
• Osmium tetroxide

• COMPATIBILITY AND INCOMPATBILITY

- Few fixatives permit the use of all stains.
- Some fixatives may act as a mordant for one group of dyes and an inhibitor for another set of stains.
SECONDARY FIXATION
- The process of placing an already fixed tissue in a second fixative in order to:
a) To facilitate and improve the demonstration of particular substances
b) To make special staining techniques possible (secondary fixative acting as mordant)
c) To ensure further and complete hardening and preservation of tissues
- Not recommended when primary fixation for specific observations is possible in the first instance
- This is done before dehydration and on deparaffinized sections before staining
- 10% formol-saline/10% formalin: commonly used primary fixatives

POST-CHROMATIZATION
- AKA: Post-chroming or Post-mordanting
- A secondary fixation whereby a primary fixed tissue is placed in aqueous solution of 2.5 – 3% potassium
dichromate for 24 hours, to act as mordant for better staining effects and to aid in cytologic preservation
of tissues.
WASHING OUT
- The process of removing excess fixative from the tissue after fixation in order to improve staining
and remove artifacts from the tissue.
Solutions that may be used:
1. Tap water – used to remove:
a. Excess Chromates from tissues fixed in Helly’s, Zenker’s and Flemming’s solutions
b. Excess Formalin
o 10% formalin is extracted more rapidly in 70% alcohol than in water
a. Excess Osmic acid/Osmium tetroxide

2. 50-70 % alcohol – used to wash out excess amount of picric acid

3. Alcoholic iodine – used to remove excessive mercury
 SPECIAL FACTORS AFFECTING FIXATION
A. RETARDED BY:
1. Size and thickness - Larger and thicker tissues require more fixative 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 NSS.
3. Presence of fats - Fatty tissues should be cut in thin sections and should be fixed longer

4. Presence of blood - Tissues containing large amount of blood (e.g. Blood vessels and spleen) should
be flushed out with saline (arterial cannulization) before fixing
5. Cold temperature - Inactivates enzymes

6. Hot temperature - Denatures enzymes

B. ENHANCED BY:
1. Size and thickness - Smaller and thinner tissues require less fixative and shorter fixation tim

2. Agitation - Fixation is accelerated when automatic or mechanical tissue processing is used.

- Autotechnicon

3. Moderate heat - Accelerates fixation but hastens autolytic changes and enzyme destruction
 IMPORTANT NOTES ON IMPROPER FIXATION
1. Failure to arrest early cellular autolysis Due to the failure to fix immediately by which one first
allowed the tissue to dry before fixing or insufficient
fixative
2. Too brittle and too hard blocks Due to prolonged fixation

3. Soft and feather-like tissues Due to incomplete fixation

4. Removal of fixative soluble substances Wrong choice of fixative

5. Enzyme inactivation and loss Wrong choice of fixative

6. Presence of artefact pigments on sections Incomplete washing of fixative

7. Shrinkage and swelling of cells in tissue blocks Due to overfixation

Incomplete fixation → Improper and incomplete

clearing and impregnation → incorrect sectioning and
staining of pathologic slides
 FACTORS INFLUENCING RATE
DECALCIFICATION OF DECALCIFICATION
 Procedure whereby calcium or lime salts are removed
a. Structure = heavily calcified
from tissues (most especially bones and teeth) tissues take longer to
 It is a form of ionization decalcify
 Done after fixation and before impregnation (before b. Temperature = heat hastens
dehydration) decalcification but increases
the damaging effect of acids
 Calcified tissues are harder to cut
on tissues
c. Concentration and Volume
Recommended temperature: Room temperature (18-30oC)
Ideal time required for decalcifying tissue: 24-48 hours
Volume: 20x the volume of the tissue • Too rapid removal of CA+2
salts may produce complete
digestion of the tissue
specimen and poor staining
capacity of the cell
DECALCIFYING AGENTS:
1. Acids
2. Chelating Agents
3. Ion Exchange Resins
4. Electrical Ionization / Electrophoresis

For routine purposes, only acids are recommended:

- Nitric acid - almost 2x as fast as formic acid
- Formic acid - provides better tissue preservation and staining
- The volume of acid decalcifying solution used should be at least 1 ounce per gram of tissue; should be
changed once or twice a day until decalcification is completed
I. ACID DECALCIFYING AGENTS
1. Nitric acid - The most common and fastest decalcifying agent used so far
- Very rapid decalcifying agent, producing minimal distortion
- Recommended for routine purposes
Recommended - It undergoes spontaneous yellow discoloration owing to
concentrations: 5-10% formation of nitrous acid (HNO2)
o This accelerates decalcification
o Also stains and damages the tissues

a. 10% aqueous Nitric Acid - Rapid in action

- Recommended for urgent biopsies and for needle and small biopsy
Decalcification time: 12-24 specimen
hours - Used for large or heavily mineralized cortical bone specimen

b. Formol-Nitric acid - Less tissue destruction than 10% aqueous nitric acid
- For urgent biopsies with good nuclear staining.
Decalcification time: 1-3 days - Yellow color imparted by nitrous acid is removed through neutralization
with 5% sodium sulfate and running tap water for 12 hours
o Yellow color formation is prevented by addition of 0.1% urea to pure
concentrated nitric acid
- This should be used inside a fume hood
c. Perenyi’s fluid - It is recommended for routine purposes.
- It decalcifies and softens tissues at the
Decalcification time: 2-7 days same time
- Maceration is avoided due to the presence
of chromic acid and alcohol
d. Phloroglucin Nitric Acid - Most rapid decalcifying agent,
recommended for urgent works
Decalcification time: 12-24 hours - Has poor nuclear staining
- Yellow color formation
2. Hydrochloric acid/ Muriatic - Inferior compared to nitric acid (slower action and greater distortion of
acid tissue)
- Good nuclear staining
- Recommended for surface decalcification of tissue blocks if used in
1% solution with 70% alcohol

Von Ebner’s fluid Formula:

36% Sat. Aq. NaCl, Conc. HCl, Distilled water
- Recommended for teeth and small pieces of bones

3. Formic acid - Moderate-acting decalcifying agent

- Recommended for routine decalcification of post-mortem research
Decalcification time: 2-7 days tissues

Disadvantage: Not suitable for urgent examination

 Aqueous Formic Acid (Formic acid + 10% Formol saline)
o Recommended for small pieces of bones and teeth
o Suitable for most routine surgical specimen and
immunohistochemical staining.

 Formic Acid – Sodium Citrate (20% Aq. Sodium citrate + 45%

Formic acid) o Recommended for autopsy materials, bone marrow,
cartilage and tissues studied for research purposes
4. Trichloroacetic acid - Does not require washing out
- Not recommended for urgent examinations (very
slow acting)

5. Sulfurous acid - Weakest decalcifying agent, suitable only for

minute pieces of bone

6. Chromic acid - Used as a fixative and decalcifying agent

- Caution: It is an environmental toxin, highly
corrosive to skin and mucous membrane and
carcinogenic

7. Citric acid – Citrate buffer - Does not produce cell or tissue distortion
II. CHELATING AGENTS

 Substances which combine with calcium ions and other salts (iron and magnesium) to form weakly
dissociated complexes and facilitate removal of Calcium salt
 The most common chelating agent: EDTA (Used as an anticoagulant and water softener)
o Commercial name: Versene, Sequestrene
EDTA + Ca2+ Insoluble non-ionized complex (Removes Ca)
III. ION-EXCHANGE RESIN
 Ion exchange resin (ammonium form of polystyrene resin) hastens decalcification
 Removes calcium ions from formic acid, thereby increasing solubility from the tissue.
 Not recommended for fluids containing mineral acids such as nitric or hydrochloric acids
IV. ELECTRICAL IONIZATION (ELECTROPHORESIS)

 Process whereby positively charged Calcium ions are attracted to a negative electrode (cathode)
and subsequently removed from the decalcifying solution.
 The time required for decalcification is thereby shortened due to the heat and electrolytic reaction.
produced in this process.
 The principle applied is similar to that of chelating agents; with the main difference that this process
utilizes electricity and is dependent upon a supply of direct current to remove calcium deposits.
- Solutions used
1. 88% Formic acid
2. Concentrated HCl
3. Distilled water
3 WAYS TO MEASURE THE EXTENT OF DECALCIFICATION
1. Physical or Mechanical Method - By touching or bending the tissue with the fingers to determine the
consistency of tissues
- By pricking the tissue with a fine needle/probe

2. X-ray/ Radiological method - Very expensive, although it is the most ideal, most sensitive and most
reliable method
- Good but not always convenient
- Not recommended for mercuric chloride-fixed tissues (radio-opacity
will interfere with the plate interpretation)

3. Chemical method/ Calcium Oxalate - Simple, reliable and convenient method recommended for routine
test purposes (still favored)
- Decalcifying fluid is changed every 24-48 hrs.

Solutions used:
1. Concentrated Ammonium Hydroxide
2. Saturated Aqueous Ammonium Oxalate

- Detection of calcium in acid solutions by precipitation of insoluble

calcium hydroxide or calcium oxalate
- Presence of cloudiness indicates the presence of Ca2+ (incomplete
decalcification
TISSUE SOFTENERS

Unduly hard tissues which are reliable to damage the microtome knives may require tissue softener, aside
from decalcification.
1. Perenyi’s fluid – act both as a decalcifying agent and tissue softener
2. 4% phenol (1-3 days)
3. Molliflex (tissues may appear swollen and soapy)
4. 2% Hydrochloric acid
5. 1% HCl in 70% alcohol
 DEHYDRATION

 A.k.a. Desiccation
 The process of removing intracellular and extracellular water from the tissue
following fixation (if decalcification is not required) and prior to wax
impregnation (clearing).
 Naturally carried out by the use of some reagents (dehydrating
agents/dehydrants) that mixes with and has a certain affinity for water so that it
may penetrate easily between the tissue cells.
 Ideal volume: not less than 10x the volume of tissue to be dehydrated
CHARACTERISTICS OF AN IDEAL DEHYDRATING SOLUTION:

1. It should dehydrate rapidly without producing considerable shrinkage or distortion of tissues.

2. It 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 DEHYDRATING AGENTS

1. Alcohol - Dehydration is best accomplished by the use of increasing grades of

alcohols, beginning with 70% (70%→95%→100%→100%→100%)
- Transfer of tissue directly from formalin to higher grades of alcohol (85% -
95%) is risky, since it could lead to distortion of tissues (shrinkage and
hardening of tissues).
- Concentrated solutions (above 80%) makes tissue hard, brittle and
difficult to cut
- Prolonged storage in lower concentrations (below 70%) tends to
macerate the tissue
- Tissue stored in 70-80% may interfere with the staining properties of the
specimen
a. Ethanol/ Ethyl alcohol - Undoubtedly the best dehydrating agent (fast acting,
mixes with water and organic solvents and penetrates
tissues easily)
- Has the advantage of not being poisonous and not very
expensive
- Should be at least 99.7% pure
b. Isopropanol/ Isopropyl alcohol - Should be used if good-grade absolute ethyl alcohol is not
easily available
c. Methanol/ Methyl alcohol - Toxic dehydrating agent - For blood and tissue films and
for smear preparations
d. Butanol/ Butyl alchohol - Utilized for plant and animal microtechniques.
2. Acetone - A cheap, rapid dehydrating agent utilized for most urgent biopsies (1/2
(CH3COCH3) to 2 hours)
- Its use is limited only to small pieces of tissues due to its extreme
volatility and flammability
- Produce considerable tissue shrinkage, not recommended routinely

3. Dioxane/ Diethylene Dioxide - Excellent dehydrating and clearing agent miscible to water, melted
paraffin, alcohol and xylol/xylene
- Expensive and toxic and extremely dangerous (main disadvantage)

4. Cellosolve/ Ethylene glycol - Dehydrates rapidly and not harmful to tissues

monoethyl ether - Toxic by inhalation, skin contact and ingestion (use propylene-based
glycol esters)
5. Triethyl phosphate - Tissues can be transferred directly after fixation & washing
- Used to dehydrate sections and smears following certain stains
6. Tetrahydofuran - Dehydrates and clears tissues since it is miscible to water and paraffin
- It is toxic if ingested or inhaled. Vapors cause nausea, dizziness and
headache.
- It is an eye and skin irritant and prolonged exposure (up to 6 months)
may cause conjunctival irritation
Additional notes!
 The temperature that will hasten alcohol dehydration and is used for tissue sections that require
urgent examination of fragmentary biopsies: 37°C
 3 additives that are added to dehydrating agents in duly hard tissues:
o 4% phenol
o Molliflex
o Glycerol alcohol mixture
 CLEARING

 AKA: “Dealcoholization”
 The process whereby alcohol or a dehydrating agent is removed from the tissue and
replaced by a fluid (clearing agent) that will dissolve the wax with which the tissue must be
impregnated.
 Clearing agent must be miscible with the dehydrating agent, paraffin wax, and mounting
medium.
 Ideal amount: not less than 10x the volume of the tissue
 Glycerin/Gum syrup: used when the tissue is cleared directly from water (frozen section)
COMMON CLEARING AGENTS Other Clearing agents:

1. Xylene/Xylol 13. Cedar oil 1 1. Terpineol

2. Phenol in alcohol
2. Toluene/Toluol 14. Creosote
3. Dioxane
3. Benzene 4. Carboxylol
4. Chloroform
5. Cedarwood oil
6. Carbon bisulfide
7. Oil of Bergamot
8. Clove oil
9. Carbon tetrachloride
10. Aniline oil 1
11. Tetrahydrofuran
12. Petroleum ether
 The word clearing is used CHARACTERISTICS OF A GOOD CLEARING AGENT:
because, in addition to removing
alcohol, many of these substances 1. It should be miscible with alcohol to promote rapid
have the property of making removal of the dehydrating agent from the tissue.
tissues transparent/translucent. 2. It should be miscible with, and easily removed by
melted paraffin wax and/or by mounting medium to
 They effect this by virtue of high
facilitate impregnation and mounting of sections.
refractive index (index of refraction) 3. It should not produce excessive shrinkage, hardening
and the resultant optical changes or damage of tissue.
when the clearing agents penetrate 4. It should not dissolve out aniline dyes.
between the highly refractile tissue 5. It should not evaporate quickly in a water bath.
elements; i.e. the RI of clearing 6. It makes tissues transparent.
agents is approximately equal to
that of the tissues.
 However, not all dealcoholization
agents can act as clearing agents
and vice versa.
COMMONLY USED CLEARING AGENTS

1. Xylene Clearing time: - An excellent and true clearing agent

½ to 2 hours (usual) Advantages:

15-30 Minutes (urgent 1. The most rapid clearing agent
biopsies) 2. Cheap and does not extract out aniline dyes
3. It is miscible with absolute alcohol and paraffin
4. Can be used with celloidin sections

Disadvantages:
5. It is highly flammable
6. When dehydration is not complete, the xylene becomes milky when the
tissue or section is added to it
7. It causes considerable hardening and shrinkage of tissues; hence, is not
suitable for nervous tissues and lymph nodes.
2. Toluene - Substitute to xylene or benzene
- It is miscible with absolute alcohol and paraffin
Clearing time: 1-2 hours - It is not carcinogenic but highly concentrated emit fumes that are toxic
upon prolonged exposure
- It acts slower than benzene and is expensive
3. Benzene - Rapid acting, recommended for urgent biopsies and routine
purposes
Clearing time: 15-60 minutes - It is miscible with absolute alcohol and paraffin
- It is highly flammable
- Carcinogenic or may damage bone marrow (Aplastic anemia; If
antibiotic: Chloramphenicol)

4. Chloroform - It is slower in action than xylene, but causes less brittleness

- It is the best of the traditional clearing agents for routine use
Clearing time: 6-24 hours - Gives the widest latitude
- It is recommended for tough (skin, fibroid and decalcified tissues)
and large tissue specimens
- Also best for nervous tissue, lymph nodes, granulation tissue, and
fetal and other delicate, highly cellular specimens, all of which tend
to become distorted and to break up on sectioning if cleared in
xylene, toluene, or benzene
- It does not make tissues transparent and it is toxic to the liver
(hepatotoxic) after prolonged inhalation
5. Cedarwood oil - Clears both Paraffin and Celloidin sections
- Recommended for CNS tissues and cytological studies (esp. Smooth
Clearing time: 2-3 days muscles and skin)
- Very expensive and it requires 2 changes in clearing solution
- Quality is not always uniform and good and is extremely slow
- It becomes milky on prolonged storage

6. Aniline oil - Recommended for clearing embryos, insects and very delicate
specimens since it clears 70% alcohol without excessive tissue
shrinkage and hardening
7. Clove oil - Causes minimum shrinkage of tissues
- It removes aniline dyes and dissolves Celloidin; Tissues
become brittle
- Its quality is not guaranteed due to its tendency to be
adulterated
- Not suitable for routine purposes because it is expensive

8. CCl4 (Carbon Tetrachloride) - Properties are very similar to chloroform but it is cheaper
- Toxic on prolonged exposure

9. Tetrahydrofuran - Dehydrates and clears at the same time since it is miscible

in both water and paraffin
10. Methyl benzoate/ Methyl Salicylate - These are slow-acting clearing agents that can be used when
double embedding techniques are required.