COLLEGE OF MEDICAL TECHNOLOGY

MLS 111 – Histopathology Lec

Module 1 - Introduction to Fresh Tissue Examination/Fixation

FRESH TISSUE EXAMINATION • Commonly used for:

Methods of tissue examination may vary according to: ✓ Rapid pathologic diagnosis during surgery
1. the structural and chemical components of the cells ✓ Diagnostic and research enzyme
2. the nature histochemistry
3. amount of the tissue to be evaluated ✓ Diagnostic and research demonstration of
4. the need for an immediate examination of a tissue soluble substances such as lipid and
structure. carbohydrates.
✓ Immunofluorescent and
METHODS OF TISSUE EXAMINATION immunohistochemistry staining
a. Teasing or Dissociation ✓ Some specialized silver stains, particularly in
• Selected tissue specimen is immersed in a watch neuropathology.
glass containing isotonic salt solution ✓ FRESH
• Dissected, separated, and examined under the
microscope Methods in Freezing Tissue: (LICA)
✓ Unstained by phase contrast or bright field (1) Liquid nitrogen
microscope (2) Isopentane cooled by nitrogen
✓ Stained with differential dyes (3) Carbon dioxide gas
b. Squash Preparation (4) Aerosol sprays
• small pieces of tissue not more than one mm in
diameter is placed in a slide and forcibly compressed 10 STEPS IN PROCESSING TISSUE
with another slide or with a cover glass 1. Fixation
• dudurugin 2. Dehydration
c. Smear Preparation 3. Clearing
• Examining sections or sediments, whereby cellular 4. Infiltration (Impregnation)
materials are spread lightly over a slide means of a 5. Embedding
wire loop 6. Trimming
• Specifically used in cytological examinations (cancer 7. Section-Cutting
diagnosis) 8. Mounting
• Pap smear, FNAB (Fine Needle Aspiration Biopsy) 9. Staining
10. Labelling
3 WAYS OF SMEAR PREPARATION
(1) STREAKING – with an applicator stick, the material FIXATION
is rapidly and gently applied in direct or zigzag line - The first and most critical step in histotechnology
throughout the slide. - a process that involves fixing or preserves FRESH tissues
(2) SPREADING – maintains cellular inter relationships from decay/decompositions, thereby preventing autolysis
of the material to be examined. It is especially or putrefaction, degeneration, and distortion of tissues.
recommended for smear preparations of fresh - The fixatives employed prevent autolysis by inactivating
sputum and bronchial aspirates and also for thick lysosomal enzymes
mucoid secretions. - Fixatives also inhibit the growth of bacteria and molds that
(3) PULL APART – useful for preparing smears of thick give rise to putrefactive changes
secretions such as serous fluid, concentrated - the most important reactions in maintaining morphology
sputum and blood smears. in the fixation of tissues for routine histopathology are
those that stabilize proteins
d. Touch Preparation (impression smear)
• has added advantage in that cells may be examined PRIMARY GOAL: preserve the morphologic and chemical
without destroying their actual intracellular integrity of the cell in as life-like a manner as possible.
relationship, and without separating them from their
normal surroundings. SECONDARY GOAL: to harden and protect the tissue from the
trauma of further handling, so that it is easier to cut and
e. Frozen Sections/RFS (Rapid frozen section) process for microscopy. Fixation allows the tissue to be more
• for rapid diagnosis, especially recommended when properly oriented in the cassette in preparation for paraffin
lipids and nervous tissue elements are to be a embedding and microtomy.
demonstrated
→ 10% Formaldehyde (commonly used), NSS

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 COLLEGE OF MEDICAL TECHNOLOGY
MLS 111 – Histopathology Lec
Module 1 - Introduction to Fresh Tissue Examination/Fixation

CHANGES compromised in order to obtain full penetration and

➢ It is important to know that a fixative will initially satisfactory fixation.
produce a number of changes to the tissues in what is - Large solid tissue, such as uterus, should be opened
usually an aqueous environment. This includes or sliced thinly to improve penetration of fixatives. In
shrinkage, swelling, and hardening of various addition, fecal matter and stomach contents can
components. inhibit the penetration of fixative, as well as damage
➢ During processing, the specimen may shrink and lose tissue during sectioning, and therefore must be
20%-30% of its volume. removed before fixation.

TWO BASIC MECHANISM IN FIXATION  OSMOLARITY

1. Additive fixation - The best results are usually obtained using slightly:
• whereby the chemical constituent of the fixative is ➢ Hypertonic solutions 400-459 mOsm
taken in and becomes part of the tissue by forming (preferred)
cross-links or molecular complexes and giving ➢ Isotonic solutions are 340 mOsm
stability to the protein
• Examples: formalin, mercury, and osmium tetroxide  CONCENTRATION
- Concentration down to the lowest level possible. Too
2. Non-additive fixation high a concentration may adversely affect the tissue
• whereby the fixing agent is not incorporated into the and produce artefact similar to that caused by
tissue, but alters the tissue composition and excessive heat
stabilizes the tissue by removing the bound water ➢ Formaldehyde is normally used as a 10%
attached to H-bonds of certain groups within the solution
protein molecule. ➢ Glutaraldehyde is normally used as a 3%
• Examples: alcoholic fixatives solution.
➢ Glutaraldehyde (0.25%) have been found to be
MAIN FACTORS INVOLVED IN FIXATION an ideal concentration for immuno-electron
 HYDROGEN ION CONCENTRATION microscopy.
- Fixation is best carried out close to neutral pH, in the - The presence of a buffer causes polymerization of
range of 6-8. Outside this range, changes may occur the aldehyde, with consequent decrease in its
that are detrimental to ultrastructural preservation effective concentration
of the tissue.
- Hypoxia of the tissue lowers the pH, so there must  DURATION OF FIXATION
be buffering capacity in the fixative to prevent - Some tissues take longer to fix than others,
excessive acidity. Acidity favors formation of depending on their structure. Fibrous organs such as
formalin-heme pigment that appears as black, uterus or intestinal tract take longer to fix than small
polarizable deposits in tissue. or loosely textured tissues such as biopsies or
- Common buffers include phosphate, bicarbonate, scrapings.
cacodylate, and veronal. - Fixation time can be cut down by using heat, vacuum,
- Commercial formalin is buffered with phosphate at a agitation or microwave.
pH of 7. - Prolonged fixation may cause shrinkage and
hardening of tissue, and may severely inhibit enzyme
 TEMPERATURE activity and immunological reactions, although
- Surgical specimens: room temperature washing of the tissue in running water considerably
- Cytological: refrigerated restores the activity of some enzymes.
- Electron microscopy and histochemistry: 0-4 C
PRACTICAL CONSIDERATIONS
Refrigeration is used to slow down decomposition if the tissue 1. SPEED
needs to be photographed and cannot be fixed immediately. • to prevent autolysis and putrefaction
• Time interval from of removal of tissues to fixation is
 THICKNESS OF SECTION important. In order to maintain tissue morphology,
- Tissue blocks should be small (e.g., 1 to 2mm for samples should be fixed immediately after removal
electron microscopy and 2 cm wide for light or death to prevent autolysis or putrefaction.
microscopy) and thin (no more than 0.4 cm for light • The longer the blood supply is interrupted, the
microscopy). These measurements should not be poorer will be the quality of tissue.

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 COLLEGE OF MEDICAL TECHNOLOGY
MLS 111 – Histopathology Lec
Module 1 - Introduction to Fresh Tissue Examination/Fixation

2. PENETRATION TYPES OF FIXATIVES ACCORDING TO

3. VOLUME COMPOSITION
• 10-25 times the volume of the tissue. A. SIMPLE FIXATIVES - made up of only one component
• Maximum effective is 20 times. substances
• The most common error in histotechnology is ▪ Formaldehyde
insufficient ratio of tissue volume to fixative volume. ▪ Glutaraldehyde

EFFECTS OF FIXATIVES IN GENERAL METALLIC FIXATIVES

1) They harden soft and friable tissues and make the ▪ Mercuric chloride
handling and cutting of sections easier. ▪ Chromate fixatives
2) They make the cells resistant to damage and distortion o Potassium dichromate
caused by hypotonic and hypertonic solutions used during o Chromic acid
tissue processing. LEAD FIXATIVES
3) They inhibit bacterial decomposition. ▪ Picric acid
4) They act as mordants or accentuators to promote and ▪ Acetic acid
hasten staining, or they may be inhibited certain dyes in ▪ Acetone
favor of another. ▪ Alcohol
5) They reduce the risk of infections during the handling and ▪ Osmium tetroxide
actual processing of tissues
B. COMPOUND FIXATIVES - made up of two or more fixatives
CHARACTERISTICS OF A GOOD FIXATIVE which have been added together to obtain the optimal
1) It must be cheap. combines effect of their individual actions upon the cells
2) It must be stable. and tissue constituents.
3) It must be safe to handle.
4) It must kill the cell quickly thereby producing minimum TYPES OF FIXATIVES ACCORDING TO ACTION
distortion of cell constituents. A. MICROANATOMICAL FIXATIVES - Those permit the
5) It must inhibit bacterial decomposition and autolysis. general microscopic study of tissue structures without
6) It must produce minimum shrinkage of tissue. altering the structural pattern and normal intercellular
7) It must permit rapid and even penetration of tissues. relationship of the tissues in question.
8) It must harden tissues thereby making the cutting of
sections easier. ✓ 10% formol slaine
9) It must make cellular components insoluble to hypotonic ✓ 10% neutral buffered formalin
solutions and render them insensitive to subsequent ✓ Heidanhain’s susa
processing ✓ Formol sublimate
10) It must permit the subsequent application of many ✓ Zenker’s sol
staining procedures to facilitate easier and more ✓ Zenker formol
profitable examination. ✓ Bouins sol
✓ Brasil’sol
FOUR MAJOR GROUPS OF FIXATIVES
a. Aldehydes - formaldehyde, glutaraldehyde B. CYTOLOGICAL FIXATIVES - are those that preserve specific
b. Oxidizing agents - osmium tetroxide, potassium parts and particular microscopic elements of the cell itself.
permanganate
c. Alcohol based fixatives – methyl alcohol, ethyl alcohol, 1) Nuclear fixatives
acetic Acid (protein-denaturing agents.) - pH of 4.6 or less, preserves nuclear structures, usually
d. Metallic Fixatives contains glacial acetic acid as their primary component
due to its affinity for nuclear chromatin
Note:
✓ The aldehydes and oxidizing act by crosslinking ✓ Flemming’s fluid
proteins. ✓ Carnoy’s fluid
✓ Metallic fixatives act by forming insoluble metallic ✓ Bouin’s fluid
precipitates like mercuric chloride and picric acid. ✓ Heidenhain’s susa
✓ The Choice of the fixative is based on tissue and ✓ Newcomer’s fluid
anticipated ancillary tests.

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 COLLEGE OF MEDICAL TECHNOLOGY
MLS 111 – Histopathology Lec
Module 1 - Introduction to Fresh Tissue Examination/Fixation

2) Cytoplasmic fixatives DISADVANTAGES

- must never contain glacial acetic acid (because it destroys  Slow fixative
mitochondira and golgi bodies of the cytoplasm), pH more  Formol saline fixed tissues tend to shrink during
than 4.6 alcohol dehydration, this may be reduced by
secondary fixation
✓ Flemming’s fluid w/o acetic acid
✓ Kelly’s fluid c. 10% Neutral Buffered Formalin or Phosphate Buffered
✓ Formalin w/o post-chroming Formalin (NBF/PBF)
✓ Orth’s fluid ▪ pH 7
✓ Muller’s fluid ▪ recommended for preservation and storage of
surgical post mortem, and research specimen
3) Histochemical fixatives ▪ FIXATION TIME: 4-24HRS
- preserve chemical constituents of cells and tissues
ADVANTAGES
✓ Formol saline (10%) ✓ prevents participation of acid formalin pigments on
✓ Absolute ethyl alcohol post mortem tissue
✓ Acetone ✓ best fixative for tissues containing iron pigment
✓ Newcomer’s fluid and for elastic fiber which do not stain well after
susa, zenker and chromate fixation.
ALDEHYDE FIXATIVES
a. Formaldehyde (Formalin) DISADVANTAGES
▪ one of the most widely used fixative  longer to prepare, time consuming
▪ must be diluted to 1:10 or 1:20 to make a 5 to 10%
formalin d. Formol Corrosive (Formol-Sublimate)
▪ stock solution 40% ▪ Recommended for routine post mortem tissues
▪ usual fixation time is 24hrs ▪ Excellent for many staining procedures including
▪ usually buffered to pH 7 with phosphate buffer silver reticulum methods.
▪ FIXATION TIME: 3-24hrs
ADVANTAGES
✓ cheap, rapidly available, easy to prepare ADVANTAGES
✓ compatible with many stains ✓ Penetrates small pieces of tissues rapidly
✓ does not over harden tissues ✓ There is no need for washing out, tissues can be
✓ penetrates tissue well transferred directly from the fixative to alcohol
✓ preserves fat, mucin, and glycogen DISADVANTAGES
 Penetration is slow
DISADVANTAGES  Forms mercuric chloride deposits
 fumes are irritating to the nose and eyes
 solutions are irritating to the skin and may cause e. Alcoholic Formalin (Gendre’s Fixative)
allergic dermatitis ▪ Use for sputum since it coagulates mucus

b. 10% Formol Saline ADVANTAGES

▪ Recommended for fixation of CNS tissues and ✓ Fixation is faster
general post mortem tissues for histochemical ✓ Can be used for rapid diagnosis
examination ✓ Can fix and dehydrate at the same time
▪ FIXATION TIME: 24hrs (35 C), 48hrs (20-25 C)
DISADVANTAGES
ADVANTAGES  Produces gross hardening of tissue
✓ penetrates fixes tissue evenly  Causes partial lysis of RBC
✓ preserves microanatomic and cytologic details with  Preservation of iron containing is poor
minimum shrinkage and distortion
✓ ideal for most staining techniques f. Glutaraldehyde
✓ preserves enzymes and nucleoproteins ▪ Made up of two formaldehyde residues, linked by
three carbon chain
▪ More expensive

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 COLLEGE OF MEDICAL TECHNOLOGY
MLS 111 – Histopathology Lec
Module 1 - Introduction to Fresh Tissue Examination/Fixation

▪ For small tissue fragments: 2.5% solution fixed for 2- - Mercuric deposits may be removed by
4hrs immersing tissues in alcoholic iodine solution
▪ For larger tissues less than 4 mm thick: fixed in 6-8hrs prior staining through a process known as
to 24hrs DEZENKERIZATION. (Done by the oxidation
with sodium to mercuric iodide which can be
ADVANTAGES subsequently treated with sodium thiosulfate)
✓ more stable effect on tissue
✓ preserves plasma proteins better 2. ZENKER-FORMOL (HELLY’ SOLUTION)
✓ produce less tissue shrinkage - Excellent in micro anatomic fixative for
✓ preserves cellular structures better pituitary gland and bone containing organs
✓ more pleasant and less irritating such as spleen and liver
- Preseves cytoplasmic granules well
DISADVANTAGES - It may produce brown pigments due to more
 expensive than 24 hrs stay in fixatives and due to RBC lysis,
 penetrates tissue slowly it can removed by immersing tissue in saturated
picric acid or sodium hydroxide
METALLIC FIXATIVES
a. Mercuric Chloride 3. HEIDENHAIN’S SUSA
▪ Most common metallic fixative - Recommended mainly for tumor biopsies be
▪ Widely sued as secondary fixative specially of the skin
▪ frequently used in saturated aqueous solutions 5- - Excellent cytologic fixative
7%. - FIXATION TIME: 3-12HRS
▪ Routine fixative of choice for preservation of cell - REMEMBER: after using, tissue SHOULD be
detail in tissue photography. Recommended for transferred directly to a high-grade alcohol
renal disease, fibrin, connective tissues and muscles (90%) to avoid undue swelling.

ADVANTAGES 4. B5 FIXATIVE
✓ Penetrates and hardens tissues rapidly and well - Commonly used for bone marrow biopsies
✓ Precipitates all protein - Rapid fixative (1-2HRS)
✓ Has greater affinity to acid dyes - Same as mercuric chloride (dezenkerization)

DISADVANTAGES b. Chromate Fixatives

 Caused marked shrinkage of cells ▪ CHROMIC ACID
 If left in fixative for more than 1-2 days tissue - Used in 1-2% aqueous sol. Usually as a
become unduly hard and brittle constituents of compound fixative
 Extremely corrosive to metals ▪ POTASSIUM DICHROMATE
- Used in 3% aqueous solution
REMEMBER: black deposits of mercury - Fixes but does not precipitate cytoplasmic
structures
REMEDY: treating the section with 0.5% iodine solution in 70% - Preserves lipids and mitochondria
ethanol for 5-10 mins. Sections are rinsed in water, decolorized ▪ REGARD’S (MULLER’S) FLUID
for 5 mins. In 5% sodium thiosulfate and washed in running - Recommended for demonstration of Golgi
water. bodies, chromatin, mitochondria, and colloid-
containing tissues
TYPES OF MERCURIC CHLORIDE - Does not preserve fats
1. ZENKER’S FLUID - Prolonged fixation blackens tissue pigments;
- made up of mercuric chloride stock solution to - REMEDY: removed by washing the tissues in
which glacial acetic acid has been added just running tap water prior to dehydration.
before use to prevent turbidity and formation ▪ ORTH’S FLUID
of a dark precipitate. - Recommended for study of early degenerative
- Recommended for fixing small pieces liver, processes and tissue necrosis
spleen, connective tissue fibers and nuclei. - Recommended for acid mucopolysaccharide
- FIXATION TIME: 12-24hrs - Demonstrates Rickettsiae and other bacteria

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 COLLEGE OF MEDICAL TECHNOLOGY
MLS 111 – Histopathology Lec
Module 1 - Introduction to Fresh Tissue Examination/Fixation

c. Lead Fixatives b. FLEMMING’S SOLUTION WITHOUT ACETIC ACID

▪ Recommended for acid mucopolysaccharides ▪ Recommended for cytoplasmic structure particularly
▪ Fix connective tissue mucin the mitochondria

PICRIC ACID FIXATIVES TRICHLOROACETIC ACID

- excellent fixative for glycogen demonstration. - Use as weak decalcifying agent
- yellow color may produce, it can be removed treatment a. Acetone
with another dye (lithium carbonate) ▪ Recommended for the study of water diffusible
- REMEDY: tissue may be placed in 70% ethyl acohol enzymes especially phosphate and lipase
followed by 5% sodiumthiosulfate and then washed in ▪ Fixing brains
running water. ▪ Diagnosing rabies

a. BOUIN’S SOLUTION b. Heat Fixation

▪ Recommended for fixation of embryos and pituitary ▪ A form of fixation, fixes the smear
biopsies
▪ Does not need washing out SECONDARY FIXATION
▪ Excellent fixative for preserving soft and delicate - Process of placing an already fixed tissue in a second
structures (eg: endometrial cutterings) Not suitable fixative in order:
for fixing kidney structures, lipid and mucus. ✓ To facilitate and improve the demonstration of
particular substances
b. BRASILS ALCOHOLIC PICROFORMOL FIXATIVE ✓ To make special staining technique possible (with
▪ Excellent fixative for glycogen secondary fixative acting as mordant)
▪ Better and lessy messy than bouin’s solution ✓ To ensure further and complete hardening and
preservation of tissues
GLACIAL ACETIC ACID
- Fixes and precipitates nucleoproteins POST CHROMATIZATION
- Solidify at 17C - A form of secondary fixation whereby primarily fixed
tissue is placed in aqueous sol of 2.5-3% potassium
ALCOHOL FIXATIVES dichromate for 24 hrs. to act as mordant for better
- Used as both fixative and dehydrating agent staining effects and to aid in cytologic preservation of
tissues.
1. METHYL ALCOHOL (100%)
- Excellent for fixing dry and wet smears, blood WASHING OUT
smear, and bone marrow smear - Process of removing excess fixative from the tissue after
2. ISOPROPHYL ALCOHOL 95% fixation.
3. ETHYL ALCOHOL - Several solutions may be used:
- Used for fixing touch preparations. ✓ Tap water – used to remove
4. CARNOY’S FLUID ✓ Excess chromates from tissues fixed in kelly’s,
- Recommended for fixing chromosomes, lymph zenkers and flemming solution
glands and urgent biopsies ✓ Excess formalin
- Used to fix brain tissues ✓ Excess osmic acid 50-70%
- most rapid fixative ✓ alcohol – used to wash out excess amount of
5. NEWCOMER’S FLUID picric acid (bouin’s solution)
- Recommended for fixing mucopolysaccharides ✓ Alcoholic iodine – used to remove excess
and nuclear proteins mercuric fixatives
- Acts as both as a nuclear and histochemical
fixative FACTORS THAT AFFECT FIXATION
- Retarded by:
OSMIUM TETROXIDE (OSMIC ACID) • Size and thickness of the specimen
a. FLEMMING’S SOLUTION • Presence of mucus
▪ Most common chrome osmium acetic acid fixative - • Presence of fat
Recommended for nuclear preparation • Presence of blood
▪ Permanently fixes fat • Cold temperature

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 COLLEGE OF MEDICAL TECHNOLOGY
MLS 111 – Histopathology Lec
Module 1 - Introduction to Fresh Tissue Examination/Fixation

- Enhanced by: - does not require washing out before

• Size and thickness of the specimen dehydration
• Agitation - recommended for teeth and small pieces
of bone
PROCESSING OF TISSUE
DECALCIFICATION 3) Formic Acid
- Procedure whereby calcium or lime salts are removed • may be used as both fixative and decalcifying
from tissues following fixations. agent.
- Recommended ratio of fluid to tissue volume for • recommended for routine decalcification of
decalcification is 20:1 postmortem research tissues.
- Calcium may be removed by the following acid: • FORMIC ACID-SODIUM CITRATE SOLUTION
✓ Chelating agents - recommended for autopsy materials,
✓ Ion exchange resins bone marrow, cartilage.
✓ Electrical ionization
4) Trichloroacetic Acid
METHODS OF DECALCIFICATION • may be used as both fixative and decalcifying
A. ACID DECALCIFYING AGENT agent.
1) Nitric Acid • recommended for routine decalcification of
• most common and the fastest decalcifying postmortem research tissues.
agent used so far. However, it inhibits nuclear
stain and destroying tissues especially in 5) Sulfurous Acid
concentrated solutions. • Is a very weak decalcifying solution suitable
only for MINUTE PIECES OF BONE
1.1. AQUEOUS NITRIC ADD SOLUTION 10%
- recommended for urgent biopsies, for 6) Chromic Acid (FLEMMING’S Fluid)
needle and small biopsy specimens to • may be used as both fixative and decalcifying
permit rapid diagnosis within 24 hrs. or agent.
less • suggest for minute bone specimens.
1.2. FORMOL-NITRIC ACID • consider as carcinogenic.
- Rapid acting
- recommended for urgent biopsies. 7) Citric Acid – Citrate Buffer Solution
- Produce yellow color • pH 4.5
- Remedy: neutralizing tissue in 5% sodium • excellent nuclear and cytoplasmic staining.
sulfate and washing in running tap water • Too slow
for at least 12 hrs. Addition of 0.1% urea • Contains chloroform as preservatives.
to pure concentrated nitric acid will also
make discoloration disappear without B. CHELATING AGENTS
affecting the efficiency of the decalcifying 1. EDTA (VERSENE)
agent • most common chelating agent
1.3. PERENYI’S FLUID
• recommended for detailed microscopic studies
- recommended for routine purposes
• very slow decalcifying agent
- nuclear and cytoplasmic staining is good.
• for small specimens: 1-3 weeks
Maceration is avoided due to the
• 6-8 weeks or longer totally decalcify dense
presence of chromic acid and alcohol.
cortical bone.
- not recommended for urgent work.
- composed of chromic acid, ethyl alcohol • excellent for immunohistochemistry or enzyme
and nitric acid. staining and electron microscopy
1.4. PHLOROGLUCIN NITRIC ACID • inactivates ALP; to restored add magnesium
- most rapid decalcifying agent chloride

2) Hydrochloric Acid C. ION EXCHANGE RESIN

• VON EBNER’S FLUID • not recommended for fluids containing nitric
- moderately rapid decalcifying agent acid or hydrochloric acid.

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 COLLEGE OF MEDICAL TECHNOLOGY
MLS 111 – Histopathology Lec
Module 1 - Introduction to Fresh Tissue Examination/Fixation

• the decalcifying agent is then added, usually 20- a. ALCOHOL

30 times of the volume of the tissue • Anhydrous copper sulfate will accelerate
• the tissue will stay in solution 1-14 days dehydration by removing water from
dehydrating fluid. A blue discoloration will
D. ELECTROPHORESIS indicate a complete dehydration.
• most rapid method 1. Ethyl alcohol
- recommend for routine dehydration of
POST DECALCIFICATION tissues
- Involves lithium carbonate to wash the tissue after the - best dehydrating agent
decalcification is complete. 2. Methyl alcohol
- Also decalcified tissue can rinse in running tap water to re - Toxic dehydrating agent
moved acids. - Used for blood and tissue film for smear
preparations
TISSUE SOFTENER’S 3. Butyl alcohol
1. Perenyi’s fluid - may act as both decalcifying and tissue - For plant and animal micro-techniques
softners - Slow dehydrating agent
2. 4% aqueous sol
3. Molliflex - tissue immersed in molliflex appear swollen b. ACETONE
and soapy. • Cheap, rapid acting dehydrating agent
• For urgent biopsies which dehydrates 30 mins
FACTORS INFLUENCE OF DECALCIFICATION to 2 hrs. Limited for only small specimen
• Concentration and Volume • Volatility and inflammable
- More concentrated, more rapidly; more harmful • Most lipids are removed from tissues with this
• Structure and Temperature dehydrating agent.
- Ratio: 20:1
- higher concentration and greater amount of fluid will c. DIOXANE (DIETHYL DIOXIDE)
increase the speed of the process. • Excellent dehydrating agent and clearing agent
• Heat • Readily miscible with water and paraffin −
• Mechanical Agitation Tends tissue ribbon poorly
• Accelerates the rate of diffusion and speeds up • Highly toxic in man and expensive
the decalcification process.
d. CELLOSOLVE (EHTYLENE GLYCOL
MEASURING EXTENT OF DECALCIFICATION MONOETHYLETHER)
• Physical or mechanical test • Cellosolve dehydrates rapidly. The tissue may
- Touching or bending the tissue with the fingers. be transferred from water or normal saline
Inaccurate method. directly to cellosolve and stored in it for months
• X – ray or radiological method without producing hardening or distortion.
- Very expensive;
- most ideal; e. TRIETHYL PHOSPHATE
- most sensitive and most reliable method. • Used to dehydrate sections and smears
• Chemical method (calcium oxalate test) following certain stains and produces minimum
- Simple, reliable and convenient method shrinkage
- Cloudiness will signify incomplete decalcification
f. TETRAHYDROFURAN (THF)
DEHYDRATION • Both dehydrates and clears tissue since it is
- Starts by placing the fixed specimen in 70% to 95% to miscible in both water and paraffin
100% ethyl alcohol.
- For delicate tissues, particularly embryonic tissues starting CLEARING
30% ethyl alcohol De-alcoholization
- The amount of dehydrating agent is should not be less - process whereby alcohol or a dehydrating agent is
than 10 times the volume of the tissue to ensure complete removed from the tissue and replaced with a substance
penetration of the tissue by the dehydrating agent. that will dissolve the wax with which the tissue to be
impregnated.

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 COLLEGE OF MEDICAL TECHNOLOGY
MLS 111 – Histopathology Lec
Module 1 - Introduction to Fresh Tissue Examination/Fixation

- Must be miscible with water, paraffin and mounting 9) METHYL BENZOATE AND METHYL SALICYLATE
medium - Double embedding technique is needed

COMMON CLEARING AGENTS IMPREGNATION

1) XYLENE (XYLOL) Process whereby the clearing agent is completely removed
- Colorless clearing agent, commonly used in histology from the tissue and replaced by a medium that will completely
laboratories. fill the cavities, thereby giving a firm consistency to the
- Miscible with absolute alcohol and paraffin specimen and allowing easier handling and cutting suitably
- It is cheap; used for celloidin sections thin sections without any damage to the tissue and its cellular
- Highly inflammable components.
- Not suitable for nervous tissues and lymph nodes
- Xylene turns milky when an incompletely dehydrated → Paraffin impregnation
tissue is immersed in it. → Celloidin impregnation
→ Gelatin impregnation
2) TOLUENE
- May be used as a substitute for xylene and benzene. a. PARAFFIN WAX IMPREGNATION
- More expensive ➢ Simplest, most common and best embedding
- Miscible with absolute alcohol and paraffin medium, used for routine processing
➢ Very rapid
3) BENZENE ➢ Prolong impregnation will cause excessive shrinkage
- Preferred by some as clearing agent in the and hardening making cutting of sections difficult
embedding process of tissues because it penetrates ➢ Not recommended for fatty tissues
and clears tissue rapidly. ➢ For routine work melting point is: 56-58 C
- Carcinogenic, may damage the bone marrow ➢ For the lab temp between: 15-18 C Melting point is:
resulting aplastic anemia 50-54 C
- Miscible with absolute alcohol ➢ For the lab temp between 20-24 C Melting point is
54-58 C
4) CHLOROFORM
- does not make tissue translucent METHODS OF PARAFFIN WAX IMPREGNATION
- recommended for tough tissues (skin, fibroid, 1) Manual processing
decalcified tissues) - Four changes of wax are required at 15 minutes
- toxic to liver interval.
- tissue tend to float in chloroform to avoid this 2) Automatic processing
wrapped the tissues with absorbent cotton gauze to - Use auto-technicon which fixes, dehydrates,
facilitate sinking of the section in solution clears and infiltrates tissues
- Miscible with absolute alcohol - 2 -3 changes of wax are required
3) Vacuum embedding
5) CEDARWOOD OIL - Involves the wax impregnation under negative
- Recommended for central nervous system tissues atmospheric pressure inside an embedding
and cytological studies (particularly smooth muscles) oven to hasten removal of air bubbles and
- Requires two changes in clearing solution clearing agent
- It makes tissue transparent - Most rapid
- Extremely slow clearing agent - Recommended in urgent biopsies

6) ANILINE OIL SUBSTITUTES FOR PARAFFIN WAX

- Recommended for clearing embryos, insects and 1) PARAPLAST
very delicate specimens - Mixture of highly purified paraffin and synthetic
plastic polymers
7) CLOVE OIL - Melting point: 56-57 C
- Unsuitable for routine clearing process - More elastic and resilient
- For bones and brains
8) CARBON TETRACHLORIDE 2) EMBEDDOL
- Properties are very similar to chloroform - Synthetic wax substitute similar to paraplast
- Melting point is: 56-58 C

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 COLLEGE OF MEDICAL TECHNOLOGY
MLS 111 – Histopathology Lec
Module 1 - Introduction to Fresh Tissue Examination/Fixation

3) TISSUE MAT - Consist of special stainless steel base mold fitted

- contains rubber with a plastic embedding ring, which later serves as
4) ESTER WAX the block holder during cutting
- Has a lowering melting point 46-48 C – 4. Tissue-tek
- Harder than paraffin - Equipped with a warm plate to manage the
5) WATER SOLUBLE WAX (CARBO WAX) impregnated specimen
- Melting point: 38-42 C or 45-46 C 5. Disposable embedding molds
- Peel-away
b. CELLOIDIN IMPREGNATION - Plastic ice trays
➢ Purified form of nitrocellulose soluble in many - Paper boats
solvents REMEMBER:
➢ Suitable for specimens with large hollow cavities ✓ Celloidin or nitrocellulose method – recommended
which tend to collapse for hard and dense tissues for embedding hard tissues.
such as bones teeth and for large tissue sections such ✓ Double-embedding method – process; in which
as whole embryo. tissue first infiltrate by celloidin and embedded in
➢ Recommended for processing neurological tissue paraffin
➢ Very slow

TWO METHODS FOR CELLOIDIN IMPREGNATION

1) Wet celloidin method
- Recommended for bones teeth large brain and
whole organs
2) Dry celloidin method
- Recommended for whole eye sections
- Does not use alcohol due to the presence of
cedarwood oil in the block.

c. GELATIN IMPREGNATION
➢ Rarely used except for histochem and enzyme
studies
➢ Used for delicate specimens and frozen tissue
➢ Water soluble
➢ Tissue should not be more than 2-3mm thick; add 1%
phenol to prevent molds
➢ Excess gelatin may remove by floating the sections to
paper and trimming them with scissors

EMBEDDING
- After impregnation, the tissue placed into a mold
containing the embedding medium and these media allow
solidifying.
- In this process orientation is very important.
- Temperature of melted paraffin is 5-10 deg C above the
melting point
- Immersed in cold or ref temp to solidify

1. Leukhart’s embedding mold

- Contains two L-shapes strips of heavy brass
2. Compound Embedding unit
- Made up of a series of interlocking plates resting on
a flat metal base
3. Plastic embedding rings and base molds

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