Inflammation and Disease Process: Name: Aldrin Ligan BSMT 2B

NAME: ALDRIN LIGAN BSMT 2B
INFLAMMATION AND DISEASE PROCESS

DEFINITION: devoted to the study of the structural, biochemical, and functional changes in cells, tissues, and organs that underlie
disease.
SIGNS VS SYMPTOMS
SYMPTOMS – subjective
 Perceived or experience by the patients
SIGNS – objective
 Measurable observations
 Physical observations
INFLAMMATION: The sum total of changes in the living tissues, in response to an injurious agent.
INFLAME – to set fire
CARDINAL SIGNS OF INFLAMMATION:
1. RUBOR (redness)
2. CALOR (heat)
3. TUMOR (swealing)
4. DOLOR (pain)
5. FUNCTIO LAESA (loss of function)
AULUS CORNELIUS CELSUS - described the first 4 cardinal sign.
RUDOLF VIRCHOW – added function laesa
CLASSIFICATION OF INFLAMMATION
ACCORDING TO DURATION:
a. Acute inflammation – early onset
 Exudative inflammation
 Usually, but not necessarily of, sudden onset
 Characterized with five cardinal signs
 Predominant cells: PMN
b. Subchronic Inflammation
c. Chronic inflammation – later onset
 Persistence of injurious agent for weeks/years
 Characterized with proliferative rather than educate response
 Predominant cells: mononuclear cells (macrophage, Lymphocytes, Plasma cells)
SYSTEM EFFECTS OF INFLAMMATION
• Fever: elevation of body temperature (by 1 to 4C), is one of the most prominent manifestations of the acute-phase
response.
• Acute-phase proteins: whose plasma concentrations may increase several hundred-fold as part of the response to
inflammatory stimuli. (CRP, fibrinogen, and serum amyloid A (SAA) protein)
• Leukocytoses: cytokines (colony stimulating factors) production of leukocytes from precursor in the bone marrow stimulate
• Other manifestations: pulse. Blood pressure; seating, rigors (shivering) chills (search for warmth), anorexia, somnolence, and
malaise
• In sever bacterial infections (sepsis): disseminated intravascular coagulation, hypotensive shock, and metabolic disturbances
including insulin resistance and hyperglycemia.
CLASSIFICATION OF INFLAMMATION
ACCORDING TO CHARACTER OF EXUDATE
TYPES OF EXUDATES:
1. Serous inflammation: when the fluid exudate resembles your serum

 Watery/serum like
 Ex. BLISTERS
2. Fibrinous: increase amount of fibrinogen
 Fibrin content of fluid exudate
3. Cattarhal: when your surface of epithelium is incased of inflammation, produces increase amount of mucous
 Cloudy mucous
 Mucus-like
4. Hemorrhagic: vascular damage
 Presence of RBC
5. Purulent: suppurative exudate
 Formation of pus
 Ex. Bacterial infection
 Yellow or greenish pus
ABNORMALITIES IN CELL GROWTH
1. Retrogressive changes: organs/tissues are smaller than normal
a. Development defects
i. Aplasia: defective development
 Failure of organ to develop after a stage

 Failure of development of embryonic tissue or cell
 Defective development.
ii. Agenesia: failure of organ or part to develop or grow
iii. Hypoplasia: incomplete development, or under development of organ or tissue.
iv. Atresia: congenital absence or closure of normal body opening or tubular structures.
b. Atrophy: shrinkage/shrinkage of cell.
i. Physiologic atrophy
ii. Pathologic Atrophy: result for some form of cellular injury.
2. Progressive Changes: Organs/ tissues are larger than normal.
a. Hypertrophy: increase in size of tissues/organs due to increase in the size of individual cells.
b. Hyperplasia: increase in size of tissues/organs due to increase in the number of cells resulting from growth of new cells.
 HYPERTROPHY: increase in size of cell/tissue

 HYPERPLASIA: increase number of cell/tissue.
3. Degenerative Changes due to Aberrations of Cellular Growth Patterns.
a. Metaplasia: “Transformation” reversible; transformation in one type of adult cell to another
b. Dysplasia “Disorder growth”: reversible; regressive alteration in adult cells manifested by variation in size, shape
& orientation, associated with chronic inflammation and protracted irritation. Used as a criterion for malignancy.
c. Anaplasia “Dedifferenation”: “Reverse differentiation” “irreversible” – marked regressive that occur to form
backward in an adult cell towards embryonize / primitive cell type.
d. Neoplasia: continuous abnormal proliferation of cells without control pathologic overgrowth of the tissue.
TUMOR BASIC COMPONENTS:
 Parenchyma: refers to the active elements of the tumor

 Stroma: refers to the connective tissue framework with lymphatic and vascular channels

DIFFERENTIATION OF TUMOR
CAPACITY TO PRODUCE DEATH
 BENIGN TUMORS: usually are non-spreading tumor.

 MALIGNANT TUMORS: “METASTASIS”, spreading from one organ to another.
DEPENDING ON HISTOLOGICAL CHARACTERISTICS:
• Medullary: where there are more cells than supporting tissue; it is soft and very malignant.
• Scirrhous (stony/hard): where there is more connective tissue than cells, where the cells are apparently trapped within
the fibrous tissue.
MESENCHYMAL/ EPITHELIAL
CONNECTIVE TISSUE
TISSUE (suffix)
(suffix)
BENIGN “OMA” “OMA”
MALIGNANT “SARCOMA” “CARCINOMA”
DEGENARATION VS. INFILTRATION
• In DEGENERATION, injury to the cell processed and results in an accumulation of metabolites, whereas, in
INFILTRATION, overloading of a healthy cell by metabolites causes the injury.
• Fatty degeneration refers to the abnormal accumulation of fat within previously injured parenchymal cells, commonly
encountered in heart, kidneys, and liver.
• Liver is the organ most commonly affected by FATTY DEGENERATION.
NECROSIS means cell death, which is due to disease or injury. It is a rapid process that brings about death of group of cells or part of
tissue or organ.
NECROBIOSIS is a slower process which refers to the physiologic death of cells.
SOMATIC DEATH refers to the death of the entire body or organism.
CAUSES OF NECROSIS:
 ISCHEMIA/HYPOXIA: loss of blood supply to an area leads to cell death due to deprivation of oxygen and nutrients.
 PHYSICAL AGENTS: mechanical trauma, extreme temperature, electric shock
 CHEMICAL AGENTS: strong acids and alkalis, therapeutic drugs
 BIOLOGICAL PRODUCTS: endotoxins and exotoxins.
 INFECTIOUS AGENTS: viruses, bacterias, fungi, parasites, or all microbios.
 IMMUNOLOGIC REACTIONS: autoimmune diseases
 NUTRIONAL IMBALANCES: protein-calorie deficiency or lack specific vitamis.
MICROSCOPIC CHANGES IN NECROSIS:
NUCLEAR CHANGES:
 Pyknosis: Reduction in size & condensation of nuclear material.
 Karyorrhexis: Segmentation & fragmentation of nucleus, whereby nuclear contents are broken up and released into the
cytoplasm.
 Karyolysis: Dissolution of the nucleus where all basophilism is lost and the nucleus disappears.
CYTOPLASMIC CHANGES:
 Cells may appear large and granular cloudy swelling later becoming more acidophilic dense and opaque
 Cell boundary is lost, with granular coagulation and fragmentation, with appearance of fat droplets in the necrotic cytoplasm.
TYPES OF NECROSIS ACCORDING TO MORPHOLOGIC CHANGES:

1. COAGULATIVE NECROSIS:
 Most common pattern/hypoxic death
 Predominated by protein denaturation with preservation of cell and tissue framework.
 Pattern is characteristic of hypoxic death and all tissues except brain.
o Necrotic tissue undergoes either:
 Heterolysis: there’s a digestion of lysosomal enzymes or invading leukocytes.
 Autolysis: there’s a digestion by its own lysosomal enzymes.

2. LIQUEFACTIVE NECROSIS:
 Occurs when autolysis or heterolysis predominates over protein denaturation
 Most common frequently seen in localized bacterial infection.
 There’s an abscess, and in the brain
 Including brain
3. FAT NECROSIS:
 Seen in adipose tissue
 There’s a lipase activation
 Chalky areas, because of fat saponification
4. CASEOUS/CHESSY NECROSIS:
 Cheesy
 Common for tuberculosis (tuberculosed lesion)
 Grossly appears soft, friable, and cheesy material
5. GANGRENOUS NECROSIS:
 Does not have specific pattern
 Coagulative necrosis
 Super impose bacterial infection that makes for a more liquefactive pattern called “WET GANGREN”.
 Very common is the hyperglycemic patient.
6. FIBRINOID NECROSIS:
 Pathologic pattern which results from antigen-antibody deposition in the blood vessels.
SOMATIC CHANGES
PRIMARY CHANGES
 Circulatory failure
 Respiratory failure
 Nervous failure/CNS failure
SECONDARY CHANGES
 Algor Mortis
o Body temperature changed
 Rigor mortis
o Stiffening of body muscles
 Livor mortis
o Purple red coloration
o Result of the blood settle under the force of gravity
 Post-mortem clot
o Moist of the body
 Desiccation
o Body starting to dry
 Putrefaction
o Decaying of the dead body
 Autolysis
o Digestion of own lysosomal enzyme
TISSUE PROCESSING AND FIXATION

TYPES OF BIOPSY SPECIMEN:
• Fine needle aspiration is the simplest, least invasive test and uses the smallest needle to simply remove cells from the area
of abnormality. This is not always adequate to obtain a diagnosis, depending on the area to be biopsied.
• A core needle biopsy removes not only cells, but also a small amount of the surrounding tissue. This provides additional
information to assist in the examination of the lesion.
• An incisional biopsy takes out even more surrounding tissue. It takes out some of the abnormality, but not all. The doctor
will slice into the lesion and remove only a portion of it. If the lesion is found to be cancerous, further surgery may be
needed to remove or excise the entire lesion.
• An excisional biopsy generally removes the entire area in question.
• Punch biopsy is considered the primary technique for obtaining diagnostic full-thickness skin specimens. It requires basic
general surgical and suture-tying skills and is easy to learn. The technique involves the use of a 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 - where small fragments of tissue are “shaved” from a surface (usually skin).
• Curettings - where tissue is scooped or spooned to remove tissue or growths from body cavity such as endometrium or
cervical canal.

METHODS OF FRESH TISSUE EXAMINATION:
1. Teasing or Dissociation – is a process whereby a selected tissue specimen is immersed in isotonic salt solution such as
normal saline or Ringer’s solution in a petri dish or watch glass, carefully dissected with a needle and separated by direct
or zigzag spread using an applicator stick. Selected pieces of the tissue are transferred carefully to a microscope slide and
mounted as a wet preparation underneath a cover glass, care being taken to avoid forming bubbles. It is either stained with
a supravital dye or examined unstained by Phase Contrast or Bright Field microscopy. It has the advantage of permitting
the cells to be examined in the living state. The use of the phase contrast microscope greatly increases the structural detail
of the cells examined in the living state, allowing movement and mitotic division to be observed. The application of
certain stains such as methylene blue can be also of great value. The preparations, however, are not permanent.
2. Squash Preparation (Crushing) is a process whereby small pieces of tissue (not more than one mm. in diameter) are placed
in a microscopic slide and forcibly compressed with another slide or with a cover glass. If necessary, a supravital stain
may be placed at the junction of the slide and the cover glass, and allowed to be absorbed by the tissue through capillary
attraction.
3. Smear Preparation – The method of preparing the smear differs depending on the nature of the material to be examined. As
a general rule, smears are made either by spreading the selected portion of the specimen over the surface of the slide with
a platinum loop. Alternatively, an apposition smear can be made using a second slide to obtain a relatively
uniform distribution of secretion. Too thin or too thick smears have to be avoided, since they make the tissues less suitable
for examination. Smears may be examined either as fresh preparations similar to that described for teased preparations, or
by using a supravital staining technique. Smear preparations can be made permanent by fixing them while still wet,
staining them to demonstrate specific structures and inclusions, and mounting the cleared specimen beneath a cover glass
with a suitable mounting medium. This is useful for preparing smears of thick secretions such as serous fluids,
concentrated sputum, enzymatic lavage samples from the gastrointestinal tract, and blood smears. This technique is
especially useful in cytological examinations, particularly for cancer diagnosis.
• Streaking -With an applicator stick or a 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. Too thin or too thick smears have
to be avoided, since they make the tissues unsuitable for examination.
• 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. It is a little more tedious than streaking, but has the
advantage of maintaining cellular interrelationships of the material to be examined. It is especially recommended for
smear preparations of fresh sputum and bronchial aspirates, and also for thick mucoid secretions.
• Pull-Apart – This is done by placing a drop of secretion or sediment upon one slide and facing it to another clean slide. The
material disperses evenly over the surface of the two slides. Slight movement of the two slides in opposite directions may
be necessary to initiate the flow of materials. The two slides are then pulled apart with a single uninterrupted motion, and
the specimen is placed under the microscope for immediate examination, or applied with vital stains.
• Touch Preparation (Impression Smear) – This is 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 by Phase Contrast microscopy or staining for light microscopic study. It
has an added advantage in that the cells may be examined without destroying their intercellular relationship.
FIXATION: The first and most critical step in histotechnology is FIXATION, a process that preserves tissues from
decay, thereby preventing autolysis or putrefaction.
• Fixation should be carried out as soon as possible after removal of the tissues (in the case of surgical pathology) or soon
after death (in the case of autopsy) to prevent autolysis
• The primary goal of fixation is to preserve the morphologic and chemical integrity of the cell in as life-like a manner as
possible
• The second goal of fixation is to harden and protect the tissue from the trauma of further handling, so that it is easier to cut
and process for microscopy.
PRACTICAL CONSIDERATION TO OPTIMIZE FIXATION OF TISSUE
• Specimens should be transferred to fixative quickly (<1 hour) after surgery as deterioration will commence with the loss of
blood supply.
• Tissues should be fixed in a sufficient volume of solution; generally in a ratio of 20:1 or at least 10:1 fixative to specimen,
for penetration to occur in the most efficient manner.
• Fixatives diluted and/or contaminated by bodily fluids (e.g. bile, blood, feces) will be reduced in concentration and must be
replaced to ensure effectiveness.
• Sufficient time must be allowed for penetration of fixative; rates of penetration vary according to fixative type.
• The size of tissue blocks should be small enough to allow adequate permeation of fixative (and subsequent processing
solutions) through the perforations in cassettes.
• Prolonged fixation may be more difficult to reverse and may also result in loss of immunohistochemical antigenicity.
• The tissue hardens upon fixation and remains in whatever shape it was fixed in. This should be considered in those cases
where the final orientation of the sections is important.
MAIN FACTORS INVOLVED IN FIXATION:
• Volume: The volume of fixative is important. Traditionally, the amount of fixative used has been 10-20 times the volume of
tissue to be fixed. The most common error in histotechnology is insufficient ratio of tissue volume to fixative volume.
• Hydrogen Ion Concentration: Fixation is best carried out close to neutral pH, in the range of 6-8. Commercial formalin is
buffered with phosphate at a pH of 7.
• Temperature: Many laboratories use tissue processors that work at 40°C for regular tissue processing. For electron
microscopy and some histochemistry, the ideal temperature is 0-4°C. Refrigeration is used to slow down decomposition if
the tissue needs to be photographed and cannot be fixed immediately.
• Thickness of section: Tissue blocks should be small (e.g. 1 to 2 mm2 for electron microscopy and 2 cm2 wide for light
microscopy) and thin (no more than 0.4 cm for light microscopy).
• Osmolality: If cells are fixed in a hypertonic solution, the cells may shrink. If the cells are fixed in a hypotonic solution, the
cells may swell and burst. For that reason, we recommend using a normal phosphate buffered saline (PBS) based fixative.
Hypertonic solutions give rise to cell shrinkage. Isotonic as well as hypotonic fixatives cause cell swelling and poor
fixation. The best results are usually obtained using slightly hypertonic solutions (400-450 mOsm; isotonic solutions are
340 mOsm). Sucrose is commonly added to osmium tetroxide fixatives for electron microscopy.
• Concentration of fixative should be adjusted down to the lowest level possible. Formaldehyde is normally used as a 10%
solution, and glutaraldehyde is normally used as a 3% solution. Low concentrations of glutaraldehyde (0.25%) have been
found to be an ideal concentration for immuno-electron microscopy
• Duration of fixation: Some tissues take longer to fix than others, depending on their structure. Fibrous organs such as uterus
or intestinal tract take longer to fix than small or loosely textured tissues such as biopsies or scrapings
EFFECTS OF FIXATIVES IN GENERAL:
• They reduce the risk of infection during handling and actual processing of tissues.
• They harden soft and friable tissues and make the handling and cutting of sections easier. This is usually accelerated by the
action of alcohol during the dehydration process.
• They make the cells resistant to damage and distortion caused by the hypotonic and hypertonic solutions used during tissue
processing.
• They inhibit bacterial decomposition.
• They increase the optical differentiation of cells and tissue components thereby rendering them more readily visible during
examination.
• They may act as mordants or accentuators to promote and hasten staining, or they may inhibit certain dyes in favor of
another (e.g.formaldehyde intensifies while osmium tetroxide inhibits hematoxylin staining).
CHARACTERISTICS OF A GOOD FIXATIVE:
• It must be cheap.
• It must be stable.
• It must be safe to handle.
• It must kill the cell quickly thereby producing minimum distortion of cell constituents.
• It must inhibit bacterial decomposition and autolysis.
• It must produce minimum shrinkage of tissue.
• It must permit rapid and even penetration of tissues.
• It must harden tissues thereby making the cutting of sections easier.
• It must be isotonic, causing minimal physical and chemical alteration of the cells and their constituents. This is not,
however, a strict rule since there are some hypotonic solutions (i.e. glacial acetic acid) producing tissue swelling, which
are being used in conjunction with hypertonic solutions (e.g. picric acid) causing shrinkage of cells, to produce a
compound which would give an optimal effect on the tissue structure.
• It must make cellular components insoluble to hypotonic solutions and render them insensitive to subsequent processing.
• It must permit the subsequent application of many staining procedures to facilitate easier and more profitable examination.
TYPES OF FIXATIVE:
Microanatomical Fixatives -are those that permit the general microscopic study of tissue structures without altering the
structural pattern and normal intercellular relationship of the tissues in question.
• 10% formal saline
• 10% neutral buffered formalin
• Heidenhain 's Susa
• Formal sublimate (formal corrosive)
• Zenker 's solution
• Zenker-formal (Kelly 's solution)
• Bouin's solution
• Brasil's solution
Cytological Fixatives - are those that preserve specific parts and particular microscopic elements of the cell itself.
➢ Nuclear Fixatives - are those that preserve the nuclear structures (e.g., chromosomes) in particular. They usually contain
glacial acetic acid as their primary component due to its affinity for nuclear chromatin. They have a pH of 4.6 or less.
• Flemming's fluid
• Carnoy's fluid
• Bouin's fluid
• Newcomer's fluid
• Heidenhain's Susa
Many fixatives have been used over the years, specifically for work with nucleic acids, but relatively few (including
mercury and chromium salts) are known to react with them chemically. Mercuric chloride has been found to react
with viruses and causes the loss of their infective power.
➢ Cytoplasmic Fixatives - are those that preserve cytoplasmic structures in particular. They must never contain glacial acetic
acid which destroys mitochondria and Golgi bodies of the cytoplasm. They have a pH of more than 4.6.
• Flemming's fluid without acetic acid
• Kelly's fluid
• Formalin with "post-chroming"
• Regaud 's fluid (Muller 's fluid)
• Orth 's fluid
For RNA, the precipitant fixatives - ethanol and acetone - give the best quantitative results using frozen tissues as the
standard.
Histochemical Fixatives - are those that preserve the chemical constituents of cells and tissues.
• Formal Saline 10%
• Absolute Ethyl Alcohol
• Acetone
• Newcomer's Fluid
SECONDARY FIXATION: is the process of placing an already fixed tissue in a second fixative in order:
• To facilitate and improve the demonstration of particular substances.
• To make special staining techniques possible (with secondary fixative acting as a mordant).
• To ensure further and complete hardening and preservation of tissues.
➢ Post-Chromatization is a form of secondary fixation whereby a primarily fixed tissue is placed in aqueous solution of 2.5-
3% potassium dichromate for 24 hours to act as mordant for better staining effects and to aid in cytologic preservation of
tissues.
WASHING OUT: is the process of removing excess fixative from the tissue after fixation in order to improve staining
and remove artefacts from the tissues. Several solutions may be used.
1. Tap water is used to remove:
a. excess chromates from tissues fixed in Kelly's, Zenker's, and Flemming's solutions
b. excess formalin
c. excess osmic acid
2. 50-70% alcohol is used to wash out excess amount of picric acid (Bouin's solution).
3. Alcoholic iodine is used to remove excessive mercuric fixatives.
METALLIC FIXATIVES:
Mercurials fix tissues through an unknown mechanism that increases staining brightness and gives excellent nuclear
detail.
MERCURIC CHLORIDE
DEZENKERIZATION
Chemically, de-zenkerization is done by oxidation with iodine to form mercuric iodide, which can be subsequently
removed by treatment with sodium thiosulfate, using the following procedure:
• Bring slides to water.
• Immerse in Lugol's iodine (5 minutes).
• Wash in running water (5 minutes).
• Immerse in sodium thiosulfate 5% (5 minutes).
• Wash in running water (5 minutes).
• Proceed with required water soluble stain
Heidenhain's Susa Solution – is recommended mainly for tumor biopsies especially of the skin; it is an excellent
cytologic fixative.
FORMULA:
• Mercuric chloride 45 gm
• Sodium chloride 5 gm
• Trichloroacetic acid 20 gm
• Glacial acetic 40 ml
• Acid Formaldehyde 40% 200 ml
• 40% Distilled water 800 ml
CHROMATE FIXATIVES
Chromic Acid - is used in 1-2% aqueous solution, usually as a constituent of a compound fixative. It precipitates all
proteins and adequately preserves carbohydrates. It is a strong oxidizing agent; hence, a strong reducing agent
(e.g. formaldehyde) must be added to chrome-containing fixatives before use in order to prevent counteracting effects and
consequent decomposition of solution upon prolonged standing.
Potassium Dichromate -is used in a 3% aqueous solution.
• It fixes but does not precipitate cytoplasmic structures.
• It preserves lipids.
• It preserves mitochondria (If used in pH 4.5-5.2, mitochondria isfixed.
• If the solution becomes acidified, cytoplasm, chromatin bodies and chromosomes are fixed but mitochondria are destroyed)
Regaud’s (Muller's) Fluid
FORMULA:
Potassium dichromate 3% 80 ml
Strong formaldehyde 40% 20 ml (To be added just before use).
Orth's Fluid
FORMULA:
Potassium dichromate 2.5% 100ml
Sodium sulfate (optional) 1gm
Strong formaldehyde 40% 10 ml (To be added just before use).
LEAD FIXATIVES
Lead fixatives are used in 4% aqueous solution of basic lead acetate. Lead oxaloacetate, a primary reaction product
precipitate for the visualization of the activity of glutamic oxaloacetic transaminase in tissue sections, is stable at a slightly
alkaline Ph.
Advantages:
1. It is recommended for acid mucopolysaccharides.
2. It fixes connective tissue mucin.
PICRIC ACID FIXATIVES
Picrates include fixatives with picric acid. Foremost among these is Bouin's solution that does almost as well
as mercurials with nuclear detail but does not cause as much hardness.
Bouin's Solution
The complementary effects of the three ingredients of Bouin’ssolution work well together to maintain morphology.
Specimens are usually fixed in Bouin’s solution for 24 hours. Prolonged storage in this acidic mixture causes hydrolysis
and loss of stainable DNA and RNA. Thorough washing after fixation is necessary.
FORMULA:
• Picric acid saturated aqueous soln. (2.1%) 75 ml
• 40% formaldehyde 25 ml
• Acetic acid glacial 5 ml
Brasil's Alcoholic Picroformol Fixative
FORMULA:
• Formaldehyde 37% 2040 ml.
• Picric acid 80 gm.
• Ethanol or isopropyl alcohol 6000 ml.
• Trichloroacetic acid 65 gm. Overnight tissue fixation by automatic processing technique may utilize 3-4 changes
of Brasil's fixative at 1/2 to 2 hours each, succeeded directly by absolute alcohol.
ADVANTAGES:
• 1. It is better and less "messy" than Bouin's solution.
• 2. It is an excellent fixative for glycogen.
GLACIAL ACETIC ACID
Acetic acid is a colorless liquid that when undiluted is also called “Glacial” Acetic Acid because it is a water-free
(anhydrous) acetic acid that freezes and solidifies at about 16°C. Acetic acid does not have much effect on proteins, other
than to enable swelling by the absorption of water. Its major effect is to precipitate DNA, which is split off from
nucleoprotein. For this reason, acetic acid is valuable for the preservation of nuclei, and is often added to fixatives
specifically to do that. Acetic acid is not used alone for fixation but is incorporated into other fixatives to form a
compound solution, most commonly at a concentration of approximately 5%.
PRECIPITATING ALCOHOL FIXATIVES
Alcohols are protein denaturants and are not used routinely for tissues because they cause too much brittleness and
hardness. The protein denaturants - methanol, ethanol and acetone - are rarely used alone for fixing blocks unless studying
nucleic acids.
Alcohol rapidly denatures and precipitates proteins by destroying hydrogen and other bonds. It must be used in
concentrations ranging from 70 to 100% because less concentrated solutions will produce lysis of cells. Ethanol (95%) is
fast and cheap. Since smears are only a cell or so thick, there is no great problem from shrinkage, and since smears are not
sectioned, there is no problem from induced brittleness. They are not good for electron microscopy, though, because they
cause tissue shrinkage.
Absolute alcohol can be used to fix and preserve glycogen, pigments, blood, tissue films and smears. The color of
the specimen can be preserved for photographic work using 80% alcohol. Sometimes the specimen is taken out from
fixative and placed in 80% alcohol to bring out some of the original color. Glycerin is also used in combination with
alcohol for this purpose. All these alcoholic fixatives contain acetic acid, which produces characteristic patterns of
coagulated nuclear chromatin, facilitating the recognition of cell types.
➢ Methyl Alcohol 100%
Advantages:
1. It is excellent for fixing dry and wet smears, blood smears and bone marrow tissues.
2. It fixes and dehydrates at the same time.
Disadvantages:
1. Penetration is slow.
2. If left in fixative for more than 48 hours, t issues may be over hardened and difficult to cut.
➢ Isopropyl Alcohol 95% - is used for fixing touch preparations ,although some touch preparations are air dried and not
fixed, for certain special staining procedures such as Wright-Giemsa.
➢ Ethyl Alcohol - is used at concentrations of 70-100%. If the lower concentrations are used, the RBC's become
hemolyzed and WBCsare inadequately preserved. It may be used as a simple fixative. It is, however, more frequently
incorporated into compound fixatives for better results.
➢ Carnoy’s Fixative
FORMULA:
• Absolute alcohol 60 ml.
• Chloroform 30 ml.
• Glacial acetic acid 10 ml.
➢ Clarke’s solution
FORMULA:
• Ethanol (absolute) 75 ml
• Acetic acid glacial 25 ml
Recommended Applications: Clarke’s solution has been used on frozen sections and smears. It can produce fair
results after conventional processing if fixation time is kept very short. It preserves nucleic acids but extracts lipids.
Tissues can be transferred directly into 95% ethanol.
➢ Newcomer's Fluid
FORMULA:
• Isopropyl alcohol 60 ml.
• Propionic acid 30 ml.
• Petroleum 30 ml.
• Ether 10 ml.
• Acetone 10 ml.
• Dioxane 10 ml.
OSMIUM TETROXIDE (OSMIC ACID) FIXATIVES
This is a pale yellow powder which dissolves in water (up to 6% at 20°C) to form a strong oxidizing solution.
Fixation with osmium tetroxide or postosmication of glutaraldehyde-fixed tissue causes the complete denaturation of
protein. Potassium permanganate and potassium dichromate are also oxidizing agents but are less reactive towards
proteins than is osmium tetroxide.
Osmium tetroxide is traditionally used in electron microscopy both as a fixative and a heavy metal stain. Osmium
tetroxide is a good fixative and excellent stain for lipids in membranous structures and vesicles
Flemming's Solution is the most common chrome-osmium acetic acid fixative used, recommended for nuclear
preparation of such sections.
FORMULA:
• Aqueous chromic acid 15 ml.
• 1% Aqueous osmium tetroxide 4 ml.
• 2% Glacial acetic acid 1 ml.
Flemming's solution without acetic acid - is made up only of chromic and osmic acid, recommended for
cytoplasmic structures particularly the mitochondria. The removal of acetic acid from the formula serves to improve the
cytoplasmic detail of the cell.
ACETONE
Acetone is not recommended as morphological fixative for tissue blocks, mainly because of its shrinkage and
poor preservation effects. Its use is reserved for the fixation of cryostat sections or for tissues in which enzymes have to be
preserved. Acetone is almost always used alone and without dilution; it fixes by dehydration and precipitation.
It is used to fix specimens at cold temperatures (0 to 4°C).
ADVANTAGES:
• It is recommended for the study of water diffusible enzymesespecially phosphatases and lipases.
• It is used in fixing brain tissues for diagnosis of rabies.
• It is used as a solvent for certain metallic salts to be used in freeze substitution techniques for tissue blocks.
DECALCIFICATION
• Removal of calcium ions from a bone or calcified tissue
• Histological process that makes them flexible and easier to cut
• BONES are the main object of decalcification
• This uses microtome to cut soft sections of bone
• Fine detail radiographs: often used to assist in the section of appropriate bone specimens for processing.
• If calcified areas in tissue are substantial, it is impossible to obtain decent sections without first decalcifying the entire
specimen.
• Alternative: apply “surface decalcification” to a paraffin block, allowing sections to be obtained where the presence
of calcium was not anticipated.
• Decalcification is a lengthy procedure.
PRINCIPLE: principle of decalcification is fairly simple. Strong mineral acids, such as 10% hydrogen chloride (HCl), or
weak organic acids, such as 5-10% formic acid (HCOOH), form soluble calcium salts in an ion exchange that moves
calcium into the decalcifying solution. The same final effect makes 14% ethylene diamino tetracetic acid (EDTA) an ideal
chelating agent that sequesters metallic ions, including calcium, in aqueous solutions.
• It is also possible to prepare bone specimens by infiltrating them with acrylic or epoxy resins which, when
polymerized, have a hardness equivalent to that of mineralized bone and hence do not require decalcification at all.
• Decalcification should be done after fixation and before impregnation to ensure and facilitate the normal cutting of
sections and to prevent obscuring the microanatomic detail of such sections by bone dust and other cellular debris.
There are three main types of decalcifying agents:
• Those based on strong mineral acids
• Those based on weaker organic acids
• Those composed of chelating agents.
The acids make up a solution of calcium ions while the chelating agents take up the calcium ions. Dilute mineral acids
(hydrochloric or nitric) or formic acid can be used effectively if the end point of decalcification is monitored carefully.
Nuclear and cytoplasmic detail are compromised if specimens are exposed for too long to acidic decalcifying agents,
which can extract RNA and remove the purine and pyrimidine bases from DNA. It is also imperative to wash the acid out
of the tissue.

ACID DECALCIFYING AGENTS
• most widely used agents for routine decalcification of large amounts of bony tissues because they are stable, readily
available, and relatively inexpensive as compared to other decalcifying agents.

STRONG MINERAL ACIDS
• Strong acids such as hydrochloric or nitric acid at concentrations up to 10% are the most rapid in action but if used
longer than necessary will rapidly cause a loss of nuclear staining and can macerate tissues.
• It is important that an appropriate end-point test is used to minimize exposure of the specimens to these agents.
• Staining by acid dyes will be less affected, although eosin can produce a deep, brick red stain without differential
shading. These effects of decalcifying agents on H&E staining can be reduced by post-decalcification and removal,
and by appropriately adjusting the staining procedure.
NITRIC ACID
• Nitric acid is the most common and the fastest decalcifying agent used so far, utilized both as a simple solution or
combined with other reagents.
• This may be used as simple aqueous solutions with recommended concentrations of 5- 10%.
• It is a very rapid decalcifying agent, producing minimal distortion and is, therefore, recommended for routine
purposes.
• It has, however, the disadvantage of inhibiting nuclear stains and destroying tissues, especially in concentrated
solutions.
• To prevent progressive tissue damage and impaired staining, combine nitric acid with formaldehyde or alcohol.
Aqueous Nitric Acid Solution 10%
FORMULA:
Concentrated nitric Acid 10 ml.
Distilled water added up to 100 ml.
DECALCIFICATION TIME: 12-24 hours

Formol-Nitric Acid
FORMULA:
Concentrated nitric acid 10 ml.
Strong formaldehyde, 40% 5 ml.
Distilled water 85 ml.
DECALCIFICATION TIME: 1-3 days

Perenyi’s Fluid
FORMULA:
Nitric acid 10% 40 ml.
Chromic acid 0.5% 30 ml.
Absolute ethyl alcohol 30 ml.
Mix shortly before use.
Chromic acid must be collected for proper disposal. DECALCIFICATION TIME: 2 - 7 days

Phloroglucin-Nitric Acid
FORMULA:
Concentrated nitric acid 10 ml.
Phloroglucin 1 gm.
Nitric acid 10% 100 ml.
(To be added after disappearance of dense white fumes formed by combining the first two ingredients.)
DECALCIFICATION TIME: 12-24 hours

HYDROCHLORIC ACID
• Hydrochloric acid (HCI) is inferior compared to nitric acid in its role as a decalcifying agent because of its slower
action and greater distortion of tissue produced on the decalcified section.
• However, it produces good nuclear staining and if used in 1% solution with 70% alcohol, may be recommended for
surface decalcification of the tissue blocks.
• Rapid proprietary solutions usually contain hydrochloric acid, whereas slow proprietary mixtures contain buffered
formic acid or formalin/formic acid
• Dilution of a proprietary HCl is not deleterious for effective decalcification or staining, and this is an option if a
strong mixture is considered too concentrated.

Von Ebner's Fluid
FORMULA:
Saturated aqueous solution of NaCl 50 ml.
36% concentrated hydrochloric acid 8 ml.

WEAK ACIDS
• such as formic acid are popular and are widely used for decalcification.
• Organic acids such as acetic and formic acid are better suited to bone marrow, since they are not as harsh.
• However, they act more slowly on dense cortical bone.
• Other acids such as trichloracetic acid (TCA) have also been used.

FORMIC ACID
• is a moderate-acting decalcifying agent which produces better nuclear staining with less tissue distortion and is safer
to handle than nitric acid or hydrochloric acid.
• It is recommended for routine decalcification of postmortem research tissues, although not suitable for urgent
examinations.
• Formic acid can be used as a simple 10% aqueous solution or combined with formalin or with a buffer.
• It is slower than the strong acid agents, but it is much gentler in action and less likely to interfere with nuclear
staining.
• Formic acid in a 10% concentration is the best all-around decalcifier.
• Formic acid is the only weak acid used extensively as a primary decalcifying agent.
• Addition of citrate probably accelerates decalcification by chelating the calcium as it is liberated from the bone.
• FORMULA:
Formic acid (Sp. grav. 1.20) 10 ml. Normal saline 10% 90 ml. DECALCIFICATION TIME: 2-7 days

Formic Acid-Sodium Citrate Solution
FORMULA:
Aqueous sodium citrate 20% 50 ml.
Formic acid 45% 50 ml.
Decalcification TIME: 3 -14 days

TRICHLOROACETIC ACID
FORMULA:
Trichloroacetic acid 5 gm.
Formal saline 10% 95 ml.
DECALCIFICATION TIME: 4- 8 days

SULFURIC ACID
• is a very weak decalcifying solution suitable only for minute pieces of bone.

CHROMIC ACID (FLEMMING'S FLUID)
FORMULA:
Chromic acid % 15 ml.
Osmium tetroxide 4 ml.
2% Glacial acetic acid 1 ml.
CAUTION: CHROMIC ACID IS AN ENVIRONMENTAL TOXIN.

CITRIC ACID-CITRATE BUFFER SOLUTION (pH 4.5)
FORMULA:
Citric acid (monohydrate) aqueous solution 7% 5.0 ml.
Ammonium citrate (anhydrous) aqueous solution 7.4% 95.0 ml.
Zinc sulfate aqueous solution. 1% 0.2 ml.
Chloroform (as preservative) - a few drops
DECALCIFICATION TIME: 6 days

DECALCIFYING AGENTS – CHELATING AGENTS
• Chelating agents are substances which combine with calcium ions and other salts (e.g. iron and magnesium deposits)
to form weakly dissociated complexes and facilitate removal of calcium salt.
• The most common chelating agent in the market is ethylene diamine tetra acetic acid (EDTA) salt, with the
commercial name of Versene, recommended only for detailed microscopic studies.
• EDTA combines with calcium, forming an insoluble nonionized complex (which is why it is also used as an
anticoagulant and water softener).
• It works by capturing the calcium ions from the surface of the apatite crystal, slowly reducing its size.
• Decalcification by EDTA takes much longer than decalcification by acids – weeks rather than days.
• The rate at which EDTA will decalcify is pH dependent. It is generally used at pH7.0. It works more rapidly
at pH10 but some tissue elements can be damaged at alkaline pH.
• EDTA does not work in formic acid with pH 3 as a decalcifier.
• Neutral EDTA, though being a slow decalcifying agent, gives excellent results for soft-tissue integrity, and best
quality of both soft-tissue and hard-tissue staining.
• The optimal pH is 7-7.6, so it is necessary to maintain this narrow window.
• EDTA works too slowly under pH 5, owing to insolubility, but over pH 8, tissue maceration starts due to alkaline
sensitive protein bonds.
• The tissue is usually placed in EDTA from 1-3 weeks for small specimens, but it may take 6-8 weeks or longer to
totally decalcify dense cortical bone.
• The solution should be changed every 3 days, and in the final stage, every day, to facilitate decalcification.

Neutral EDTA
EDTA disodium salt 250 gm
Distilled water 1750 ml
Bring to pH 7.0 by adding sodium hydroxide (about 25 gm will be needed).
Neutral EDTA acts slowly but causes little tissue damage. Conventional stains are largely unaffected.

OTHER TECHNIQUES FOR INCREASING THE EFFICIENCY OF DECALCIFICATION
• Sonication with EDTA has been successfully used to accelerate decalcification of trephine specimens for subsequent
molecular analysis.
• Ion-exchange resins have been incorporated into some decalcification protocols. They are added to the
container holding the decalcifier and take up the ionized calcium, thereby maintaining the effectiveness of the acid.

ION EXCHANGE RESIN
• Ion exchange resin (ammonium form of polystrene resin) hastens decalcification by removing calcium ions from
formic acid-containing decalcifying solutions, thereby increasing solubility from the tissue.
• It is not recommended for fluids containing mineral acids such as nitric acid or hydrochloric acid.

ELECTROPHORESIS (ELECTRICAL IONIZATION)
Electrophoresis is a process whereby positively charged calcium ions are attracted to a negative electrode and
subsequently removed from the decalcifying solution. The time required for decalcification is thereby shortened due to the
heat and electrolytic reaction produced in the process. The principle 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 the calcium
deposits.

Solution Used for Electrolytic Decalcification
Formic acid 88% 100 ml.
Concentrated hydrochloric acid 80 ml.
This method is satisfactory for small bone fragments, processing only a limited number of specimens at a time.
Good cytologic and histologic details are, however, not always preserved in tissues that have been electrically decalcified.


FACTORS INFLUENCING THE RATE OF DECALCIFICATION
CONCENTRATION:
• The concentration of active agent will affect the rate at which calcium is removed.
• In general, more concentrated acid solutions decalcify bone more rapidly, but are more harmful to the tissue.
• High concentrations and greater amount of fluid will increase the speed of the process.
• Rapid depletion of an acid or chelator by their reaction with calcium can be avoided by using large volumes of fluid
compared with the volume of tissue, and by changing the solution several times during the decalcification process.
• The recommended ratio of fluid to tissue volume for decalcification is 20 to 1.
FLUID ACCESS:
• As with fixation, a fresh decalcifier should have ready access to all surfaces of the specimen.
• This will enhance diffusion and penetration into the specimen and will facilitate solution, ionization and removal of
calcium.
• Decalcification may be hastened by suspending the tissue in decalcifying solution for greater fluid access.
SIZE AND CONSISTENCY:
• Increase in size and consistency of tissues will require longer periods for complete decalcification.
• Dense bone tissues usually require up to 14 days or longer in order tocomplete the process.
• In such cases, the solution should be changed daily to ensure better penetration and to test for the degree of
decalcification.
AGITATION:
• Gentle agitation may increase the rate of decalcification.
• Mechanical agitation and moving of the tissue in solution usually influences fluid exchange, accelerates the rate
of diffusion and speeds up the decalcification process.
• Gentle fluid agitation is achieved by low-speed rotation, rocking, stirring or bubbling air into the solution.
• Sonication vigorously agitates both specimen and fluid, and may cause disruption of tissue, with formation of cellular
debris on the floor of container.
TEMPERATURE:
• Increased temperature will hasten decalcification, but it will also increase the damaging effects of acids on tissue.
• At 37°C, there will be impaired nuclear staining of Van Gieson's stain for collagen fibers.
• At 55°C, the tissue will undergo complete digestion within 24-48 hours.
• Microwave, sonication and electrolytic methods produce heat, and must be carefully monitored to prevent excessive
temperatures that damage tissue.
• Conversely, lower temperature decreases reaction rates.
• The optimum temperature so far recommended is the room temperature range of 18°C -30°C.

DETERMINING THE END POINT OF DECALCIFICATION
• Prolonged decalcification of tissue is liable to prevent hydrolysis and lead to maceration and destruction of tissue
components which are poorly stained.
• Overdecalcification, particularly with the strong acid decalcifiers, spoils the staining of basophilic elements such as
cell nuclei and in certain circumstances can cause maceration of the softer tissue elements.
• On the other hand, when the tissue is allowed to stay in the decalcifying agent for a very short period of time,
decalcification may be incomplete thereby interfering with the normal cutting of sections and staining of specimens.

THERE ARE SEVERAL METHODS TO CHECK IF THE ENDPOINT OF DECALCIFICATION HAS BEEN
REACHED:
• Physical tests require manipulation, bending, probing or trimming of the specimen to “feel” for remaining calcified
areas. While this method may be successful in experienced hands it is generally considered to be unreliable.
• A simple chemical test can be applied when some acid decalcifiers are used (particularly formic acid). The
decalcifying fluid is usually changed every 24 -48 hours and the chemical test is performed on the discarded fluid.
• The best method, particularly with large specimens such as femoral heads, is to X-ray the specimen. This is a very
expensive although the 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. A good-
quality X-ray will clearly reveal tiny residual calcium deposits and allow further treatment if required. It is an
excellent method for following the process of decalcification of large specimens such as femoral heads. It is,
however, not recommended for mercuric chloride-fixed tissues due to the latter's characteristic radio-opacity which
will interfere with the correct interpretation of the plate.

Treatment following decalcification and prior to processing: Various methods for neutralizing residual
acid decalcifier before processing have been published, including extensive washing in tap water or the application of
alkaline solutions.

SURFACE DECALCIFICATION
• This is a method of dealing with small unexpected deposits of calcium that may be encountered in paraffin blocks.
• When the paraffin-embedded block has been trimmed, the tissue surface may reveal small foci of calcification and
may cause resistance or a "grating" sensation when sectioned with a microtome knife.
• If this is encountered, the block can be removed from the chuck and placed face down on a pad of cotton or gauze
saturated with 10% hydrochloric acid for approximately one hour.
• This surface treatment will allow the decalcifier to penetrate a small distance into the block and dissolve the calcium.
• The block can then be thoroughly rinsed in water to remove residual acid, chilled and sectioned.
• Careful realignment of the block will be required because the decalcifier will penetrate a very small distance into the
block allowing only a couple of sections to be taken.
• The staining properties of the tissue will be affected after this procedure, so that allowances in staining need to be
made to achieve optimum results.

TISSUE SOFTENERS
• Unduly hard tissues which are liable to damage the microtome knives may require tissue softeners, aside from
decalcification. Perenyi's fluid may act both as a decalcifying agent and tissue softener
• To soften unduly hard tissues, selected portions are left in the fluid for 12-24 hours and dehydrated in the usual
manner; or the cut surface of the block may be submerged in the fluid for 1-2 hours before sectioning, to facilitate
easier cutting of tissues.
• Washing out and immersion of fixed tissues in 4% aqueous phenol solution for 1-3 days may also cause considerable
tissue softening and easier sectioning of blocks without producing marked deleterious effects and tissue distortion.
• Other substances which may be used as tissue softeners are Molliflex, 2% hydrochloric acid, or 1% hydrochloric acid
in 70% alcohol. Tissues immersed in Molliflex may appear swollen and soapy. This does not, however, affect the
normalizing and subsequent staining of tissue sections.

DEHYDRATION
Process of removing intercellular and extracellular water from the tissue following fixation and prior to wax impregnation
is known as "dehydration”, and the solutions utilized to make this possible are called "Dehydrating Agents".
• Drying is the removal of water by evaporation from a solid, semi-solid or liquid. Solid tissues should NEVER be
allowed to air dry
• Dehydration involves slow substitution of the water in the tissue with an organic solvent.
Most dehydrating agents are strong organic solvents that bring about some shrinkage and extraction of cell components.
To minimize these effects, dehydrating agents are used in a graded series for short periods of time, and water is gradually
replaced so that violent osmotic changes do not produce distortions.

CHARACTERISTICS OF AN IDEAL DEHYDRATING
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.
• As a general rule, whatever dehydrating agent is used, the amount in each step should not be less than 10 times the
volume of the tissue in order to ensure complete penetration of the tissue by the dehydrating solution.
• It is also important to keep the dehydration times as brief as possible to minimize the risk of extracting cellular
constituents
• Almost any water miscible, anhydrous fluid can be used as a dehydrating agent providing that it does not damage the
tissue proteins and is also miscible with the fluids to be used subsequently.

COMMONLY USED DEHYDRATING AGENT ARE:
1. ALCOHOL
• Ethyl alcohol (ethanol) is the alcohol recommended for routine dehydration of tissues. It is a clear, colorless,
flammable fluid.
• It is considered to be the best dehydrating agent because it is fast-acting, it mixes with water and many organic
solvents, and it penetrates tissues easily.
• It is not poisonous and not very expensive.
Methyl alcohol is a toxic dehydrating agent, primarily employed for blood and tissue films and for smear preparations.
Butyl alcohol, which is utilized in plant and animal micro-techniques, is a slow dehydrating agent, producing less
shrinkage and hardening than ethyl alcohol and is recommended for tissues which do not require rapid processing.
It is not advisable to transfer fixed tissues directly from water or aqueous fixative directly into absolute ethanol. Doing so
causes a rapid removal of water which can distort the appearance of more delicate cells and structures. It is advisable to
remove water gently and allow the tissue to slowly adjust to its removal. The more delicate the tissue, the more gently this
should be done, but there is no hard and fast rule.
• dehydration starts by placing the fixed specimen in 70% ethyl alcohol in water, progressing through 95% ethyl
alcohol to 100% ethyl alcohol.
• For delicate tissues, particularly embryonic tissues, dehydration starting with 30% ethanol is recommended.
• Concentrated alcohols (95% or absolute) tend to harden only the surface of the tissue while the deeper parts are not
completely penetrated. This will result in a relatively unequal impregnation of tissue with consequently poor cutting
of sections. To avoid this, 70% or lower concentrations of alcohol, gradually increased to 95%, are used.
• A very concentrated solution (above 80%) makes tissues hard, brittleand difficult to cut.
• Prolonged storage in lower concentrations of alcohol (below 70%) tends to macerate the tissue.
• The tissue may be stored in 70-80% alcohol, although not for very long periods of time, since this may later interfere
with the staining properties of the specimen.
Although the tissue reaches the final stage of dehydration in 100% ethanol, it’s not possible to proceed straight to wax
embedding-- ethanol and wax don’t mix! This is where ‘clearing’ comes in. The term ‘clearing’ refers to the property of
the solvents used- -when they have a relatively high refractive index and when tissue is immersed in them, the tissue
becomes transparent and clear.
FOR TISSUE PREPARATION: one to two hours in each solution should be adequate.
To ensure complete removal of water during dehydration, use at least two changes of 100% ethanol of at least one
half hour each. Never leave tissues in 95 or 100% ethanol more than a total of 2 hours or the tissues will harden. Tissues
can be stored in 70% ethanol at any time during an interruption in the routine.
✓ A temperature of 37°C will hasten dehydration time and is especially used for tissue sections that require urgent
examinations such as fragmentary biopsies.
✓ To insure complete dehydration, a layer of anhydrous copper sulfate, about 1/4 inch deep is placed in the bottom of
the container and covered with filter paper. This will accelerate dehydration by removing water from the
dehydrating fluid.
✓ A blue discoloration of copper sulfate crystals will indicate full saturation of dehydrating fluids with water. Alcohol
is then discarded and changed with a fresh solution.

ETHANOL (ETHYL ALCOHOL)
BOILING POINT OF 78.3 DEGREE CELSIUS
ADVANTAGES:
✓ Nontoxic
✓ Miscible in all proportions with water
✓ Little shrinkage if graded alcohols are used
✓ Can be used on eyes and embryos, if graded alcohols are used Fast acting
✓ Still considered best dehydrating solution
✓ Reliable
✓ Appears to cause less extraction of cellular components in general than other agents
✓ Inexpensive and easily obtained
DISADVANTAGES:
✗ Expensive
✗ Long periods in absolute ethanol will cause excessive shrinkage and hardening
✗ May be difficult to obtain
✗ May have prohibitive taxes that necessitate troublesome book-keeping
✗ Extracts methylene blue and other thiazine dyes from sections
✗ Extracts more lipids than acetone
✗ May cause more shrinkage of specimen
✗ May react with an unreduced 0s04 remaining in specimen
✗ Only slightly miscible with most resins

BUTANOL (BUTYL ALCOHOL)
ADVANTAGES:
✓ Less shrinkage and hardening than with ethyl
✓ Excellent for slow processing
✓ Miscible with paraffin
DISADVANTAGES:
✗ Odorous
✗ Slow-acting
✗ Long periods of infiltration necessary
✗ Dehydrating power low

TERTIARY BUTANOL (BUTYL ALCOHOL)
ADVANTAGES:
✓ Universal solvent—acts as dehydrating and clearing agent
✓ May be used in staining series as a dehydrating agent
✓ Mixes with water, ethanol, xylene, and paraffin in all
DISADVANTAGES:
✗ Odorous
✗ More expensive than butanol
✗ Primary infiltration must be done in half tertiary butanol and half paraffin, prior to paraffin impregnation
✗ Reagent tends to solidify at room temperature or below 25° C

ISOPROPANOL (ISOPROPYL ALCOHOL)
ADVANTAGES:
✓ Excellent substitute for ethanol
✓ Less shrinkage and hardening than ethanol
✓ No government restrictions on its use
✓ Sufficiently water-free to use in place of absolute ethanol
✓ Lillie considers it “the best all- around substitute for ethyl alcohol”
✓ Less expensive than tax-free alcohol
DISADVANTAGES:
✗ Cannot be used in the celloidin technic since nitrocellulose is insoluble in it • Cannot be used for preparing staining
solutions, since dyes are not soluble in it

PENTANOL (AMYL ALCOHOL)
BOILING POINT OF 128 DEGREE CELSIUS
ADVANTAGES:
✓ Miscible with 90% alcohol, toluene and xylene
✓ Dissolves paraffin wax
DISADVANTAGES:
✗ Toxic
✗ Cannot be used in poorly ventilated rooms
✗ Not miscible with water

2. ACETONE
• cheap, rapid-acting dehydrating agent utilized for most urgent biopsies which it dehydrates in 1/2 to 2 hours.
• is a clear, colorless fluid that mixes with water, ethanol and most organic solvents.
• more miscible with epoxy resins than alcohol, but is highly flammable and requires considerable care in handling.
• It is rapid in action but penetrates tissues poorly and causes brittleness in tissues that are placed in acetone for
prolonged period of time.

ADVANTAGES:
✓ Rapid dehydrating agent
✓ Less expensive than ethanol
✓ Does not extract methylene blue and other dyes from stained sections
✓ May cause less shrinkage of specimen than ethanol
✓ Not reactive with 0s04 remaining in specimen.
✓ Miscible with most embedding resins.
DISADVANTAGES:
✗ Requires a clearing agent
✗ Volume must be 20 times that of the tissue
✗ Best processing requires a graded series of a mixture of acetone and xylene before one can go into paraffin
✗ Needs good ventilation
✗ Evaporates rapidly
✗ Flammable
✗ Absolute acetone is easily contaminated with water, resulting in complete dehydration.
✗ Uranyl acetate and phosphotungstic acid are only soluble in dilute solutions of acetone.

3. DIOXANE (DIETHYL DIOXIDE)
REFRACTIVE INDEX: 1.42
BOILING POINT: 101.5 DEGREE CELSIUS
• is an excellent dehydrating and clearing agent readily miscible in water, melted paraffin, alcohol and xylol.
• produces less tissue shrinkage as compared to alcohol dehydration.
• Tissues can be left in this reagent for long periods of time without affecting the consistency or staining properties of
the specimen. Because dioxane is miscible with both water and paraffin, tissues may be placed directly into the
solution after washing out. However, tissue sections dehydrated with dioxane tend to ribbon poorly.
MAIN DISADVANTAGE: Aside from being expensive, dioxane is also extremely dangerous. Its vapor produces a
cumulative and highly toxic action in man; hence, it should not be used routinely.

ADVANTAGES:
✓ Universal solvent—it dehydrates and clears
✓ Miscible with water, alcohol, xylene, and paraffin
✓ Does not harm tissue over long time periods
✓ Faster dehydrant than ethanol
DISADVANTAGES:
✗ Needs large volume for dehydration
✗ Costs about for times more than does absolute alcohol
✗ Must be used in well-ventilated rooms
✗ Cumulatively toxic
✗ Odorous
✗ Distorts tissue-containing cavities

4. CELLOSOLVE (ETHYLENE GLYCOL MONOETHYL ETHER)
BOILING POINT: 156.4 DEGREE CELSIUS
• Cellosolve dehydrates rapidly
CAUTION: Ethylene glycol ethers are combustible at 110-120°F and are toxic by inhalation, skin contact and ingestion.
Following exposure, the reproductive, fetal, urinary and blood systems are particularly vulnerable to their toxic side
effects.
• If it cannot be avoided, propylene-based glycol ethers should be used instead of ethylene-based glycol ethers.

ADVANTAGES:
✓ Rapid dehydrating agent
✓ Tissue may remain in it for months without injury
✓ Avoids distortion and does not require graded dilutions
DISADVANTAGES:
✗ Expensive
✗ Rapidly absorbs water from the air
✗ Requires clearing agent

5. TRIETHYL PHOSPHATE
BOILING POINT: 215 DEGREES CELSIUS
• it removes water very readily and produces very little distortion and hardening of tissue.
• It is soluble in alcohol, water, ether, benzene, chloroform, acetoneand xylene.
• It is used to dehydrate sections and smears following certain stains and produces minimum shrinkage.

ADVANTAGES:
✓ May be used in routine paraffin technic
✓ Displaces water readily with slight distortion
✓ Does not harden tissue excessively
✓ May be used as a dehydrating solution in the staining sequence
✓ Soluble in alcohols, benzene, toluene, xylene, ether, chloroform

6. TETRAHYDROFURAN (THF)
• is a reagent that both dehydrates and clears tissues since it is miscible in both water and paraffin
• It can dissolve many substances including fats and is in itself misciblewith lower alcohols, ether, chloroform, acetone,
benzene and xylene.
• may be used for demixing, clearing and dehydrating paraffin sections before and after staining.
• causes less shrinkage and easier cutting of sections with fewer artifacts.
• It does not dissolve out aniline dyes.
In fact, most staining procedures give improved results with tetrahydrofuran.
• THF is toxic if ingested or inhaled.
• Vapors cause nausea, dizziness, headache and anesthesia.
• It is an eye and skin irritant, and prolonged exposure (up to 6 months) may cause conjunctival irritation. Because of
this and its rather offensive odor, processing with THF should be done in a well- ventilated room.
Although Teflon gloves may be suitable, the use of THF should be avoided if possible, as there is no practical
way to absolutely protect skin against contact.

ADVANTAGES:
✓ Miscible in all proportions with water, ether, chloroform, acetone, and the hydrocarbons xylene, toluene, and
benzene
✓ Rapid without excessive shrinkage and hardening
✓ Low toxicity; low fire and explosion hazard
✓ Not toxic
✓ Better results than most universal solvents
✓ Solvents of mounting media
DISADVANTAGES:
✗ Odorous- should be used in well-ventilated room
• Evaporates rapidly
✗ Dyes are not soluble in tetrahydrofuran
DEHYDRATING AGENTS FOR ELECTRON MICROSCOPY
• Tissue processing for transmission electron microscopy (TEM) is commonly accomplished using ethanol as a
dehydrating solvent and propylene oxide as a transition fluid.
Both solvents have some undesirable properties:
➢ ethanol solubilizes lipids
➢ propylene oxide is completely miscible with embedding resins and, because of its low viscosity, it can infiltrate
tissues readily and reduce the viscosity of embedding resin mixtures.
However, it is highly flammable, volatile, toxic, and potentially carcinogenic.
It is very reactive even at low temperatures, may combine with reactive groups in cells, and may cause certain
cytochemical and staining reactions.

ACETONITRILE
• is a good substitute for propylene oxide.
• reported to be non-carcinogenic, less toxic and not as flammable as propylene oxide.
• It is freely miscible with water, alcohols, acetone, and epoxy resins.
• does not interfere with epoxy polymerization; and the resulting cured resins have excellent cutting quality and beam
stability
• Acetonitrile is also an excellent dehydrating agent whose use does not necessitate modification of current techniques.
• Most importantly, the low solubility of phospholipids in acetonitrile limits the loss of membrane lipids and, hence,
leads to a better preservation of tissue features.
• It is also used as a dehydrating agent for cells prepared for Scanning Electron Microscopy (SEM).
CLEARING
Clearing (de-alcoholization) is 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 (e.g. paraffin) or used as
the medium on which the tissue is to be mounted (e.g. Canada balsam).
• Aside from removing alcohol, a clearing agent must also be miscible with Canada balsam and other resins that are
used for mounting sections.
• This stage in the process is called “clearing” because many (but not all) clearing agents impart an optical clarity or
transparency to the tissue due to their relatively high refractive index.
• This change in appearance is often used as an indication of the effectiveness or completeness of the clearing process.
• Because of the high refractive indices of most reagents used for de-alcoholization, tissues, particularly embryos and
parasites, become transparent so that the internal structures become visible to the naked eye
Another important role of the clearing agent is to remove a substantial amount of fat from the tissue which otherwise
presents a barrier to wax infiltration.

The most commonly used clearing agent for this purpose is xylene.
• Glycerin and gum syrup are used when the tissue is to be cleared directly from water, as in a frozen section. No de-
alcoholization is involved in this process.

CHARACTERISITCS OF A GOOD CLEARING AGENT:
• It should be miscible with alcohol to promote rapid removal of the dehydrating agent from the tissue.
• It should be miscible with, and easily removed by melted paraffin wax and/or by mounting medium to facilitate
impregnation and mounting of sections.
• It should not produce excessive shrinkage, hardening or damage of tissue.
• It should not dissolve out aniline dyes.
• It should not evaporate quickly in a water bath.
• It make tissues transparent.

➢ Clearing fluids with a low boiling point are generally more readily replaced by melted paraffin, although chloroform
which has a lower boiling point than xylene in fact takes longer than the latter to clear.
➢ Viscosity also affects the speed of penetration of the clearing agent.
➢ Prolonged exposure to most clearing agents causes the tissue to become brittle and therefore more difficult to cut.

A. XYLENE (XYLOL)
• Xylene is a colorless clearing agent that is most commonly usedin histology laboratories.
• Clearing time is usually 1/2 to 1 hour. It is used for clearing, both for embedding and mounting procedures.
• generally suitable for most routine histologic processing schedules of less than 24 hours, and when the tissue
block size is less than 5 mm. in thickness.
• reasonably cost effective and works well for short-term clearing of small tissue blocks
• one of the routinely used chemical in histology and pathology laboratories because of its vital role in the
paraffin wax tissue processing method.
• mostly used as a clearing agent during tissue processing and as a dewaxing agent during staining
• It is also used in cover slipping, in cleaning tissue processors, as solvent to remove synthetic immersion oil
from the microscope objective and in recycling of used slides.

ADVANTAGES:
• It is the most rapid clearing agent, suitable for urgent biopsies which it clears within 15-30 minutes.
• It makes tissues transparent.
• It is miscible with absolute alcohol and paraffin.
• It does not extract out aniline dyes.
• For mounting procedures, it does not dissolve celloidin and can, therefore, be used for celloidin sections.
• It evaporates quickly in paraffin oven and can, therefore, be readily replaced by wax during impregnation and
embedding.
• It is cheap.
DISADVANTAGES:
• It is highly inflammable and should be appropriately stored.
• If used longer than 3 hours, it makes tissues excessively hard and brittle.
• It causes considerable hardening and shrinkage of tissues; hence, is not suitable for nervous tissues and lymph nodes.
• Xylene becomes milky when an incompletely dehydrated tissue is immersed in it.
• Xylene may irritate eyes, nose and respiratory tract. It can be absorbed through the skin and cause dermatitis. At high
concentrations, it is toxic and narcotic.

B. TOLUENE
• better at preserving tissue structure and is more tolerant of small amounts of water left behind in the tissues
than xylene
• However, toluene is more expensive than xylene and more toxic, so toluene is less commonly used.
• Toluene may be used as a substitute for xylene or benzene for clearing both during embedding and mounting
processes.
• Time recommended for clearing is 1 -2 hours.

ADVANTAGES:
• It is miscible with both absolute alcohol and paraffin.
• It acts fairly rapidly and is recommended for routine purposes.
• Tissues do not become excessively hard and brittle even if left in toluene for 24 hours.
• Clears overnight.
• It is not carcinogenic.
DISADVANTAGES:
• It is slower than xylene and benzene.
• It tends to acidify in a partially filled vessel.
• Highly concentrated solutions will emit fumes that are toxic upon prolonged exposure.
• It is more expensive.

C. BENZENE
• Benzene is preferred by some as clearing agent in the embedding process of tissues because it penetrates and
clears tissues rapidly
• It used to be a popular routine clearing agent until recently when its highly carcinogenic properties were
recognized.
• use for clearing purposes is therefore strongly discouraged.

ADVANTAGES:
• It is rapid acting, hence is recommended for urgent biopsies (15-60 minutes) and routine purposes.
• It volatilizes rapidly in paraffin oven and is therefore easily eliminated from the tissue.
• It is miscible with absolute alcohol.
• It does not make tissues hard and brittle.
• It causes minimum shrinkage.
• It clears overnight.
DISADVANTAGES:
• It is highly flammable.
• If a section is left in benzene for a long time, considerable tissue shrinkage may be observed. Hence, tissues should be
transferred to paraffin wax as soon as possible.
• Excessive exposure to benzene may be extremely toxic to man and may become carcinogenic or it may damage the
bone marrow resulting in aplastic anemia. If ever benzene is to be used for clearing, the laboratory should be well-
ventilated.

D. CHLOROFORM
• Chloroform, when used for clearing of tissues during the embedding process, is slower in action than xylene,
but causes less brittleness.
• Thicker tissue blocks, even those up to I cm. in thickness, can be processed.
• However, tissues placed in chloroform do not become translucent.
ADVANTAGES:
• It is recommended for routine work (6-24 hours).
• It is miscible with absolute alcohol.
• It is recommended for tough tissues (e.g. skin, fibroid and decalcified tissues) for nervous tissues, lymph nodes and
embryos because it causes minimum shrinkage and hardening of tissues.
• It is suitable for large tissue specimens.
• It is not inflammable.
DISADVANTAGES:
• It is relatively toxic to the liver after prolonged inhalation; this may be prevented by adequate room ventilation.
• Wax impregnation after chloroform clearing is relatively slow.
• It does not make tissues transparent.
• It is not very volatile in paraffin oven; hence, it is difficult to remove from paraffin sections. It may even produce
considerable deterioration of the wax.
• Its vapor may attack the rubber seal used in vacuum impregnating bath.
• Complete clearing is difficult to evaluate.
• Tissues tend to float in chloroform; this may be avoided by wrapping the tissues with absorbent cotton gauze to
facilitate sinking of the section in solution.
• It evaporates quickly from a water bath

E. CEDARWOOD OIL
• Cedarwood oil is used to clear both paraffin and celloidin sections during the embedding process.
• It is especially recommended for central nervous system tissues and cytological studies, particularly of smooth
muscles and skin.
• It requires two changes in clearing solution. Clearing is usually complete in 2-3 days.

ADVANTAGES:
• It is very penetrating.
• It is miscible with 96% alcohol which it removes readily.
• It clears celloidin in 5-6 days.
• It causes minimal shrinkage of tissues.
• Tissues may be left in oil indefinitely without considerable damage and distortion.
• It does not dissolve out aniline dyes.
• It does not harden tissues.
• It does not interfere too seriously with paraffin penetration if it is not completely removed.
• Clearing with cedarwood oil often improves cutting of the sections.
DISADVANTAGES:
• It is an extremely slow clearing agent, hence, it is not recommended for routine purposes.
• It is slightly slower in penetrating than benzene.
• It is hard to eliminate from the tissues in paraffin bath, making the wax impregnation process very slow. This may be
improved or hastened by transferring the specimen from oil to benzene for 1/2 hour before finally placing the tissue
in wax.
• Quality is not always uniform and good. Tissues cleared in cedarwood oil initially float before gradually staying to
the bottom as clearing proceeds. Hence, the tissue may dry out before it is completely cleared. This can be
prevented by superimposing absolute alcohol on the surface of the clearing agent. Once saturated, the specimen
should then be transferred to a fresh solution of cedarwood oil.
• Cedarwood oil becomes milky upon prolonged storage and should be filtered before use.
• Cedarwood oil that has been previously used to clear acetic-alcohol fixed tissues may produce crystals with a melting
point of approximately 35°C and therefore interfere with adequate clearing of tissue. The solution must be heated to
200°C in order to dissolve the crystals and restore the solution to its normal state.
• It is very expensive.

F. ANILINE OIL
• This is not normally utilized as a routine clearing agent but it is recommended for clearing embryos, insects and
very delicate specimens, due to its ability to clear 70% alcohol without excessive tissue shrinkage and
hardening.

G. CLOVE OIL
• This reagent causes minimum shrinkage of tissues. However, its quality is not guaranteed due to its tendency to
become adulterated. Wax impregnation after clearing with clove oil is slow and difficult. Tissues become
brittle, aniline dyes are removed, and celloidin is dissolved. All of these, in addition to the expensiveness of
the solution, make it unsuitable for routine clearing purposes.

H. CARBON TETRACHLORIDE
• Carbon Tetrachloride may be used in clearing tissues for embedding. Its properties are very similar to that of
chloroform although it is relatively cheaper. Its disadvantage is the same as that of chloroform. It produces
considerable tissue hardening, and is dangerous to inhale on prolonged exposure due to its highly toxic
effects.

I. TETRAHYDROFURAN
• Tetrahydrofuran is superior to ordinary dehydrating and clearing agents due to its ability to perform two
processes at the same time, thereby shortening the total processing time and allowing more time for fixation.
It is non-toxic but has offensive odor and should be used in a well-ventilated room.

J. DIOXANE
• Dioxane is miscible both with water and paraffin. It is used primarily when time is important because the
tissues may be embedded with paraffin within 4 hours after fixation. The tissues are transferred to dioxane
straight from Bouin's fluid or a formalin fixative. The dioxane is changed 3 times within 4 hours and the
tissues are transferred directly to paraffin (3 changes are made in a total of 90 minutes). Dioxane causes
greater shrinkage than xylene does. In addition, it is dangerous. Fumes of dioxane are toxic to human
especially to the liver.

OTHER XYLENE SUBSTITUTES:
TERPENES: are isoprene polymers found in essential oils originally derived from plants, though some are now
synthesized.
• They are the earliest transition solvents to be used in histology and include turpentine and oils of bergamot,
cedarwood, clove, lemon, oreganum and sandalwood.
• Many terpenes clear tissues and celloidin sections from 80%-95% alcohol, render tissues transparent and have a
slow gentle non-hardening action
• Terpenes are moderately effective solvents, but they too are considered toxic. Solvents in this class also dry
slowly, leave an oily residue on slides and are relatively expensive.
LIMONENE: a volatile oil found in citrus peels which goes by several trade names.
• It is a natural oil found in the skins of citrus fruits, such as lemons or oranges, and in cooking is usually referred to as
lemon or orange zest.
• obtained industrially by the steam distillation of orange peel which is a byproduct of the orange juice industry.
• It is a clear, colorless fluid with a distinctly citrus aroma, not unpleasant to most people, although some do not like it.
• often sold as a xylene replacement and some technologists substitute it for xylene in other uses, but this is not
universally successful.
ORANGE OIL BASED CLEARING AGENT: offer the clearing action with the lowest hazard rating of all xylene
alternatives.
• It is excellent for preserving fine tissue structure, and can often be used in place of xylene with no alteration of
protocol.
• In using a product containing orange oils, it is important to use a product which has been rigorously purified then
stabilized.
• Orange oils that are neither pure nor stable can break down to produce compounds which will interfere with staining
procedures.
CHLORINATED HYDROCARBONS: can be effective solvents, but they are considered toxic chemicals, posing
serious health risks.
COCONUT OIL: is an efficient substitute for xylene, as it is non-hazardous, less expensive and causes less shrinkage of
the tissue.
• It can be used as a de-alcoholization agent in the histopathological laboratory, without losing the quality of the
histological details.
• The only drawback associated with coconut oil, is its tendency to get solidified at a lower temperature. However, this
can be overcome by performing the clearing procedure in an incubator, maintaining the required temperature.
BLEACHED PALM OIL: Substitution of the conventional xylene with bleached palm oil as a clearing agent during
tissue processing and as a dewaxing agent during staining gives good tissues, sections and histological slides.
• In addition, bleached palm oil is nontoxic, nonhazardous, nonflammable, bio-degradable, economic, easy to handle,
and readily available.
IMPREGNATION AND EMBEDDING

Impregnation (Infiltration) is the process whereby the clearing agent is completely removed from the tissue and replaced
by a medium that will completely fill all the tissue cavities and give a firm consistency to the specimen.
• This allows easier handling and cutting of suitably thin sections without any damage or distortion to the tissue and its
cellular components.
Embedding (Casting or Blocking) is the process by which the impregnated tissue is placed into a precisely arranged
position in a mold containing a medium which is then allowed to solidify. Ideally, an infiltrating and embedding medium
should be:
• soluble in processing fluids
• suitable for sectioning and ribboning
• molten between 30°C and 60°C translucent or transparent; colorless
• stable homogeneous
• capable of flattening after ribboning
• non-toxic
• odorless
• easy to handle
• inexpensive

FOUR TYPES OF IMPREGNATION AND EMBEDDING MEDIUM:
1. PARAFFIN WAX:
• Paraffin is the simplest, most common and best embedding medium used for routine tissue processing.
• is a polycrystalline mixture of solid hydrocarbons produced during the refining of coal and mineral oils.
• solid at room temperature but melts at temperatures up to about 65°C or 70°C.
• most common for histological use being about 56°C to 58°C
• The traditional advice with paraffin wax is to use this about 2°C above its melting point.

ADVANTAGES:
✓ Thin individual serial sections may be cut with ease from the majority of tissues without distortion.
✓ The process is very rapid, allowing sections to be prepared within 24 hours
✓ Tissue blocks and unstained mounted sections may be stored in paraffin for an indefinite period of time after
impregnation without considerable tissue destruction.
✓ Because formalin-fixed, paraffin-embedded tissues may be stored indefinitely at room temperature, and nucleic
acids (both DNA and RNA) may be recovered from them decades after fixation, they are an important resource for
historical studies in medicine.
✓ Many staining procedures are permitted with good results.
DISADVANTAGES:
✗ Overheated paraffin makes the specimen brittle.
✗ Prolonged impregnation will cause excessive tissue shrinkage and hardening, making the cutting of sections
difficult.
✗ Inadequate impregnation will promote retention of the clearing agent. Tissues become soft and shrunken, and tissue
blocks crumble when sectioned and break up when floated out in a water bath
✗ Tissues that are difficult to infiltrate, e.g. bones, teeth, brains and eyes, need long immersion for proper support;
otherwise, they will crumble on sectioning. Prolonged immersion in paraffin, on the other hand, is not advisable.
✗ Paraffin processing is not recommended for fatty tissues. The dehydrating and clearing agents used in the process
dissolve and remove fat from the tissues.

After being completely cleared, the tissue is submerged in two or more changes of melted paraffin wax, either in
a paraffin oven or in an incubator which has been regulated at 55-60°C. The duration and number of changes
required for thorough impregnation of tissue depends on:
• Size and type of tissues: Longer time is required for thicker tissues.
• Use of vacuum imbedding: Vacuum reduces the time required for complete impregnation.
• Clearing agent employed

Three ways by which paraffin wax impregnation and embedding of tissues may be performed:
BY MANUAL PROCESSING:
• At least four changes of wax are required at 15 minutes intervals in order to insure complete removal of the clearing
agent from the tissue.
• The specimen is then immersed in another fresh solution of melted paraffin for approximately 3 hours
to insure complete embedding or casting of tissue.

AUTOMATIC PROCESSING:
• This method makes use of an automatic tissue processing machine (i.e., Autotechnicon) which fixes,
dehydrates, clears and infiltrates tissues, thereby decreasing the time and labor needed during the processing of
tissues.
• This results in a more rapid diagnosis with less technicality.
PRECAUTIONS WITH AUTOMATIC PROCESSING:
✓ frequency with which fluids are changed depends on the number and sizes of the tissues processed
✓ presence of any odor in the clearing agent during final paraffin wax bath indicates that the paraffin wax needs to be
changed.
✓ Dehydrating fluids should be changed frequently since dehydration is the most critical stage of tissue processing and
inadequate dehydration is difficult to correct once the tissue is in paraffin
✓ first 100% ethanol bath should be discarded, and the others moved down, so that the final bath has fresh 100%
ethanol after two complete processing runs of loads of at least three-quarters capacity
✓ clearing agent and the dilute ethanols should be changed at least once a week.
✓ To avoid spillage, fluid and wax containers must be filled to the appropriate level and correctly located in the
machine.
✓ ax accumulating on any surface or beaker leads must be removed and any spillage should be wiped away.
✓ Wax bath thermostats should be set at least 3 degrees above the melting point of the wax, and timing should be
checked when loading the machine, especially if the machine is equipped with a delay mechanism.

VACUUM EMBEDDING:
• involves wax impregnation under negative atmospheric pressure inside an embedding oven.
• It reduces the time when tissues are subjected to high temperatures thus minimizing heat-induced tissue hardening.
• It facilitates complete removal of transition solvents and prolongs the life of wax by reducing solvent contamination.
• Vacuum hastens the removal of air bubbles and clearing agent from the tissue block, thereby promoting a more rapid
wax penetration of the tissue.
• This technique is particularly recommended for urgent biopsies, for delicate tissues such as lung, brain, connective
tissues, decalcified bones, eyes, spleen and central nervous system
• Vacuum infiltration requires a vacuum infiltrator or embedding oven, consisting of wax baths, fluid trap and vacuum
gauge, to which a vacuum of up to 760 mm Hg is applied using a water or mechanical pump.
• With vacuum embedding, the time required for complete impregnation is reduced by 25% -75% of the normal time
required for tissue processing.
• The tissue is not over-exposed to heat; brittleness, shrinkage and hardening of tissues consequent to overheating is
therefore prevented.

SUBSTITUTE FOR PARAFFIN WAX
PARAPLAST:
• is a mixture of highly purified paraffin and synthetic plastic polymers, with a melting point of 56-57°C.
• more elastic and resilient than paraffin wax thereby permitting large dense tissue blocks such as bones and brain to be
cut easily with the same result as in double embedding.
• It is soluble in common clearing agents and follows the same time schedule for paraffin impregnation, and does not
tend to crack like other paraffin wax substitutes.
• Paraplast with a melting point of 56 to 58 oC is recommended.
• During the winter, 54 to 56 oC Paraplast may be used if the tissue is cut in a cool room.
• During the summer it may be necessary to use 60 to 63 oC, although this is to be avoided if possible in order to not to
"cook" the tissue. "Cooked" tissue does not section well or, if it does, it does not stain well and most details are
destroyed.
EMBEDDOL:
• is synthetic wax substitute similar to Paraplast with a melting point of 56-58°C.
• It is less brittle and less compressible than Paraplast.
• Bio/aid is a semisynthetic wax recommended for embedding eyes.
• Tissue Mat is a product of paraffin, containing rubber, with the same property as Paraplast
ESTER WAX:
• has a lower melting point (46-48°C), but it is harder than paraffin.
• It is not soluble in water, but is soluble in 95% Ethyl Alcohol and other clearing agents; hence, it can be used for
impregnation without prior clearing of the tissue.

WATER SOLUBLE WAXES:
• are plastic polymers, mostly polyethylene glycols with melting points of 38-42°C or 45-56°C.
• The most commonly used is Carbowax, a polyethylene glycol containing 18 or more carbon atoms, which appears
solid at room temperature.
o It is soluble in and miscible with water; hence does not require dehydration and clearing of the tissue
For routine processing, four changes of Carbowax, one each in 70% and 90% and 2 times in I 00% concentration,
at a temperature of 56°C are used, at 30 minutes, 45 minutes and 1 hour (with agitation), respectively. Specimens
are then embedded in fresh Carbowax at 50°C and rapidly cooled in a refrigerator.
o Carbowax is very easily dissolved in water. Hence care must be taken to avoid contact of the block with water
or ice.
o Adding soap to water or using 10% Polyethylene Glycol 900 in water will reduce tissue distortion and promote
flattening and "floating out" of sections.
DIMETHYL SULPHOXIDE (DMSO):
• added to proprietary blends of plastic polymer paraffin waxes reduces infiltration times and facilitates thin sectioning.
• DMSO scavenges residual transition solvent and probably alters tissue permeability by substituting for or removing
bound water thus improving infiltration

CELLOIDIN IMPREGNATION:
• is a purified form of nitrocellulose soluble in many solvents, suitable for specimens with large hollow cavities which
tend to collapse, for hard and dense tissues such as bones and teeth and for large tissue sections of the whole
embryo.
• It is supplied in thin (2%), medium (4%) or thick (8%) solutions of cellulose dissolved in equal parts of ether and
alcohol.
• This is used mainly for preparing soft tissue sections of mixed consistency such as eyes and brain.
• No heat is required, and the resultant block has a rubbery consistency which gives good support to the tissues.
DISADVANTAGE: inability to cut thin sections, storage of blocks in alcohol and speed of technique (which can take
several weeks or months).

ADVANTAGES:
✓ It permits cutting of tissue sections which are thicker than in paraffin wax, and is recommended for processing of
neurological tissues.
✓ Its rubbery consistency allows tissue blocks that are either very hard or of varying consistency, to be cut without
undue distortion.
✓ Dense tissues which are hard to infiltrate (e.g. bones and brain) and specimens which tend to collapse easily due to
air spaces (e.g. eyes) are supported better, thereby avoiding the crumbling of tissues during sectioning. When eye
sections are embedded by the paraffin method, the retina may be detached from the harder tissues (e.g. sclera and
choroid) that encircle it. The cedarwood oil used in the dry celloidin technique helps to soften the brittle layers.
✓ It does not require heat during processing; hence, producing minimum shrinkage and tissue distortion especially for
cutting large bone sections. It is, therefore, recommended in cases when minimum shrinkage is required and when
frozen section technique cannot be done.
DISADVANTAGES:
Celloidin impregnation is very slow (lasting for several days or weeks).
✗ Very thin sections (less than I 0 µ) are difficult to cut.
✗ Serial sections are difficult to prepare.
✗ Vapor of the ether solvent is very flammable; hence, it should never be used near an open flame.
✗ Photomicrographs are difficult to obtain.
✗ It is very volatile and therefore must be kept in bottles with ground-glass stoppers to prevent evaporation.

2 METHODS USED FOR CELLOIDIN IMPREGNATION OF TISSUE:
WET CELLOIDIN:
• Recommended for bones, teeth, large brain sections and whole organs.
DRY CELLOIDIN:
• preferred for processing of whole eye sections.
• The principle and procedure of this method is similar to wet celloidin method, except that 70% alcohol is not used for
storage before cutting.
• The dry method does not make use of alcohol due to the presence of cedarwood oil in the block.
NITROCELLULOSE
• Low Viscosity Nitrocellulose (L.V.N.) is another form of celloidin soluble in equal concentration of ether and
alcohol, with a lower viscosity, allowing it to be used in higher concentrations and still penetrate tissues rapidly.
• It forms a harder tissue block and makes cutting of thinner sections possible
• The tendency of tissues to crack may be prevented by adding plasticizers (e.g. oleum ricini or castor oil) when
embedding chrome-mordanted tissues
• Low viscosity nitrocellulose is more explosive than celloidin and should therefore be handled with care

GELATIN IMPREGNATION
• rarely used except when dehydration is to be avoided and when tissues are to be subjected to histochemical and
enzyme studies.
• used as an embedding medium for delicate specimens and frozen tissue sections because it prevents fragmentation of
tough and friable tissues when frozen sections are cut.
• It is water-soluble, and does not require dehydration and clearing, although fixatives (such as 10% formalin) should
still be washed out by running water whenever indicated.
• It has a low melting point and does not cause over-hardening of tissues by heating.

EMBEDDING
• After impregnation, the tissue is placed into a mold containing the embedding medium and this medium is allowed to
solidify
Ideally the embedding medium should match the tissue type in strength and hardness. If the embedding medium is too
soft for the material, the tissue will not be supported, and sections will be torn or shredded. If the medium is too hard
for the tissue, sections will be brittle and will shatter.
ORIENTATION: Process by which a tissue is arranged in precise positions in the mold during embedding, on the
microtome before cutting, and on the slide before staining.

SEVERAL TYPES OF BLOCKING-OUT MOLDS
1. LEUCKHART’S EMBEDDING MOLD
• consists of two L-shaped strips of heavy brass or metal arranged on a flat metal plate and which can be moved to
adjust the size of the mold to the size of the specimen.
• Blocks produced are even, with parallel sides, and with a fairly shapedinitial setting of the wax.
• It is recommended for routine use, although, too slow and cumbersome for use in a busy laborator

2. COMPOUND EMBEDDING UNIT
• made up of a series of interlocking plates resting on a flat metal base, forming several compartments
• It has the advantage of embedding more specimens at a time, thereby reducing the time needed for blocking.

3. PLASTIC EMBEDDING RINGS AND BASE MOLD
• consist of a special stainless steel base mold fitted with a plastic embedding ring, which later serves as the block
holder during cutting.
TISSUE TEK: is equipped with a warm plate to manage the impregnated specimen, and a cold plate at -5°C for rapid
solidification of the block. It consists of a white plastic cassette mold with detachable, perforated stainless steel hinge and
Snap-On lid, used to hold the tissue specimen through-out fixation, dehydration, clearing and wax impregnation.
ADVANTAGE OF TISSUE TEK: ease of use, less paraffin wax needed, faster embedding, firmly attached tissue and
holder, and permanent identification. It produces easier orientation when resectioning of tissue is required, and blocks can
be filed immediately after sectioning.

4. DISPOSABLE EMBEDDING MOLDS
A. PEEL AWAY
▪ disposable thin plastic embedding molds, available in 3 different sizes, are simply peeled off one at a time, as
soon as the wax has solidified, giving perfect even block without trimming. It may be placed directly in the
chuck or block holder of the microtome.
B. PLASTIC ICE TRAYS
▪ such as those used in ordinary refrigerators may be recommended for busy routine laboratories.
▪ Each compartment may be utilized for embedding one tissue block, which may then be removed by bending the
plastic tray once the wax has solidified or by smearing the inner mold with glycerin or liquid paraffin before
embedding.

C. PAPER BOATS
▪ normally utilized for embedding celloidin blocks but are equally useful for paraffin wax blocks.
▪ They have the advantage of being cheap and easy to make.
▪ They provide easy and accurate identification of specimen, thereby avoiding confusion and interchange of
tissue blocks.
▪ Rapid embedding of small or large volume of individual specimen is possible, since paper molds can be made
to suit any size of tissue.

CELLOIDIN OR NITROCELLULOSE EMBEDDING METHOD
• used to be recommended for embedding hard tissues such as bones and teeth, and for large sections of whole organs
like the eye, since the delicate layers of the eyeball are difficult to keep i ntact when other media are used.

DOUBLE EMBEDDING
• process by which tissues are first embedded or fully infiltrated with a supporting medium such as agar or
nitrocellulose, then infiltrated a second time with paraffin wax in which they are subsequently embedded.
• This is used to facilitate cutting of large blocks of dense firm tissues like the brain.
• They are also recommended for making small sections of celloidin blocks.

PLASTIC (RESIN) EMBEDDING
• provided superior results for light microscopic studies, particularly in hard tissues such as undecalcified bone and for
high resolution light microscopy of tissue sections thinner than the usual 4-6 µm, such as renal biopsies a n d bone
marrow biopsies.
CLASSIFFIED INTO:
➢ EPOXY: embedding plastics are made up of a carefully balanced mixture of epoxy plastic, catalysts and
accelerators
o ARALDITE
o EPON
o SPUR
➢ POLYESTER PLASTICS: were originally introduced for electron microscopy in the mid- 1950s, but have been
superseded by more superior epoxides, and are now seldom used.
➢ ACRYLIC PLASTICS: made up of esters of acrylic or methacrylic acid, and are used extensively for light
microscopy
o Acrylic plastics based on methyl methacrylate (MMA) are also widely used because of its hardness as the ideal
embedding medium for undecalcified bone and is widely used for bone histomorphometry and bone marrow
hematopathology.

PRACTICAL CONSIDERATIONS:
✓ Specimen should only be processed under an operational fume hood.
✓ Processing is best achieved if the specimen is agitated continuously on a roller mixer.
✓ Small aliquots of benzoyl peroxide should be dried carefully away from direct heat and sunlight as it is potentially
explosive. It is important that no water is present before dissolving the catalyst (2 minutes) in the infiltrating
solution. It must be completely dissolved in the infiltrating solution, and this may take up to 30 minutes.
✓ The acrylic plastic mixes are best prepared only in the quantity required, preferably using a large glass vial. It is
advisable to measure the quantities volume by weight.
✓ Any waste solutions containing plastic components must be handled and discarded in accordance with
local and legal requirements.
MICROTOMY
MICROTOMY: process by which processed tissue, most commonly a paraffin embedded tissue, is trimmed and cut into
uniformly thin slices or "sections" to facilitate studies under the microscope
• basic instrument used is a microtome that is capable of cutting a section at a predetermined thickness by sliding the
block into a cutting tool, usually a steel knife, glass or diamond blade, which is fixed and attached to the machine.

3 ESSENTIAL PARTS:
BLOCK HOLDER: where the tissue is held in position.
KNIFE CAREER AND KNIFE: for actual cutting of tissue sections.
PAWL, RATCHET FEED WHEEL AND ADJUSTMENT SCREWS: to line up the tissue block in proper position
with the knife, adjusting the proper thickness of the tissue for successive sections.

5 KINDS OF MICROTOMES:
ROCKING (CAMBRIDGE) MICROTOME:
• for cutting serial sections of large blocks of paraffin embedded tissues.
• invented by Paldwell Trefall in 1881
• simplest among the different types of microtomes
• Cambridge rocking microtome, available in two sizes, has been used to cut small and large blocks of paraffin tissues
• It is theoretically not recommended for serial sections since tissues are cut in slightly curved planes.
• not currently favored by most laboratories because of the restrictions in size of tissue block that can be cut, and the
difficulty of reorienting the block.
ROTARY (MINOT) MICROTOME:
• for cutting paraffin embedded sections.
• invented by Minot in 1885-86 to cut paraffin embedded tissues, and is currently the most common type used for both
routine and research laboratories, especially for sectioning paraffin-embedded tissues.
• It is heavier and more stable than the rocking microtome, is more complex in design and construction, and is
therefore more expensive.
• It may be used for cutting large blocks of tissues although results are better when the sliding microtome is used

SLIDING MICROTOME:
• for cutting celloidin embedded sections.
• developed by Adams in 1789.
2 TYPES:
BASE SLEDGE:
• Base-Sledge microtome is favored in laboratories where very hard tissue or large blocks are usually sectioned.
• It was originally designed for cutting sections of very large blocks (whole brain)
• Sections are cut in a perfectly flat plane, thereby making excellent serial tissue sections
• It is comparatively heavier and more stable than the ordinary sliding microtome
• The angle of the knife is adjustable.
STANDARD SLIDING MICROTOME:
• block remains stationary while the knife is moved backward and forward during the process of sectioning.
• developed mainly for cutting celloidin embedded tissue blocks and is inherently more dangerous because of the
movable knife, which makes it difficult to attach knife guards.
BOTH:
• knife can be set obliquely for celloidin sections or straight for large refractory paraffin blocks, cutting both large and
small tissues with ease
• it is especially recommended for cutting extremely hard and rough tissue blocks.
• It is the most dangerous type of microtome due to the movable exposed knife.
• A slow but very steady motion is therefore required to manipulate the instrument.

FREEZING MICROTOME:
• for cutting unembedded frozen sections.
• invented by Queckett in 1848.
• It is used to cut undehydrated thin to semi-thin sections of fresh, frozen tissues, especially in instances when
rapid diagnosis is required, when histological demonstration of fat is needed, when certain neurological
structures are to be studied, and when sensitive tissue constituents to be studied are damaged or destroyed by
heat.
• this type will give the best results and is used almost universally
• freezing microtome is equipped with a stage upon which tissue can be quickly frozen using either liquid carbon
dioxide, from a cylinder, or a low temperature recirculating coolant.
• The cutting action of the freezing microtome differs from those described previously as in this case the knife is
moved whilst the tissue block remains static, same as sliding microtome.
CRYOSTAT OR COLD MICROTOME:
• for cutting frozen sections
• cryostat provides a means of preparing thin sections of fresh frozen tissues especially for fluorescent antibody
staining techniques or histochemical enzyme studies.
• cryostat provides a means of preparing thin sections of fresh frozen tissues especially for fluorescent antibody
staining techniques or histochemical enzyme studies.
• It is most commonly used for rapid preparation of urgent tissue biopsies for intraoperative diagnosis
• It is often housed in the frozen section room close to the operating room to allow direct consultation between surgeon
and pathologist.
ULTRATHIN MICROTOME:
• for cutting sections for Electron Microscopy
• equipped with a glass or gem grade diamond knife is used to cut very thin sections (typically 60 to 100 nanometer) of
tissue embedded in epoxy resin.
• Sections are stained with an aqueous solution of an appropriate heavy metal salt and examined with a transmission
electron microscope (TEM)
• used with its glass knife or an industrial grade diamond knife to cut semi-thin sections prior to thin sectioning
• Thin sectioning for the TEM is often done with a gem quality diamond knife.

CARE OF MICROTOMES:
✓ all the accumulated paraffin and small pieces of tissues must be brushed away with a soft brush and not allowed to
stay in the microtome
✓ parts should be wiped with xylol.
✓ Prolonged and continuous application of the painted parts with xylene should, however, be avoided since this
reagent is capable of removing the paint
✓ Movable portions should be oiled thoroughly to prevent rusting
✓ microtome must always be covered when not in use, to prevent accumulation of dust and other dirt which may later
on interfere with the normal sectioning of tissues.
✓ microtome should be placed on a stable bench, away from air drafts, doorways and passing staff.
✓ Always remove the knife or blade before cleaning. The knife holder can easily be removed to facilitate access for
cleaning. No fluid must enter the inside of the instrument during cleaning
✓ When cleaning the blade avoid dragging anything along the cutting edge. Even cellulose fibers can cause damage to
the blade.
✓ Have the instrument inspected at least once a year by a qualified service technician.
SAFETY MEASURES:
✓ It is very important that staff are not distracted when using the microtome because of the risks of injury from
extremely sharp blades. It is preferable to have non-slip flooring in the vicinity of microtomes because, inevitably,
wax fragments will find their way onto the floor where they can produce a slippery surface.
✓ Use forceps or brush instead of fingers to pick up sections or wax fragments from blade or block face.
✓ Use hand wheel lock when changing blocks. The knife or blade should be removed from the microtome when the
instrument is left unattended or when cleaning the instrument

MICROTOME KNIVES:
Trimming and section-cutting are done with a microtome knife, which is available in three basic types or shapes:
1. PLANE-CONCAVE KNIFE (USUALLY 25 MM. IN LENGTH):
• One side of the knife is flat while the other is concave
• Less concave sides are recommended for cutting celloidin-embedded tissue blocks on a sliding microtome
• More concave sides are used to cut paraffin sections on base-sledge, rotary or rocking microtome
2. BICONCAVE KNIFE (USUALLY 120 MM. IN LENGTH):
• with both sides concave, recommended for cutting paraffin - embedded sections on a rotary microtome
3. PLANE-WEDGE KNIFE (USUALLY 100 MM. IN LENGTH):
• have both sides straight,
• recommended for frozen sections or for cutting extremely hard and tough specimens embedded in paraffin
blocks, using a base sledge type or sliding microtome.

HONING AND STROPPING:
HONING (SHARPENING):
• Involves the removal of gross nicks on the knife edge (Coarse Honing) to remove blemishes, and grinding the cutting
edge of the knife on a stone (Honing Proper) to acquire an even edge
• The degree of sharpness is proportional to the fineness of the abrasive used in sharpening
• This procedure makes use of a hone, a natural sharpening stone or hard grinding surface (carborundum), which serves
to remove nicks and irregularities on the knife edges
TYPES OF HONES:
▪ BELGIUM YELLOW: for manual sharpening when cutting edge has been rendered blunt or nicked
➢ This type usually gives the best result.
▪ ARKANSAS: gives more polishing effect than the Belgium Yellow.
▪ FINE CARBORUNDUM: is much coarser than the first two types and is used only for badly nicked knives
followed by either one of the first two knife sharpeners.

PRECAUTIONS DURING HONING:
✓ hone should be long enough (about 8" x 3") to allow the whole length of the knife edge to be sharpened in a single
stroke and wide enough to sufficiently support and prevent the rocking of the knife.
✓ hone should be lubricated with warm soapy water or fine oil before using. It is then washed, preferably with water,
to remove all metal particles that may have been collected during the process. The washing fluid used must flow
rapidly enough so that the metal chips are removed between strokes and a clean hone is presented every time.
✓ pressure on the knife should be gentle and steady to keep it from rocking.
✓ number of strokes usually amounts to 20-30 times in each direction, depending upon the condition of the knife.
✓ Badly nicked knives require greater and longer honing than less irregular knives.
✓ hone should be cleaned before, during, and after use
✓ After its use, the hone must be washed with warm soapy water, dried, and kept in a box to protect it from dust while
it is not in use
✓ After honing, wipe off the oil or soap from the knife with xylene. Then strop it thoroughly.

TROPPING
STROPPING: process whereby the "burr" formed during honing is removed and the cutting edge of the knife is polished.
• purpose of stropping is to polish and sharpen the cutting edge, while that of honing is to remove the irregularities
from the knife
• The procedure is the reverse of honing.
• The knife is first fitted with its appropriate knife back, then laid obliquely on the strop and with the cutting edge
behind, (EDGE LAST) is pushed backward and drawn forward in a TOE TO HEEL direction.
• Around 40- 120 double strokes are usually required
• In the case of plane-wedge or Minot knives, the knife is turned around at the end of each stroke so as
to sharpen each surface alternately
• For plane concave knives, only the concave surface should be stropped.

PRECAUTIONS:
✓ Knife should always be wiped clean with a soft cloth before and after a series of stropping strokes and before
changing from a coarse to a fine strop to remove particles which may have been taken off the knife
✓ After stropping is satisfactorily completed, the knife edge is then oiled or greased to prevent it from rusting
✓ Then, the knife is kept covered in a suspension box to prevent the settling of dust and grit on its surface, causing
damage to the knife edge
✓ The knife should not be allowed to rest on its sides since this may also damage the cutting edge.
✓ Pressure during the first stropping strokes should be quite light, since the natural compressibility of the leather is
what actually does the work.
✓ Only a gentle pressure should be applied while the knife is held steady on the strop, since a slip may cut the strop
and damage the cutting edge
✓ Speed in stropping should be avoided.
✓ One full second should be allowed for each stroke to avoid injury to the strop and the knife.
✓ Leather strops are usually dry and require oiling before they are used.
✓ Strops are usually treated with vegetable oil (e.g. castor oil) applied into the back of the strop, NOT the surface.
✓ The strop should not be used for at least 24-48 hours after treatment.
✓ Too much oil will make the stropping surface slippery and will render the procedure unsatisfactory
✓ To remove excessive oil from the strop, its surface is scraped with a blunt instrument, e.g. the back of the knife.
✓ Mineral oil is not recommended and should NEVER come in contact with a strop since it will tend to blister and
destroy the leather
✓ Stropping surfaces should be firm and not loose, to prevent the turning of the knife's edge
✓ Wax must not be allowed to come in contact with the strop
DISPOSABLE BLADES:
Sharpening (honing) and polishing (stropping) are no longer common practice in most modern laboratories
because of the availability of disposable knives that are cheaper to use than conventional steel knives.
GLASS KNIVES:
• generally used for trimming and semi-thin sectioning of tissue blocks for electron microscopy
• Glass knives should be prepared and stored in dust-free boxes with lids, just before use, to avoid contamination.
DIAMOND KNIVES:
• used to cut any type of resin block for electron microscopy.
• Diamond knives are brittle and expensive, but very durable, and the cutting edge must be kept clean to make it
cut longer and to avoid damage during sectioning.

OTHER EQUIPMENT:
In addition to the microtome and the microtome knife, the following items are also required during the process of
sectioning:
WATERBATH:
1.
• The thermostatically controlled type is preferable
• The temperature of the water should be between 5 and 10°C below the melting point of the paraffin wax
DRYING OVEN OR HOT PLATE:
2.
• designed for drying tissue section on slides.
• With a temperature setting at the melting point of the wax no obvious damage is done to the sections and drying is
complete in 30 minutes.
• hot plate may also be used instead of a drying oven
• For more delicate tissues such as brain, a lower drying temperature is used to avoid splitting and cracking of the
section due to excessive heat
• In such cases, 37 oC for 24 hours or longer is recommended.
FORCEPS (fine pointed or curved) and squirrel hair brush:
3.
• These tools are needed for handling sections during cutting, and for removing folds and creases on the sections during
"floating out" in water bath.
CLEAN SLIDES:
4.
• For routine work, 76 x 25 mm. slides that are 1.0 -1.2 mm thick are usually preferred because they do not break
easily.
• Frost-ended slides are generally used, where the identification number of the section can be inscribed with a pencil.
SLIDE RACK:
5.
• made on the assumption that regular slides have been used.
• Larger size of slides are used for sections of eyes or CNS tissues when these will not fit on the regular.

The quality of sections cut on a microtome suffer badly from several (avoidable) causes. Things to avoid include:
• fecal material in intestine, especially in the colon where this material is very hard; hair is particularly bad - it can be
removed using a razor blade or clippers.
➢ be careful during dissections; sutures, thread or staples should be removed from the specimen prior to cutting
with the knife.
CUTTING SECTIONS
SECTIONING: process whereby tissues are cut into uniformly thin slices or "sections" with the aid of a microtome, to
facilitate the studies under the microscope.

GENERAL TYPES OF TISSUE SECTIONS:
PARAFFIN SECTIONS: for paraffin embedded tissue blocks which may be cut by rocking and rotary microtome
CELLOIDIN SECTIONS: for celloidin embedded tissues which are usually cut by means of the sliding microtome.
FROZEN SECTIONS: which may be cut from tissues that have been fixed and frozen with CO2 or for fresh or fixed
tissues frozen with the cryostat.

PARAFFIN SECTIONS:
TRIMMING: the wax block is removed from the mold; the identification number is noted, and the excess wax is cut off
from the block to expose the tissue surface in preparation for actual cutting.
COARSE TRIMMING: s done on the microtome at approximately 30 microns at a time until the entire tissue surface is
exposed.
FINE TRIMMING: may be done by either setting the thickness adjuster at 15 mm or by advancing the block using the
coarse feed mechanism
• knife is usually tilted at 0-1 5° angulation on a microtome to allow a clearance angle between the cutting facet and the
tissue block
• Biconcave knives require smaller clearance angles than wedge-shaped knives.

COLD WAX: provides better support for the harder elements in a specimen allowing thinner sections to be obtained.
Place the blocks on a cold plate or a cold wet surface for a few minutes (such as the surface of melting ice).
FLOTATION: should expand the section to its original dimensions and ensure that it is completely flat.
• temperature will need to be 5 - 9 ˚C below the melting point of the wax
CELLOIDIN EMBEDDING
• is a slow process, usually taking weeks, and does not produce sections as thin as those produced by
paraffin embedding
• The advantage of celloidin embedding is that it completely avoids the use of heat at any stage.
• The disadvantages are the longer time to cut, the thickness of the sections, the necessity for staining to be done on
free floating section, the inconvenience of having to store the blocks in sealed jars with tight lids to prevent
complete evaporation of 70% ethanol, the resulting restrictions on the type of staining methods that may be used.
• he slow method of hardening the block allows the increasing concentration of celloidin to get into the block and give
additional support to the tissue
• Hardening of the celloidin block may be hastened by placing a small open container of chloroform under the bell jar
• The chloroform will saturate the atmosphere and harden the celloidin without further evaporation.
• As the block hardens the celloidin will shrink.
• To avoid dehydration and shrinkage, section cutting is usually done wet, which means that the block is lubricated
with a fluid, usually 60-70% ethanol, and is not allowed to dry out.
o This makes section cutting somewhat messy and quite a bit slower than the dry sectioning used with paraffin
• Celloidin sections do not come off in ribbons and tend to roll up during cutting, and moistening the block and section
with alcohol by means of a camel hair brush will serve to flatten the sections on the knife.

Inflammation and Disease Process: Name: Aldrin Ligan BSMT 2B

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Inflammation and Disease Process: Name: Aldrin Ligan BSMT 2B

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NAME: ALDRIN LIGAN BSMT 2B

INFLAMMATION AND DISEASE PROCESS

 Perceived or experience by the patients

INFLAME – to set fire

CARDINAL SIGNS OF INFLAMMATION:

AULUS CORNELIUS CELSUS - described the first 4 cardinal sign.

RUDOLF VIRCHOW – added function laesa

a. Acute inflammation – early onset

 Usually, but not necessarily of, sudden onset

 Characterized with five cardinal signs

 Predominant cells: PMN

c. Chronic inflammation – later onset

 Persistence of injurious agent for weeks/years

 Characterized with proliferative rather than educate response

 Predominant cells: mononuclear cells (macrophage, Lymphocytes, Plasma cells)

SYSTEM EFFECTS OF INFLAMMATION

ACCORDING TO CHARACTER OF EXUDATE

1. Serous inflammation: when the fluid exudate resembles your serum

ABNORMALITIES IN CELL GROWTH

1. Retrogressive changes: organs/tissues are smaller than normal

i. Aplasia: defective development

 Failure of organ to develop after a stage

ii. Agenesia: failure of organ or part to develop or grow

iii. Hypoplasia: incomplete development, or under development of organ or tissue.

b. Atrophy: shrinkage/shrinkage of cell.

ii. Pathologic Atrophy: result for some form of cellular injury.

2. Progressive Changes: Organs/ tissues are larger than normal.

 HYPERTROPHY: increase in size of cell/tissue

3. Degenerative Changes due to Aberrations of Cellular Growth Patterns.

a. Metaplasia: “Transformation” reversible; transformation in one type of adult cell to another

TUMOR BASIC COMPONENTS:

 Parenchyma: refers to the active elements of the tumor

CAPACITY TO PRODUCE DEATH

 BENIGN TUMORS: usually are non-spreading tumor.

DEPENDING ON HISTOLOGICAL CHARACTERISTICS:

MALIGNANT “SARCOMA” “CARCINOMA”

DEGENARATION VS. INFILTRATION

• Liver is the organ most commonly affected by FATTY DEGENERATION.

NECROBIOSIS is a slower process which refers to the physiologic death of cells.

SOMATIC DEATH refers to the death of the entire body or organism.

MICROSCOPIC CHANGES IN NECROSIS:

 Pyknosis: Reduction in size & condensation of nuclear material.

TYPES OF NECROSIS ACCORDING TO MORPHOLOGIC CHANGES:

TISSUE PROCESSING AND FIXATION

IMPREGNATION AND EMBEDDING

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