Fixation

• 1. First and most critical step

• 2. If fixation is not adequate, the other
processes that follow will also be inadequate.
• 3. Inadequate fixation, poor diagnosis.
• 4. Primary aim is to preserve the morphology
and chemical integrity of the cell.
Fixation
Preserves:
a. Shape
b. Structure
c. Intercellular Relationship
d. Chemical Constituents

Prevents:
a. Degeneration
b. Decomposition
c. Putrefaction
d. Distortion of tissues
Fixation
• Harden and protect the tissue from trauma of
further handling

Neutral buffered
• Common Fixative
formaldehyde or
Formalin

Electron Microscopy,
Specific Fixation
Immunochemistry,
Procedures
Histochemistry
To Preserve the Tissue

• Fixation preserves the tissue by stopping all cellular

activities .

• -Leaving the tissue specimen in air will cause it to dry

out and result to distortion.
• -Leaving the tissue in water (a hypotonic solution)
will cause the cell to swell.
• -Strong salt (hypertonic solution) will cause the cell
to shrink.
To prevent Breakdown of Cellular Elements

• *Tissue deprive in oxygen and nutrition will lead to

degradative chemical process or cell death.
• -lysosomes “suicide sac”

• *Post mortem decomposition “Autolysis”

- Cells contains hydrolytic enzymes within the lysosomes

• Fixations prevents autolysis by:

- inactivating the lysosomal enzymes
- chemically altering
- stabilizing and making the tissue insoluble
- protects from further decomposition (putrefaction)
2 Mechanisms in Fixation

• 1. ADDITIVE FIXATION
- Chemical constituents are taken in and becomes
part of the tissue.
- Form cross-links and stabilize proteins
ex. Formalin, Mercury, Osmium Tetroxide

2. NON-ADDITIVE FIXATION
- Fixing agent is not incorporated into the tissue
- Removing of water; to form new cross-links
ex. Alcoholic Fixatives
MAIN FACTORS INVOLVED IN FIXATION
• 1. Hydrogen Ion Concentration
• pH : 6 and 8
• 2. Temperature
• Surgical specimen : Room temp
• Tissue processors : 40oC
• Electron microscopy : 0-4oC

• Note: fixations are more rapid at higher temperatures

• Formalin at 60oC is used for urgent biopsy specimens
• Formalin at 100oC is used to fix tissues with tuberculosis

• Note: TEMP  DISTORTION

• 3. Thickness of section
• 1-2 mm2 = Electron microscopy
• 2 cm2 = Light microscopy
• 0.4 cm = Light microscopy (thin sections)

• Note!:
*Large solid tissue should be opened and sliced thinly
– e.g. uterus

*Brain tissue  10% Formalin 2-3 weeks

• 4. Osmolality
• Hypertonic = Shrinkage
• Hypotonic & Isotonic = Swell and poor fixation
• Best: Slightly Hypertonic solutions
•  400-450 mOsm
• 340 mOsm = Isotonic solutions

• Electron Microscopy = Sucrose + Osmium Tetroxide

• 5. Concentration
• 10% Formaldehyde
• 3% Glutaraldehyde

• 0.25% Glutaraldehyde = immunoelectron

• microscopy

• Note!
• Presence of BUFFER causes polymerization of the
aldehyde  effectiveness
• 6. Duration of Fixation
• 2-6 hours = Primary fixation in buffered formalin from
the time the specimen is obtained.

Note!:
Fixation can remain over the weekend without much
adverse effect.

Prolonged fixation = shrinkage

hardening of tissue
inhib. enzyme activity & immunological
reactions

Electron Microscopy = 3 hours

 Practical Considerations of Fixation
• 1. Speed
• 2. Penetration
• 1 mm/hour
• Note! Time of fixation varies with the types of tissue
• 3. Volume
• Traditionally = 10-25x the volume of the tissue
• 20x the tissue volume = maximum effectiveness
• 4. Duration of Fixation
• Fibrous organs (uterus, intestinal tract) take longer than small
loosely textured tissues (biopsies, scrapings).
• Fixation can be improved by the ff:
• -Heat -Vacuum
• -Agitation -Microwave
Miscellaneous Consideration
• 1. Recommended size: 2 cm2 and no more than 4
mm thick
• 2. Most tissue can be cut and trimmed without prior
fixation, except for the brain.
• 3. Brain must be fixed before “grossing” or
“sectioning”
• 4. Position or shape desired should be maintain
– eg. piece of intestine strecthed out on the piece of
paper and placed in proper container before
pouring fixative over it
• 5. Refrigeration = slow down decomposition
• Note!

• Cells from different parts of the body die at different

time intervals

• Brain cells, deteriorate very quickly

• Bone marrow continues to undergo mitosis up to 30

minutes after death when refrigerated
Effects of Fixatives in General
• 1. Harden soft and friable tissues
• 2. Make handling and cutting of sections easier
• 3. Make the cells resistant to damage and distortion
• 4. Inhibit bacterial decomposition
• 5. Increase optical differentiation of cells
• 6. They act as mordants or accentuators
• 7. Reduce the risk of infections

CELL MORDANT STAIN

Characteristics of a good Fixative
• 1. It must be cheap
• 2. It must be stable
• 3. It must be safe to handle
• 4. It must kill the cell quickly
• 5. It must inhibit bacterial decomposition and autolysis
• 6. It must produce minimum shrinkage of tissue
• 7. It must permit rapid and even penetration of tissues
• 8. It must harden tissues
• 9. It must isotonic (however)
• 10. It must make cellular components insoluble to
hypotonic solutions
• 11. Permit application of many staining procedures
Types of Fixative According to Action and
Composition
• I- According to COMPOSITION

• A. Simple Fixatives – are made up of only one component substance

1. Aldehydes
a. Formaldehyde
b. Glutaraldehyde
2. Metallic Fixatives
a. Mercuric chloride
b. Chromate fixatives
- Potassium dichromate
- Chromic acid
c. Lead fixatives
d. Picric acid
e. Acetic acid
f. Acetone
g. Heat
B. Compound Fixatives – made up of two or more fixatives
Types of Fixative According to Action and
Composition
• II – According to ACTION

• A. Microanatomical Fixatives – permit general

microscopic study of tissue structures w/out altering
the structural pattern and normal intercellular
relationship of the tissues.
1. 10% Formol saline
2. 10% Neutral buffered formalin
3. Heidenhain’s Susa
4. Formol sublimate (formol corrosive)
5. Zenker’s solution
6. Zenker-formol (Kellly’s solution)
7. Bouin’s solution
8. Brasil’s solution
• B. Cytological Fixatives – Preserve specific parts
and particular microscopic elements of the cell
itself

1. Nuclear Fixatives
-preserve the nuclear structures
-contain glacial acetic acid
- pH 4.6 or less
eg.
-Flemming’s fluid
-Carnoy’s fluid
-Bouin’s fluid
-Newcomer’s fluid
-Heidenhain’s Susa
• 2. Cytoplasmic Fixatives
• - preserve cytoplasmic structures
• - never contain glacial acetic acid
• - pH > 4.6

• e.g .
• - Flemming’s fluid w/out acetic acid
• - Kelly’s fluid
• - Formalin with “post-chroming”
• - Regaud’s fluid (Muller’s fluid)
• - Orth’s fluid
• 3. Histochemical Fixatives
• - Preserve the chemical constituents of cells
and tissues.

• e.g.
• - Formol Saline 10%
• - Absolute Ethyl Alcohol
• - Acetone
• - Newcomer’s Fluid
• Note!
• Lipid Fixation
• -Mercuric chloride Preservation of
• -Potassium dichromate lipids in cryostat

• *Phospholipids are fixed with aldehydes

• *Formaldehydes react with unsaturated fatty acids

• *Baker’s formol-calcium: used to preserved phospholipids

• *Imidazole osmium tetroxide: for improved ultrastructural

demonstration of lipids

• *Digitonin: for cholesterol demonstration

• Note!
• Carbohydrate Fixation

• *Alcoholic fixatives: recommended for

glycogen fixation

• *Human skin
• Alcoholic formaldehyde or neutral buffered
formaldehyde
• Note!
• *Protein Fixation
Neutral buffered formol Amino
saline or formaldehyde vapor Acid

*Glycogen Fixation
Rossman’s fluid or Cold absolute alcohol
 most useful glycogen fixatives
• A- Aldehydes Fixatives
1. Formaldehyde
2. 10% Formol Saline
3. 10% NBF (Neutral Buffered Formalin)
BEST GENERAL TISSUE FIXATIVE
4. Formol-Corrosive (Formol-Sublimate)
5. Glutaraldehyde = Preserves plasma protein
better
6. Formol-calcium = for the preservation of lipids
7. Karnovsky paraformaldehyde – Electron
glutaraldehyde Cytochemistry
8. Acrolein
• Formaldehyde (Formalin)

• -produced by oxidation of methanol

• - 10% formalin
• -soluble in water
• -40% pure stock solution is unsatisfactory
• - formaldehydes concentrates  overharden
• - pH 7 = phosphate buffer
• -Fixation Time: 24 hours
• Advantages:
• a. easy to prepare
• b. compatible with many stains
• c. it penetrates tissues well
• d. preserves fats and mucin
• e. preserves glycogen
• f. it preserves but does not precipitate
• proteins
• g. it allows natural tissue colors to be
• restored
• Disadvantages
• a. Fumes are irritating to the nose and eyes, causes
sinusitis, allergic rhinitis or excessive lacrimation.

• b. It is a soft fixative and does not harden some

cytoplasmic structures

• c. If unbuffered:
• -reduces both basophilic and eosinophilic
• staining
• -forms abundant brown pigment granules
• on blood-containing

• d. Prolonged fixation
• -bleaching of the specimen
• -loss of natural tissue color
• -dispersal of fat from the tissues into the fluid
• FORMALDEHYDE

Paraformaldehyde Formic Acid

-white crystalline ppt +
-removal: Hgb in blood
1. 10% methanol
2. Filtration Acid Formaldehyde
Hematin
-Brown or black
crystalline
-removed by:
1. Lilies Mtd
2. Kardasewitch
3. Sat. alcohol picric acid
4. 1% KOH in 80%
alcohol
• -Bleaching of tissues may be prevented by changing
the fluid fixative every three months
•
• - 70% Alcohol- restore the natural color of tissue

• Note!!
• Magnesium carbonate use as buffer or
• Calcium carbonate neutral agent to
formic acid
• 10% Formol-Saline

• -simple microanatomical fixative

• -saturated Formaldehyde + 10% NaCl
• -recommended for central nervous tissues and
general post-mortem tissues

• Fixation I Time: 24 hrs at 35 deg Celcius

• 48 hrs at 20-25 deg Celcius
• Advantages:
• -it preserves enzymes and nucleoproteins
• -do not over harden the tissue therefore
• facilitates dissection of the specimen
• -ideal for silver impregnation
•
• Disadvantages:
• -Slow fixative
• -Metachromatic reaction of amyloid is
• reduced
•
• 10% Neutral Buffered Formalin or Phosphate-
Buffered Formalin
• -pH 7
• -recommended for preservation and storage of
surgical, post-mortem and research specimens
 Advantages:
• a. similar to formol-saline
• b. prevents ppt of acid formalin pigments on post-
mortem tissue
• c. best fixative for tissues containing iron pigments

• Disadvantages:
• a. Longer to prepare & time consuming
• b. Positivity of mucin to PAS is reduced
• c. reactivity of myelin in Weigert’s iron hematoxylin
stain is reduced

• Fixation Time: 4 – 24 hours

• Formal-Corrosive (Formal-Sublimate)
• -recommended for routine post-mortem tissues

• Advantages:
• -penetrates small tissue rapidly
• -excellent for many staining procedures
• -brightens cytoplasmic and metachromatic stains better than
formalin alone
• -Cytological structure and blood cells are well preserved
• -no need for washing out
• -fixes lipid (neutral fats and phospholipids)
• Disadvantages
• -penetration is slow (< 1 cm thick)
• -forms mercuric chloride deposits
• -do not allow frozen section
• -inhibit tissue decalcification

• Fixation Time: 3-24 hours

• Alcoholic Formalin (Gendre’s) Fixative
• -Post-fixation with phenol-formalin for 6 hours enhance
immunoperoxidase studies on the tissues and electron
microscopy
•
• Advantages:
• -fixation is faster
• -rapid diagnosis because it fixes and dehydrates
• -preserves glycogen
• -micro-incineration technique
• -used to fix sputum (coagulates mucus)

• Disadvantages:
• -produces gross hardening of the tissue
• -causes partial lysis of RBC
• -poor iron preservation
• Glutaraldehyde
• -made up of two formaldehyde residues
• -2.5% solution is used for small tissue fragments
• Fixed in 2-4 hours at RT
• -4% solution is recommended for larger tissues less than 4 mm
thick fixed in 6-8 hours up to 24 hours

• Advantages:
• -stable effect on tissues
• -preserves plasma proteins better
• -less tissue shrinkage
• -recommended for enzyme histochemistry & EM
• -it does not cause dermatitis and less irritating to the nose

• Disadvantages :
• -more expensive
• -penetrates tissues more slowly
• -tends to make tissue more brittle
• B- Metallic Fixatives

• 1. Mercuric Chloride
• -most common metallic fixative
• -may produce black deposits on tissues (except SuSa)
•
• a. Zenker’s Fluid (w/ glacial acetic acid)
• b. Zenker’s Formol (Helly’s Sol’n)
• c. Heidenhain’s Susa – for tumor biopsy of
• the skin
• d. Schaudinn’s fluid
• e. Ohlmacher’s Fluid
• f. Carnoys-Lebrun Fluid
• g. B-5 Fixative
•
• Mercuric Chloride
• -used as a secondary fixative
• -penetrates poorly and shrinkage of tissues
• -0.5% iodine solution in 70% ethanol for 5-10 minutes to
remove black ppt deposit

• Advantages:
• -hardens tissues rapidly and well
• -it precipitates all proteins
• -greater affinity to acid dyes
• -trichrome staining is excellent
• -permits brilliant metachromatic staining of cells

• Disadvantages:
• -It is inert to fats and lipids
• -lysis of RBC
• -remove much iron from hemosiderin
• Zenker’s Fluid
• -made up of mercuric chloride stock sol’n
• -added with glacial acetic acid
• *glacial acetic acid: prevent turbidity and dark precipitation
• -recommended for fixing small pieces of liver, spleen, connective
tissue fibers and nuclei

• -Fixation Time: 12-24 hours

• Advantages:
• -Stock sol’n w/o disintegration
• -Recommended: Trichrome staining
• -May act as a mordant

• Disadvantages:
• -penetration is poor
• -not stable after addition of Acetic acid
• -does not permit cutting of frozen section
• Zenker-Formol (Helly’s Sol’n)

• Advantages:
• -excellent microanatomic fixative for pituitary gland, bone
marrow and spleen and liver

• Disadvantages:
• -similar to Zenker
• -brown pigments are produced after 24 hours
•
• Fixation time: 12-24 hours
• Heidenhain’s Susa
• -recommended for tumor biopsies especially of the
skin
• -excellent cytologic fixative
• -after using Heidenhain’s Susa, transferred the tissue
to a high grade alcohol to avoid undue swelling.

• Fixation Time: 3-12 hours

• B-5 Fixative
• -commonly used for bone marrow biopsies
• -prior to use, add 1 cc 40% formalin + 10 cc of B-5
• 2. Chromate Fixatives
•
• a. Chromic acid
• b. Potassium dichromate
• c. Regaud’s (Moller’s)
• d. Orth’s Fluid – for Rickettsia and other
• bacteria
• – for study of early
• degenerative process

•
• Chromic Acid
• - 1-2% aqueous sol’n
• - ppt all proteins and preserves CHO
• -strong oxidizing agent & strong reducing agent

• Potassium Dichromate
• -3% aqeuous sol’n
• -preserves lipids
• -preserves mitochondria (pH: 4.5 – 5.2)
• -Acidified: cytoplasm, chromatin and
chromosomes are fixed but mitochondria are
destroyed)
• Regards (Muller’s) Fluid
• -recommended for chromatin, mitochondria, golgi
bodies,RBC, colloid-containing tissues
• -deteriotrates and darken on standing due to acidity
• -Does not preserve fats
• -Fixation time: 12-48 hours

• Orth’s Fluid
• -recommended for study of early degenerative
processes and tissue necrosis
• -demostrate rickettsiae
• -it preserves myelin better than buffered formalin
• -Fixation Time: 36-72 hours
• 3. Lead Fixatives
• -are generally for ACID MUCOPOLYSACCHARIDES
• -4% aqueous sol’n
• -it takes up CO2 to form insoluble lead carbonate
• remove by: 1. Filtration
2. Acetic acid
• C. Picrate Fixatives
• - highly explosive when dry
• - will produce excessive yellow staining of tissues
• - picrates are formed upon protein;
• -precipitates in water; hence tissues must
• first rendered insoluble by direct
• immersion in 70% ETOH
• - picrate fixatives MUST NEVER be washed
• in water before dehydration
• -Lithium carbonate  removal of yellow color

• a. Bouin’s Solution – for fixation of embryos

• b. Brasil’s Alcoholic Picroformol – less messy than
• Bouin’s
• Bouins Sol’n
• -Recommended for fixation of embryos and pituitary
biopsies
• -Does not need washing out
• -Fixation Time: 6-24 hours

• Brasil’s Alcoholic Picroformol Fixative

• -Excellent fixative for glycogen
• -Better and less “messy” than Bouin’s solution
• D. Glacial Acetic Acid
• -Fixes nucleoprotein
• -Solidifies at 17 deg. Celcius
• -It causes tissue to swell
• -Use to counteract the shrinkage produced by other
fixatives (eg. mercury)
• E. Alcoholic Fixatives
• -destroying hydrogen bond
• -general disadvantage: Polarization
• Polarization: glycogen granules move towards
the poles or ends of cells.
• -Lower conc.  RBC lysis
• 70-100% must be used!

• -Absolute alcohol  fix glycogen, pigments,

blood, tissue films and smears.

•
Alcoholic Fixatives

• a. Methanol – blood smears and BM tissues

• b. Ethanol – Cytologic smear
• c. Carnoy’s fluid – MOST RAPID FIXATIVE
• (fixation time: 1 – 3 hours)
• d. Alcoholic Formalin (Gendre’s Fixative)
• - useful in preserving sputum
• e. Newcomer’s fluid
• Methyl Alcohol 100%
• -excellent for dry and wet smears
• -fixes and dehydrates
• -penetration is slow

• Isoprophyl Alcohol
• -fixing touch preparations

• Ethyl Alcohol
• -70-100%
• -lower concentrations will lyzed the RBC
• -do not fix glycogen
• Fixation time: 18-24 hours
• Carnoy’s Fluid
• -recommended for chromosomes, lymph glands and
urgent biopsies and brain tissues
• -use in the diagnosis of rabies
• -considered the most rapid fixative

• Newcomer’s Fluid
• -recommended for mucopolysaccharides and nuclear
proteins
• -better rxn in Feulgen stain than Carnoy’s
• F. Osmium Tetroxide Fixatives

• - should be kept in a dark-colored, chemically clean

• bottle to prevent evaporation and reduction by
• sunlight or organic matter
• - inhibits hematoxylin and makes counterstaining
• difficult

• *Produces black precipitates when exposed to sunlight

• (Osmic oxide)
• *Prevention!: add saturated aqueous mercuric chloride
• *Remedy: Black osmic oxide crystals may be
• dissolved in cold water
• *Precaution: may cause conjunctivitis or blindness
•
• a. Flemming’s Solution
• b. Flemming’s w/o acetic acid
• Flemming Sol’n
• -common chrome-osmium acetic acid fixative
• -recommended for nuclear preparation
• -excellent for nuclear structures
• -permanently fixes fats
• Fixation Time: 24-48 hours

• Flemming’s Sol’n w/o acetic acid

• -for cytoplasmic structures
• -chromic and osmic acid
• Fixation Time: 24-48 hours
• G. Trichloroacetic Acid
• -ppt proteins
• -weak decalcifying agent
•
• VIII- Acetone:
• -used for the Dx of Rabies
• -recomm. for water diffusible enzymes (e.g. lipase)
•
• IX- Heat Fixation
• a. Direct flaming fixation
• b. Microwave fixation
• (optimum temp. 45 – 55oC)
• *underheating – poor sectioning
• *overheating – (above 65oC)
-vacuolation
• -overstained cytoplasm
• -pyknotic nuclei
• Secondary Fixation
• -process of placing a fixed tissue in a second fixative
• -to improve demonstration
• -for special staining techniques

• Post-Chromatization
• -form of secondary fixation
• -2.5-3% potassium dichromate (mordant)

• Washing-Out
• -process of removing excess fixative from the tissue after fixation
• 1. Tap H2O: remove excess chromate,
• formalin and osmic acid
• 2. 50-70% Alcohol: remove excess picric
• acid (Bouin’s)
• 3. Alcoholic Iodine: remove excess mercuric
• fixatives
• Fixative for E.M.
• 1. Glutaraldehyde
• 2. Platinic chloride (PtCl3)
• 3. Platinic Chloride-formalin
• 4. Gold chloride
• 5. Osmium tetroxide
• 6. 10% NBF
• Factors Enhanced the Fixation
• 1. Size and Thickness of Tissue
• 2. Agitation
• 3. Moderate Heat (37-56oC)

• Factors Affect Fixation

• 1. Size and thickness of Tissue
• 2. Mucus
• 3. Fat
• 4. Blood

• Note!
• NSS used to wash tissue with mucus and blood
• Decalcification
• -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

• -The recommended ratio of fluid to tissue volume for decalcification

is 20:1

• -Heat will serve to hasten decalcification BUT it also increases the

damaging effects on tissues

• -At 37oC = impaired nuclear staining of Van Gieson’s stain for

collagen fibers

• -At 55oC = tissue will undergo complete digestion within 24-48

hours
• -Optimum temperature = RM TEMP (18-30oC)
• -The ideal time required for decalcifying tissue is 24-
48 hours
• -Dense bone tissues usually require up to 14 days or
longer in order to complete the process
• Decalcifying Agent
• -used to remove calcium and lime salts following
fixation.

• Calcium may be removed by the ff:

• 1. Acids
• 2. Chelating Agents
• 3. Ion exchange resins
• 4. Electrical ionization
• ACID DECALCIFYING AGENTS
• 1. Nitric Acid
• 2. Hydrochloric acid
• 3. Formic acid
• 4. Trichloroacetic acid
• 5. Sulfurous acid
• 6. Chromic acid
• 7. Citric acid
 1. Nitric Acid – MOST COMMON & FASTEST!

Disadvan.: progressive tissue damage

 combined with formaldehyde or alcohol

• a. Perenyi’s fluid – acts as BOTH

• tissue softener and decalcifying agent
• b. Phloroglucin-Nitric Acid – most rapid
• decalcifying agent
• c. Aqueous Nitric Add Solution 10%
• d. Formol-Nitric Acid
• 2. Formic Acid -BOTH fixative and decalcifying
agent

• -5% formic acid is considered to be the

• “BEST GENERAL DECALCIFYING AGENT”
• -Formic acid is recommended for small pieces of
bones and teeth

• 3. Hydrochloric Acid
• -for surface decalcification of the tissue blocks

• a. Von Ebner’s fluid – recommended for

• teeth and small pieces of bones
• 4. Trichloroacetic Acid
• -suitable only for small spicules of bone
• -do not require washing out (90% alcohol)

• 5. Sulfurous Acid
• -very weak decalcifying agent, suitable for minute
pieces of bone

• 6. Chromic Acid
• -both fixative and decalcifying agent
• -minute bone spicules

• 7. Chelating Agents
• -EDTA
• Extent of Decalcification
• 3 ways to measure extent of decalcification
• 1. Physical/Mechanical Test – inaccurate
• 2. X-ray or Radiological Mtd – very expensive,
• “MOST IDEAL, MOST SENSITIVE, MOST
• RELIABLE”
• 3. Chemical Mtd – simple, reliable,
• recommended for routine purposes
• Tissue Softener
• -for unduly hard tissues that may damage
• the microtome knives
•
• a. 4% aq. Phenol
• b. Molliflex
• c. 2% HCl
• d. 1% HCl in 70% alcohol
• Dehydration
• -Aim: To remove fixative and water from the
tissue and replacing them with dehydrating fluid in
preparation for impregnation.

• -Dehydrating fluids are generally used in

• increasing strengths
•
• Ex. 70% ETOH 95% ETOH 100% ETOH
• Delicate tissue: 30% ETOH

• -Dehydrating agents: Tissue = 10:1

• Characteristics of an Ideal Dehydrating Sol’n
• 1. Dehydrate rapidly
• 2. It should not evaporate very fast
• 3. It should be able to dehydrate even fatty
• acids
• 4. It should not harden tissues excessively
• 5. It should not be toxic to the body
• 6. It should not be a fire hazard

• Commonly Used Dehydrating Agent Are:

• 1. Alcohol
• 2. Acetone
• 3. Dioxane 4 – cellosolve
• 4. Triethyl phosphate
• 5. Tetrahydrofuran
• Commonly Used Dehydrating Agents:
• 1. Alcohol – MOST COMMON

• a. Ethanol = for routine dehydration of

• tissues
• “BEST DEHYDRATING AGENT”
• b. Methyl Alcohol = employed for blood
• and tissue films
• c. Butyl Alcohol = Utilized in plant and
• animal microtechniques
• d. Industrial Methylated Spirit (denatured
• alcohol) = ethanol + small amt. of methanol
• e. Isopropyl Alcohol
• 2. Acetone
• - BOTH fixative and dehydrating agent
• - rapid acting dehydrating agent for urgent biopsies for 1
½ and 2 hours.

• 3. Dioxane (Diethylene dioxide)

• -BOTH dehydrating and clearing agent
• -Expensive and tend the ribbon poorly in sectioning

• 4. Cellosove (Ethylene glycol monoethyl ether)

• -BOTH dehydrating and clearing agent
• -CAUTION: Ethylene glycol are combustible at
• 110-120 C
• -Toxic by inhalation, skin contact
• Note: Propylene-based glycol = Ethylene-based glycol
• 5. THF (Tetrahydrofuran)
• -BOTH dehydrating agent and clearing agent
• - dissolves fats

• 6. Triethyl phosphate
• -soluble to alcohol, chloroform,water, ether,
benzene, acetone and xylene

• Additives to Dehydrating Agents:

• 1. 4% phenol + each 95% ETOH baths
• Note! Phenol serves as tissue softener
•
• 2. Anhydrous copper sulfate
• Clearing
• (de-alcoholization)
• -process whereby alcohol or a dehydrating agent
is removed from the tissue.
• Make microscopic tissue prep transparent

• Characteristics of a good Clearing Agent

• 1. Miscible w/ alcohol and paraffin and
• mounting medium
• 2. It should not produce excessive shrinkage,
• hardening and damage of tissue
• 3. Do not dissolve out aniline dyes
• 4. Do not evaporate quickly
• 5. Make the tissue transparent
• Common Clearing Agents
• 1. Xylene
• 2. Toluene
• 3. Benzene
• 4. Chloroform
• 5. Cedarwood oil
• 6. Aniline oil
• 7. Clove oil
• 8. Carbon tetrachloride
• Xylene (xylol)
• -colorless
• -clearing time ½ to 1 hr
• -both embedding and mounting

• Advantages:
• -most RAPID clearing agent
• -makes tissue transparent
• -miscible to alcohol and paraffin
• Disadvantages:
• -highly inflammable
• -become milky
• Toluene
• -substitute for xylene or benzene
• -both embedding and mounting
• -1-2 hrs

• Advantages:
• -miscible both alcohol and paraffin
• -tissues do not become exces. Hard & brittle
• -not carcinogenic
• Disadvantages:
• -slower than xylene and benzene
• -more expensive
• Benzene
• -recommended for urgent biopsies
• -misicible with alcohol
• -does not make the tissue brittle and hard
• -causes minimum shrinkage
• -carcinogenic
• Chloroform
• -during embedding process
• -slower action than xyline but less brittleness
• -thicker tissue blocks; > 1 cm
• -tissue do not become translucent

• Advantages:
• -recommended for tough tissue
• -miscible w/ abs alcohol
• -not inflammable
• Disadvantages:
• -toxic to liver
• -evaporates very quickly
• -does not make tissue transparent
• Cedarwood oil
• -used both paraffin and celloidin section
• -recommended for CNS tissues
• -2-3 days

• Aniline oil
• -recommended for embryos, insect and delicate tissues.

• Clove oil
• -causes minimum shrinkage

• Carbon tetrachloride
• Methyl benzoate and methyl salicylate
• -slow acting clearing agent
• -double embedding techniques are required
• IMPREGNATION and EMBEDDING

• IMPREGNATION (Infiltration)
• -process of completely removing the clearing
agent
• -replaced by a medium that will completely fill
all the tissue cavities
• -allowing easier cutting and handling
• EMBEDDING
• -process by which the impregnated
tissue is placed into precisely
arranged position in a mold
containing a medium
• Note!
• The medium used to infiltrate the
tissue is usually the same medium
utilized for embedding
Types of impregnating and embedding media:
1. Paraffin wax
2. Celloidin (colloidin)
3. Gelatin
4. Plastic
• Paraffin Wax
• -SMB!

• Advantages:
• 1. Serial sections are cut with ease and majority w/out undue distortion
• 2. Very rapid
• 3. Tissue blocks and unstained can be stored in paraffin for an indefinite
period of time
• 4. Many staining procedures are permitted

• Disadvantages:
• 1. Overheated paraffin makes tissue brittle
• 2. Prolonged impregnation will cause tissue shrinkage
• 3. Inadequate impregnation promotes clearing agent retention (tissues
become soft and shrunken, crumble and break –up)
• 4. Tissues difficult to infiltrate need long immersion for proper support
• 5. Not recommended for fatty tissues
•
• Paraffin Wax
• -melt in paraffin oven or an incubator
• -should be regulated at 55-60 oC
• -common waxes melting points:
• a. 45 oC
• b. 52 oC
• c. 56 oC
• d. 58 oC
• Laboratory Temperature
• 20-24 oC  54-58 oC
• 15-18 oC  50-54 oC
• Note!
• HARD tissues require wax with a HIGHER melting point
than SOFT tissues
•
• 3 Ways of Impregnation and Embedding of
tissues

• 1. By Manual Processing
• 2. By Automatic Processing
• 3. By Vacuum Embedding
• Manual Processing
• -4 changes of wax for 15 minutes each
intervals
Fixation
10% Buffered Formalin 24 hours
Dehydration
70% Alcohol 6 hours
95% Alcohol 12 hours
100% Alcohol 2 hours
100% Alcohol 1 hour
100% Alcohol 1 hour
Clearing
Xylene 1 hour
Xylene 1 hour
Impregnation
Paraffin wax 15 minutes
Paraffin wax 15 minutes
Paraffin wax 15 minutes
Paraffin wax 15 minutes
Embedding
Paraffin wax 3 hours
• Automatic Processing
• -processing machine (autotechnicon)
• -2 to 3 changes
12 Processing Steps
* 10 1 liter glass beakers
* 2 thermostatically wax baths
* Transfer arm (clock schedules)
-Agitation of fluid
*Electrical clock
• Factors Affecting Paraffin Wax Impregnation

• 1. Nature and size of the tissues

• Note!
• *Larger and denser tissue blocks  longer period of time
and frequent changes of wax

• 2. Type of clearing agents

• Note!
• *Benzene and Xylene are easily removed
• *Chloroform and Cedarwood oil are difficult to remove
and require frequent wax changes

• Note!
• *Addition of benzene increased the displacement of
cedarwood oil w/ less tissue shrinkage
• Vacuum Embedding
• wax impregnation under negative
• atmospheric pressure inside an
• embedding oven
• Recommended for:
• 1. Urgent biopsies
• 2. Delicate Tissues (Lungs, Brain, CT)
• 3. Decalcified bones
• 4. Eyes
• 5. Spleen
• 6. CNS
• Reduced from 25-75% normal impregnation time
• Note!
• 1. Prolonged treatment of melted paraffin
causes:
• a. shrinkage
• b. hardening of tissues
• c. difficult to cut
• 2. Infiltration of overheated paraffin ( ↑60oC)
• a. shrinkage
• b. hardening of tissues
• c. destroy lymphoid tissues completely
•
• Note!
• 3. Paraffin wax must be pure; free from:
• a. Dust
• b. Water droplets
• c. Other foreign materials
• 4. Paraffin may used twice only
• 5. Fixed microtomes  hard wax and higher m.p.
• 6. Heavier microtome knives  harder paraffin
• wax than lighter
• Substitutes for Paraffin Wax
• 1. Paraplast
• -Highly purified and synthetic plastic polymers
• -w/ a m.p. of 56-57oC
• -more elastic and resilient than paraffin
• -large dense tissue blocks (bones and brain)
• -prevents ice crystals formation
• -no deposits after staining
• -soluble in common clearing agents
• -same time schedule like paraffin
• -do not crack
• Embeddol: synthetic wax w/ m.p. of 56-58oC
• Bioloid: semisynthetic wax, recomm. for eyes
• Tissue Mat: product of paraffin, containing rubber
• w/ the same property as paraplast
• 2. Ester wax
• -m.p. of 46-48oC
• -harder than paraffin
• -not soluble to water
• -soluble to 95% ETOH and other clearing agents
• Note!
• it is used w/ heavy duty microtome such as:
• a. sliding microtome
• b. sledge microtome
• 3. Water Soluble Waxes
• -polyethylene glycol
• -w/ m.p. of 38-42oC or 45-56oC
• -Carbowax: most commonly used
• -soluble and miscible w/ water
• -does not require dehydration and clearing
• -does not remove neutral fats and lipids
• -suitable for enzyme histochemical studies
• -cytological details are excellently preserved

• Note! Carbowax
• a. easily dissolved in water
• b. difficult to float out mount due to its extreme solubility in
water, dehydrating and clearing agents
• c. adding soap or 10% Polyethylene glycol to water reduce
tissue distortion and promote flattening and “floating out”
of sections.
• Celloidin Impregnation
• -purified form of nitrocellulose
• -suitable for large hollow cavities, hard and dense tissues such as
bones and teeth
• -for large sections of embryo

• Advantages:
• 1. permits cutting which are thicker than in paraffin wax
• 2. its rubbery consistency allows the tissue to be cut w/o undue
distortion
• 3. Dense tissues w/c are hard to infiltrate are supported better
• 4. It does not require heat during processing
• Disadvantages:
• 1. very slow
• 2. very thin sections (↓10 um) are difficult to cut
• 3. vapor of ether are very inflammable
• 4. Photomicrographs are difficult to obtain
• 5. Very volatile
• 2 Methods of Celloidin Impregnation
• 1. Wet Celloidin
• 2. Dry Celloidin

• 1. Wet celloidin
• recommended for bones, teeth, large brain
• sections and whole organs
• Note!
• When ball of the fingers leaves no mark on the surface of
the tissue block the processed is considered to be complete
• 2. Dry celloidin
• preferred for whole eye sections; used of Gilson’s
mixture
• Note!
Does not require alcohol due to the presence of
cedarwood oil in the bock
• Nitrocellulose Method
• -another form of celloidin
• -w/ lower viscosity allowing penetration of the
tissue rapidly
• -makes cutting of thinner sections possible
• Gelatin Impregnation
• -rarely used except when dehydration is to be
avoided or the tissues are subjected
histochemical and enzyme studies
• -prevents fragmentation of tissues when used in
frozen section and as embedding medium
• -soluble to water
• -does not require dehydration and clearing
• Note!
• -tissues should not more than 2-3 mm thick
• -1% phenol: prevent the growth of molds
• -Impregnating medium is 25 times the volume of
the tissue
• Embedding
• -after impregnation the tissue is placed into a
mold containing the embedding medium and
allowed to solidify.
• -5 to 10oC above the m.p.
• -cooled rapidly at -5oC

• Orientation: process by w/c tissue is arranged in

precise positions in the mold during embedding

• Note!
• The surface of the section to be cut should be
placed parallel to the bottom of the mold
•
• Types of Blocking-Out Molds
• 1. Leuckhart’s Embedding Mold

• 2. Compound Embedding Unit

• 3. Plastic Embedding Ring and Base Mold
• 4. Disposable Embedding Molds
• a. Peel-Away

• b. Plastic Ice Tray

• c. Paper boat
• Plastic (Resin) Embedding
• -provides superior results for light microscopic
studies, (hard tissues)

• Plastic Classification
• 1. Epoxy
• 2. Polyester or acrylic
• Epoxy Embedding Plastics
• -made up of balanced mixture of:
• a. epoxy plastics
• b. catalysts
• c. accelerator

• 3 types of epoxy plastics

• 1. Bisphenol A (Araldite)
• 2. Glycerol (Epon)
• 3. Cyclohexene dioxide (Spurr)

• -hydrophobic
• -oxidation of peroxide
• -carcinogenic (Vinylcyclohexane dioxide)
• Polyester Plastic
• -introduced for electron microscopy (mid 1950’s)

• Acrylic Plastics
• -made up of esters of acrylic or methacrylic acids
• -used for light microscopy
• -Polyglycol Methacrylic  extremely hydrophilic
• allowing many staining mtds to be applied
• -Methyl Methacrylic  used for undecalcified
• bone and other tissue
• Microtomy
• -Process by which the
processed tissue is trimmed
and cut unto uniformly thin
slices or “sections” to
facilitate studies under the
microscope.
•
• -Microtome
• capable of cutting a section
at a predetermined
thickness
• 3 Essential Parts of Microtome
• 1. Block Holder
• - where the tissue is held in position

• 2. Knife Carrier and Knife

• - for actual cutting of tissue sections

• 3. 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
 TISSUE
HOLDER

KNIFE
CARRIER AND
KNIFE
• 5 Kinds of Microtomes
•
• 1. Rocking Microtome
• -for cutting serial sections of large blocks of
• paraffin embedded tissues
• 2. Rotary Microtome
• -for cutting paraffin embedded
• sections
•
• 3. Sliding Microtome
• -for cutting celloidin embedded sections
• 4. Freezing Microtome
• -for cutting unembedded frozen sections
• 5. Ultrathin Microtome
• -for cutting sections for electron microscopy
•
•
• Rocking (Cambridge) Microtome
• -inveted by Paldwell Trefall (1881)
• -Simplest microtome
• -Consist of: a. Heavy base
• b. Two arms
•
Lower Arm Upper Arm
Resting on pivots and Carrying the block holder on
supporting one end by means of screw,
column and attached the connected to a lever by a
micrometer screw at the piece of nylon thread
base of which is found the
ratchet wheel
• Cambridge Rocking Microtome
• -tissue cut in 10-12 u thickness
• - available in two sizes
• a. Small blocks of paraffin tissues
• b. Large blocks of paraffin tissues
• -theoretically not recommended for serial
• sections
• -not favored in most laboratories because of
the restrictions in size
•
•
•
• Rotary Microtome
• -invented by Minot in 1885-86
• -most common type used for both routine and
• research laboratories
• -Electrically Driven Rotary Microtome
• also now available and can be ideally used
• when to produce ribbons for serial sections
• Rotary Microtome
• -operated by rotation of the flywheel
• *causing reciprocal motion of the knife
• over the block
• -thickness of section is regulated by ratchet
feed wheel
• -Pawl: used to come in contact with the
ratchet wheel w/c in turn rotates the
micrometer screw
• -Adjustment screws: make the block parallel
to the microtome knife at all planes
• -The knife and the block holder are upward
and in vertical motion allowing excellent serial
sections to be cut
• Rotary Microtome
• -it is heavier and more stable than rocking
microtome
• -more complex in design and construction
• -maybe used in cutting large blocks of tissues
although results are better when the sliding
microtome is used
• The knife is placed in a blade-up position
• Sliding Microtome
• -developed by Adams in 1789
• -2 Types of Sliding Microtome
• a. Base-Sledge Microtome
• b. Standard Sliding Microtome
• Base-Sledge Microtome
• -consists of 2 movable pillars holding
• the adjustable knife clamps, allowing
• the knife to be set at an angle for
• cutting celloidin sections.
• -the chunk or block holder can be moved
• backward and forwards under the knife
• -favored for hard and large blocks
• sectioning.
• -sections are cut flat, allowing
• excellent serial sections
•
•
• Standard Sliding Microtome
• -the block remains stationary while knife is
moved backward and forward.
• -mainly developed for cutting celloidin
embedded tissue blocks
• -more dangerous because of the movable
knife

• *Both these machine, the knife can be set at

an angle for cutting celloidin
• *Recommended for cutting extremely hard
and rough tissue blocks
• Freezing Microtome
• -invented by Queckett in 1848
• -the stage for block holder is hollow and
perforated around its perimeter
• -w/ flixible lead pipe thru CO2 passes from a
cylinder
• -lever operated valve allows the release of
rapid and intermittent burst of CO2
• -a cooling device for lowering the temperature
of the knife to facilitate sectioning
• Freezing Microtome
• -Used to cut:
• a. undehydrated tissues for rapid diagnosis
• b. histological demonstration of fats
• c. for certain neurological structures
• d. for sensitive tissue constituents
• damaged by heat
• Cryostat or Cold Microtome
• -is a refrigerated apparatus used for fresh
tissue microtomy
• -consist of microtome (rotary microtome) kept
inside a cold chamber w/c maintained the
temperature between -5 to -300C (ave. -200C )
• by an adjustable thermostat.
• -Thermostat: capable of freezing fresh tissues
w/in 2-3 minutes
• -Cutting sections of 4 micra
• Cryostat or Cold Microtome
• -Provides sections for fresh tissue examination
• esp. Fluoroscent Antibody Staining techniques
or Histochemical enzymes studies
• -used for intraoperative diagnosis
• Ultrathin Microtome
• -Primarily used for cutting tissue sections at
0.5 micra for electron microscopy
• -The knife used to cut ultrathin sections is
consist of broken plate glass
• -The specimen used is small, fixed w/ osmium
tetroxide and embedded in plastic
• Care of Microtome
• After Sectioning
• 1. Brushed away all the accumulated
• paraffin and small pieces of tissue w/ soft
• brush
• 2. The parts should be wiped w/ xylol
• 3. Movable portions should be oiled to
• prevent rusting
• 4. Microtome must always be covered
• when not in use
• Microtome Knives
• Trimming and section-cutting are done w/ a
microtome knife.

• Three Basic Types or Shapes

• 1. Plane-Concave Knife
• 2. Biconcave Knife
• 3. Plane-Wedge Knife
• Plane-Concave Knife (25 mm in length)
• -One side of the knife is flat while other is
• concave.
• -Less concave sides are recommended for
• cutting Celloidin-embedded tissue blocks
• on a sliding microtome
• -More concave sides used to cut paraffin
• sections on base-sledge, rotary or rocking
•
• Biconcave Knife (usually 120 mm in length)
• -w/ both sides concave
• -recommended for cutting paraffin-
• embedded sections on a rotary microtome

• Plane-Wedge Knife (usually 100 mm in

length)
• -both sides straight
• -recommended for frozen sections or
cutting extremely hard and tough
specimens embedded in paraffin blocks
• -using a base-sledge type or sliding
• microtome
• “Bevel Angle”
• -the angle formed between cutting edge
• -27o to 32o
• Honing and Stropping
• -process where badly nicked knives w/
blunted ends, jagged edges must undergo
sharpening in order to prevent gross
irregularities and tearing of tissue sections.

• Sharpening of knife involves two stages:

• 1. Honing
• 2. Stropping
• Honing
• -removal of gross nicks on the knife (coarse
• honing) to remove blemishes and grinding
• the cutting edge of the knife on a stone
• Hone: a natural sharpening stone or hard grinding surface
(carborundum) serves to remove nicks and irregularities of
the knife edges
• Types of Hones:
• 1. Belgium Yellow – for manual sharpening when cutting
edge has been rendered blunt or nicked. This type give the
• best result.
•
• 2. Arkansas-gives more polishing effect than Belgium
Yellow
• 3. Fine carborundum-much coarser than the first two, used
only for badly nicked knives followed by either the two
• Lubrication: a. Mineral
• b. Clove oil
• c. Xylene
• d. Liquid paraffin or soapy
• water

• Note!
• Honning
• - Edge First, w/ a Heel to Toe direction
• - Minot or plane wedge knife: turned over so as
to sharpen the other surface every 10-20 strokes.
• Stropping
• -process whereby the “burr” formed during
honing is removed and the cutting edge of the
knife is polished.

• Note!
• Edge last, Toe To Heel direction
• Precautions Observed in Stropping
• A. Speed should be avoided, one full second
• should be allowed
• B. Leather strops are dry and require oiling
• C. Strops usually treated w/ vegetable oil
• (e.g. Castrol oil)
• D. Strops should not be used 24-48 hours after
• treatment
• E. Mineral oil is not recommended and should
• never come in contact since it will tend to
• blister and destroy leather
• F. Never wipe the paper or cloth on the knife,
Knife should wipe on the paper or cloth
• Disposable Blades
• -can cut 2-4 micra thick sections

• Magnetic knives
• -suitable for use in the cryostat

• Glass Knives
• -used for trimming and semi-thin sectioning of
tissue blocks for electron microscopy

• Diamond Knives
• -are used to cut any type of resin block for
electron microscopy
• Paraffin Sections
• “Sectioning” is a process whereby tissues are cut
into uniformly thin slices or “sections”
• -microtome
• Three General Types of Tissue Sections
• 1. Paraffin Sections – paraffin embedded tissue
blocks w/c may be cut by rocking and rotary
microtome
• 2. Celloidin Sections – for celloidin embedded
tissues w/c are usually cut by means of the sliding
microtome
• 3. Frozen Sections – w/c may be cut from tissues
that have been fixed and frozen w/ CO2 or for
fresh or fixed tissues frozen w/ cryostat
 Faults Reason Remedy
Brittle or hard tissue Prolonged fixation Tissue may be softened by
Prolonged dehydration soaking in small dish or bowl
Prolonged clearing containing water with detergent,
Prolonged paraffin infiltration phenol or Molliflex
Overheated paraffin oven
Drying out of tissue before actual
fixation
Clearing agent turns milky as soon Water not completely removed Repeat dehydration with absolute
as tissue is placed in it due to incomplete dehydration alcohol, then clear again
On trimming, tissue smells of Clearing agent not completely Block is trimmed down nearest to
clearing agent removed due to insufficient the tissue. Remaining wax is
impregnation melted on embedding oven and
paraffin impregnation is repeated,
changing the paraffin at least once
before blocking
Tissue is opaque, section cutting is Isufficient clearing Repeat clearing; If object has
difficult due to presence of already been embedded, prolong
alcohol clearing up to 12 hours, then re-
embed
Tissue shrinks away from wax Insufficient dehydration, Repeat the whole procedure
when trimmed therefore incomplete clearing and
impregnation
 Faults Reason Remedy
Tissue is soft when block is Incomplete fixation Repeat the whole procedure
trimmed

Air holes found on tissue Incomplete Impregnation Repeat impregnation

during trimming

On trimming, wax appears Contaminated wax Re-embed in freshly filtered wax

crystalline Block not cooled rapidly enough

Paraffin block, after cooling, is Insufficient paraffin impregnation Repeat paraffin impregnation, the
moist and crumbles re-embed

Sections fail to form ribbons Surfaces and Edges of the block are Re-trim the block
not parallel Re-adjust and re-orient the block
Horizontal surface of the block is not Coat horizontal edges of the block
parallel to the knife with wax of lower melting point
Knife is tilted too much Reduce the tilt
Sections are too thick Readjust the thickness of the
Knife is dull sections
Hone and strop
Sections roll up on cutting so Knife is blunt Sharpen the knife
that they adhere and get Tilt of knife is too great Reduce the tilt
broken against the knife edge Knife edge is dirty Clean the knife edge
 Faults Reason Remedy
Ribbon is curved, crooked or Blunt of dull spot on the knife, Adjust the knife so that the knife
uneven instead of straight producing an irregular knife edge edge will present a uniformly
Edges of the block are not parallel sharp edge to the block, or,
but round or wedge shape sharpen.
Knife is not parallel to the block Re-trim the block
Paraffin is impure Readjust knife and block
Repeat impregnation using pure
wax..
Sections are compressed, Knife is blunt or dull Re-sharpen the knife
wrinkled, or jammed Paraffin block is warm and soft Cool the block on ice water until
Knife edge is coated with paraffin firm
Sections are too thin Clean the knife edge
Microtome set screw is loose Readjust thickness of section
Tilt of knife is too vertical Tighten the screw
Reduce tilt
Sections are squashed (width of Bevel of knife is lost due to Re-sharpen, using a knife back or
each section is less than that of incorrect sharpening automatic knife sharpener
block)
A hole is formed in the sections Bubble of dirt formed in the Re-embed in freshly filtered wax if
embedding medium necessary
Hard spot in tissue due to calcium Once embedded in paraffin wax,
decalcification is impractical; use a
 Faults Reason Remedy
Sections of unequal thickness are Tilt of knife is too great or bevel is Reduce the tilt
produced not cleared, hence object is Tighten the screw
compressed against the knife edge Cut blocks into smaller fragments
Clamp set screw on knife or block Soften the blocks in detergent or
holder is loose phenol
Blocks are too large
Blocks are too hard

Sections adhere to the knife or Static electricity due to low Breathe out or blow gently on the
other parts of the microtome atmospheric humidity block and knife to breakup static
Knife edge is dirty electricity, or boil water in the
Knife edge is dull room to increase the humidity
Knife tilt is too great Clean the knife edge
Sharpen the knife
Reduce the tilt

Ribbon is split or lengthwise Nicks or damage on the knife edge Sharpen the knife
vertical scratches are seen on Dirty embedding Re-embed in filtered wax
sections Knife edges is dirty Clean knife edge with xylene
Tilt of knife is too great Reduce the tilt
 Faults Reason Remedy
Sections are lifted from the knife Knife tilt is too great Reduce the tilt
on upstrokes Knife is dull Sharpen the knife
Paraffin is too soft or room Cool paraffin wax in ice water
temperature is warm

Resistance is felt on the lower part Tilt of knife is too small, paraffin Increase the tilt
of the section during cutting block is therefore compressed
against the base of the knife
towards the end of stroke

Horizontal or parallel lines or Knife edge vibrates due to Treat with phenol during
furrows across the section hardness of tissue processing or collodionize
(“Chatters”) are seen, forming Tilt of knife is too great Reduce the tilt
thin and thick zones

Section cut is sometimes thin, Knife is blunt Sharpen the knife

sometimes thick Knife is not clamped properly Adjust the knife
Tilt of knife is too great Reduce the tilt
Knife or block holder is loose Tighten adjusting and locking
Knife tilt is too small that block is screws
compressed by bevel and section Increase the tilt
is not cut
 Faults Reason Remedy
Knife makes a hard metallic Tilt of knife is too slanted or too Re-adjust the angulation of the
scraping or ringing sound on big knife
backstroke, when section is cut Tissue is too hard Take fresh block treated with
Knife blade is too thin phenol during processing

Frozen tissue crumbles and comes Freezing is not adequate Refreeze the tissue block
off the block holder when cut

Frozen tissue chips into fragments Tissue is frozen too hard Warm the tissue with the fingers
when cut
• Principle Of Staining

• Staining – is the process of applying dyes on

the sections to see and study the architectural
pattern of the tissue and physical
characteristics of the cells.
EOSIN

HEMATOXYLIN
• 3 Major Groups

• 1. Histological Staining
• 2. Histochemical Staining
• 3. Immunohistochemical Staining
• Histological Staining
• -Process whereby the tissue constituents are
demonstrated in sections by direct interaction with a
dye or staining solution, producing coloration of the
active tissue component.
• a. micro-anatomic stains
• b. bacterial stains
• c. specific tissue stains

• Histochemical Staining (Histochemistry)

• -various constituents of tissues are studied thru
chemical reactions that will permit microscopic
localization of a specific tissue substance.
• a. Perl’s Prussian Blue  Hemoglobin
• b. Periodic Acid Schiff  CHO
• Immunohistochemical Staining
• -combination of immunologic and
histochemical techniques that allow
phenotypic markers to be detected and
demonstrated under the microscope, using a
wide range of polyclonal or monoclonal,
fluorescent labeled or enzyme-labeled
antibodies.

•
• Methods of Staining

• 1. Direct Staining
• -process of giving color to the sections by
using aqueous or alcoholic dye solutions
• e.g. methylene blue & eosin

• 2. Indirect Staining
• -the action of the dye is intensified by
adding another agent or MORDANT
 MORDANT
-serve as a link between the tissue
TISSUE MORDANT STAIN and the dye

Ex. 1.Potassium alum w/ hematoxylin

in Ehrlich’s Hematoxylin
2. Iron in Weigert’s Hematoxylin

TISSUE ACCENTUATOR STAIN

ACCENTUATOR
-accelerates or hastens the speed of the staining reaction by
Increasing the staining power and selectivity of the dye
Ex. 1. Potassium hydroxide in Loeffler’s methylene blue
2. Phenol in Carbol thionine and carbol fuchsin
• Progressive Staining
• - Process whereby tissue elements are stained
in a definite sequence and applied for a
specific periods of time
• - it is not washed or decolorized

• Regressive Staining
• - The tissue is first overstained to obliterate
the cellular details and the excess stain is
removed or decolorized from unwanted parts
of the tissue, until the desired intensity of
color is obtained
• Differentiation (Decolorization)
• -selective removal of excess stain from the tissue during
regressive staining

• Note: Primary Stain = Basic Dye  Differentiator = Acid Dye

Primary Stain= Acid Dye  Differentiator = Alkaline
Alcohol = acts differentiator for both Basic and Acid Dye
Note: A mordant can act as a differentiating agent
Metachromatic Staining
-use of specific dyes w/c differentiate particular substances w/ a
color that is different from that of the stain itself

-tissue components combine w/ these dyes form a different color

-belongs to the thizine and triphenylmethane group

ex. 1. Methyl violet 6. Methylene blue
2. Cresyl blue 7. Thionine
3. Safranin 8. Toluidine blue
4. Bismark brown 9. Azure A and B
5. Basic fuchsin
Note!
*All metachromatic dyes are cations or basic
• Counterstaining
• -application of a different color or stain to provide
contrast and background to the staining of the
structural components to be demonstrated
CYTOPLASMIC STAINS
RED YELLOW GREEN
1. Eosin Y 1. Picric Acid 1. Light green SF
2. Eosin B 2. Orange G 2. Lissamine green
3. Phloxine B 3. Rose Bengal
NUCLEAR STAINS
RED BLUE
1. Neutral red 1. Methylene Blue
2. Safranin 0 2. Toluidine Blue
3. Carmine 3. Celestine
4. Hematoxylin
• Metallic Impregnation
• -specific tissue elements are demonstrated not
by stains, but by colorless solutions of metallic
salts
Reduced by Tissue
• Matallic Salts Black deposits
• Ammoniacal silver Reduced by Argentaffin cells Black Dip.

• Vital Staining
• -is a selective staining of living cells constituents
• Example: Janus green  Mitochondria

• Note!
• Nucleus of living cells is resistant to vital stains
• Intravital Staining
• -Injection of dye into any part of the animal
body.
• -Producing specific colorization of certain cells
• Examples:
• 1. Lithium
• 2. Carmine
• 3. India ink
• Supravital Staining
• -used to stain living cells immediately after
removal from the living body
• Examples:
• 1. Neutral Dye – best vital dye
• 2. Janus green – recommended for
• mitochondria
• 3. Trypan blue
• 4. Nile blue
• 5. Thionine
• 6. Toluidine blue
• Stains and Staining Solutions
• 1. Natural dyes
• a. Hamatoxylin
• b. Cochineal dyes
• c. Orcein
• d. Saffron
• 2. Synthetic (Artificial) dyes
• a. aniline
• b. coal tar dyes
• c. etc.
•
• Hematoxylin
• -”Hematoxylin campechianum”
• -is not a stain
• -hematin: coloring agent
• -Ripening: oxidation of hematoxylin
• Natural ripening : 3-4 months
• Artificial ripening: hydrogen peroxide
• potassium permanganate
• sodium perborate
• sodium iodate
• mercuric oxide
• *over-ripening
• Cochineal Dyes
• -female cochineal bug (Coccus cacti)
• -treated w/ alum  Carmine
• -powerful chromatin and nuclear stain for
fresh material and smear prep.

• Carmine + Picric acid Neuropathological

• (Picrocarmine) studies

• Carmine + Aluminum chloride GLYCOGEN!

• (Best’s Carmine)
•
• Orcein
• -vegetable dye (lichens)
• -colorless
• -treated w/ ammonia + air  Blue or Violet
• -weak acid
• -used to stain Elastic fibers
• -(litmus) used mainly as indicator
• Synthetic Dyes
• -”Coal Tar Dyes”
• -Chromophores – are substances w/ definite
atomic groupings and capable of producing
visible color. (Benzene  Chromogens)
• -color impart to the tissue is not permanent

• -Chromogen + Auxochrome  Dye

• (coloring property) (dyeing property)
Dye Classification
• Acid Dyes
Basic Dyes
• Neutral Dyes
• 1. Acid Dyes
• e.g.: Acid fuchsin
• Picric acid (fix, differentiate or stain)
• 2. Basic Dyes
• e.g.: Methylene blue (both indicator and dye)
• 3. Neutral Dyes
• e.g.: Romanosky dyes
• Giemsa’s stain
• Irishman’s stain  Leukocyte
• differentiatiom
•
Common Stains
• Hematoxylin

• Alum Iron
– Aluminum hematoxylin
(recom. For progressive stainning)
ex. Ehrlich Hematoxylin (used for regressive)
Harris Hematoxylin (exf. Cytology & sex chromosomes)
Cole’s Hematoxylin
Mayer’s Hematoxylin (repened w/ sodium iodate)
Common Stains
• Hematoxylin

• Alum Iron
- used only for differential and
regressive
ex. Weigert’s Hematoxylin
Heidenhain’s Hematoxylin
Common Stains
Eosin  Cytoplasmic stain
Commonly used as counterstain in H &E
Eosin Y (most common)
Other Stains Used

• Acid Fuchsin-Picric Acid (Van Gieson’s Stain)

– is a mixture of picric acid and fuchsin for the
demonstration of connective tissues.

• Acridine Orange
– is a basic acridine fluorochrome which permits
discrimination between dead and living cells
– giving fluorescence for DNA and a red
fluorescence for RNA.
• Acridine Red 3B
– Is used to demonstrate deposits of calcium salts
and possible sites of phosphatase activity.

• Alcian Blue
– Is a complex, water-soluble phthalocyanin dye,
similar to chlorophyll, which stains acid
mucopolysaccharides by forming salts linkages
with them.

• Aniline Blue
– Is a cytoplasmic stain used for counterstaining of
epithelial sections.
• Basic Fuchsin
– Is a plasma stain utilized also for deep staining of
acid-fast organisms, for mitochondria, for
differentiation of smooth muscles with the used
of picric acid
– It is main constituents of Feulgen’s (DNA) and
Schiff’s reagent for the detection of aldehydes ,
Van Gieson’s solution for connective tissues,
mucin, and for elastic tissue staining.

• Bismarck Brown
– Is used as a contrast stain for Gram’s technique, in
acid fast and Papanicolau method, and for staining
diphtheria
• Carmine
– Is usually combined with aluminum chloride to
stain glycogen ( Best Carmine Solution)

• Mayer’s Carmalum Solution

– Is a mordanted dye acting as a basic dye and
staining acidic substances.

• Celestine Blue
– resistant to strong acid dyes
– recommended for routine staining of fixed
sections, giving a good nuclear definition when
used in conjunction with alum hematoxylin.
• Congo Red
– Is best known as an indicator, but may be utilized
as a stain for axis cylinders embryos.
– it is used as a 4% aqueaous solution in Krajian’s
method of staining elastic tissues, amyloid and
myelin

• Crystal Violet
– Is a nuclear or chromatin stain used for staining
amyloid in frozen sections and platelets in blood .

– Crystal + Methyl + Dexterin = Gentian Violet

violet violet
• Giemsa Stain
– is used for staining blood to differentiates
leukocytes

• Gold sublimate
– Is the stained used for metallic impregnation ,
made up of gold chloride and mercuric chloride

• Iodine
– It is used for microscopic study of starch granules.
– It is used for removal of mercuric fixative artifact
pigments
– Reagents to alter crystal and methyl violet so that
they may retained by certain bacteria and fungi.
• Janus Green B
– Is used for demonstrating mitochondria during
intravital staining.

• Malachite Green
– It is used as a contrast stain for staining ascaris
eggs and erythrocytes, and as a bacterial spore
stain.
– It is also used both as a decolorizer and as a
counter stain

• Methyl Green
– Stains chromatin green in the presence of an acid.
– It gives false positive reaction with certain
secretions as mucin.
• Methylene Violet
– is a metachromatic dye formed whenever
methylene blue is heated in fixed alkali carbonate
– coloring nuclei of leukocytes reddish-purple in the
presence of methylene blue.

• Neutral Red
– Is a basic dye recommended for observing cell
granules and vacuoles of phagocytic cell.

• Night Blue
– Used as a substitute for carbol fuchsin in acid-fast
staining.
• Orcein
– Is an excellent stain for elastic fibers (Tanzer Unna
Orcein Method)
– especially recommended in dermatological studies

Osmium Tetroxide
– Fixative
– Used to stain fats
– Fats reduces Osmium tetroxide  Osmium dioxide
(Black)
Picric Acid
– Contrast stain to acid fuchsin
– Demonstration of connective tissue
– Cytoplasmic stain
– Counterstain to violet
– Fixative
– Decalcifying agent
• Prussian Blue
- Utilized to manufacture paints
- Microanatomical color contrast
- Demonstration of circulatory system by injection

• Rhodamine B
– Used with osmic acid to fix and stain blood and
glandular

• Silver Nitrate
– Is used in 10% aqueous solution to prepare
various dilutions to be used in identification of
spirochetes, reticulum and other fiber stains
• Toluidine Blue
– Is a nuclear stain for fixed tissues, used as a
substitute for thionine in fresh frozen tissue
sections.
– It is recommended for staining of Nissl granules
or chromophilic bodies

• Victoria Blue
– Is used for demonstration of neuroglia in frozens
sections.
• Lysochromes (Oil Soluble Dyes)
• Sudan Black
• - most sensetive of the oil soluble dyes
• - greater affinity to phospholipids

• Sudan Black B
• - stains Phospholipids and neutral fats
• do not stain crystalline cholesterol and free fatty acids

• Sudan IV
• - does not color phospholipids, lipid droplets
• - recommended for triglycerides

• Sudan III
• - good as a fat stain for CNS tissues
• ADHESIVES AND MOUNTING MEDIA
• -After Cutting:
• Floated out
• (Bubbles accumulation)
• -smooth teasing needle
• -pulling the ribbon
• Fishing Out
• (Drained)
• Fixed to slides
• -37 C incubator overnight
• -placing in a wax oven (56-60 C
• for 2 hrs)
-drying w/ hotplate (45-55 C) for
• 30-45 mins
• -Using ADHESIVES
• Adhesives
• 1. Mayer’s Egg Albumin
• Formula: Egg white 50 cc
• Glycerine 50 cc
• Thymol 100 mg

• 2. Dried Albumin
• 3. Gelatin (1%)
• 4. Gelatin-formaldehyde mixture
• 5. Starch Paste
• 6. Plasma
• 7. Poly-L-Lysine
• 8. APES ( 3-aminopropylthriethoxysilane)
• Mounting Medium
• -Protects the stained section from getting
scratched, bleaching and deterioration
• - Permanent keeping
• Facilitate easy handling
• Storage
• -Prevent distortion of image

• - 1.518 glass refractive index

• Characteristic of Good Mounting Medium
• 1. To avoid distortion of image (1.518)
• 2. Miscible w/ xylene and toluene
• 3. It should not dry quickly
• 4. It should not crack or produce artefactual
granularity upon drying
• 5. It should not dissolve out or fade tissue
secretions
• 6. It should not cause shrinkage and distortion of
tissues
• 7. It should not leach out any stain or affect
staining
• 8. It should not change in color or pH
• 9. It should set hard, thereby producing
permanent mounting of sections
• Mounting Medium Main Groups
• -Aqueous Media
• -Resinous Media

• 1. Aqueous Media
• a. Water
• b. Glycerin
• c. Farrant’s Medium
• d. Apathy’s Medium
• e. Brun’s Fluid
• 2. Resinous Mounting Media
• a. Canada Balsam
• -recom. for whole mounts and thick sections)
• b. DPX (1.532)
• -recom. for small tissue secretions but not
• whole mounts
• c. XAM (1.52)
• -synthetic resin mixture in xylene
• d. CLARITE (1.544)
• -synthetic resin w/c is soluble to xylene
• e. PERMOUNT
• f. H.S.R.
• g. Clearmount
• Ringing
• -process of sealing the margins of the
coverslip to prevent escape of fluid or semi-
fluid mounts

• Ex. Kronig
• Duroflex
• Stains for Carbohydrates
• 1. Periodic Acid Schiff (PAS)
• 2. Best Carmine  Highly Specific for Glycogen
• 3. Langhan’s Iodine  Oldest stain, obsolete
•
• Mucin
• Acid Mucopolysaccharides
• 1. Toluidine blue and Azure A
• 2. Uranyl nitrate-Azure Method
• 3. Alcian blue
• 4. Colloidal iron technique
• 5. Aldehyde fuchsin stain
• 6. Mucicarmine stain (south gate mucicarmine)
• 7. Fluorescent acridine orange technique
•
•
• *Southgate Mucicarmine Technique
• -useful for encpsulated fungi
• e.g. C. neoformans

• Note!
• Mucoproteins and Glycoproteins = (+) PAS

• Mucoproteins and Glycoproteins = (-) Alcian blue

• Mucopolysaccharides = (+) Alcian blue

• Staining of Fats or Lipids
• 1. Simple Stain (Neutral fats)
• Esters of fatty acids w/ alcohol
• Triglycerides
• -Sudan Black B
• -Sudan IV
• -Oil Red O
• 2. Compound Lipids
• Fatty acids + alcohol + Phosphorus or Nitrogen
• a. Phospholipid
• -Sudan Black B
• -Acid Hematin
• b. Glycolipids
• -Sudan Black B
• -PAS
• 3. Derived lipids
• Sudan Black Method
• Results:
• Lipids – Blue Black
• Nuclei – Red

• Sudan IV (Scharlach R)
• Results:
• Lipids – Red
• Nuclei – Blue/Black

• Oil Red O in Dextrin

• Results:
• Fats – Brilliant Red
• Nuclei – Blue
• Nile Blue Sulfate Method for Fats
• Results:
• Neutral Fats – Pinkish red
• Cholesterin esters – Light red
• Cerebrosides – Light blue
• Fatty acids and soap – Deep blue to violet
• Staining of Proteins, Enzymes and Nucleic
Acids
• Nucleic Acids

• 1. Feulgen Technique  DNA

• (most reliable and specific)
• 2. Methyl Green-Pyronin Technique  RNA
• Differential staining:
• -Methyl green  DNA
• -Pyronin  RNA

• Results:
• DNA – Green or Blue Green
• RNA – Red-rose; granules (dark-rose-red)
• Plasma cell cytoplasm – Purple
• Nucleic Acid
• 3. Fluorescent Staining for DNA and RNA

• 4. In-Situ Hybridization
• -most sensitive technique for identifying DNA
• 5. PCR
• Staining of Connective Tissue
• 1. Loose Connective Tissue
• 2. Adipose Tissue
• 3. Dense Connective Tissue
• 4. Cartilage tissue
• Reticulin Connective Tissue
• 1. Silver Impregnation Technique
• a. Gomori’s Silver Impregnation Stain
• results:
• Reticulin fibers – Black

• Collagen
• 1. Van Gieson’s Stain  Pink or deep red
• 2. Masson’s Trichrome Stain  Blue
• 3. Mallory’s Aniline Blue Stain  Red
• 4. Azocarmine Stain
-Amyloid & Colloid  Deep blue
-Nuclei  Red
• 5. Krajian’s Aniline Blue Stain
• Elastic Fibers
• 1. Weigert’s Elastic Tissue Stain  Dark blue or Blue
• Black
• 2. Taenzer-Unna Orcein Method  Dark brown
• 3. Verhoeff’s stain  Black
• 4. Gomori’s Aldehyde-Fuchsin Stain
• 5. Krajian’s Method  Bright red
• RBC  Orange-yellow
• Basement Membrane
• 1. Periodic Acid Schiff (PAS)
• Fibrin
• 1. Mallory’s Phosphotungstic Acid Hematoxylin
• -Blue black
• 2. Martius, Scarlet Blue technique
• Collagen  Blue
• Fibrin  Red
• Erythrocytes  Yellow
• Amyloid
• 1. Gram’s Iodine Stain  purple or blue color
• 2. Congo Red Method  deep pink to red color
• 3. Metachromatic Staining
• 4. Induced Fluorescence Staining w/ Thioflavine
•  Silver blue fluorescence
• 5. Krajian stain
• 6. Lieb’s Crystals Violet
• Staining of Muscles and Bone

• Bone
• 1. Modified Gomori’s Trichrome Stain
• 2. Rapid Gomori Trichrome Stain for Frozen Muscle
• Tissue
• 3. Mallory’s Phosphotungstic Acid Hematoxylin
• (PTHA) Myelin  Blue
• Muscle  Blue
• 4. Lissamine Fast Red –Tartrazine Method For
• Muscles and Bone
• Muscles  Red
• Bone
• 1. Schmorl’s Picro-Thionin Method
• Bone matrix  Yellow or Brownish-yellow
• Staining of Bone Marrow and Blood Elements

• 1. Rapid Toluidine-Eosin Stain for Glycol

• Methacrylate Sections
• -Eosinophilic granules  Red
• -Basophilic granules  Blue
• and Mast cells
• 2. Romanosky Stains (Methylene Blue/Azure B and
• Eosin, dissolved in acetone-free methanol)
• 3. Wright’s Stain (Methylene Blue is polymerized w/
• Sodium bicarbonate and dissolved in Methyl alcohol)
• 4. Giemsa Stain
• 5. Wright-Giemsa or Jenner-Giemsa Stain
• 6. May-Grunwald-Giemsa Stain
• 7. Peroxidase Reaction for Myeloid Cells
•
• Staining of Central Nervous Tissue
• 1. Bielschowsky’s Technique
• For: Neurons
• Axons
• Neurofibrils
• 2. Bodian’s Stain
• For: Nerve fibers and Nerve Ending
• -demonstration of Neuritic plaques and
• neurofibrillary tangles for the diagnosis of
• Alzheimer’s disease
• 3. Sevier-Munger Technique
• For: Neutral Tissues
• - demonstration of Neuritic plaques and
• neurofibrillary tangles for the diagnosis of
• Alzheimer’s disease
• H&E
• Cresyl Fast Violet
• Toluidine blue Nissl Bodies
• Methylene blue
• Thionine

• Cajal’s Gold Sublimate  Astrocytes

• Modified PTAH Stain  Reactive Astrocytes
• Luxol Fast Blue –PAS-Hematoxylin Myelin
• Weil’s Method Sheath
• Masson Fontana Technique  Melanin and
• Argentaffin

• Von Kossa’s Silver Nitrate Method  Calcium

• Lindquist’s Modified Rhodanine Technique
•  copper
***ALL IS WELL***