HISTOPATH LABORATORY 3.

The best microtome for ophthalmology and large

neuropathology section
TOPIC 1: MICROTOMY Disadvantages
MICROTOMES - It is the main instrument by which we 1. Difficult to get thin section
2. large slides are required
cut the embedded tissue in the paraffin block as thin section.
Sliding microtome
 Rotary microtome • knife Is static
 Rocking microtome • block moves horizontally over the knife
 Base sledge microtome Advantages
 Sliding microtome 1. Large sections can be cut
 Cryomicrotome 2. Mainly used for celloidin-embedded tissue.
 Ultramicrotome 3. Simpler design and easy maintenance.
4. Brain sections can be cut better by this type of
 Laser microtome
microtome.
Disadvantages
Rotary microtome 1. The knife may glide in case of hard tissue and
• Most commonly used microtome in routine lab may jump.
• Cutting blade is kept in horizontal position 2. Long knives are difficult to sharpen
Cryomicrotome
• Block containing tissue moves up and down
with help of rotary handle attached with the • Used for: cutting tissue for frozen sample
microtome • Sample is made hard in liquid nitrogen and then
Advantages cut by the microtome in the chamber that
1. Good-quality 2–3-μm-thin section is possible. contains liquid nitrogen
2. Heavy and stable automated rotary microtome Advantages
reduces health hazard and gives the best-quality 1. To get rapid section for rapid diagnosis
section. 2. To study nerve biopsy
3. Good tissue ribbon production. 3. To study enzyme histochemistry
4. Easy-to-cut various types of tissue: firm, fragile, Disadvantages
small biopsy, etc 1. It needs continuous supervision to maintain the
Disadvantages temperature.
1. Expensive. 2. Freezing artefact is often seen
2. Unsuitable to cut large block. 3. Very expensive instrument.
3. Knife faces up and so may be dangerous to the 4. fixed tissue is very difficult to cut.
technical staff Ultramicrotome
Rocking microtome • used for: cutting ultrathin sections for
• AKA: Cambridge rocking microtome transmission electron microscopy
• The word “rocking” is used as there is a rocking • section are cut between 40 and 100 nanomicron
action of the microtome like arm movement thickness with the help of glass knife or diamond
• The knife sis static knife
• Block of the tissue moves in a rocking motion • The tissue is at first trimmed to make small
(arc-like movement) block of 1 × 1 mm size, and then the section is
• One of the oldest designs of the microtome cut by this ultramicrotome with the help of
optical microscope.
• The microtome can cut thin section with ribbons
and is ideal for serial section • The cut section is allowed to float on the water
hold by a boat and then finally picked up on a
Advantages
metal grid.
1. Thin section
Laser microtome
2. Easy to operate
3. Low-cost instrument and reliable • Ultramodern microtome
Disadvantages • The laser beam is used to cut the biological
1. Tissue is curved and the microtome does not section w/o any processing
provide flat section. MICROTOME KNIFE
2. As the microtome is of light weight so vibration
 The microtome knife is important to cut uniform
may occur
and thin section of tissue
 Made of steel
Base sledge microtome
Plano concave (Profile A)
• Block is fixed in a static position within a steel
 Originally these knives were used for cutting
carriage
celloidin-embedded tissue
• Best for large tissue sample or hard tissue
 One side of the knife is plain and the other side
• The tissue sections are usually thick (more than is concave
10um)  This is a very sharp knife and is used for cutting
Advantages soft tissues.
1. Hard tissue can be cut.  However, presently these knives are less
2. Large tissue sample can be cut. frequently used
Biconcave (Profile B) ANGLES OF KNIFE
 Concave on both sides
 Used for rocking microtome
 The concavity of the knife is often difficult to
identify
 a less rigid type of knife and often vibrates
during cutting
Wedge (Profile C)
 plain on both sides
 most widely used knife for routine microtomy
 compatible with different type of microtome
 easy to sharpen
Tool edge (Profile D)
 resembles chisel
 both sides of the knife are plain • Rake angle
 the cutting edge is steep  It is the angle between the upper bevel of
 The tool edge knife is mainly used to cut the the knife and the perpendicular line drawn
hard tissue such as decalcified bone from the surface of the block
 Knife is difficult to sharpen and is not  Increased rake angle makes the section
recommended presently cutting easier
• Angle of clearance
DISPOSABLE KNIFE  It is the angle between the lower bevel of
the knife and the surface of the block. It is
• Low profile blade - used to cut small biopsy or usually around 5°
soft large tissue.  The angle of clearance is related with the
• High profile blade - used to cut firm to relatively friction between the block and knife
hard tissue such as the uterus, heart, etc.  Lower the angle of clearance, the less will
be the compression on the block
Advantages
• Bevel angle
1. Easy to replace within seconds
2. No need to sharpen  This is the angle in between the two
3. The overall cost of disposable blade system is planes of the knife.
low as there is no need of any knife sharpener or • Cutting edge
abrasive powder to sharpen the knife  This is the straight line formed by the
Disadvantages intersecting of the two planes of the bevel
1. They are not very rigid like ordinary knife, and
therefore vibration effect may be seen MICROTOME KNIFE SHARPENING

Manual method:
MATERIALS USED IN KNIFE
• Honing
• Conventional Knife  Abrasive grindings are used to sharpen the
 made of very high-quality carbon or steel knife
material that are usually tempered from  Light oil can be used to lubricate the stone
the edge to inside one third of the width before use.
 desired hardness is between 400 and 900  Iron oxide, aluminum oxide and silicon
Vickers hardness scale carbide are usually used as abrasive
 The handle of the knife is hold by one
• Diamond Knife hand, and the other hand holds the front
 This is a costly knife end of the knife.
 Used to cut epoxy resin blocks in electron  The knife is pushed forward and
microscopy diagonally over the slab several times

• Glass knife • Stopping

 used for ultramicrotomy to cut very hard  It helps to polish the cutting edge of the
tissue knife that is already sharped by honing
 The cutting edge of the knife is parallel to and also to remove any “burr” formed
one surface of the glass. during honing.
 Strop is made of leather and it should be
free from any dust or grit.
 • Automatic knife sharpener 1. Trimming the tissue
 Presently automatic knife sharpener is • needed to expose the tissue piece within
available in the market. the paraffin wax for cutting
 The knife is placed horizontally on the • The block is fixed in the chuck of the
surface of the rotating plate made of glass microtome, and the paraffin is cut till the
or copper coated with abrasive agents. tissue is fully exposed
• Adequate caution should be taken not to
FACTORS INVOLVED IN CUTTING overcut tissue as this may produce various
1. Temperature – lowering the temperature artefactual changes.
facilitates section cutting 2. Cooling the block
2. Angle of rake - Higher rake angle helps in smooth • the blocks are kept for cooling for 15–
flow of ribbons. Lower rake angle is used for hard 20 min.
tissue • this will help to maintain the same
3. Consistency of tissue - Soft tissue is cut at a slow consistency of the paraffin and tissue
rate than the hard tissue. • helps in easy cutting
3. Cutting proper
SECTIONING THE PARAFFIN BLOCK • The block is fixed in the chuck of the
1. Microtome with blade microtome
2. Water bath • The cutting surface of the block should be
3. Paraffin block with embedded tissue to cut 4 parallel to the knife
4. Ice tray • The angle of clearance should be only 2–
5. A blunt forceps or camel brush 5° to have good section
6. Slide rack with slides • The tissue in the block is cut by gentle,
smooth and slow stroke. The ribbon-like
WATER BATH (Floatation chamber) tissue sections are produced
• The tip of the ribbon is held by forceps,
• used to float the tissue after cutting
and the end part of the ribbon is removed
• temperature should be 10 °C below the melting
from the knife edge by brush
point of the embedded paraffin wax and is usually
• In case of any difficulty to get the flat
kept in 40–50 °C.
section, the cutting surface should be
• It is necessary to prevent formation of any air
gently warmed by warm water.
bubbles within the water bath
4. Floating the ribbon
• For adequate floating, the tissue, one can add a few
• The ribbon of the tissue is floated in the
drops of alcohol or little amount of detergent. This
water bath, and this makes the tissue flat
reduces the surface tension of the water and tissue
and removes any wrinkling of the tissue
floats smoothly
• In case of temperature variation in the
BLUNT FORCEPS AND CAMELL HAIR BRUSH bath, the air bubbles may be formed that
may rupture the tissue
• Blunt forceps - helps to manipulate the floating 5. Picking up the tissue
tissue section • The slide is placed vertically within the
• Camel hair brush - is used to clean the blade water bath in front of the tissue, and when
the tissue is touched, the slide is
SLIDE RACK WITH CLEAN GLASS SLIDE
withdrawn vertically from the water
• The clean slides are kept in the slide rack. • To prevent any mix-up, the water bath
• The slides can be already labelled by diamond should be cleaned immediately after
pencil or on the frosted side by lead pencil. cutting each block
• Alternatively, this can be marked after lifting the 6. Drying the section
tissue section. • The slide containing the picked-up section
is kept in slide rack.
ADHESIVE • The slides are now kept in hot oven to get
dry.
• Mayer’s egg albumin and glycerol
• The temperature of the oven should be
• Poly-l-lysine
slightly more than the melting point of the
• 3-Aminopropyltriethoxysilane
paraffin.
• Permanent positively charged slides

STEPS OF TISSUE PROCESSING

TOPIC 2: MOUNTING DISADVANTAGES

• The main function of the mounting media is to give • Yellow staining after some time.
a protective cover over the smear and to make a • Takes time to dry.
permanent bond between the coverslip and the • The basic dyes are poorly preserved
slides
• Mounting medium should have following SYNTHETIC RESIN
properties:
• DPX is the most widely used synthetic resin with a
 The same refractive index of the coverslip refractive index 1.523. It is called DPX as it
and glass slide. The refractive index of the contains:
mounting medium should be 1.52–1.54.
 D = distyrene
 Mounting media should be colourless.
 P = plasticizer (tricresyl phosphate)
 It should quickly dry and stick to the slide.
 X = xylene
 It should resist contamination particularly • DPX is colourless and preserves the stains very
the growth of microbes.
well. It dries also very quickly.
 It should not react with the stain or tissue.
• DPX should be used liberally over the slide, and
 It should be miscible with clearing agent. the excess
 A neutral pH to prevent fading of the • DPX should be wiped off from the coverslip
stain. margin.
Clearing Agent DISADVANTAGE
Xylene (dimethyl benzene)
• Colorless • It frequently retracts from the margins of the
• Refractive index should be the same as the coverslip. This can be prevented by adding a
mounting media and coverslip plasticizer that makes a mesh with plastic.
• It gives transparent cytoplasm
AQUEOUS MEDIA
Warning: toxic
• used when the stains are affected by dehydration by
Mounting media alcohol or clearing by xylene, e.g. fat stain by
AIM: to give a protective cover over the smear and to Sudan black, needs aqueous media.
make a permanent bond between the coverslip and the
slides

Ideal mounting media: • Glycerine-glycerol: This is the commonly used

• The same refractive index of the coverslip and aqueous media for temporary mounting.
glass slide (1.52–1.54). • Polyvinyl alcohol: This is alternative to glycerine
• Colourless. jelly and is often used in immunofluorescence or
• It should quickly dry and stick to the slide. frozen section of lipid stains.
• Resist the growth of microbes.
• No reaction with the stain or tissue APPLICATION OF MOUNTING MEDIUM
• Miscible with clearing agent.
• Put one to two drops of mounting medium on the
• A neutral pH to prevent fading of the stain.
middle of the tissue section over the slide.
• Low viscosity.
• Select an appropriate coverslip for the section.
Types of mounting media • Rapidly invert the slide over the coverslip.
• Neutral resins: such as Canada balsam and • Mounting medium slowly spreads under the
Euparal coverslip.
• Synthetic resins: DPX
• Aqueous media: glycerine, polyvinyl alcohol,
etc.

NATURAL RESIN

• commonly used neutral resin is Canada balsam

• The refractive index of it is 1.523. Canada balsam
is well soluble in xylene and is made as
 Canada balsam: 60 g
 Xylene: 100 ml
CAUTIONS: • The second change of xylene will also raise the
refractive index of the glass slide, thereby reducing
• Too small amount of mounting medium: Air light refraction during microscopic examination.
bubbles may appear.
• The stained section may be left in xylene for an
• Too much amount of mounting medium: It may indefinite period of time until it is finally mounted
spread beyond the edges of coverslip, and the on the slide.
sample may also float
• Sections may float off the slide during staining if
COVERSLIP the slides are dirty or greasy, or if the sections have
not been left in the paraffin oven long enough to
• Clear glass of 0.130–0.170 mm thickness dry and be fixed in the slide. Sections must be left
• Plane surface and straight margin in the oven for a minimum of 30 minutes before
• Sufficiently wide to cover the smear they are finally stained to avoid such problems.

TOPIC 3: STAINING HISTOLOGICAL STAINING

PRINCIPLES OF STAINING • process whereby the tissue constituents and general

relationship between cell and tissue are
• Staining is the process whereby tissue components demonstrated in sections by direct interaction with
are made visible in microscopic sections by direct a dye or staining solution, producing coloration of
interaction with a dye or staining solution. the active tissue component.
• Cells may be stained to highlight metabolic
processes, to differentiate between live and dead METHODS OF STAINING
cells in a specimen, to demonstrate the relationship
Direct staining:
between internal and external structures of the
cells, and to identify different types of cells • process of giving color to the sections by using
• Histologic stain - is the purified form of a coloring aqueous or alcoholic dye solutions
agent or crude dye that is generally applied in an • only one dye is used, which is washed away after
aqueous solution. 30–60 seconds, prior to drying and examination.
• The great majority of routine histology is done with • The molecules that make up basic dyes have a
hematoxylin and eosin (H&E) staining, because it positive charge. This is important because the cell
is quick, cheap and informative. wall and cytoplasm of bacterial cells have a
negative charge.
STAINING OF PARAFFIN SECTIONS
 The positively charged dye is attracted to
• Paraffin wax the negatively charged cells, enhancing
 poorly permeable to most staining the ability of the stain to stick to and color
solutions and should therefore be removed the cells. Methylene blue is a classic
from the section prior to staining. example of a simple stain.
 This is usually done by immersing the • This blue stain will color all cells blue, making
paraffin section in a solvent (e.g. xylene) them stand out against the bright background of the
two times, at 1-2 minutes duration each, light microscope.
for sections up to 10 micron thick
Indirect staining:
• Xylene
 not miscible with aqueous solutions and • process whereby the action of the dye is intensified
low graded alcohol, and should therefore by adding another agent or a MORDANT which
be subsequently removed with absolute serves as a link or bridge between the tissue and the
alcohol, followed by descending grades of dye, to make the staining reaction possible.
alcohol to prevent damage and detachment • The mordant combines with a dye to form a
of sections colored "lake", which in turn combines with the
 The alcohol is then finally replaced with tissue to form a "tissue mordant-dye-complex" that
water before actual staining of section is is rendered insoluble in ordinary aqueous and
performed. alcoholic solvents
 Such procedure is the exact reverse of • A mordant may be applied to the tissue before the
impregnation and may be summed up by stain, or it may be included as part of the staining
the phrase "Sections to Water". technique, or it may be added to the dye solution
itself.
• After staining, the section is again dehydrated with
increasing grades of alcohol and cleared with two ACCENTUTATOR
changes of xylene to prepare the section for
• not essential to the chemical union of the tissue and
mounting, since most mountants are miscible in
the dye. It does not participate in the staining
xylene.
reaction, but merely accelerates the reaction.
PROGRESSIVE STAINING METACHROMATIC STAINING

• process whereby tissue elements are stained in a • entails the use of specific dyes which differentiate
definite sequence, and the staining solution is particular substances by staining them with a color
applied for specific periods of time or until the that is different from that of the stain itself
desired intensity of coloring of the different tissue (metachromasia)
elements is attained • the actual staining process may involve immersing
• Once the dye is taken up by the tissue, it is not the sample (before or after fixation and mounting)
washed or decolorized. The differentiation or in dye solution, followed by rinsing and
distinction of tissue detail relies solely on the observation.
selective affinity of the dye for different cellular
elements. METALLIC IMPREGNATION

REGRESSIVE STAINING • process where specific tissue elements are

demonstrated, not by stains, but by colorless
• the tissue is first overstained to obliterate the solutions of metallic salts which are thereby
cellular details, and the excess stain is removed or reduced by the tissue, producing an opaque, usually
decolorized from unwanted parts of the tissue, until black deposit on the surface of the tissue or bacteria
the desired intensity of color is obtained • Specific tissue elements are demonstrated, not by
• Routine Hematoxylin and Eosin (H&E) staining is stains, but by colorless solutions of metallic salts
the most common method utilized for which are thereby reduced by the tissue, producing
microanatomical studies of tissues, using the an opaque, usually black deposit on the surface of
regressive staining which consists of overstaining the tissue or bacteria
the nuclei, followed by removal of superfluous and • A metallic impregnating agent is different from a
excessive color of the tissue constituent by acid stain in that it is not absorbed by the tissue, but is
differentiation. held physically on the surface as a precipitate or as
a reduction product in certain tissue components.
DIFFERENTIATION (DECOLORIZATION)
• The most valuable metals for this purpose are gold
• selective removal of excess stain from the (gold chloride) and silver (silver nitrate).
tissue during regressive staining in order that a
VITAL STAINING
specific substance may be stained distinctly
from the surrounding tissues. • selective staining of living cell constituents,
• differentiates or distinguishes between types of demonstrating cytoplasmic structures by
bacteria is termed as a differential staining phagocytosis of the dye particle (cytoplasmic
technique phagocytosis), or by staining of pre-existing
cellular components (true vital staining), as in the
DIFFERENTIAL STAINING
staining of mitochondria by Janus green.
• uses more than one chemical stain to better • Vital stains are excluded by the living cells but
differentiate between various microorganisms or taken up by the already dead cells as in the vital
structures/cellular components of a single organism staining of reticulo-endothelial system with trypan
• This is usually done by washing the section in blue, or propidium iodide for eukaryotic cells
simple solution (e.g. water or alcohol), or by the • The usual purpose is to reveal cytological details
use of acids and oxidizing agents. In general, if the that might otherwise not be apparent; however,
primary stain used is a basic dye, the differentiation staining can also reveal where certain chemicals or
is carried out by an acid solution, while alkaline specific chemical reactions are taking place within
medium is used for differentiation after applying an cells or tissues.
acidic dye
INTRAVITAL STAINING
• Alcohol acts as a differentiator for both basic and
acidic dyes, probably by simply dissolving out the • done by injecting the dye into any part of the
excess dye animal body (either intravenous, intraperitoneal or
• Differential staining is also used to detect subcutaneous
abnormalities in the proportion of different white • producing specific coloration of certain cells,
blood cells in the blood. The process or results are particularly those of the reticulo-endothelial
called a WBC differential. This test is useful system.
because many diseases alter the proportion of • Common dyes used:
certain white blood cells  Lithium
• Gram staining uses two dyes: Crystal violet and  Carmine
Fuchsin or Safranin  India ink.
SUPRAVITAL STAINING 8. Differentiate in 1% acid-alcohol (1ml concentrated
HCl to 99 ml. of 80% ethyl alcohol) for 10-30 sec.
• a method of staining used in microscopy to monitoring the changes in color microscopically
examine living cells that have been removed from until only the nuclei are stained.
an organism 9. Rinse in tap water
• Those that enter and stain living cells are called 10. Blue in ammonia water (average of 5 minutes) or
supravital stains 1% aqueous lithium carbonate until the sections
• due to their toxic interaction inside a living cell, appear blue (about 30 seconds).
when supravital stains enter a living cell, they 11. Wash in running water for 5 minutes.
might produce a characteristic pattern of staining 12. Counterstain with 5% aqueous eosin for 5 minutes.
different from the staining of an already fixed cell If alcoholic eosin is used, the time can be reduced
• dilute solutions: ranging from 1:5,000 to 1:50,000 to 30 seconds or 1 minute.
• common dyes used: 13. If aqueous eosin is used, wash and differentiate in
 Neutral red -probably the best vital dye. tap water under microscope control until the nuclei
 Janus green-especially recommended for appear sharp blue to blue black and the rest of the
mitochondria tissue appear in shades of pink. If alcoholic
 Trypan blue -one gram of dye is solution is used, differentiate with 70% alcohol.
dissolved in 100 ml. of sterile distilled 14. Dehydrate, clear and mount.
water to be used immediately; it is
FROZEN SECION STAINING
dangerous to allow the suspension to stand
for more than one hour, because it is likely • Frozen sections mounted on the slides may be
to become toxic to the cell. stained as in paraffin sections although the duration
 Nile blue of staining is usually shorter
 Thionine • Sections may be mounted in an aqueous medium
 Toluidine blue directly from water if necessary. Frozen sections
may be stained by picking up sections on
HEMATOXYLIN AND EOSIN (H & E) STAINING
albuminized slides and drying them quickly or by
• corner stone of tissue-based diagnosis. The process simple direct staining on a wet slide with an eye
stains thin tissue sections so that pathologists can dropper
visualize tissue morphology
PRECAUTIONS IN STAINING
• The process uses a hematoxylin dye to stain cell
nuclei (and other parts) blue and an eosin dye to • Stains on the skin should be avoided
stain other structures pink or red.  Stains may be effectively removed from
• Routine H&E staining plays a significant role in the skin by prompt topical application of
tissue-based diagnosis by coloring otherwise 0.5% acid alcohol, followed by rinsing
transparent tissue sections, and allowing cell with tap water.
structures including the cytoplasm, nucleus, and • Failure of staining may be due to paraffin, fixative,
organelles and extra-cellular components to be or decalcifying solution that has not been
clearly visible under the microscope thoroughly washed out and removed. Early fixation
• Histology laboratory- all specimens are initially in alcohol before paraffin embedding may have
stained with H&E and additional stains are only been incorrect, for which no remedy can be made
ordered if additional information is needed to • Impurities found in the dye or in the water solvent
provide a more detailed analysis. will affect not only the solubility of the dye but
even the intensity of the staining reaction,
ROUTINE H&E STAINING
necessitating purification and filtering of the dye.
Fixation: Most fixatives can be used except osmic acid Stains that have already been deteriorated should
solutions which inhibit hematoxylin be replaced
• If, after staining, sections are fuzzy and do not
Procedure: appear clear under the microscope, xylol should be
1. 1. Clear paraffin embedded sections in first xylene replenished. There may be water in the absolute
bath for 3 minutes alcohol, moisture in the coverslip, or too much egg
2. Transfer to second xylene bath for 2 to 3 minutes. albumin on the slide, thereby obliterating the image
3. Immerse in first bath of absolute ethyl alcohol for 2 of the stained tissue
minutes • Failure of sections to remain on the slide during
4. Transfer to a bath of 95% ethyl alcohol for 1 or 2 staining could have been due to a dirty or oily slide.
minutes
5. Rinse in running water for 1 minute
6. Stain with Harris alum hematoxylin for 5 minutes
(Ehrlich's hematoxylin requires 15-30 minutes)
7. Wash in running tap water to remove excess stain.
COLLODIONIZATION OF SECTIONS (e.g., proteins) and phenotypic markers under the
microscope.
• Paraffin ribbons containing air bubbles, torn or • widely used in the diagnosis of abnormal cells such
inadequately infiltrated sections are likely to float as those found in cancerous tumors, in the
from the slide when deparaffinized and stained localization of biomarkers and differentially
• They are more firmly attached by coating the slide expressed proteins in different parts of a biological
with dilute (thin) celloidin solutions, a process tissue, and in the detection of specific molecular
known as collodionization, which is also markers that are characteristic of particular cellular
recommended for sections that will be subjected to events such as proliferation or cell death
strong alkaline or acid solutions and for tissues that (apoptosis)
contain glycogen for demonstration • antibody can also be tagged with a fluorophore,
• Procedure: such as fluorescein or rhodamine
 Deparaffinize in xylene • Commercially produced antibodies most frequently
 Dehydrate thru absolute alcohol originate from mice, and less frequently from
 Dip individual slides in Coplin jar rabbits
containing dilute ether alcohol solution. • The degree of autolysis or putrefaction, the
 Dip in dilute ether solution of celloidin selection of fixation medium, fixation duration,
(thin celloidin). incubation period, and concentration of the selected
 Hold slide on one end for 1/2 to 1 minute antibodies can be crucial factors that can affect the
to drain or until the section begins to results of immunohistochemical staining protocols.
whiten around the edges. • immunohistochemical techniques are based on
 Wipe off the back of the slide and place in antigen–antibody bindings, which can be affected
80% alcohol for 3-5 minutes to harden the by inappropriate fixative selection and duration
celloidin. • The current recommendation for
 Stain as desired immunohistochemical techniques is a maximum of
4% neutral buffered formaldehyde solution and, for
RE-STAINING OF OLD SECTIONS
some antibodies, fixation time can be up to a
• Old, bleached or faded sections may be re- maximum of 48h.
stained: the slide is usually immersed in xylene for
STAINS AND STAINING SOLUTIONS
24 hours, or gently heated until the mounting
medium begins to bubble 1. Natural dyes – obtained from plants and animals,
• The coverslip may then be removed by lifting it previously utilized for dyeing of wool and cotton.
with a dissecting needle  cochineal dyes, logwood dyes, and
• The section is placed in xylene for up to 24 hours vegetable extracts
to remove the remaining balsam and then brought 2. Synthetic (artificial) dyes - e.g., aniline or coal tar
down to water dyes
• It is placed in a 0.5 potassium permanganate
NATURAL DYES
solution for 5-10 minutes, rinsed in tap water and
subsequently immersed in 5% oxalic acid for 5 1. HEMATOXYLIN
minutes or until the section is decolorized • natural dye derived by extraction from the core or
• After washing it again in running tap water for the heartwood of a Mexican tree known as
another 5 minutes, the section may then be re- "Hematoxylin Campechianum”
stained with the appropriate staining technique. • most valuable staining reagent used by the
HISTOCHEMICAL STAINING (HISTOCHEMISTRY) cytologist
• Hematoxylin itself is not a true basic dye
• process whereby various constituents of tissues are • The active coloring agent is hematin, which is
studied thru chemical reactions that will permit formed by the oxidation of hematoxylin, a process
microscopic localization of a specific tissue known as "ripening."
substance  This is usually accomplished by exposing
• Chemical ions such as calcium, molecules such as the substance to air and sunlight, thereby
bile pigments, and biopolymers such as cellulose, oxidizing hematoxylin (natural ripening).
DNA and specific enzymes are among the tissue  slow and takes as long as 3-4 months
components that can be identified using
histochemical staining techniques. MORDANTS

IMMUNOHISTOCHEMICAL (IHC) STAINING • substances that combine with the tissue and the
staining solution, forming a "bridge" that allows
• combination of immunologic and histochemical staining reaction to take place.
techniques using a wide range of polyclonal or
monoclonal, fluorescent labeled or enzyme-labeled
antibodies to detect and demonstrate tissue antigens
ALUM HEMATOXYLIN STAINS SYNTHETIC DYES

• recommended for progressive staining of tissues, • AKA: “Coal Tar Dyes”

and are usually counterstained with Eosin, Congo • since they were originally manufactured from
Red and Safranin substances that have been taken from coal tar
• Both the Ehrlich’s solution and the Harris’solution • They are derived from the hydro-carbon benzene
contain Alum Hematoxylin. (C6H6), and are collectively known as Aniline
• Rapid ripening of Ehrlich’s reagent, however, is Dyes.
brought about by the addition of Sodium Iodate;
while Harris solution is ripened with Mercuric CHROMOPHORES
Chloride. • substances with definite atomic groupings and are
IRON HEMATOXYLIN COMPOUNDS capable of producing visible colors.
• These are different from the dyes in that any color
• used only for differential or regressive staining, that they impart to the tissue is not permanent and
using Acid-Alcohol as a differentiating agent. An can, therefore, be easily removed
example of an Iron Hematoxylin compound is • Before a chromogen can properly be called a dye, it
Weigert’s Stain using Iron (Ferric) Chloride. must have the property of retaining its color in the
tissue
COPPER HEMATOXYLIN SOLUTIONS
 This property is acquired by the addition
• are utilized for the study of spermatogenesis. of an auxochrome - an auxiliary radical or
substance which imparts to the compound
H & E STAINING PROTOCOL the property of electrolytic dissociation
• A dye, therefore, should consist of a chromophore
• used frequently in histology to examine thin
and an auxochrome group attached to a
sections of tissue.
hydrocarbon benzene ring.
• Hematoxylin stains cell nuclei blue
• The coloring property is attributed to the
• Eosin stains cytoplasm, connective tissue and other
chromophore, and the dyeing property to the salt-
extracellular substances pink or red.
forming auxochrome.
 Eosin is strongly absorbed by red blood
cells, coloring them bright red.

1. ACID DYE
• where the active coloring substance is found in the
2. COCHINEAL DYES
acid component, and the inactive base, e.g. acid
• old histologic dye extracted from the female
fuchsin, is usually the sodium salt of a sulfonate of
cochineal bug (Coccus Cacti)
rosaniline
• treated with alum to produce the dye: carmine
• ex: picric acid
• widely used as a powerful chromatin and nuclear
• Picric acid - is outstanding in the sense that it is
stain for fresh material and smear preparations
the only substance so far that can fix, differentiate
• When combined with picric acid (picrocarmine), it and stain tissue all by itself
is extensively used in neuropathological studies;
 It may be employed as a counterstain to
and when combined with aluminum chloride
basic cytoplasmic stains, to acid fuchsin in
(Best's carmine stain), it is used for the
Van Gieson's connective tissue staining, or
demonstration of glycogen
to crystal violet for the microscopic study
of fungi.

3. ORCEIN 2. BASIC DYE

• vegetable dye extracted from certain lichens which • where the active coloring substance is found in
are normally colorless a basic component that combines with the acid
• when treated with ammonia and exposed to air, radical
produce blue or violet colors. • ex: methylene blue
• It is a weak acid, is soluble in alkali, and is mainly • Tissues fixed with mercuric chloride and
used for staining elastic fibers formaldehyde usually favor staining with basic
• Litmus is also obtained from lichens, treated with dyes
lime and soda, and exposed to ammonia and air
• not used as a cytological stain because of its poor 3. NATURAL DYES
staining property. It is instead, used mainly as an • formed by combining aqueous solutions of
indicator. acid and basic dyes, capable of staining
cytoplasm and nucleus simultaneously and
differentially.
• Because they are made up of large molecular
complexes, neutral dyes are insoluble or barely
soluble in water, but they are usually soluble in
alcohol.

• Ex: Romanowsky, giemsa, Irishman