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ADHESIVE & MOUNTING MEDIA . ➔ Materials like glass become totally invisible if immersed in a
Mounting solution of the same refractive index.
➔ is the last step in tissue processing that results in a ➔ Refractive index is important because it governs the
permanent histological preparation suitable for microscopy contrast between the cellular detail and the background,
➔ Bubbles accumulating under the ribbon may be removed and also the transparency of the observed sample against
with a smooth teasing needle, care being taken not to tear the bright field of the microscope.
the section.
➔ Bubbles may also be removed by pulling the ribbon very Characteristics of a good mounting medium:
gently across the long edge of a glass slide held below the 1. It should be colorless and transparent.
section in the water bath 2. It should be freely miscible with xylene and toluene.
3. It should not dry to a non-stick consistency and harden
➔ After draining, the sections are fixed to the slides. relatively quickly.
➔ This can be done either by: 4. It should protect the section from physical damage and
○ leaving the slides in a 37°C incubator overnight, chemical activity (oxidation and changes in pH).
○ by placing the slides in a wax oven at 56° to 60°C 5. It should be resistant to contamination (particularly
for 2 hours microorganism growth).
○ by drying the slides on a hot plate at 45° to 55°C 6. It should not cause shrinkage and distortion of tissues.
for 30 to 45 minutes. 7. It should not leach out any stain or affect staining.
8. It should not change in color or pH.
➔ For more delicate tissues like the CNS tissue or brain, a 9. It should be compatible with the adhesive in use.
longer drying time at lower temperature (e.g. 37°C for 24
hours or longer) is recommended to avoid splitting and Mounting media may be divided into two main groups:
cracking of the section due to excess heat. 1. Aqueous Media
2. Resinous Media
ADHESIVE .
➔ An adhesive is a substance which can be smeared on to AQUEOUS MOUNTING MEDIA
the slides so that the sections stick well to the slides. ● Aqueous mounting medium are used for mounting sections
➔ The choice of slide and adhesive will be influenced by the from distilled water when the stains would be decolorized or
staining methods to be subsequently applied. removed by alcohol and xylene as would be the case with
➔ Adhesives are not necessary for routine staining, provided most of the fat stains (Sudan methods) or for
that the slides are clean and free from grease. However, metachromatic staining of amyloid
they are essential for methods that require exposure of
sections to acids and alkalis (especially ammoniacal silver Following are examples of common aqueous mounting
solutions) during staining. media:
➔ If clean grease-free slides are used and sections are 1. Water
adequately dried, the sections will not float off during 2. Glycerin - provides greater visibility if slightly diluted with
staining and adhesive will not be necessary. water

Glycerin - Glycerin jelly is the standard mounting

There are still certain instances when sections may float from
Jelly medium used when dehydration and
the slide:
clearing with xylene cannot be made (as
1. For urgent cryostat sections to be submitted for
in fat stains).
immunocytochemistry
- Pure glycerin has the highest index of
2. For central nervous system tissues
refraction and thus provides the best
3. For tissues containing blood clot
viewing and may be optimal for critical or
4. For tissues which have been decalcified
irreplaceable material, because old
5. When sections are to be subjected to high temperatures
material
Albumin = One disadvantage of using is that it retains some of
Farrant’s Medium
the stain and gives a dirty background.

Apathy’s - Refractive Index 1.52

Thymol resistant organisms growing in the adhesive = have
Medium - This medium is used for methylene
been known to contaminate gram-stained sections and cause
blue-stained nerve preparations and as a
confusion during microscopic examination.
general purpose aqueous mountant.
- It is one of the most useful aqueous
MOUNTING MEDIUM .
mountants for fluorescent microscopy,
➔ a mounting medium is usually a syrupy fluid applied
between the section and the coverslip after staining, setting
Canada - It is a transparent, almost colorless
the section firmly, preventing the movement of the coverslip.
Balsam oleoresin that adheres firmly to glass
➔ It protects the stained section from getting scratched, to
and sets to a hard consistency without
facilitate easy handling and storage of the slides, and to
granulation.
prevent bleaching or deterioration due to oxidation, thereby
- recommended for whole mounts and for
preserving the slides for permanent keeping.
thick sections because it does not shrink
much. It sets hard without granulation; it
➔ Mounting media are often chosen for a specific refractive
is, however, quite expensive.
index (R.I.), which can enhance specimen details or make
them invisible.
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PROGRESSIVE STAINING
- mounting in it implies the use of a
➔ Progressive staining is the process whereby tissue
sequence of dehydration
elements are stained in a definite sequence, and the
staining solution is applied for specific periods of time
DPX - Refractive Index 1.532
➔ The differentiation or distinction of tissue detail relies solely
(Dibutyl - recommended for small tissue sections
on the selective affinity of the dye for different cellular
Phthalate but not for whole mounts because of
elements.
and Xylene) shrinkage produced on drying; hence, it
should be used in excess amounts.
REGRESSIVE STAINING
- It is a colorless, neutral medium in which
➔ With this technique, the tissue is first overstained to
most standard stains are well preserved.
obliterate the cellular details, and the excess stain is
removed or decolorized from unwanted parts of the tissue,
XAM - Refractive Index 1.52
➔ consists of overstaining the nuclei, followed by removal of
- It dries quickly without retraction, and
superfluous and excessive color of the tissue constituent by
preserves stains well
acid differentiation.

Clarite
Differential Staining
➔ uses more than one chemical stain to better differentiate
between various microorganisms or structures/cellular
Mountants for immunochemical staining components of a single organism.
● The choice of mounting medium following immunochemical ➔ also used to detect abnormalities in the proportion of
staining is largely dictated by the label (and in the case of different white blood cells in the blood.
enzymatic labels, the chromogen) used to visualize the
antigen. METALLIC IMPREGNATION .
● Aqueous mounting medium is generally suitable for all ● Metallic Impregnation is a process where specific tissue
enzymatic label/chromogen combinations and fluorescent elements are demonstrated, not by stains, but by colorless
labels. solutions of metallic salts
● A metallic impregnating agent is different from a stain in that
Ringing it is not absorbed by the tissue, but is held physically on the
➔ is the process of sealing the margins of the cover-slip to surface as a precipitate
prevent the escape of fluid or semi-fluid mounts and
evaporation of mountant, to fix the coverslip in place, and to STAINS AND STAINING SOLUTIONS .
prevent sticking of the slides upon storage. HEMATOXYLIN
➔ most valuable staining reagent used by the cytologist
➔ not a true basic dye.
PRINCIPLES OF STAINING . ➔ The active coloring agent is hematin, which is formed by
➔ Staining is the process whereby tissue components are the oxidation of hematoxylin, a process known as
made visible in microscopic sections by direct interaction "ripening."
with a dye or staining solution. ➔ oxidizing agents such as hydrogen peroxide, mercuric
➔ Most cells are colorless and transparent, and therefore oxide, potassium permanganate, sodium perborate or
histological sections have to be stained in some way to sodium iodate
make the cells visible. The same is true of components of ➔ Ripened hematoxylin is seldom used alone due to its
the extracellular matrix. inherent low affinity for the tissue itself
➔ The main reason why cells are stained is to enhance
contrast and visualization of the cell or certain cellular
Mordants are substances that combine with the tissue
components under a microscope.
and the staining solution
➔ Cells may be stained to highlight metabolic processes, to
differentiate between live and dead cells in a specimen, to
Alum are recommended for progressive staining of
demonstrate the relationship between internal and external
hematoxylin tissues, and are usually counterstained with
structures of the cells, and to identify different types of cells.
stains Eosin, Congo Red and Safranin.

Direct Staining:
Iron used only for differential or regressive
➔ Direct staining is the process of giving color to the sections
hematoxylin staining
by using aqueous or alcoholic dye solutions.
compounds
➔ In simple (or direct) staining only one dye is used, which is
washed away after 30–60 seconds, prior to drying and
Copper utilized for the study of spermatogenesis.
examination.
hematoxylin
solutions
Indirect Staining:
➔ Indirect staining is the process whereby the action of the
Hematoxylin used frequently in histology to examine thin
dye is intensified by adding another agent or a MORDANT
and eosin sections of tissue.
(H&E)
staining
protocol
MLS 114 - LAB

COCHINEAL DYES Harris Hematoxylin

➔ Cochineal dye is an old histologic dye carmine ➢ Harris hematoxylin is a good regressive stain that may
➔ widely used as a powerful chromatin and nuclear stain for either be used immediately or stored for future use, since it
fresh material and smear preparations remains stable for a long time (about 6 months).
➔ Picrocarmine ➢ widely used for routine nuclear staining, in exfoliative
➔ extensively used in neuropathological studies; cytology, and for staining of sex chromosomes

ORCEIN Cole’s Hematoxylin

➔ produce blue or violet colors. ➢ Cole's hematoxylin is another alum hematoxylin solution
recommended for routine purposes

SYNTHETIC DYES “Coal Tar Dyes”

Mayer's Hematoxylin
Chromophores are substances with definite atomic
➢ This is an alum hematoxylin that is chemically ripened with
groupings and are capable of producing visible colors.
sodium iodate.
Simple benzene compounds which contain such substances
➢ used as a regressive stain
are known as chromogens. auxochrome
➢ useful as a progressive stain
➢ also used in instances when acid-alcohol differentiation
Acid Dyes - Picric acid is outstanding in the sense that
might destroy or decolorize the stained cytoplasmic
it is the only substance so far that can fix,
components
differentiate and stain tissue all by itself.
- Van Gieson's connective tissue staining
Iron Hematoxylin Solutions
- acidophilic
➢ regressive staining
➢ Two main iron hematoxylin solutions are employed for
Basic - An example of a basic nuclear stain is
routine work in the laboratory: Weigert's Solution, using
Dyes methylene blue
- Tissues fixed with mercuric chloride and ferric ammonium chloride, and Heidenhain's solution,
formaldehyde usually favor staining with using ferric ammonium sulfate (iron alum) as mordants
basic dyes
Regaud’s Hematoxylin for Mitochondria
Neutral formed by combining aqueous solutions of acid ➢ the most permanent and the simplest is Regaud's
Dyes and basic dyes modification of iron hematoxylin on sections of material
fixed in potassium dichromate and formalin and
Common Staining Solutions subsequently mordanted in dichromate.
HEMATOXYLIN
➔ Hematoxylin is the staining solution most commonly used Heidenhain’s Hematoxylin
for routine histologic studies ➢ a popular cytological stain, especially for the study of
➔ can be considered as a basic dye mitosis.
➔ actually a dye called hematin ➢ It can be used after almost any fixative.
➔ Eosin is an acidic dye ➢ Heidenhain's solution is a cytological stain recommended
➔ This means that the nucleus, and parts of the cytoplasm for regressive staining of thin sections.
that contain RNA stain up in one color
Phosphotongstic Acid Hematoxylin (PTAH)
Aluminum Hematoxylin Solutions ➢ The color of the solution ranges from reddish-brown to
➢ Aluminum (alum) hematoxylin stains are recommended for purple
progressive staining of tissues ➢ Staining is usually progressive
➢ The alum hematoxylins can also be used for regressive Eosin - one of the most valuable stains used
staining for differentially staining connective
➢ “Blueing” tissues and cytoplasm.
➢ Aluminum salts give a blue lake - an acid dye and the terms acidophilic,
➢ The two main alum hematoxylin solutions employed are oxyphilic and eosinophilic are often
Ehrlich's hematoxylin and Harris hematoxylin solutions used interchangeably
➢ Alum or potassium aluminum sulfate, when used as the
Yellowish - most commonly used
mordant, usually dissociates in an alkaline solution
(Eosin Y) - readily soluble in water, less in alcohol,
➢ For blueing of alum-hematoxylin -stained sections, warm available in both aqueous and alcoholic
(40° to 50°C) solutions
➢ Blueing with ammonia, lithium carbonate or Scott's Tap
Water Substitute has more rapid action Eosin B The two dyes are interchangeable

Ehrlich's Hematoxylin Romanowsky - are all based on a combination of

➢ As hematoxylin solution becomes oxidized, the color of the Stains eosinate (chemically reduced eosin)
solution will change from purplish to deep red, while the and methylene blue (sometimes with its
oxidation products azure A and azure B
pungent odor of acetic acid will be replaced by a pleasant
- variants include Wright's stain, Jenner's
aroma. stain, Leishman stain and Giemsa stain
➢ Glycerin acts as a stabilize - preferred over H&E for inspection of
➢ Ehrlich's hematoxylin is not an ideal stain for frozen blood cells because different types of
sections. leukocytes
MLS 114 - LAB

OTHER STAINS

Acid Fuchsin-Picric Acid a mixture of picric acid and acid fuchsin for demonstration of connective tissues.
(Van Gieson’s Stain)

Acid Fuchsin (Masson Stain) - used to stain collagen, smooth muscle, or mitochondria.
- used as the nuclear and cytoplasmic stain in Mallory's trichrome method.

ACRIDINE ORANGE - is a basic acridine fluorochrome which permits discrimination between dead and living cells,
giving green fluorescence for DNA and a red fluorescence for RNA.
- It is a nucleic acid selective fluorescent cationic dye useful for cell cycle determination

ACRIDINE RED 3B used to demonstrate deposits of calcium salts and possible sites of phosphatase activities.

ALCIAN BLUE - is a complex, water-soluble phthalocyanin dye, similar to chlorophyll, which stains acid
mucopolysaccharides by forming salt linkages with them.
- an excellent stain because it is simple, it produces a striking blue color, and it is resistant to
various counterstaining procedures.
- more specific for connective tissue and epithelial mucin

ALIZARIN RED S The alizarin method is also used on the Dupont ACA analyzer to measure serum calcium
photometrically

ANILINE BLUE is a cytoplasmic stain used for counterstaining of epithelial sections.

BASIC FUCHSIN - a plasma stain utilized also for deep staining of acid fast organisms, for mitochondria, for
differentiation of smooth muscles with the use of picric acid.
- main constituent of Feulgen's and Schiff's reagent for the detection of aldehydes, of Van Gieson's
solution for connective tissues, mucin,

COLEMAN'S FEULGEN
REAGENT

BISMARCK BROWN is used as a contrast stain for Gram's technique,

CARMINE - is used as a chromatin stain for fresh materials in smear preparations.

- slightly soluble in water at a neutral reaction
- can be used for determining the presence of certain metal ions, such as aluminum

CARMALUM is a mordanted dye acting as a basic dye and staining acidic substances

CELESTINE BLUE an oxazine dye used as an alternative to iron hematoxylin nuclear stain, producing a strong and
precise nuclear stain that is resistant to decolorization

CONGO RED is best known as an indicator, but may be utilized as a stain for axis cylinders in embryos

CRYSTAL VIOLET is a nuclear or chromatin stain used for staining amyloid in frozen sections and platelets in blood.

ETHIDIUM BROMIDE intercalates and stains DNA, providing a fluorescent red-orange stain

GIEMSA STAIN consists of a mixture of methylene-blue and eosin, and it is used for staining blood to differentiate
leukocytes. It is mostly used on methanolfixed blood films

GOLD SUBLIMATE is the stain used for metallic impregnation, made up of gold chloride and mercuric chloride.

- is probably the oldest of all stains, originally used for microscopic study of starch granules.
- It stains amyloid, cellulose, starch, carotenes and glycogen.
- It is widely used for removal of mercuric fixative artefact pigments, and as a reagent to alter
crystal and methyl violet so that they may be retained by certain bacteria and fungi.

Gram's Iodine is used to Gram's Iodineand differentiate bacteria. For example, staphylococci, streptococci and
pneumococci are gram-positive and stain a deep blue, whereas coliforms and Neisseria are
gram-negative and stain pink

Lugol's solution or Lugol's Lugol’s solution is a brown solution that turns black in the presence of starches and can be used as a
iodine cell stain, making the cell nuclei more visible

JANUS GREEN B is used for demonstrating mitochondria during intravital staining.

MLS 114 - LAB

MALACHITE GREEN has sometimes been used for staining erythrocytes is a weakly basic dye 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 counterstain

MASSON’S TRICHROME is (as the name implies) a three-color staining protocol

METHYL GREEN stains chromatin green in the presence of an acid. It gives false positive reactions with certain
secretions such as mucin. Methyl green is used commonly with bright-field microscopes

METHYLENE BLUE is a common basic nuclear stain employed with eosin to provide marked differentiation of various
structures in the tissue. It usually contains some azures or methylene violet

"Polychroming" involves the oxidation of methylene blue, resulting in loss of methyl groups and
leaving lower homologues of the dye (azures)

Mallory's Phloxine Methylene originally known as EosinMethylene Blue (EMB) method

Blue Stain

METHYLENE VIOLET is a metachromatic dye formed whenever methylene blue is heated in fixed alkali or alkali carbonate,

NILE RED (also known as Nile blue oxazone) is formed by boiling Nile blue with sulfuric acid.

OIL RED O is a dye that is more soluble in fat than in water or alcohols, hence it is used as a stain for neutral
lipids

ORCEIN is an excellent stain for elastic fibers, and is especially recommended in dermatological studies due to
its ability to demonstrate the finest and most delicate fibers in the skin

OSMIUM TETROXIDE Osmic Acid or Osmium Tetroxide (OsO4 ) is a selective stain for unsaturated lipids and for lipoproteins
such as myelin

PERIODIC ACID SCHIFF is an all-around useful stain for many things. It stains glycogen, mucin, mucoprotein, glycoprotein,
(PAS) basement membranes, capsules, and blood vessels

PHOSPHOTUNGSTIC ACID is a common negative stain for viruses, nerves, polysaccharides, and other biological tissue materials.

PICRIC ACID is employed as a contrast stain to acid fuchsin, for the demonstration of connective tissue (Van
Gieson's stain), as a cytoplasmic stain in contrast to basic dyes

PRUSSIAN BLUE is an insoluble colored salt of ferric ferrocyanide (an iron cyanide compound) normally utilized for the
manufacture of paints

RHODAMINE B is used with osmic acid to fix and stain blood

SAFRANIN (or Safranin O) is a nuclear stain. It produces red nuclei, and is used primarily as a counterstain

SILVER NITRATE is used in 10% aqueous solution to prepare various dilutions

TOLUIDINE BLUE is a nuclear stain for fixed tissues, used as a substitute for thionine in fresh frozen tissue sections

VAN GIESON STAIN binds to collagen in the extracellular matrix, staining it pink

VICTORIA BLUE is used for demonstration of neuroglia in frozen sections.

VON KOSSA STAIN is a silver reduction method that demonstrates phosphates and carbonates

WRIGHT STAIN causes blood cells to exhibit four major staining properties that allow the cell types to be distinguished

Sudan Black Sudan Black is a stain that colors fat droplets black and is the most sensitive of the oil soluble dyes. It
possesses two secondary amino groups per molecule, making it a slightly basic dye which may cause
non-specific staining.

Sudan IV Sudan IV (Scharlach R) is different from Sudan Black because it has no secondary amino group and it
does not color phospholipids or the fine lipid droplets

Sudan Ill Sudan III was the first Sudan dye to be introduced into histochemistry. It is also fat soluble, and is
good as a fat stain for central nervous system tissues, giving a less deep and lighter orange stain
compared to the darker staining Sudan IV