A TERM PAPER ON

STAINING PROCEDURE
Submitted to the department of Human Anatomy, SHHT,
Federal University of Technology Akure (FUTA)

Submitted to
Dr. Olawuyi Solomon

GROUP LIST
IROEGBU JOY DUBEM HUA/15/3268

FAMOOFO OLUWASEUN HUA/15/3259

OGUNMOLA ESTHER HUA/15/3276

OGUNSOLA ISRAEL HUA/15/3277

ADEPOJU ABIB HUA/15/3228

ABIADE AFEEZ HUA/15/3216

NWACHUKWU PAULINUS HUA/15/32

ADEBOLA WILLIAMS HUA/15/32

BAMISAYE MOBOALJI HUA/15/3252

OJO INIOLUWA HUA/15/3278

ADELE OLAMIPOSI HUA/15/3225

OLAGUNJU ISRAEL HUA/15/3280

TABLE OF CONTENTS
 Introduction
 Definition
 How stains works
 Acidic and basic dyes
 Heamatoxylin
 Eosin
 Convectional histological staining
 Trichromatic staining
 Lysochromes
 Accentuators
 Reference
INTRODUCTION.
While conventional histological staining methods have been established for decades,
some for more than a century, immunohistochemical techniques are not yet routinely used
in forensic diagnostics. They are used, however, when specific problems occur. In such
cases, depending on the problem, routine diagnostics may be supplemented with specific
microscopic techniques, including electron microscopy, laser scanner microscopy, and laser
micro dissection techniques, in order to isolate single cells or cell groups. For important
routine diagnostics, established standard histological staining methods are discussed here.
Basic information on immunohistochemical techniques and on the best-practice use of
immunohistochemical and other methods are mentioned only briefly and therefore do not
substitute reference to the specialist literature.
DEFINITION
Staining is employed to give both contrast to tissue as well as highlighting particular
features of interest in a tissue.
HOW STAIN WORK
The components involved in histological staining are dyes and proteins. The
fundamental process involved is the chemical bonding between the carboxyl groups of one
and the amino groups of the other. The commonest bonds involved are ionic bonds,
although there are exceptions especially in the case of nuclear staining of DNA
Dyes are colored, ionizing, aromatic organic compounds, based on the structure of
benzene. Color is not an objective phenomenon, it is the human detection and perception of
electromagnetic radiation. Visible wavelengths 380-750.
When some of the wavelengths found in white light are absorbed, then we see what
is left over as colored light. The color that we see is referred to as the complementary color
of the color that was removed. For instance, if the red rays are removed from white light, the
color we detect is blue-green
ACID AND BASIC DYES
Most histologic dyes are classified either as acid or as basic dyes. An acid dye exists
as an anion (negatively charged) in solution, while a basic dye exists as a cation (positive
charge). For instance, in the hematoxylin-eosin stain (H&E), the hematoxylin-metal complex
acts as a basic dye. The eosin acts as an acid dye.
Any substance that is stained by the basic dye is considered to be basophilic; it
carries acid groups which bind the basic dye through salt linkages. When using
hematoxylin, basophilic structures in the tissue appear blue. A substance that is stained by
an acid dye is referred to as acidophilic; it carries basic groups which bind the acid dye.
With eosin, acidophilic structures appear in various shades of pink. Since eosin is a widely
used acid dye, acidophilic substances are frequently referred to as eosinophilic.
HEAMATOXYLIN
EOSIN

CONVENTIONAL HISTOLOGICAL STAINING

Conventional histological staining methods, including stain selection for specific
situations, have long been established. Descriptions of the most frequently used staining
methods are shown in (Table 2.1).
stains Presented structures

Haematoxylin–eosin Acidophilic cytoplasm is red, basophil

(H&E) staining nuclei are blue, erythrocytes are red
Alcian blue Detection of acid mucopolysaccharides
Azan staining (azo Connective tissue staining (red): azo
carmine and aniline blue) carmine
stains cell nuclei, erythrocytes, fibrin,
fibrinoid, acidophilic cytoplasm, epithelial
hyaline; Aniline blue (blue): collagen fibers,
fibrous hyaline, basophil cytoplasm, mucus
Best’s carmine stain Classified as a glycogen stain, but is not
specific; also stains mucus, fibrin, gastric
glands, and mast cell granules
Elastika van Gieson (EvG) Combined staining of collagen fibers (red)
and elastic fibers according to Weigert
(black and brown); cytoplasm, musculature,
amyloid, fibrin, and fibrinoid (yellow)
 Elastin staining according Stains elastic fiber violet black
to Weigert
Iron stain (Prussian blue reaction) Stains trivalent iron, in particular
hemosiderin;
detection of iron deposits
Gomori’s stain Argyrophilic reticular fibers (silver)
Grocott’s stain Ideal fungal stain: fungal conidia, fungal
fibers stain black
Mallory’s stain Trichromatic stain; collagen and reticular
connective tissue is light-blue, nuclei are
red, smooth musculature is violet, striated
musculature orange-red, mucus is blue
PAS (periodic acid- Stains carbohydrates, in particular
Schiff’s reagent) glycogen,
purple-red (magenta) and epithelial mucin
Periodic acid – silver Stains basal membranes, Alzheimer’s
plaques, and fungi black
Toluidine blue Detects striation of muscle fibers and
metachromatic substances

TRICHROMATIC STAINING
Staining methods which use two or more acid dyes of contrasting colors to selectively color
different basic tissue components. Most commonly they are used to demonstrate connective
tissue, often in contrast to smooth muscle, but may also be used to emphasize fibrin in
contrast to erythrocytes.
In the trichromatic stains, use is made of dye competition. For instance, acid fuchsine and
picric acid are used in Van Gieson's trichrome stain. In the picric acid-fuchsine mixture, the
small picric acid molecule reaches and stains the available sites in muscle before the larger
fuchsine molecules can enter. Used by itself, acid fuchsine has no difficulty in staining
muscle

LYSOCHROMES
Lysochrome is the technical name for what are more plainly called fat stains, dyes such as
Sudan III, oil red O, and Sudan black
The basis for these dyes coloring fats is that they dissolve into it. From another perspective,
the fat is a solvent for the dye. The Lysol part of lysochrome has the meaning of solution,
and the chrome part refers to color. The word therefore means dissolve colourer

ACCENTUATORS
An accentuator is any chemical which facilitates the staining process. Accentuators fall into
three groups: pH, salts and phenol

REFERENCES
Ananian V, Tozzo P, Ponzano E, Nitti D, Rodriguez D, Caenazzo L (2010)
Tumoural specimens for forensic purposes: comparison of genetic alterations in
frozen and formalin-fixed paraffin-embedded tissues. Int J Legal Med
125(3):327–332, Epub 2010 Apr 6
Bankfalvi A, Navabi H, Bier B, Böcker W, Jasani B, Schmid W (1994a) Wet
autoclave pretreatment for antigen retrieval in diagnostic immunohistochemistry.
J Pathol 174:223–228
Bankfalvi A, Riehemann K, Öfner D, Checci R, Morgan JM, Piffko J, Böcker W,
Jasani B, Schmid KW (1994b) Feuchtes Autoklavieren. Pathologe 15:345–349
Ben-Ezra J, Johnson DA, Rossi J (1991) Effect of fixation on the amplification of
nucleic acids from paraffin-embedded material by the polymerase chain
reaction. J Histochem Cytochem 39:351–354