Stain/certified fixed orthodontic courses by Indian dental academy

INDIAN DENTAL ACADEMY
Leader in continuing Dental
Education
www.indiandentalacademy.com

LEARNINGOBJECTIVES
At the end of the journal club the learner should
be able to
• Define staining
• Classify stains
• Vital stains
• Structure of TB
• Its principle
• Applications
• Method of staining and interpretation

REFRENCES
• Carleton’s: Histological technique, 5th Ed., Oxford
University Press.
• CFA Culling: Cellular pathology technique, 4th Ed.,
Butterworth’s Publication.
• John D. Bancroft: Theory and practice of
histological techniques, 5th Ed., Churchill
Livingstone.
• Toluidine blue uptake in potentially malignant
oral lesions in vivo: Clinical and histological
assessment;Sergio Gandolfo et al;Oral oncologywww.indiandentalacademy.com

• Diagnostic efficacy of Lugol’s iodine and
Toluidine blue in oral premalignant and
malignant lesions; Kamarthi Nagaraju et al; Indian
Journal of Dental Reaserch;2010.
• Advanced Diagnostic Aids in Oral Cancer; KMK
Masthan et al; Asian Pacific J Cancer Prev,2012;
13, 3573-3576.
• Supravital Staining: It’s Role In Detecting Early
Malignancies;Mahesh Chandra Hegde,
Panduranga M et al:Indian Journal of
Otolaryngology and Head and Neck Surgery Vol.
58, No. 1, January-March 2006.
• 06-Cell Patholgy – Ch 06; Staining theory.

STAINING
• Staining may be loosely defined as treating the
tissues or cells with a reagent or series of reagents
so that they acquire a colour, usually no particles
of dye are visible and the stained elements are
transparent.

THEORIES OF STAINING
ELECTROVALENT BONDING:
• Ionic bonding involves electrostatic attraction
between oppositely charged ions.
• One ion is a fixed ion in the tissue section and the
other is the dye ion.
• Anionic (negatively charged) dyes will bind to
cations (positively charged) in the tissue, and
cationic dyes will bind to tissue anions.

Acidic Stains
Basic Stains

HYDROGENBONDING
• It is a force of attraction between a hydrogen atom
in one molecule and a small atom of high
electronegativity in another molecule.
• Thus, it is an intermolecular force, not an
intramolecular force.
• The hydrogen bond has a very limited range and
will only form if the two interacting groups are
brought sufficiently close together.

VanderWaals’ forces
• These are short-range forces and will only have an
effect if the two atoms are between about 0.12 and
0.2 nm apart.
• They are active when there is dipole-dipole
interaction.

COVALENTBOND
• These are very strong bonds and are not easily
broken once formed.
• They are important in the attachment of dyes to
antibodies in immuno-fluorescence.

CLASSIFICATIONOF STAINS
STAINS
CATIONIC ANIONIC SPECIAL

Cationic Dyes
i. Hematoxylins
ii. Azures
iii. Methylene blue
iv. Toluidine blue

Anionic Dyes
i. Aniline blue: blue
ii. Eosin: pink-red
iii. Fast green: green
iv. Orange G: orange
v. Picric acid: yellow

Special Stains and Reactions
i. Elastic stains
ii. Lipid stains
iii. Metachromatic stains
iv. Silver stains
v. Vital stains
vi. Romanowsky dyes.

VITALSTAINS
• A technique in which harmless dye is used to
stain the living tissues for microscopical
observation.
• It can be divided into
INTRAVITAL
SUPRAVITAL

INTRAVITAL
• Intravital staining in the living body (in vivo) .
• The stain may be injected into a living animal
and the stained tissues are removed and
examined.

SUPRAVITAL
• Supravital staining outside the body usually
applied to slide preparation of detached cell.
• The living tissue may be removed directly and
subsequently stained.

INTRODUCTION
• Toluidine blue is an acidophilic metachromatic
dye that selectively stains acidic tissue
components .
• It is also known as tolonium chloride.

• TB has been used as a vital stain to highlight
potentially malignant oral lesions and may
identify early lesions.
• TB may stain malignant epithelia of the mucous
membrane in vivo, whereas normal tissue failed
to retain the dye.
• It can outline the full extent of dysplastic
epithelium or carcinoma prior to excisions.

• Loss of heterozygosity may be detected in TB-
stained lesions. TB-stained tissue may appear
dark royal blue or pale royal blue color

HISTORY
• TB was discovered by William Henry Perkin in
1856.
• TB was first applied for in vivo staining by Reichart
in 1963 for uterine cervical carcinoma in situ.
• Neibel ,Chomet and Shedd first used TB for the
detection of premalignant and malignant lesions of
the oral cavity .

STRUCTURE
• TB also known as methylanaline or
aminotoluene.
• It basically has 3 isoforms, namely, ortho-
toluidine, para-toluidine, and meta-toluidine.

ISOFORMS OF TB
2-amino-1-
methylbenzene
3-amino-1-
methylbenzene
4-amino-1-
methylbenzenewww.indiandentalacademy.com

PRINCIPLE
• TB stains tissues based on the principle of
metachromasia.
• The dye reacts with the tissues to produce a
color different from that of the original dye and
from the rest of the tissue.

METACHROMASIA
• Majority of the dyes stain tissues in differing
degrees of intensity of the same color.
• However , certain tissue components, in the
presence of certain basic dyes, will stain a color
other than that of the dye.
• Such staining reaction is known as metachromasy
and the tissue is said to exhibit metachromasia and
the dye as a metachromatic dye.www.indiandentalacademy.com

• Metachromasia was discovered in 1875 by Cornil,
Jurgens, and Ranvier.
• Among the principal tissue components that exhibit
metachromasia are mucin, cartilage, and mast cell
granules.
• The dyes are mainly of thiazine group, thionine,
TB, azure A, azure B, methyl violet, safranin, and
acridine orange.

PRINCIPLE
• It is a phenomenon whereby a dye may absorb
light at different wavelengths depending on its
concentration and surroundings
• The dye has the ability to change its color
without changing its chemical structure.
• The physical changes that bring about this color
change is dye aggregation.

• Metachromasia is attributed to stacking of dye
cations at the sites of high density of anionic
groups in the tissue.
• Stacking shortens the wavelength of maximum
absorption, a hypsochromic shift.
• So that the maximum wavelength in the spectrum
of the transmitted light is longer.
• This makes the observed color red instead of
blue. www.indiandentalacademy.com

• The dye exists in a normal monomeric
(orthochromatic) form to a potential polymeric
(metachromatic) form.
• The negative charges on the chromotropes attract
the positively charged polar groups on the dye
leading to dye-to-dye aggregation.
• There are three forms of metachromasia alpha (α),
beta (β), and gamma (g) giving a range of colors.

Alpha (α)Beta (β),Gamma(g)

TYPE COLOR STRUCTURE TISSUE
α(alpha) Blue Monomeric Hyaluronic acid
β(beta) Purple Dimer & trimer Pectin
 (gamma) Red Polymeric Mast granules

• The color absorption shifts to shorter wavelengths,
leaving only the longest wavelengths to be seen.
• This is believed to represent polymerization of the
dye.
• The greater the degree of polymerization, the
stronger is the metachromasia.
• Metachromasia requires water between dye
molecules to form the polymer.

• TB is a small weakly hydrophilic cationic dye.
• Attached to DNA or RNA, this dye has a blue color.
• Attached to GAGs in mast cell granules or cartilage
matrix, the dye displays a purple metachromatic
color.
• TB is typically applied from weakly acidic aqueous
solutions.
• DNA, RNA, and GAG are polyanionic, whereas
most proteins are polycationic.www.indiandentalacademy.com

APPLICATIONS
• Connective tissue mucins stains purple to red,
while the background is stained blue.
• Acid mucosubstances (sulphated and
carboxylated) are metachromatic, where as neutral
mucins are orthochromatic.

• These tissues contain highly electronegative
polymers which concentrates the dye molecules
in close proximity to one another.
• These include chondroitin sulphate, heparin,
keratin sulphate & hyaluronic acid.

• Mast cell granules stain purple in color due to
the presence of heparin and histamine.
• Amyloid stain blue but under polarized light they
give a bright red birefringence.
• It is metachromatically stained by methyl violet,
and orthochromatically by standard toluidine
blue method

• The methyl violet is a mixture of tetra, penta, and
hex methyl pararosaniline and coloration of
amyloid is by selective affinity for one of these
colored fractions, hence polychromasia would be
more suitable explanation for this staining.

Endocrine cells:
• Many endocrine cells exhibit masked
metachromasia.
• This characteristic can be unmasked by prior
treatment of tissue sections by hot acid
hydrolysis by HCl.
• This releases carboxyl groups from polypeptides
which are then free to react with and change the
color of basic dyes such as toluidine blue.
• Endocrine cell granules stain – purple to red.

• Corneybacterium diphtheria, stains red violet color.
• These granules contain polymerized inorganic
polyphosphate responsible for metachromasia.
• Toluidine blue or methylene blue granules stain –
red violet color contrasting with the blue staining of
the bacterial protoplasm.
• Helicobacter stains dark blue against a variably
blue background. The concentration of TB used is
1%

TBin MALIGNANCY
• TB is used based on the fact that dysplastic and
neoplastic cells may contain quantitatively more
nucleic acids than normal tissues.
• Also, malignant epithelium may facilitate easy
penetration of the dye.
• The other proposals about the uptake of TB include
the high density of nuclear material, loss of cell
cohesion, and increased mitosis.www.indiandentalacademy.com

PREPARATIONOF TBSTAIN
A 100 mL
of 1% TB
pH =4.5
10 mL of
1% acetic
acid
1 gm TB
powder
86 mL
distilled
water
4.19 mL
absolute
alcohol

PROCEDUREFORTB STAINING
Patient is asked to rinse his mouth
twice with water for 20 seconds
1% acetic acid is given for 20
seconds
1% TB solution is then applied for 20
seconds
1% acetic acid is given for 20
seconds
Water rinse for 20 seconds

INTERPRETATION
Light blue staining is considered doubtful.
A dark blue (royal or navy) stain is considered positive.
No colour absorbed by the lesion, it is taken as a
negative stain www.indiandentalacademy.com

SENSITIVITY ANDSPECIFICITY
STUDY SENSITIVITY SPECIFICITY
Mashburg et al; 1980 86-100 44-100
Rosenberg et al;
1989
93.5% to 97.8 73.3% to 92.9
Epstein et al; 1992 92.5 63.2
Jhonson et al; 1996 100. 62
Lingen et al; 2008 78-100 31-100

CONCLUSION
• Early detection and timely intervention is the
essence of any cancer treatment protocol.
• Supravital staining with 1% toluidine blue is
useful in the early detection of malignancies.
• TB stain is of value due to its high sensitivity but
is reduced in specificity due to the potential of
false positive results in benign lesions.

• It is useful in high risk populations to enable
earlier detection.
• It assists in selecting the best site for biopsy.
• It is very useful in the developing countries like
India because of the cost effectiveness and easy
technique.
• The test is sensitive, simple, noninvasive and
highly cost effective procedure.

PROS
• The chemistry of the
TB dye was
explained.
• The applications of
TB were well
explained.
• The principle of
metachromasia was
well explained.
CONS
• The theories of
staining were not
given.
• The principle behind
the application of TB
was not given in
detail.

Stain/certified fixed orthodontic courses by Indian dental academy

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