STAINS & STAINING

Faculty: Dr. Rakesh Sharda

 OBJECTIVES OF STAINING

Improves visibiltiy by greater contrast between the

organism and the background,
differentiate various morphological types (by shape, size,
arrangement, etc.),
determine the staining characteristic of organism and, at
times, direct diagnosis of disease, and
demonstrate the purity of culture.
observe certain structures (flagella, capsules, endospores,
etc.),
 STAINS AND DYES
➢ A dye is a general-purpose coloring agent,
whereas a stain is used for coloring biological
material.
➢ A stain is an organic compound containing a
benzene ring plus a chromophore and an
auxochrome group.
➢ chromophore is a chemical group that imparts
color to benzene.
➢ auxochrome group is a chemical compound
that conveys the property of ionization of
chromogen (ability to form salts) and bind to
fibers or tissues.
 REQUIREMENTS FOR STAINING
Stain – Majority of the stains used for staining bacteria are of the
basic type as nucleic acid of bacterial cells attract the positive
ions, e.g. methylene blue, crystal violet. Acidic stains are used for
background staining.
Mordant – It is a chemical that forms an insoluble complex with
the stain and fixes it or causes the stain to penetrate more deeply
into the cell. These are used in indirect staining. For example,
Gram’s iodine in Gram staining and phenol in Ziehl Neelson’s
staining.
Accentuater – It is a chemical which when added to a stain to
make the reaction more selective and intense. For example,
potassium hydroxide added in Loeffler’s methylene blue.
Decolorizer – It is a chemical used to remove the excess stain in
indirect regressive staining. For example, ethanol in Gram’s
staining.
 Types of staining techniques

Simple staining Differential staining

(Use of of single stain) (Use of two contrasting stains)

Direct Indirect Separation Visualization

(Positive) (Negative) into groups of structures
1. Gram stain 1. Flagella stain
2. Acid fast 2. Capsule stain
stain 3. Spore stain
SIMPLE STAINING
 SIMPLE STAINING
➢ A staining method that uses only a single dye that which
does not differentiate between different types of organisms
➢ There is only a single staining step and everything is
stained with the same color.
➢ Simple stains are used to stain whole cells or to stain
specific cellular components.
➢ Types of simple staining:
1. Direct / Positive staining : stain object
2. Indirect / Negative staining: stain background
 Direct staining (Positive staining)
➢A simple staining technique that stains the bacterial cells in
a single color.
➢Many of the bacterial stains are basic chemicals; these
basic dyes react with negatively charged bacterial
cytoplasm (opposite charges attract) and the organism
becomes directly stained
➢Examples are methylene blue, crystal violet, and basic
fuchsin.
 Indirect staining (Negative staining)
➢ In this staining process, instead of ells background is
stained.
➢ Here, an acidic dye like nigrosin or Indian ink is
used. Acidic stain carries a negative charge and repelled
by the bacteria, which also carry a negative charge on their
surface. Hence, an acidic dye do not stain bacteria,
➢ Instead, it forms a deposit around the organism, leaving the
organism itself colorless or transparent upon
examination.
 STAINING:
GENERAL TECHNIQUE

SMEAR AIR DRY HEAT FIX STAIN LOOK

 Importance of fixing the smears

“Fixation accomplishes three things:

(1) it kills the organisms;
(2) it causes the organisms to adhere to the slide; and
(3) it alters the organisms so that they more readily accept stains
(dyes).
DIFFERENTIAL STAINING
GRAM’S STAINING
 The Gram Stain
In the late 1800’s, Christian Gram observed that some
genera of bacteria retained a dye-Iodine complex when
rinsed with alcohol, while other genera were easily
decolorized with alcohol and could be then visualized by
a contrasting counter stain.

This staining procedure defines two bacterial groups:

those which retain the primary dyes (“Positive by
Gram’s Method” or “Gram-Positive”) and those which
are easily decolorized (“Negative by Gram’s Method” or
“Gram-Negative”). This is the starting point for
bacterial identification procedures.
 The Gram Stain
➢ The difference in dye retention is dependent on such
physical properties as thickness, density, porosity, and
integrity of the bacterial cell wall, as well as its
chemical composition.
➢ Gram-Positive bacteria have thick, dense, relatively
non-porous walls, while Gram-Negative bacteria have
thin walls surrounded by lipid-rich membranes.
➢ Some non-bacterial organisms with thick cell walls (e.g.,
some yeasts) also stain Gram-Positive.
➢ Gram-Positive bacteria which have lost wall integrity
through aging or physical or chemical damage may
stain Gram-Negative.
 GRAM POSITIVE
Lipoteichoic acid Peptidoglycan-teichoic acid

Cytoplasmic membrane

Cytoplasm

GRAM NEGATIVE Porin

Lipopolysaccharide

Outer Membrane Braun lipoprotein

Inner (cytoplasmic) membrane

Cytoplasm
 Gram staining – Requirements
• Gram-staining is a four part procedure.
• The specimen is mounted and heat fixed on a slide before
proceeding to stain it.
• The reagents required are:
➢Crystal Violet (the Primary Stain)
➢Iodine Solution (the Mordant)
➢Decolorizer (ethanol)
➢Safranin (the Counter stain)
➢Water (preferably in a squirt bottle)
 Gram staining – Procedure
1 The bacteria are first stained with the basic dye crystal violet (primary
stain). Both gram-positive and gram-negative bacteria become directly
stained and appear purple after this step.
2 The bacteria are then treated with gram's iodine solution (mordant). This
allows the stain to be retained better by forming an insoluble crystal
violet-iodine complex, called as ‘iodine lake’. Both gram-positive and
gram-negative bacteria remain purple after this step.
3 Gram's decolorizer, a mixture of ethyl alcohol and acetone, is then
added. This is the differential step. Gram-positive bacteria retain the
crystal violet-iodine complex while gram-negative are decolorized.
4 Finally, the counter stain safranin (also a basic dye) is applied. Since the
gram-positive bacteria are already stained purple, they are not affected
by the counter stain. Gram-negative bacteria, that are now colorless,
become directly stained by the safranin. Thus, gram-positive appear
purple, and gram-negative appear pink.
ACID FAST STAINING
Structure of an Acid-Fast Cell Wall
 Acid fast staining - theory
➢ Once stained the acid fast bacterial cells resist
decolorization with acidified organic solvents, e.g acid
alcohol and are therefore called ACID FAST.
➢ Acid fast staining property of the genus, Mycobacteria,
depends upon their lipid-rich cell walls which are relatively
impermeable to various basic dyes unless the dyes are
combined with phenol.
➢ The exact method by which the stain is retained is unclear
but it is thought that some of the stain becomes trapped
within the cell and some forms a complex with the mycolic
acids. This is supported by the finding that shorter chain
mycolic acids or mycobacterial cells with disrupted cell
walls stain weakly acid-fast, e.g. Nocardia
Acid-Fast Stain of Mycobacterium tuberculosis in
Sputum

Note the reddish acid-fast bacilli among the blue normal flora and
white blood cells in the sputum that are not acid-fast.
SPECIAL STAINING
CAPSULE STAINING
➢Capsule staining is diagnostically useful since it is
a virulent factor(e.g. pneumococci).
➢Bacterial capsules are non-ionic, so neither acidic
nor basic stains will adhere to their surfaces.
➢Capsules are demonstrated either by negative
staining (Nigrosin or India ink) or by special
staining, e.g. Hiss’ method, Anthony’s method
 Hiss Method
• The capsule is non-ionic in nature so it doesn't get stain by a
acidic stain but a basic stain, such as crystal violet, stains
the cell as well as the capsule.
• This is followed by treatment with hypertonic solution -
20% Copper sulphate solution, which serves dual role of
both the decolorizing agent and counter stain.
• Copper sulphate solution, being hypertonic, causes diffusion
of stain towards outer surface of cell.
• After drying of slide, the stain which is not passed from the
capsular layer during diffusion retains in the capsular layer.
Copper sulphate then decolorizes the capsule.
• Capsule appears as a faint blue halo around a purple cell.
ENDOSPORE STAINING
 Endospore staining
➢Spores are normally impervious to stains.
➢Under the light microscope endospores have a high light
refractivity indicative of high protein content.
➢Endospores can be stained by:
➢ Modified Zeihl-Nelson's method using 0.25-0.5% sulphuric acid
as decolorizing agent,
➢ Barthelomew-Mittwar’s method
➢ Schaeffer-Fulton stain technique.
 Schaeffer-Fulton method
• Malachite green is used to stain the endospores (primary
stain)
• The malachite green is forced to permeate the spore wall
by heating (mordant).
• Washing with water remove stain from vegetative cells, but
not from spore wall.
• The endospores thus retain the primary dye while the
vegetative cells lose the primary stain and take the red color
of secondary stain (safranin).
Endospore stain of Bacillus megaterium

Note green endospores within pink bacilli