STAINING TECHNIQUES

I UG
What is staining technique?

Staining is a technique used in

microscopy to enhance contrast in the
microscopic image.
Stains and dyes are frequently used in
biology and medicine to highlight structures
in biological tissues for viewing, often with
the aid of different microscopes.
Staining is a technique used to enhance
contrast in samples, generally at the
microscopic level.

Biological staining is also used to mark cells in

flow cytometry, and to flag proteins or nucleic
acids in gel electrophoresis.

Staining and fluorescent tagging can serve

similar purposes.
 What is stain and dye?
A stain is a mixture of dyes that give a contrast to
the different components of a tissue on a
microscopic slide while a dye is a chemical reagent
that highlights a specific entity in the sample.
What is the purpose of bacteria staining?
Living bacteria are almost colorless, and do not present
sufficient contrast with the water in which they are
suspended to be clearly visible.
The purpose of staining is to increase the contrast between
the organisms and the background so that they are more
readily seen in the light microscope.

Bacteria are microscopic organisms.

They are mostly colorless and to visualize them to
study their structure, shape and other structural
characteristics.
 TYPES OF STAINS
• ACIDIC
Negatively charged acid radicals imparts color in eosin, acid
fuchsine, Indian ink.

• BASIC
Positively charged basic radicals combines with negatively
charged particles in cytoplasm and gives color.
e.g., Haematoxillin, Methylene blue, Crystal violet.

• NEURTAL
Both positively and negatively charged imparts different colors to
different components.
e.g., Geimsa’s stain and Leishman’s stain.
 STAINING TECHNIQUES
POSITIVE STAINING
Where the actual cells are themselves colored and appear
in a clear background.

• Simple staining: a stain which provides color contrast but

gives same color to all bacteria and cells.
e.g., Methylene blue and Crystal violet.
• Differential staining: a stain which imparts different colors
to different bacteria is called differential stain (which
contains more than one stain).
e.g., Gram’s stain, Acid fast staining and Endospore
staining.
• NEGATIVE STAINING :

Where the cells remain clear (uncolored) & the

background is colored to create a contrast to aid in
the better visualization of the image.
Nigrosin
Indian ink
 BACTERIAL SMEAR PREPARATION
SMEAR : is a distribution of bacterial cells on a slide for the
purpose of viewing them under the microscope.

METHOD :
o Aseptically a small sample of the culture is spread over a
slide surface.
o This is then allowed to air dry.
o The next step is heat fixation to help the cells adhere to
the surface.
o The smear is now ready for staining.
 SMEARING TECHNIQUES

• HEAT FIXATION
a) Pass air into dried smears through a flame two or
three times. Do not over heat.
b) Allow slide to cool before staining.
 What is simple staining?
Simple staining involves directly staining the
bacterial cell with a positively charged dye in
order to see bacterial detail, in contrast to
negative staining where the bacteria remain
unstained against a dark background.

“Only one stain is used to study microbes”

 SIMPLE STAINING
METHYLENE BLUE:
 It is generally the most useful, it shows the characterstics
morphology of polymorphs, lymphocytes and other cells more
clearly than do stronger stains such as gram stain or dilute carbol
fuchsin.
• In contrast to the blue staining of most structures by
methylene blue, the violet component stains acidic cell
structures red-purple.
• Stain for 10-25 seconds and wash well with water.
• Over staining must be avoided, as this is an intense stain,
and prolonged application colors the cell protoplasm in
addition to nuclei and bacteria.
 REQUIREMENTS

• Methylene blue stain

• Distilled water
• Compound microscope
• Cedar wood oil
• Fixed smear- culture
 PROCEDURE
• Make a thin smear on a slide.
• Heat fixes the smear by passing the slide 2-3 times gently over the
Bunsen flame with the smear side up.
• Pour Methylene blue over the smear and allow it to stand for 2
minutes.
• Wash the stained smear with water and air dry it.
• Observe the smear first under low power (10X) objective, and then
under oil immersion (100X) objective of the microscope.
• Observe the presence of organisms and also the cellular content of
sample.
Image of simple staining
Questions?

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 GRAM‘s STAINING
• Gram staining is most widely used differential
staining in microbiology.
• It differentiates the bacteria into two
groups: Gram positive and Gram
negative.
• Gram Positive Bacteria: They have a thick
cell wall of peptidoglycan and other polymers
• Peptidoglycan consists of interweaving filaments
made up of alternating N-acetylmuramic acid and
N-acetylglucosamine monomers.
• Gram Negative Bacteria: They have an outer membrane
of phospholipid and bacterial lipopolysaccharides outside
of their thin peptidoglycan layer.
• The outer membrane protects gram negative bacteria
againsts penicillin and lysozymes.

Is Gram staining cheap?

Gram staining is cheap, effective, quick, and relatively

easy to interpret. Its most useful application is in the clinical
setting.
 PROCEDURE OF GRAM STAINING
• It consists of four steps:
Primary staining: the smear is covered Crystal Violet, for
1 minute and washed with water.
Mordanting: it is then covered with Gram’s Iodine, kept for
1 minute, and washed with water.
Decolorization: the smear is covered with alcohol and is
washed with water immediately.
Counter staining: the smear is then covered with safarnin,
kept for 30 seconds and washed with water.
 Observe under microscope.
What does iodine do in a Gram stain?
Iodine fixes the crystal violet into the cell wall of the bacteria by
working as a mordant.
 RESUL
• T
Bacteria that manage to keep
the original purple dye have
only got a cell wall-they are
called Gram-positive.
• Bacteria that lose the original
purple dye and can therefore
take up the second red dye
have got both cell wall and cell
membrane-they are called
Gram-negative.
 How does Gram staining help identify bacteria?
Gram staining differentiates bacteria by the chemical
and physical properties of their cell walls.
Gram-positive cells have a thick layer of peptidoglycan
in the cell wall that retains the primary stain, crystal
violet.
Why is safranin used in Gram
staining?
The safranin is also used as a counter-
stain in Gram's staining.
In Gram's staining, the safranin
directly stains the bacteria that has
been decolorized. With safranin
staining, the gram-negative bacteria
can be easily distinguished from
gram-positive bacteria.
 ENDOSPORE STAINING
• Spores are highly resistant inactive forms.
• The morphology of bacterial endospores is best
observed in unstained wet flims under the phase
contrast microscope, where they appear as large ,
refractile, oval or spherical bodies within the bacterial
mother cells or else free from the bacteria.
• Different staining are available for staining of spores.
 Endospores staining is the type of staining to
recognize the presence spore in bacterial
vegetative cells.
The bacterial endospores need a staining
which can penetrate wall thickness of spore
bacteria.
A method of endospores staining is Schaeffer
Fulton method that used Malachite Green.
How does Endospore staining work?
Because of their tough protein coats made of keratin,
spores are highly resistant to normal staining procedures.
The primary stain in the endospore stain procedure,
malachite green, is driven into the cells with heat.
 PROCEDURE
• Flims are dried and fixed with minimal flaming.
• Place the slide over a beaker of boiling water, resting it on the
rim with the bacterial flim uppermost.
• When, within several seconds, large droplets have condensed
on the under side of slide, flood it with 5% aqueous solution
of Malachite green and leave it for 1 minute while the water
continues to boil.
• Wash in cold water.
• Treat with 0.5% safranin and 0.05% basic fuchsin for 30
seconds.
• Wash and dry.
• This method colors the spores green and vegetative
bacilli red.
ENDOSPORE STAINING
 Questions?

REF:

• Prescott L.M, Harely T.P and Klein D.A, Microbiology, 7th edition P:30-52, Brown
Publishers.

• www.biotechnika.com

Thanks