PADMASHREE INSTITUTE OF

MANAGEMENT AND SCIENCES

TOPIC: STAINING
TECHNIQUES
SUBMITTED BY:
SHYLESH MURTHY I A
M.SC BT, 1ST SEM
DEPT. OF BIOTECHNOLOGY, PIMS
STAINING TECHNIQUES
 INTRODUCTION

Staining is an auxiliary technique used in microscopy.

Stains and dyes are frequently used in biology and
medicine to highlight structure in biological tissues.
Stains may be used to define and examine bulk tissues,
cell populations or organelles with individual cells.
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, malachite green, 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 an cells.
e.g., Malachite green 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 dried smears through a flame two or three times. Do not
over heat.
b) Allow slide to cool before staining.

• METHANOL FIXATION
a) Place air dried smears in a coplin jar with methanol for one
minute. Alternatively, flood smear with methanol for one minute.
b) Drain slides and allow to dry before staining.
 SIMPLE STAINING
LOEFFLER’S 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.
POLYCHROME METHYLENE BLUE:
 This is made by allowing Loeffler’s methylene blue to ‘ripen’
slowly.
 The slow oxidation of the methylene blue forms a violet
compound that gives the stain its polychrome properties .
 The ripening takes 12 months or more to complete, or it
may be ripened quickly by the addition of 1.0% potassium
carbonate to the stain.
• In contrast to the blue staining of most structures
by methylene blue, the violet component stains
acidic cell structures red-purple.
DILUTE CARBOL FUCHSIN
• Made by diluting Ziehl-Neelsen’s stain with 10-20
times its volume of water.
• 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

• Loefflers methylene blue

• Dilute Carbol Fuchsin
• Distilled water
• Compound microscope
• Cedar wood oil
• Fixed smear
 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 Loeffler’s Methylene blue over the smear and allow it to stand for
3 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.
• Observe the presence of organisms and also the cellular content of
sample.
 GRAM 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.
 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.
 RESULT

• 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.
 ACID-FAST STAINING
• The acid fast staining is a modification of Ehrlich’s (1882)
method also known as Ziehl-Neelsen stain.

• It is used for bacilli belonging to the genus Mycobacterium

especially Mycobacterium tuberculosis, Mycobacterium
laprae and also for Nocardia.

• Acid fastness of the acid-fast bacilli is attributed to the

presence of unsaponifiable wax fraction called mycolic acid
in their cell wall.

• Basic requirements:

 Primary and mordant staining with strong carbol fuchsin

(red).
 PROCEDURE
• Make a smear. Air dry. Hear fix.
• Flood smear with Carbol-Fuchsin stain.
• Steam for 5 minutes. Add more of the stain as needed.
• Cool slide for 5 minutes. Wash with distilled water.
• Flood with acid alcohol (leave for 15 seconds).
• Tilt the slide 45⁰ over the sink and add acid alcohol drop
wise until the red color stops streaming from the smear.
• Rinse with distilled water.
• Add Loeffler’s methylene blue stain (Counter stain).
Leave the stain on smear for 15-20 seconds.
• Rinse the slide and let it dry.
• Use oil immersion objective to view.
 RESULT

• The stained smear are contains pink colored

slender rod shaped structures are seen with
curved ends.
• The smear is positive for acid fast bacilli.
 CAPSULE STAINING
• The capsules serves as protective material by
slowing down or preventing penetration of
chemicals and body juices.
PRINCIPLE
• Chemically, the capsular material is a
polysaccharide, a glycoprotein or a polypeptide.
• Capsule staining is more difficult then other
types of differential staining procedures because
he capsular material are water soluble and may
be dislodged and removed with vigorous
washing.
• The capsule is non-ionic, so that the dyes
 PROCEDURE
• For positive staining of smears:
• Make a smear from colony of S.pneumoniae on
a clean grease free glass slide, and allow it to
air dry.
• Flood the smear with Crystal Violet and allow it
to stain for 5-7 minutes.
• Wash the smear with 20% copper sulphate
solution and dry it.
• Observe the smear first under low power(10X)
objective, and then under oil immersion (100X)
objective.
For negative staining of smears:
• Take a clean grease free glass slide.
• Put a large loopful of undiluted Indian ink on
the slide.
• Then add a small loopful of liquid bacterial
culture to the Indian ink and emulsify.
• Take a clean, grease free cover slip and place
on the ink drop and press it down, so that the
flim becomes very thin and thus pale in color.
• Observe the wet flim under high power (40X)
objective.
• The capsule in negative staining method is
 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.
 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
 SUMMARY
 Staining is technique used in microscopy to enhance
contrast in the microscopic image.
 Stain is substance that adheres to a cell, giving the
cell color.
 Stains are classified as Simple stain, Differential
stain and special stain.
 Gram staining is used to differentiate bacterial
species as Gram-Positive and Gram-Negative based
on the chemical and physical properties of cell wall.
 Acid fast staining technique or Ziehl-Neelsen stain
divides bacteria into acid-fast and non-acid-fast and
this is used in diagnosis of tuberculosis and Leprosy.
 CONCLUSION

Every bacterial cell is individually colorless and hence

NOT VISIBLE under the light microscope. So, to enable the
person to visualize its physical features – shape, size,
arrangement, etc the bacterial cells are stained with
specific dyes (stains).
In bacteriology (or Microbiology), we make use of various
staining procedures each having specific set of stains
(dyes) –
Gram’s staining – Crystal violet, Iodine and Safranin
Capsule staining – Nigrosin and Indian ink
Spore staining- Malachite green and Safranin.
 REFERNCE

• Prescott L.M, Harely T.P and Klein D.A,

Microbiology, 7th edition P:30-52, Brown
Publishers.
• www.biotechnika.com
• www.studymode.com