DYES , STAINS &

Reactions
(MICROBIOLOGY)
A PRESENTATION BY
MUHAMMAD BILAL
Introduction

 Bacteria are microscopic organisms. They are also colorless for the most part. In
order to visualize them to study their structure, shape and other structural
characteristics, it becomes necessary to make them more easily visible.
 All the biological stains are prepared from dyes, which have been manufactured
for special purpose that means to study the anatomical details of blood cells and
tissue microscopically.
Dyes

The dye used to denoted powder and the stain to denotethe solution of one
or more dyes. The dyes are classifiedinto two groups:
 1) Natural dyes
 2) Synthetic dyes
Natural Dyes

Natural dyes are prepared or extracted from natural sources such as

 hematoxylin(It is a compound extracted from the heartwood of the logwood tree
(Haematoxylum campechianum),
 litmus(Litmus is a water soluble mixture of different dyes extracted from
lichens),
 orcein(Orcein, also archil, orchil, lacmus and Natural Red 28, are names for dyes
extracted fromseveral species of lichen, commonly known as "orchellaweeds",
found in various parts of the world), carmine,safranin.
Synthetic Dyes

Synthetic dyes are manufactured or prepared

or sometimes referred as coal tar
dyes.Synthetic dyes are manufactured
from organic molecules.
 Examples of Synthetic Dyes
 Fast green.
 Picric acid.
 Orange G.
 Oil red O.
 Eosin Y.
 Light green SF.
Stains
Stain is a dye used to color the living or dead organelles.
TYPES:
ACIDIC: Negatively charged acid radicals imparts color in eosin, acid fuchsine,
malachite green, nigrosin, Indian ink.
BASIC: Positively charged basic radicals combines with negatively charged particles
in cytoplasm and gives color. Ex: Haematoxillin, methylene blue, crystal violet,
gention violet.
NEUTRAL: Both positively and negatively charged impartsdifferent colors to
different components.Ex: Geimsa's stain, Leishman's stain, Wright's stain.
Staining Methods

 Simple staining: A stain which provides color contrast but gives same color to all
bacteria and cells .Ex: methylene blue, Polychrome methylene blue, Diluted carbol
fuchsin.
 Differential Staining: A stain which imparts different colors to different bacteria is
called differential stain (which contains more than one stain). Ex: Gram's stain, Acid
fast staining, Special stains.
1. POSITIVE STAINING: - where the actual cells are themselves colored and
appearing a clear background.

2. NEGATIVE STAINING: where the cells remain clear (uncolored) and the
background is colored to create a contrast to aid in the better visualization of the
image. Examples are India ink and nigrosin
Bacterial Smear Preparation

Smear is a distribution of bacterial cells on a slide for the purpose of viewing them
under the microscope.
Method:-
 Aseptically a small sample of the culture is spread over a slide surface.
 This is then allowed to air dry.
 The next step is heat fixation to help the cells adhere to the slide surface.
 The smear is now ready for staining.
Smear Fixation

1. Heat fixation
 Pass air-dried smears through a flame two
or three times. (Do not overheat)
 Allow slide to cool before staining.
2. Methanol fixation
 Place air-dried smears in a staining tray
with methanol for one minute.
Alternatively, flood smear with methanol
for 1 minute
 Drain slides and allow to dry before
staining.
Simple Staining
INTRODUCTION
 Simple staining is a method of staining in which bacteria are stained by using a
single stain.
 Simple staining is also called as monochrome staining or positive staining.
 Examples of simple stain are Methylene blue, Safranin, Malachite green, and
crystal violet etc.
 In simple staining procedure cell are uniformly stained.
 The simple stain can be used as a quick and easy way to determine cell shape,
size and arrangements of bacteria. True to its name, the simple stain is a very
simple staining procedure involving single solution of stain.
Principle

 Any basic dye such as methylene blue, safranin, or crystal violet can be used to
color the bacterial cells.
 These stains will readily give up a hydroxide ion or accept a hydrogen ion, which
leaves the stain positively charged. Since the surface of most bacterial cells and
cytoplasm is negatively charged, these positively charged stains adhere readily to
the cell surface. After staining, bacterial cell morphology (shape and
arrangements) can be appreciated.
 The purpose of simple staining is to study the morphology and arrangement of
bacterial cells. The most commonly used basic stains are methylene blue, crystal
violet.
Requirements

Loefflers Methylene blue

Dil. 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 side 2-3 times gently over the Bunsenflame
with the smear side up.
 Pour Loeffler's methylene blue over the smear and allow it to stand for 3minutes.
 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.
Left: Cocci in Cluster; Right: Bacilli
Differential Staining
Gram Staining Technique

Gram staining is most widely used differential staining in Microbiology. Gram

staining differentiates the bacteria into 2 groups:
 Gram positive.
 Gram negative.
In 1884, Danish scientist "Hans Christian Gram" developed the technique while
working with "Carl Friedlander" in the morgue of the city hospital in Berlin.
Principle

 When the bacteria is stained with crystal violet and fixed by mordant, some
bacteria retain the primary stain while some are decolorized by alcohol.
 Gram-positive bacteria have thick layer of peptidoglycan and low lipid content.
So, ethanol cannot remove the crystal violet-iodine complex bound to cell wall
and bacteria appears blue or purple in color.
 Gram-negative bacteria have thin layer of peptidoglycan and high lipid content.
The decolorizer ethanol, dissolves the lipids in the cell wall and allows crystal
violet-iodine complex to leached out of the cell. So, bacteria appear red in color
when stained with safranin.
APPLICATIONS OF GRAM
STAINING
 Rapid presumptive diagnosis of diseases such as Bacterial meningitis.
 Selection of Empirical antibiotics based on Gram stain finding.
 Selection of suitable culture media based on Gram stain finding.
 Screening of the quality of the clinical specimens such as sputum that should
contain many pus cells & few epithelial cells.
 Counting of bacteria.
 Appreciation of morphology & types of bacteria in clinical specimens.
Gram Positive and Negative Bacteria
Reagents used in Gram Staining

 Primary stain:
Crystal Violet iodine
 Mordant:
Iodine
 Decolorizer
Alcohol and Acetone(95%)
 Counter stain:
Safranin
Procedure: It consists of four steps
 Primary staining: The smear is covered with 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.
 Decolourisation: The smear is covered with alcohol and is washed with water
immediately.
 Counter staining: The smear is then covered with safranin, kept for 30 seconds
and washed with water.
 Using filter paper the slide is gently blotted to dry. Place a drop of cedar wood
oil/Liquid paraffin on the smear.
 Adjust the microscope for increased light by raising the condenser, and the slide is
examined under the oil immersion objectives using the plane mirror
Results

 Bacteria that manage to keep the original

purple dye have only got a cell wall - they
are calledGram positive.
 Bacteria that lose the original purple dye
and can therefore take up the second red
dye have got both a cell wall and a cell
membrane - they are called Gram
negative.
ACID FAST STAINING

Is a differential staining
used to differentiate acid fast and non acid fast bacteria.
used to identify acid-fast organisms such as members of the genus Mycobacterium.
Also known as Ziehl-Neelsen method
 first introduced by Paul Ehrlich later modified by two German doctors
bacteriologist Franz Ziehl (1859-1926) and the pathologist Friedrich Neelsen
(1854-1898).
What are acid fast bacteria?

Those which have a high content of mycolic acids in their cell walls.
wax-like, nearly impermeable cell walls;
contain mycolic acid and large amounts of fatty acids,
waxes, and complex lipids.
 are highly resistant to disinfectants and dry conditions.
 Due to their waxy cell wall components, Mycobacterium are acid fast; that is, they
retain the red dye, carbol fuchsin, after rinsing with acid solvents.
Principle
 Because the cell wall containing mycolic acid is so waxy and resistant to most
compounds, acid-fast organisms require a special staining technique.
 The primary stain used in acid-fast staining, carbol fuchsin, is lipid-soluble and
contains phenol, which helps the stain penetrate the cell wall. This is further
assisted by the addition of heat.
 The smear is then rinsed with a very strong decolorizer, which strips the stain
from all non-acid-fast cells but does not permeate the cell wall of acid-fast
organisms.
 The decolorized non-acid-fast cells then take up the counterstain.
 Acid fast bacteria will be red, while nonacid fast bacteria will stain green.
Requirements

Materials
 Slides
 Sample
 Inoculating loop
 Bunsen burner
 Cotton
 Microscope
Reagents
 Primary Stain: Carbol-fuchsin.
 Decoloriser: (HCl+Ethanol)
 Counterstain: Methylene blue.
Preparation of Reagents

Primary Stain: 0.3% Carbol-fuchsin:

 Dissolve 50 g phenol in 100 mL ethanol (95%).
 Dissolve 3 g Basic fuchsin in the mixture and add distilled water to bring the
volume to 1 L.
Decolorization Solution: (HCl+Ethanol)
 Add 30 mL hydrochloric acid to 1 L of 95% denaturedalcohol.Cool and mix well
before use.
Counterstain: 0.3% Methylene blue
 Dissolve 3 g methylene blue in 1 L distilled water.
Procedure
 Take a clean grease free slide.
 Prepare the smear from provided sample.Air dry and heat fixed the smear.
 Place a small strip of filter paper over the top of smear and place the slideover a
boiling hot water bath on a mesh surface.
 Cover the filter paper with the primary stain, carbol fuchsin.
 Leave the slide on the water bath for 5 minutpes or more. Note-Continue to apply
stain if the filter paper begins to dry.
 Remove the filter paper and rinse the slide with water until the solution runs clear.
 Run acid-alcohol decolorizer over the slide for approximately 10 to 15 seconds.
 Rinse the slide with water.Cover the smear with the counterstain, methylene blue
 Gently rinse the slide with water.
 Blot the slide dry with bibulous paper.
 Observe under microscope.A.B10 of 15Clip slide 10for 1 minute.
Observation under Microscope

Acid fast: Red, straight or slightly curved

rods, occurring singly or in small groups,
may appear beaded
 Non-acid fast: Blue color; In addition,
background material stain blue.
Special Staining
Flagella Staining

 Most motile bacteria move by use of flagella (flagellum), threadlike appendages

extending from the plasma membrane and cell wall.
 They are about 15 or 20 µm long. Flagella are so thinthey cannot be observed
directly with a bright-fieldmicroscope, but must be stained with specialtechniques.
Types

Bacterial species often differ distinctively in

their patterns of flagella distribution.
Monotrichous bacteria: (trichous means
hair) have one flagella; if it is located at an
end, it is said to be a polar flagellum. Ex:
Vibrio cholera.
Lophotrichous bacteria have a cluster of
flagella at one or both ends. Ex:
Pseudomonas
 Amphitrichous bacteria: (amphi means
“on both sides”) have a single flagella at
each pole. Ex: Spirillum.
Flagella stain by using Leifson’s
method:
 The Leifson's stain is made up of tannic acid,basic fuschin stain and alcohol.
 When we treat Leifson's stain with cell the tannic acid get attach to the flagella
and alcohol get evaporated.
 After evaporation of alcohol the thickness of flagella isincreased due to deposition
of tannic acid.
 Where as Basic fuschin stain the Flagella.
 After this give a gentle stream of water wash treatment to a slide.
 Now treat the slide with 1% methylene blue treatment for 1 minute.0Clip slide
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Observation

 • After observation under microscope we can observe that flagella appear red in
colour and bacterial cell appear blue in colour.
Spore Staining

The morphology of bacterial endospores is best observed in unstained wet films

under the phase contrast microscope, where they appear as large, refractile, oval or
spherical bodies within a bacterial mother cells or else from the bacteria.
If spore-bearing organisms are stained with ordinary dyes, or by Gram’s stain, the
body of the bacillus is deeply colored, whereas the spore is unstained and appears as
clear area in the organism.
 This is the way in which spores are most commonly observed.
If desired, however, it is possible by vigorous
staining procedures to introduce dye into the
substance of the spore.
 When thus stained, the spores tends to
retain the dye after treatment with
decolorizing agents, and in this respect
behaves similarly to the tubercle bacillus,
but is more weakly acid-fast.

Spore of mold in mold in bread

Principle

 The spores are thick walled structures and very resistant to physical and chemical
agents.
 The spores have a capacity to survive for long periods even inunfavourable
environmental conditions
 The heat resistance by spores is due to the high contentcalcium- dipicolinic acid.
 The spores are differentially stained using special procedures that help dye to
penetrate the spore wall.
Procedure
 Films are dried and fixed with minimal flaming.
 Place the slide over a beaker of boiling water, resting it on the rim with the
bacterial film uppermost.
 When, within several seconds, large droplets have condensed on the underside of
the slide, flood it with 5% aqueous solution of malachite green and leave to act for
1 min while the water continues to boil.
 Wash in cold water.
 Treat with 0.5% safranine or 0.05% basic fuchsin for 30 seconds.
 Wash and dry. This method colors the spores green and the vegetative bacilli red.
Lipid granules are unstained.
Observation
Capsule Staining

 Some bacteria secrete a prominent slimy or gummy material on their surface

usually polysaccharide make up the capsule. It is not essential for life, but may
serve as a reserve food.
 It provides protection against dehydration and also against phagocytosis.
 The chemical composition of capsule varies according to organism but usually
consist of polysaccharides e.g., glucose, galactose, amino sugars.
 Capsule are colorless and have low refractive index and so are difficult to observe
without a special staining technique.
 Moreover the capsule is non-toxic, hence cannot be stained in the usual manner.
 Techniques like negative staining can be used to demonstrate the capsule.
Procedure

 Placed a drop of Manevali's solution 1 on a clear glass slide.

 Sterilised the nichrome loop, cooled it and just touched to the growth of organism
on the slant.
 Mixed the culture on loop with a drop of Manevali's solution 1on the slide.
 The drop was spread into a thin film. The film was allowed to dry completely.
 The smear was covered with Manevali's solution II and allowed to react for 1
min.
 The excess stain was discarded and dried in air.
 Observe under oil immersion objective.
Observation
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