Download Staining Bacteria

BACTERIAL
STAINING
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What is a Stain
• A stain is a substance that adheres to a cell,
giving the cell color.
• The presence of color gives the cells
significant contrast so are much more visible.
• Different stains have different affinities for
different organisms, or different parts of
organisms
• They are used to differentiate different
types of organisms or to view specific parts
of organisms
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Why we should be Stain
Bacteria
•Bacteria have nearly the same refractive
index as water, therefore, when they are
observed under a microscope by wet mount
they are opaque or nearly invisible to the
naked eye.
•Different types of staining methods are
used to make the cells and their internal
structures more visible under the light
microscope.
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stains composes from
• Stains are a mixture of chromogen and
auxochrome
• Chromogen = benzene derivative +
chromophore (coloring agent)
• Auxochrome: give +ve or –ve charge to
the chromogen
• The ionized stain is capable of binding
to cell structures with opposite charges.
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• Basic stains are cationic; when ionized, the
chromogene exhibits a positive charge. Basic
stains bind to negatively charged cell
structures like nucleic acids. Methylene blue,
crystal violet and carbolfuchsin are common
basic stains.
• Acidic stains are anionic; when ionized, the
chromogen exhibits a negative charge.
Acidic stains bind to positively charged cell
structures like proteins. Picric acid, eosin
and nigrosin are common acidic stains.
• Bacteria are slightly negatively charged at
pH 7.0
• Basic dye stains bacteria
• Acidic dye stains background
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• There are three type of staining in
Microbiological lab.
• 1. Simple stain.
• 2. Differential Stain: (Gram stain , Acid
fast Stain)
• 3. Special stain: (Capsular stain,
Endospore stain, Flagellar stain).
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Simple stain
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Simple stain
• Used to show the
general structures
(shape and
arrangement) of
some bacteria.
• Aqueous or alcohol
solution of single
(usually basic) stain
is used in this
procedure
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procedure
1. Obtain broth cultures of the bacteria.
2. Using an inoculating loop, remove a loopful of
suspension from one of the tubes. Remember
to use sterile technique.
3. Smear the bacteria across the center of the
slide with the loop. If the bacterial
suspension is very thick, add a drop of water
and mix the bacteria and the water on the
slide.
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4. Allow the smear to completely air dry.
• Air dry first to prevent lysis (boiling)
5. Heat-fix the smear by quickly passing the
slide through a Bunsen burner flame three
times. This causes partial melting of the
cell walls and membranes of the bacteria,
and makes them stick to the slide. Do not
overheat the slide as this will destroy the
bacteria.
Heat Fixing
•
•
•
•
Kill.
Stops autolysis.
Adherence to slide.
Increase dye taking
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6. Cover the smear with a few drops of one of the
stains. Allow the stain to remain for the following
periods of time:
Carbolfuchsin- 15-30 seconds.
Methylene blue- 1-2 minutes.
Nigrosin- 20-60 seconds.
. The stain can be poured drop by drop on the slide
7. Gently rinse the slide by holding its surface parallel
to a gently flowing stream of water.
8. Gently blot the excess water from the slide with
bibulous paper. Do not wipe the slide. Allow the slide
to air dry.
Observe the slide under the microscope with air
and oil lenses.
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Simple Stains
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Bacterial shape arrangement
- Cocci
-
Clusters (group).
- Bacilli
-
Chains.
-
Pairs (diploids).
-
No special arrangement.
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Differential stain
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Gram stain
• Differential stain (Hans Christian Gram, a
Danish doctor ). He developed a new
method to stain bacteria so they can be
visible in specimen samples.
• Differentiate bacteria into two large
groups (the Gram Positive and the Gram
negative)
• Gram status is important in medicine; the
presence or absence of a cell wall will
change the bacterium's susceptibility to
some antibiotics
• Almost all bacteria are described by their
Gram stain characteristics.
• Based on differences of Cell wall
structures
Reagents for Gram Stain
• Crystal Violet (purple).
• Primary stain; positive stain
• Stains cell wall purple
• Iodine
• Mordant
• Combines with primary stain to form an insoluble complex that
gets trapped in bacterial cell wall
• Ethanol
• Decolorizer
• CV-I complex washed out of Gram negative organisms because it
cannot be trapped by lippopolysaccharide layer; flows right
through outer membrane
• Safranin (pink)
• Counterstain
• Simple positive stain that provides contrasting dye for
decolorized cells (Gram negative)
• Stains all cells, but only the negative ones actually appear pink.
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Gram Stain colors
Primary stain:
Color of
Color of
Gram +
cells
Gram –
cells
Purple
Purple
Purple
Purple
Purple
Colorles
s
Purple
Red
Crystal violet
Mordant:
Iodine
Decolorizing agent:
Alcohol-acetone
Counterstain:
Safranin
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Gram Staining Procedure
(after air drying and heat fixation)
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Gm-ve bacilli
Gm+ve cocci
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Errors during staining
• Never ever used old culture.
• Never ever used sample for
patient take antibiotic.
• Time of Decolorizer:
• Over: G + see as G -.
• Low: G- see as G +.
• Time of fixation:
• Over: G + see as G -.
• Low: no sample on slide.