STAINING MICROORGANISMS
An overview of staining
STAINING
Coloring organism with a dye
Microorganisms must be FIXED to microscopic
slide first
Kills microbe
 Attaches organism to the slide
 Preserves various parts with minimal distortion
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Steps to fix:
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SMEAR is placed on slide and allowed to dry
Passed through Bunsen burner several times
Stain then applied and washed off
STAINING
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Stains are salts composed of positive and negative
ions (one is colored and is called a chromophore)
Basic dyes-color is positive ion
Acidic dyes- color is negative ion
Bacteria are slightly negatively charged at pH 7.0, so
basic dye is attracted more to them. Includes:
crystal violet
methylene blue
malachite green
safranin
STAINING
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Acidic dyes not attracted to bacteria because
stain’s negative ion is repelled
Used to stain background instead (some bacteria
are colorless). This is called negative staining
 Great when fixing is not possible because only
background is stained
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STAINING
TYPES OF STAINING:
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Simple
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Differential
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Gram Stain
Acid Fast
Special
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Negative staining for capsules
Endospore Staining
Flagella Staining
STAINING
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Simple
Sometimes an additive
called a mordant.
It increases uptake
of the dye.
STAINING
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Differential
Gram Stain (developed
in 1884)
Procedure:
1. apply heat. this fixes
smear; slide is covered with
purple dye (primary stain)
2. dye is washed off
and the mordant, iodine, is
placed and washed off
3. slide washed with
alcohol (decoloring agent)
4. slide is re-stained with
safranin (red dye is a
counterstain)
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STAINING
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Differential
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Acid Fast
Binds strongly to
waxy material in cell walls
Procedure:
1. red dye carbolfuchsin is
applied to a fixed smear and slide
is heated
2. slide is cooled and washed
with water and then acid-alcohol
3. If colorless, slide is
counterstained with methylene
blue
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STAINING
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Special Stains
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Negative Staining for capsules
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Organism is placed in solution with India ink or nigrosin
and then contrastained with safranin
STAINING
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Endospore (spore staining)
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Most common stain is Schaeffer-Fulton endospore
stain
Malachite green (primary stain) is applied to heat-fixed
smear and heated to steaming for 5 min
 Washed with water
 Next safranin (counterstain)
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STAINING
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Flagella Staining
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Uses a mordant and carbolfuchisin