Staining Microorganisms
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Transcript Staining Microorganisms
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
Steps to fix:
SMEAR is placed on slide and allowed to dry
Passed through Bunsen burner several times
Stain then applied and washed off
STAINING
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
safranine
STAINING
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
STAINING
TYPES OF STAINING:
Simple
Differential
Gram Stain
Acid Fast
Special
Negative staining for capsules
Endospore Staining
Flagella Staining
STAINING
Simple
Sometimes an additive
called a mordant.
It increases uptake
of the dye.
STAINING
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)
STAINING
Differential
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
STAINING
Special Stains
Negative Staining for capsules
Organism is placed in solution with India ink or nigrosin
and then contrastained with safranin
STAINING
Endospore (spore staining)
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)
STAINING
Flagella Staining
Uses a mordant and carbolfuchisin