Transcript Gram Staining Method

Gram Staining
Method
Mr. Beyer
Individual Supplies
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Microscope Slide
Clothes Pin
Innoculating Loop
Paper Towels
Shared Supplies
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Crystal Violet Stain
Safranin Red Stain
Gram’s Iodine
Ethyl Alcohol (denatured)
Water Bottle
Rinsing Basin
Alcohol Burner
Wax Pencil
Matches
Microscope
Create a Staining Area On Your
Microscope Slide
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Obtain a clean microscope slide
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Draw a circle on your slide with wax pencil
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Warming the slide will make the wax pencil
work better when drawing your circle
Create a Suspension of Bacteria
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Place 1-2 drops of water inside the circle
on your microscope slide
Use the inoculating loop to obtain a tiny
sample of bacteria from your bacteria
culture
Add the bacteria sample to the water on
your microscope slide, mixing to create a
suspension of bacteria
Heat Fixing Bacteria to the
Microscope Slide
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Pass your microscope slide through the
flame until all of the water evaporates
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This will leave your bacteria stuck or “fixed”
to the microscope slide
Primary Staining of Gram Positive
Bacteria
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Attach the clothes pin to one end of your
slide
Place 2-3 drops of Crystal Violet Primary
Stain inside your circle (on your bacteria)
Leave the stain on the bacteria sample for
one minute
Rinse stain off with water (gently)
Mordant (Sealer)
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Add the Gram’s Iodine to your bacteria
sample inside the circle
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Leave Gram’s Iodine on your sample for
one minute
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Rinse off with water
De-Colorizing
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Add ethyl alcohol to the bacteria
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Let the alcohol set, then rinse with water
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Continue de-colorizing until the crystal violet stain
is no longer released from your sample.
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You should just be able to see a hint of purple at
this point
Counter-Staining Gram Negative
Bacteria
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Add 2-3 drops of Safranin Red Stain to
your bacteria sample inside the circle on
your slide
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Leave safranin red on your bacteria sample
for one minute
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Rinse with water, blot or air dry
Viewing Bacteria
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View under oil immersion lens of microscope
(1000x magnification)
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Sketch in color,
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Label bacteria as gram (+) or gram (-)
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Identify shape and arrangement of bacteria
Gram Positive Coccus
Gram Positive Streptococcus
Gram Positive Rods (Bacillus)
Gram Positive
Gram Positive Staphylococcus
Gram Negative Bacillus
Gram Negative
Gram Negative vs. Gram Positive
Negative
Positive
Gram (+) and (-) Cell Structure