7) List the steps of the Gram stain procedure

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Gram staining is process of differentiating gram positive and gram negative organism by their cell wall composition.

The reagents are:

Crystal Violet (the Primary Stain)
Iodine Solution (the Mordant)
Decolorizer (ethanol)
Safranin (the Counterstain)
Water

STEP 1:

stabilize the slide
Cover completely the entire slide with crystal violet and waits for 1 minute
After wash slide for with water. The slide should appear blue-violet can observed with the naked eye.

STEP 2:

Cover slide with the iodine solution for 1 minute
Rinse the slide with water for few seconds.
At this time the slide should still be blue-violet.

STEP 3:

This step involves addition of the decolorizer, ethanol.
Add the ethanol dropwise until the blue-violet color is no longer emitted from specimen.
Rinse with the water for 5 seconds.

STEP 4

Applying with counterstain, safranin for 1 minute to allow the bacteria to incorporate the saffranin.

Gram positive cells will retain little or no counterstain and will remain blue-violet in appearance.
Gram negative bacteria take on a pink color and are easily distinguishable from the Gram positives.
Again, rinse with water for 5 seconds to remove any excess of dye.

After that blot the slide gently with paper to air dry before viewing it under the microscope.

gladiator answered 1 year ago

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