Gram Staining 

The Gram stain is a fundamental technique in microbiology used to differentiate bacterial cells into two major groups: Gram-positive and Gram-negative, based on differences in their cell wall structure.

Here's a basic protocol for performing a Gram stain:

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Materials Needed:

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Bacterial culture (overnight culture or fresh culture)
Microscope slides
Bunsen burner or alcohol burner
Inoculating loop or bacteriological loop
Crystal violet stain
Gram's iodine solution (Gram's mordant)
Ethanol or acetone-alcohol solution (decolorizing agent)
Safranin stain
Microscope with oil immersion lens (100x objective)
Lens paper or wipes
Bibulous paper (blotting paper)
Coverslips

Procedure:

Preparation of Smear:
a. Place a small drop of water on a clean microscope slide.
b. Using a sterile inoculating loop, transfer a small amount of the bacterial culture onto the drop of water on the slide.
c. Mix the bacteria and water to create a thin, even smear. Allow it to air dry.

Heat Fixation:
a. Pass the slide with the smear through the flame of a Bunsen burner or alcohol burner 2-3 times to heat-fix the bacteria. This helps the bacteria adhere to the slide and denatures the proteins.

Staining:
a. Flood the smear with crystal violet stain and let it sit for about 1 minute.
b. Rinse the slide gently with water to remove excess stain.
c. Flood the smear with Gram's iodine solution and let it sit for about 1 minute. This forms a crystal violet-iodine complex that gets trapped in Gram-positive cells.

Decolorization:
a. Tilt the slide and gently add the ethanol or acetone-alcohol solution drop by drop to the smear, while tilting the slide to let the solution flow across the smear.
b. Continue decolorizing until the runoff is almost colorless. Be cautious, as over-decolorization can result in false-negative results for Gram-negative bacteria.

Counterstaining:
a. Rinse the slide with water to remove the decolorizer.
b. Flood the smear with safranin stain and let it sit for about 1 minute. Safranin stains the decolorized

Gram-negative cells pink.

Rinse and Drying:
a. Rinse the slide gently with water to remove excess safranin.
b. Blot the slide gently with bibulous paper to remove excess water.

Mounting and Observation:
a. Allow the slide to air dry completely before moving to the next step.
b. Place a drop of immersion oil on the smear.
c. Observe the stained smear under the microscope using the oil immersion lens (100x objective).

Microscopic Examination:
a. Observe the slide under oil immersion and focus on a representative area of the smear.
b. Identify and distinguish Gram-positive (purple/blue) and Gram-negative (pink/red) bacteria.

Cleaning Up:
a. Dispose of the slide properly, following your laboratory's biohazard waste disposal protocols.

Always follow proper laboratory safety guidelines and sterile techniques while working with bacterial cultures and staining reagents. The protocol might need adjustments based on specific bacterial strains and staining reagents.