Gram Staining Method: Step-by-Step
Purpose:
Gram staining is a technique used to classify bacteria into two major groups: Gram-positive
and Gram-negative, based on differences in their cell wall structure.
Steps in Gram Staining:
1. Prepare a smear
o Place a small drop of water on a clean glass slide.
o Using a sterile inoculating loop, pick a small amount of bacteria and mix it
into the water.
o Spread to make a thin smear and allow it to air dry.
2. Heat-fix the smear
o Pass the dried slide quickly through a flame 2–3 times (smear side up).
o This kills the bacteria and fixes them to the slide.
3. Stain with crystal violet (Primary stain)
o Cover the smear with crystal violet and leave for 1 minute.
o Rinse gently with water.
4. Add iodine solution (Mordant)
o Flood the smear with iodine solution and wait 1 minute.
o Iodine helps the dye bind more strongly to Gram-positive cells.
o Rinse with water.
5. Decolorize with alcohol or acetone
o Gently add alcohol or acetone for 10–15 seconds only.
o This step removes the crystal violet from Gram-negative bacteria.
o Rinse immediately with water to stop the reaction.
6. Counterstain with safranin
o Apply safranin for 30–60 seconds.
o This stains the now colorless Gram-negative cells pink.
o Rinse with water and gently blot dry with tissue or filter paper.
7. Examine under a microscope
o Use oil immersion (100x objective) for clear viewing.
o Gram-positive bacteria will appear purple, and Gram-negative bacteria
will appear pink/red.
Results Interpretation:
Type of Bacteria
Cell Wall Structure
Color After Staining
Gram-Positive Thick peptidoglycan layer
Purple
Gram-Negative Thin peptidoglycan + outer membrane Pink/Red