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Gram staining : It is based on the composition of their cell wall.Their cell wall is typically rich with peptidoglycan and lacks the secondary membrane and lipopolysaccharide layer found in Gram-negative bacteria (red).Gram staining uses crystal violet to stain cell walls, iodine as a mordant, and a fuchsin or safranin counterstain to mark all bacteria.Do not stain well with simple stains required (heat) o Prepare a smear of the bacteria o Flood the smear with malachite green o Do not allow the stain to completely evaporate.Preparation of smears Fixation of smears Staining of smears o Cover the smear with crystal violet for 1 minute.Examination of smears Spore staining Spore: structure that can survive for long periods of time in inert state.

Gram staining : It is based on the composition of their cell wall. Gram staining
uses crystal violet to stain cell walls, iodine as a mordant, and a fuchsin or safranin
counterstain to mark all bacteria. Gram status is important in medicine; the
presence or absence of a cell wall changes the bacterium's susceptibility to some
antibiotics.
Gram-positive bacteria stain dark blue or violet. Their cell wall is typically rich
with peptidoglycan and lacks the secondary membrane and lipopolysaccharide
layer found in Gram-negative bacteria (red).
Preparation of smears
Fixation of smears
Staining of smears
o Cover the smear with crystal violet for 1 minute.
o Pour it off then wash with water.
o Add iodine solution to the smear for 1 minute.
o Gently wash with water.
o Decolorize by 95% alcohol and rock the slide from side to side and pour it
off. Reapply alcohol till no violet color comes off.
o Wash with water
o Counterstain with safranin for 1 min.
o Wash with water.
o Place the film at angle to air dry or dry with filter paper.
Examination of smears
Spore staining
Spore: structure that can survive for long periods of time in inert state. Produced
when environmental conditions are poor.
Do not stain well with simple stains required (heat)
• Prepare a smear of the bacteria
• Flood the smear with malachite green
• Do not allow the stain to completely evaporate.
• Remove from heat and allow slides to cool
• Once the slides are cool (important) rinse with water
• Flood the sample with safranin (30-60 seconds)