Download Gram staining

Differential staining
Lab 7
Abeer Saati
Differential staining
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).
Gram staining
1-Preparation of smears
2-Fixation of smears
3-Staining of smears
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Cover the smear with crystal violet for 1 minute.
Pour it off then wash with water.
Add iodine solution to the smear for 1 minute.
Gently wash with water.
Decolorize by 95% alcohol and rock the slide from side to side
and pour it off. Reapply alcohol till no violet color comes off.
Wash with water
Counterstain with safranin for 1 min.
Wash with water.
Place the film at angle to air dry or dry with filter paper.
Gram staining
1- Preparation of smears:
o Put a drop of water on the middle of the slide using the
inoculating needle.
o With the sterile needle, collect bacteria from agar surface
by touching the bacterial growth.
o Rub the tip of the needle on the glass slide in the drop of
water in a circular motion till you get homogenous
smear.
o Allow the smear to dry.
Gram staining
4-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)
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Prepare a smear of the bacteria
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Flood the smear with malachite green
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Do not allow the stain to completely evaporate.
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Remove from heat and allow slides to cool
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Once the slides are cool (important)  rinse with water
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Flood the sample with safranin (30-60 seconds)
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Rinse the slide  blot dry  observe under microscopy. 10x, 40x, 100x (oil
immersion).
• Spore: (green)
• Vegetative cell: (red)
Thank You