Scope
& Purpose
:
Gram staining procedure -The purpose of this STP is to
provide written guidelines to identify and differentiate the organism depending
upon their composition cell wall.
Requirements:
- Crystal violet stain.
- Decolorizer (Alcohol: Acetone, 1:1)
- Gram’s Iodine
- Clean glass slide.
- Cedar wood oil.
- Carbol fuschin / Saffranin stain.
Procedure:
- Place a loopful of sterile distilled water on a clean dry slide.
- Flame the loop, allow it to cool and aseptically remove a part of growth from the culture / colony.
- Emulsify the growth in the loopful of distilled water on the slide.
- Spread the suspension over an area of above ½ inch approx.
- Again flame the loop & keep on stand.
- Allow the slide to dry to form a film wither by air neat fix the seam by panning by low flame.
- Cover the above fixed film with crystal violet stain for 1 min.
- Rinse with tap water and apply Gram’s iodine for 1 min.
- Again rinse with tap water and blot dry.
- Decolorize with alcohol until only a faint violet colour remains on the slide.
- Again rinse with tap water and blot dry.
- Stain with carbol fuchsin / saffranin for 2-3 min.
- Rinse with tap water and blot dry and observe under oil immersion.
Interpretation:
1. Gram positive organisms : Stain
blue or Violet.
2. Gram negative organisms : Stain
red or pink.
