Gram Stain. Principle of staining technique:. Primary stain:- Crystal Violet Mordant(fixes the dye):- Iodine Decolorizing agent:- Alcohol/Acetone Counter stain;- Safranin. Why do gram-positive cells keep their color while gram- negative cells lose it?.
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Principle of staining technique:
Why do gram-positive cells keep their color while gram- negative cells lose it?
Due to the difference in the structure of the cell wall.
Gram-negative cells have peptidoglycan (less)+ NO teichoic acid+ large amount of lipids
Alcohol dissolves the lipids , forms large pores that leak the dye leading to loss of CV/Iodine complex.
Gram-positive cells have peptidoglycan + teichoic, no pores are formed by alcohol and the dye is retained.
+ve: Retention of the crystal violet/
-ve: Loss of the crystal violet/ Iodine complex
6. Wash gently
7. Cover the film with Iodine leave for(2 min),discard
8.Add fresh Iodine leave for (1min)
9. Wash gently
10. Decolorize by covering the film using Alcohol /Acetone for 5 sec .
11. Wash gently
12. Cover film with Safranin
leave for (5 min)
13. Wash gently
14. Leave to dry
15. Add Oil
16. Use Oil Immersion lens
Stains are added on cool slides
Don’t dry by heat after staining