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Principle of staining technique:

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:

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  1. Gram Stain Principle of staining technique: • Primary stain:- Crystal Violet • Mordant(fixes the dye):- Iodine • Decolorizing agent:-Alcohol/Acetone • Counter stain;- Safranin

  2. 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/ Iodine complex -ve: Loss of the crystal violet/ Iodine complex

  3. Gram stain Gram +ve Gram –ve

  4. Gram stain Procedure: • 1.Shake the suspension very well. • 2.Transfere Aseptically 3-4 loopfuls to • a clean glass slide marked from below. • 3.Air or heat drying • 4.Heat fixation • 5.Cover film with CV leave for(30 sec)

  5. Gram stain Procedure: 6. Wash gently 7. Cover the film with Iodine leave for(2 min),discard 8.Add fresh Iodine leave for (1min)

  6. Gram stain Procedure: 9. Wash gently 10. Decolorize by covering the film using Alcohol /Acetone for 5 sec .

  7. Gram stain Procedure: 11. Wash gently 12. Cover film with Safranin leave for (5 min)

  8. Gram stain Procedure: 13. Wash gently 14. Leave to dry 15. Add Oil 16. Use Oil Immersion lens

  9. 3. Streak the agar (cont.) • Gram stain Precautions Avoid excessivedecolorization Avoid insufficientdecolorization Stains are added on cool slides Don’t dry by heat after staining

  10. Staphylococcus aureus

  11. Escherchia coli

  12. Sarcinalutea

  13. Staphylococcus aureus Sarcina lutea

  14. Bacillus subtilis

  15. Mycobacterium pheli

  16. Candida albicans

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