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Experiment 2

Experiment 2. Gram Stain and Acid-fast Stain. G +. G -. Staining simple stain (only one dye) differential stain (more than one dye). Gram stain Acid-fast stain. Gram stain. Danish physician Hans Christian Gram (1853-1938). Procedure & Principle of Gram Stain.

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Experiment 2

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  1. Experiment 2 Gram Stain and Acid-fast Stain

  2. G+ G- • Staining • simple stain (only one dye) • differential stain (more than one dye) Gram stain Acid-fast stain

  3. Gram stain Danish physician Hans Christian Gram (1853-1938)

  4. Procedure & Principle of Gram Stain Prepare a smear, air dry ↓flame 3 times to fix Crystal violet, 1 min Primary stain ↓wash Gram’s iodine,1 min Mordant Stain ↓wash Ethanol 95%, 0.5 min Decolorization ↓ wash Safranin, 1 min Counterstain ↓ wash Dry slide with bibulous paper ↓ Observation (10× lens →40× lens → oil-immersion lens)

  5. Significance of Gram Stain • Differentiate bacteria:G+ /G- • Helpful to select sensitive antibiotics. • identify different virulentfactors of bacteria. • G+: exotoxin • G-: endotoxin.

  6. Gram Stain2 students/group Materials: 1. Your Plate culture of S. aureus and E. coli 2. Crystal violet solution 3. Gram’s iodine solution 4. Ethanol, 95% 5. Safranin solution 6. Normal saline(NS) 7. Slides and bibulous paper

  7. Acid-fast Stain • Mycobacterium possess thick waxy cell walls. They can inhibit the penetration of dye molecules. • Once stained, these bacteria resist decolorization by acid alcohol------- "acid-fastness" • Acid-fastness+: M. leprae→leprosy M. tuberculosis→ tuberculosis M.avium-intracellulare → a cause of deathin some AIDS victims

  8. Acid-fast Non acid-fast a smear (prepared by teachers) ↓heat fix Carbolfuchsin, 8-10min Primary Stain ↓wash Acid Alcohol, 0.5-1min Decolorization ↓wash Methylene Blue,1minCounterstain ↓wash Dry with bibulous paper ↓ Observation • Acid-fast Stain Procedure2 students/group BCG B.subtitis BCG is a form of tuberculosis vaccine that is a freeze-dried preparation of a live and attenuated bovine organism. • Result Acid-fast bacteria: Red • Non acid-fast bacteria: Blue

  9. Acid-fast Stain2 students/group Materials: • Bacterial smears B.subtitis slides1 slide/group BCG slides1 slide/group • Carbolfuchsin • Acid Alcohol • Methylene Blue • Normal saline(NS) • Slides and bibulous paper

  10. Experiment 3 Observation of Bacterial Morphology

  11. Observation of Bacterial Morphology

  12. Staphylococci

  13. Streptococci

  14. Streptococcus pneumoniae

  15. Escherichia coli

  16. Bacillus anthrax

  17. Vibrio cholera

  18. Streptococcus pneumoniae

  19. Proteus

  20. Clostridium tetani

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