1. Experiment 2 Gram Stain and Acid-fast Stain

2.  Staining - simple stain (only one dye) - differential stain (more than one dye) G+G+ G-G- 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 virulent factors of bacteria. G + : exotoxin G - : endotoxin.

6. 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 Gram Stain 2 students/group

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 death in some AIDS victims

8.  Acid-fast Stain Procedure 2 students/group a smear (prepared by teachers) ↓heat fix Carbolfuchsin, 8-10min Primary Stain ↓wash Acid Alcohol, 0.5-1min Decolorization ↓wash Methylene Blue, 1min Counterstain ↓wash Dry with bibulous paper ↓ Observation Result Acid-fast bacteria: Red Non acid-fast bacteria: Blue Acid-fast Non acid-fast B.subtitis BCG BCG is a form of tuberculosis vaccine that is a freeze-dried preparation of a live and attenuated bovine organism.

9.  Acid-fast Stain 2 students/group Materials:  Bacterial smears B.subtitis slides 1 slide/group BCG slides 1 slide/group  Carbolfuchsin  Acid Alcohol  Methylene Blue  Normal saline (NS)  Slides and bibulous paper

10. Observation of Bacterial Morphology Experiment 3

11. Observation of Bacterial Morphology ContentsSlidesFocus on Bacterial shape Coccus Staphylococcus Streptococcus S. pneumoniae Shape, size, arrangement Bacillus E.coli B.subtitis SpirillumV.cholerae Special structure CapsuleS. pneumoniae Size & relationship with bacteria SporeC. tetaniSize & position FlagellaProteus Shape, number & position

12. Staphylococci

13. Streptococci

14. Streptococcus pneumoniae

15. Escherichia coli

16. Bacillus anthrax

17. Vibrio cholera

18. Streptococcus pneumoniae

19. Proteus

20. Clostridium tetani