1. PHT 226 Lab # 3 Gram’s stain (mixture) Acid fast stain Spore stain

2. Staining of Bacteria Types of staining technique:- Types of staining technique:- Simple staining (use of a single stain) (use of a single stain) Differential staining (use of two contrasting stain) (use of two contrasting stain) For visualization of morphological shape & arrangement. shape & arrangement. IdentificationVisualization of structure Gramstain Acid fast stain stain SporestainCapsulestain

3. Smearing out of the sample

4. Smear Fixation

5. Principle of Differential Stains * Application of the primary stain. * Decolourization. *Application of the counter-stain.

6. Acid Fast Stain Acid fast bacteria (ex; Mycobacteria) are difficult to be stained by simple or Gram’s stain, because they have a high lipid content in their cell walls which prevent the penetration of ordinary aniline dyes. Acid fast bacteria (ex; Mycobacteria) are difficult to be stained by simple or Gram’s stain, because they have a high lipid content in their cell walls which prevent the penetration of ordinary aniline dyes. Once these organism are stained, they resist decolorization even with a very strong decolorizing agent such as acid-alcohol. Once these organism are stained, they resist decolorization even with a very strong decolorizing agent such as acid-alcohol.

7. Acid Fast Stain e.g., Ziehl-Neelsen Stain AFS is an important diagnostic value in identifying pathogenic members of genus Mycobacterium such as M. tuberculosis and M. leprea. AFS is an important diagnostic value in identifying pathogenic members of genus Mycobacterium such as M. tuberculosis and M. leprea. Materials:- Materials:- Culture of M. phelei Culture of M. phelei Conc. carbol fuchsin (primary dye) Conc. carbol fuchsin (primary dye) Acid-alcohol (decolorizing agent) Acid-alcohol (decolorizing agent) Methylene blue (counter stain) Methylene blue (counter stain)

8. Ziehl-Neelsen Stain 4 5 6 4 5 6 7 1 2 3 1 2 3

9. Acid Fast Stain Procedure:- Procedure:- Carbol fuchsin \\\\ 5 min alcohol 30-60 sec MB 1 min

11. Results Name of Stain: Acid fast stain Shape: beaded bacilli Arrangement: Tree shaped Colour: red Name of microorganism: M.phelei

12. The Spore Stain Some bacteria form resistant bodies in the cell known as endospores. Some bacteria form resistant bodies in the cell known as endospores. Bacterial spores are highly resistant to physical & chemical agents & are not easily stained by routine staining. Bacterial spores are highly resistant to physical & chemical agents & are not easily stained by routine staining. Heat is required in spore staining to promote the penetration of the dye into the spore. Heat is required in spore staining to promote the penetration of the dye into the spore. Once the spores stained they resist the decolorization. Once the spores stained they resist the decolorization.

13. The Spore Stain Materials :- Materials :- Culture of B. subtilis Culture of B. subtilis Malachite green (primary stain) Malachite green (primary stain) Safranine (counter stain ) Safranine (counter stain )

14. Spore Stain of Bacillus subtilis Name of Stain: Spore stain Shape: bacilli Arrangement: Chains Colour of spores: green Colour of vegetative cells: red Name of microorganism: B. subtilis

15. Choose the correct statement: The decolourizing agent in Gram's stain: a- 20% H2So4. b- 3% Hcl. c- 95% ethyl alcohol. d- 5% H2So4. The primary stain in Ziehl- Neelsen stain : a- Diluted carbol fuchsin. b- Methyl violet. c- Methylene blue. d- Strong carbol fuchsin. Choose the correct statement: The decolourizing agent in Gram's stain: a- 20% H2So4. b- 3% Hcl. c- 95% ethyl alcohol. d- 5% H2So4. The primary stain in Ziehl- Neelsen stain : a- Diluted carbol fuchsin. b- Methyl violet. c- Methylene blue. d- Strong carbol fuchsin.

16. Regarding Gram negative bacteria, all the following statements are correct except : a- They are not decolourized with alcohol. b- They are decolourized with alcohol. c- They take the counter stain (dil. carbol fuchsin). d- They appear pink in colour. Regarding Gram positive bacteria, all the following statements are correct except: a- They retain the primary stain (methyl violet). b- They are decolourized with alcohol. c- They are not decolourized with alcohol. d- They appear violet in colour. Regarding Gram negative bacteria, all the following statements are correct except : a- They are not decolourized with alcohol. b- They are decolourized with alcohol. c- They take the counter stain (dil. carbol fuchsin). d- They appear pink in colour. Regarding Gram positive bacteria, all the following statements are correct except: a- They retain the primary stain (methyl violet). b- They are decolourized with alcohol. c- They are not decolourized with alcohol. d- They appear violet in colour.

17. II – Give reason: a- Gram negative bacteria appear pink in colour. b- Gram positive bacteria appear violet in colour. c- Mycobacterium tuberculosis cannot be stained with Gram's stain. III – Compare: - Simple stains & differential stains. II – Give reason: a- Gram negative bacteria appear pink in colour. b- Gram positive bacteria appear violet in colour. c- Mycobacterium tuberculosis cannot be stained with Gram's stain. III – Compare: - Simple stains & differential stains.

18. Thank You