PHT 226PHT 226Lab # 3Lab # 3

Gram’s stain (mixture)Gram’s stain (mixture)

Acid fast stain Acid fast stain

Spore stainSpore stain

Staining of BacteriaStaining of Bacteria

Types of staining technique:-Types of staining technique:-

Simple stainingSimple staining (use of a single stain)(use of a single stain)

Differential stainingDifferential staining (use of two contrasting stain)(use of two contrasting stain)

For visualization of For visualization of morphologicalmorphological

shape & arrangement.shape & arrangement.

IdentificationIdentification Visualization Visualization of structureof structure

Gram Gram stainstain

Acid fastAcid fast stainstain Spore Spore

stainstainCapsuleCapsule

stainstain

Smearing out of the sampleSmearing out of the sample

Smear FixationSmear Fixation

Principle of Differential StainsPrinciple of Differential Stains

* Application of the primary * Application of the primary stain.stain.

* Decolourization.* Decolourization.

*Application of the counter-*Application of the counter-stain.stain.

Acid Fast StainAcid Fast Stain

Acid fast bacteriaAcid fast bacteria (ex; Mycobacteria)(ex; Mycobacteria) are difficult to be stained by simple are difficult to be stained by simple or Gram’s stain, because they have a or Gram’s stain, because they have a high lipid content in their cell walls high lipid content in their cell walls which prevent the penetration of which prevent the penetration of ordinary aniline dyes.ordinary aniline dyes.

Once these organism are stained, Once these organism are stained, they resist decolorization even with a they resist decolorization even with a very strong decolorizing agent such very strong decolorizing agent such as acid-alcohol. as acid-alcohol.

Acid Fast StainAcid Fast Staine.g., Ziehl-Neelsen Staine.g., Ziehl-Neelsen Stain

AFS is an important diagnostic AFS is an important diagnostic value in identifying pathogenic value in identifying pathogenic members of genus members of genus Mycobacterium such as Mycobacterium such as M.M. tuberculosistuberculosis and and M. leprea.M. leprea.

Materials:- Materials:- Culture of Culture of M. phelei 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)

Ziehl-Neelsen Ziehl-Neelsen StainStain

4 5 64 5 6

77

1 2 31 2 3

Acid Fast StainAcid Fast Stain Procedure:-Procedure:-

Carbol fuchsin\\\\

5 min

alcohol

30-60 sec

MB

1 min

ResultsResults

Name of Stain: Acid fast stain

Shape: beaded bacilli

Arrangement: Tree shaped

Colour: red

Name of microorganism: M.phelei

The Spore Stain The Spore Stain

Some bacteria form resistant bodies Some bacteria form resistant bodies in the cell known as endospores.in the cell known as endospores.

Bacterial spores are highly resistant Bacterial spores are highly resistant to physical & chemical agents & are to physical & chemical agents & are not easily stained by routine not easily stained by routine staining.staining.

Heat is required in spore staining to Heat is required in spore staining to promote the penetration of the dye promote the penetration of the dye into the spore.into the spore.

Once the spores stained they resist Once the spores stained they resist the decolorization.the decolorization.

The Spore StainThe Spore Stain

Materials :-Materials :-Culture of B. subtilisCulture of B. subtilisMalachite green (primary Malachite green (primary stain)stain)

Safranine (counter stainSafranine (counter stain))

Spore Stain ofSpore Stain ofBacillus subtilisBacillus subtilis

Name of Stain: Spore stain

Shape: bacilli

Arrangement: Chains

Colour of spores: green

Colour of vegetative cells: red

Name of microorganism:

B. subtilis

      Choose the correct statementChoose the correct statement::

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

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

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

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