Download 226 lab 3

PHT 226
Lab # 3
Gram’s stain (mixture)
Acid fast stain
Spore stain
Staining of Bacteria

Types of staining technique:-
Simple staining
(use of a single stain)
For visualization of
morphological
shape & arrangement.
Differential staining
(use of two contrasting stain)
Identification
Gram
stain
Visualization
of structure
Acid fast
stain Spore
stain
Capsule
stain
Smearing out of the sample
Smear Fixation
Principle of Differential Stains
* Application of the primary stain.
* Decolourization.
*Application of the counter-stain.
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.

Once these organism are stained, they resist
decolorization even with a very strong
decolorizing agent such as acid-alcohol.
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.

Materials:Culture of M. phelei
 Conc. carbol fuchsin (primary dye)
 Acid-alcohol (decolorizing agent)
 Methylene blue (counter stain)

1
4
2
3
5
6
7
Ziehl-Neelsen Stain
Acid Fast Stain

Procedure:-
5 min
alcohol
Carbol
MB
fuchsin
\\\\
30-60
sec
1 min
Results
Name of Stain: Acid fast stain
Shape: beaded bacilli
Arrangement: Tree shaped
Colour: red
Name of microorganism:
M.phelei
The Spore Stain




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.
Heat is required in spore staining to
promote the penetration of the dye into the
spore.
Once the spores stained they resist the
decolorization.
The Spore Stain

Materials : Culture
of B. subtilis
 Malachite green (primary stain)
 Safranine (counter stain)
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
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.
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.
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.
Thank You