Experiment 2
Gram Stain and Acid-fast Stain
Staining
- simple stain (only one dye)
- differential stain (more than one dye)
Gram stain
Acid-fast stain
G+
G-
Gram stain
Danish physician
Hans Christian Gram
(1853-1938)
Procedure & Principle of Gram Stain
Prepare a smear, air dry
↓flame 3 times to fix
Crystal violet, 1 min
↓wash
Gram’s iodine, 1 min
↓wash
Ethanol 95%, 0.5 min
↓ wash
Safranin, 1 min
↓ wash
Primary stain
Mordant Stain
Decolorization
Counterstain
Dry slide with bibulous paper
↓
Observation
(10× lens →40× lens → oil-immersion lens )
Significance of Gram Stain
 Differentiate bacteria: G+ / G-
 Helpful to select sensitive antibiotics.
 identify different virulent factors of bacteria.
• G+: exotoxin
• G-: endotoxin.
Gram Stain
2 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
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
 Acid-fast Stain Procedure
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
2 students/group
Acid-fast
BCG
Non acid-fast
B.subtitis
BCG is a form of
tuberculosis vaccine
that is a freeze-dried
preparation of a live
and attenuated
bovine organism.
 Acid-fast Stain
2 students/group
Materials:
 Bacterial smears
B.subtitis slides
BCG slides
 Carbolfuchsin
 Acid Alcohol
 Methylene Blue
 Normal saline (NS)
 Slides and bibulous paper
1 slide/group
1 slide/group
Experiment 3
Observation of Bacterial Morphology
Observation of Bacterial Morphology
Contents
Coccus
Bacterial
shape
Special
structure
Slides
Staphylococcus
Streptococcus
S. pneumoniae
Focus on
Shape, size,
arrangement
Bacillus
E.coli
B.subtitis
Spirillum
V.cholerae
Capsule
S. pneumoniae
Size & relationship
with bacteria
Spore
C. tetani
Size & position
Flagella
Proteus
Shape, number &
position
Staphylococci
Streptococci
Streptococcus pneumoniae
Escherichia coli
Bacillus anthrax
Vibrio cholera
Streptococcus pneumoniae
Proteus
Clostridium tetani