STAINS FOR THE MICROBIAL ORGANISMS
PREPARED BY MR. ABHIJIT DAS
BACTERIA
GRAM’S STAIN
Gram's Stain:
A differential staining technique to classify bacteria.
Ø Principle:
o Gram-positive
bacteria have a thick peptidoglycan layer that retains the
primary crystal violet stain.
o Gram-negative
bacteria have a thinner peptidoglycan layer and an outer
membrane, losing crystal violet and taking up the counterstain.
Ø Steps:
o Apply
crystal violet (primary stain).
o Add
iodine (mordant) to form a complex with the stain.
o Decolorize
with alcohol/acetone.
o Counterstain
with safranin.
Ø Results:
o Gram-positive:
Purple.
o Gram-negative:
Pink
ZIEHL-NEELSEN
STAIN
Ziehl-Neelsen Stain:
A special stain to identify acid-fast bacteria, such as Mycobacterium
tuberculosis.
Ø Principle:
o Acid-fast
bacteria have mycolic acid in their cell
walls, making them resistant to decolorization by
acid-alcohol.
Ø Steps:
o Primary
Stain: Apply carbol fuchsin (red) and heat to help
penetration.
o Decolorization:
Use acid-alcohol to remove stain from non-acid-fast organisms.
o Counterstain:
Apply methylene blue.
Ø Results:
o Acid-fast
bacteria: Red.
o Non-acid-fast
bacteria: Blue.
FITE
ACID-FAST STAIN FOR LEPROSY
Fite Acid-Fast Stain:
A modified acid-fast staining method used to detect Mycobacterium leprae
(causative agent of leprosy).
Ø Principle:
o M.
leprae has a lipid-rich cell wall that retains carbol
fuchsin after gentle decolorization.
o This
method uses a milder decolorizing agent to preserve the fragile structure of M.
leprae.
Ø Steps:
o Primary
Stain: Apply carbol fuchsin with gentle heating.
o Decolorization:
Use a diluted acid-alcohol solution to avoid over-decolorization.
o Counterstain:
Apply methylene blue or hematoxylin.
Ø Results:
o Acid-fast
bacilli (M. leprae): red.
o Background:
Light blue.