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Bacteria & It’s Staining
Abhishek D. Chouhan
M. Pharm*(Industrial Pharmacy)
Bacteria
A bacteria contains as outer cell envelope which consists of
• Outer cell wall
• Inner cytoplasmic membrane cytoplasm is present inside
the cell envelope.
• In the cytoplasm, there are inclusions such as
ribosomes, granules, vacuoles and DNA. The bacterial
cell as a whole may be enclosed in a capsule. Some
bacteria may flagellate which are used for locomotion
and fimbriae which are used for adhesion. The flagella
and fimbriae are appendages which protrude from the
cell surface.
Bacteria based on Shape
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Cooci (Coccus)
Rod-shaped or Bacillus
Vibrio or comma
Spiral or Helical
Spirochetes (also helical & spiral but cell
flexible)
Cocci (Coccus)
Rod shaped or Bacillus
Vibrio or Comma
Spiral or Helical
Spirochetes
Some are also as
• Filamentous
• Star-shaped
• Stalked
• Some able to change the shape due to
environment which known as Pleomorphic.
• E.g. Rhizobium, Corynebacterium.
Bacteria on the basis of flagella
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Atrichous (Pasteurella)
Monotrichous (Pseudomonas)
Lophotrichous (Spirillum undulla)
Amphitrichous (Nitrosomonas)
Peritrichous (Salmonella typhosa)
Bacteria on the basis of flagella
Bacteria on the basis of use of
oxygen
• Aerobic – Organism which can grow in
presence of oxygen.
• Anaerobic – Organism which can grow in
absence of oxygen.
• Facultative aerobic – Organism which
can grow in both.
Staining of bacteria
• Staining is an auxiliary technique used in microscopy to enhance contrast
in the microscopic image. E.g.
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Acridine orange
Bismarch brown
Carmine
Coomassie blue
Cresyl violet
Eosin
Ethidium bromide
Acid fuchsine
Iodine
Malachite green
Methyl green
Methylene blue
Safranin
Types of staining technique
Simple staining (use only
Monochrome staining
one stain and useful for
(Positive staining)
observing the morphological
features of the bacterial
Negative/indirect (relief)
cells)
staining
Use of single stain to color
the bacteria
Differential staining (which Gram staining
differentiates two kinds of
microbes)
Positive are purple &
negative are red in color.
Acid fast stain
In this background is
stained and cell remains
colorless
Ziehl Neelsen method
Auramine method
There are also some other techniques but above mentioned is more practically and
useful.
Gram staining
(By Dr. Hans Christian Gram in 1884)
• Crystal violet is added as primary stain. It colors the
cytoplasm of all cells purple.
• Iodine is then used as mordant, an agent that binds the dye
to the cell and helps resist decolorization. It combines with
crystal violet to form an insoluble complex inside the cell.
• A decoloursing agent (alcohol or a mixture of acetone &
alcohol) is then added. The purple dye complex is retained
by gram positive organisms but is readily removed from gram
negative cells. Gram-negative cells will therefore be colorless
at this stage.
• The red dye safranin is applied as a counterstain. This
stains gram negative bacteria red while gram positive cells
remain purple.
Gram staining procedure
Application of
purple dye
Application of
iodine (mordant)
Alcohol wash
(decolorization)
Application of safranin
(counterstain)
Acid fast stain
• Procedure• Fixed smear is stained by hot Carbol fuchsin for 10 minutes and
washed in tap water.
• Smear is decolorized by 1% HCl in 70% alcohol or by 20% H2SO4
for half to one minute. Decolorized is continued with 70% neutral
alcohol for 2-3 minutes until the smear is pinkish on washing.
M.leprae, however, should be strictly decolorized by 5% H2SO4
only as it is less acid fast.
• The smear is counterstained with 2% methylene blue or
malachite green for 2 minutes and washed in water.
• Observation• Acid fast bacilli appear red in blue background of pus cells and
epithelial cells.
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