Staining
Staining
Staining
Staining of Bacteria
Bacteria cells are almost colorless and
transparent
A staining technique is often applied to the
cells to color them
Their shape and size can be easily
determined under the microscope.
Principle of staining
Stains combine chemically with the
bacterial protoplasm.
Commonly used stains are salts:
Basic dyes: colored cation + colorless
anion
e.g. methylene blue (methylene blue
chloride)
MB+ + ClAcidic dyes: colored anion + colorless
cation
e.g. eosin ( Na+ + eosin-).
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basic dyes
Differential staining
For visualization of
morphological
shape & arrangement.
Identificatio
n
Gram
stain
Visualization
of structure
Acid fast
stain Spore Capsule
stain
stain
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Smear Preparation:
Preparation and Fixation of Bacteria for
Staining.
Objective:
To kill the microorganism & fix them to the
slide to prevent them from being washed out
during the process of staining.
Definition:
It is the use of single basic dye to
color the bacterial organism.
e.g. methylene blue,
crystal violet,
safranin.
All bacteria take the color of the dye.
Cocci
Bacilli
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Arrangements
Cocci
Irregular Clusters
Staphylococci
Tetrads
Micrococci
Chains or Pairs
Streptococci
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Results
Type of staining:
Name of stain:
Shape of cells:
Arrangement of cells:
Color:
Name of m.o:
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Simple Staining
Type of staining:- Simple
Stain
Name of dye:- Methylene
blue
Shape of cells:- bacilli
Arrangement of cells:strain
Color:- Blue
Name of m.o:- Bacillus
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Simple Staining
Type of staining:- Simple
Stain
Name of dye:- Methylene blue
Shape of cells:- cocci
Arrangement of cells:clusters
Color:- Blue
Name of m.o:Staphylococci
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Simple Staining
Type of staining:- Simple
Stain
Name of dye:- Crystal violet.
Shape of cells:- cocci
Arrangement of cells:clusters
Color:- Purple
Name of m.o:Staphylococci
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Gram Stain:
It is the most important
differential stain used in
bacteriology because
it classified bacteria
into two major groups:
a)Gram
positive:
Appears violet after
Grams stain
b) Gram
negative:
Appears red after
Grams stain 17
Crystal violet
Iodine
Alcohol
Safranin
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Gram +ve
S.aureus
Gram ve
E.coli
Step 3: Decolorization
(Aceton-Alcohol)
Step 3: Decolorization
(Aceton-Alcohol)
Grams +ve
Bacteria
Grams +ve
Bacteria
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Gram-positive bacteria
Have a thick peptidoglycan layer surrounds the cell.
The stain gets trapped into this layer and the bacteria
turned purple.
Retain the color of the primary stain (crystal violet)
after decolorization with alcohol
Gram-negative bacteria
have a thin peptidoglycan layer that does not retain
crystal violet stain.
Instead, it has a thick lipid layer which dissolved
easily upon decoulorization with Aceton-Alcohol.
Therefore, cells will be counterstained with safranin
and turned red.
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Gram Stain
Materials:Cultures of Staphylococcus aureus,
Candida albican,
Bacillus subtilis,
E.coli
Gram stain:
Crystal violet (primary stain)
Grams iodine (mordant)
Acetone-alcohol (decolorizing agent)
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Safranin (counter stain)
Results:
Shape: Cocci
Arrangment: irregular
clusters
Colour: Violet
Grams reaction: Grams +ve
Name of microorganism:
Staphylococci
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Results:
Shape: Oval
Arrangment: Single
Colour: Violet
Grams reaction: Grams +ve
Name of microorganism:
Candida
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Results:
Shape: Bacilli
Arrangment: Chains
Colour: Violet
Grams reaction: Grams +ve
Name of microorganism:
Bacillus
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Results:
Shape: Rods
Arrangment: Single
Colour: red
Grams reaction: Grams ve
Name of microorganism:
Gram negative
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Negative staining
(Indirect staining with acidic
dye)
The negative staining technique does not
stain the bacteria due ionic repulsion.
but stain the background.
The bacteria will appear colorless against
a dark background.
Negative staining
Candida
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Negative staining
Staphylococci
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Negative staining
Bacillus
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ACID-FAST STAINING:
(Ziehl-Neelsen stain)
M.tuberculosis.
High lipid content makes decolorisation very
difficult extraordinary property.
Principle:
Acid fast(resist) Property of Mycobacterium
species - once this bacteria stained with primary
dye difficult to decolorise with acid.
This property due to Mycolic acid in cell wall.
M.tuberculosis also Alcohol fast
Staining reagents:
1.Strong carbol fuchsin primary stain
2.20% sulphuric acid/3% Hcl decoloriser
acid-fast property.
3. 95% alcohol- decoloriser- alcohol fast
property
4. Methylene blue/ Malachite greencounterstain.
Note:
5% sulphuric acid for M.leprae.
1% sulphuric acid for Nocardia species.
Procedure:
1. Strong carbol fucshin-heat till steam rises allow 5-10 min to
act (alternately leave it 10-15 min cold staining method)
wash.
2. Decolorise with acid-alcohol mixture till get a faint pink colour
in the smear (take 3-5 min) wash.
3. Methylene blue/Malachite green 2 min wash.
4. Allow to dry and focuss under microscope.
Result:
Pink bacilli Acid fast bacteria/bacilli
SPECIAL STAIN:
CAPSULE STAIN:
Negative stain:
1.Drop of Nigrosin ink+ indian ink
2. Bacterial culture ( 1-2 colonies)
3. Spread evenly and air-dry.
4. Look for unstained structures against
stained background.
spore
Bacterial cell
spore
Bacterial cell
Spore stain
Procedure
First of all take two hours old flagellated
cell culture slant and add two to three
drops of sterile distill water in the slant
with the help of sterile pipette.
Note that the distill water is added slowly
without disturbing the growth of cells.
After addition of distill water incubated the
slant for 20 minutes.
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Procedure
Then take a drop of suspension from the slant and place
the drop on a clean slide which is kept in slanting
position.
The drop should flow slowly from one end of slide to
other end to avoid folding of flagella on cell.
Allow smear to air dry here we dont use heat fixation
treatment .
After air drying the slide is flooded with Leifsons stain till
a thin film of shinny surface appear.
After this give a gentle stream of water wash treatment to
a slide.
Now treat the slide with 1 % methylene blue treatment for
1 minute.
Give the slide water wash treatment ,air dry and observe
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under oil immersion lens.
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