226 PHT
Lab #2
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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.
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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+ + Cl Acidic dyes: colored anion + colorless
cation
e.g. eosin ( Na+ + eosin-).
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Bacterial cells are slightly negatively
charged ( rich in nucleic acids bearing
negative charges as phosphate groups)
→ combine with positively charged
basic dyes
Acidic dyes do not stain the bacterial
cell → can stain the background
material with a contrasting color.
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Types of staining techniques
Simple staining
Differential staining
(use of a single stain)
(use of two contrasting stains
separated by a decolorizing agent)
For visualization of
morphological
shape & arrangement.
Identification
Gram
stain
Visualization
of structure
Acid fast
stain Spore
stain
Capsule
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.
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Smear preparation
S
Fixation
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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.
Objective:To show the morphological shapes and
arrangement of bacterial cells.
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Simple Staining
Procedure:-
MB
1-2 min
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Basic Shapes of Bacteria
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
Gram’s stain
b) Gram negative:
Appears red after Gram’s
stain
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Crystal violet
↓
Iodine
↓
Alcohol
↓
Safranin
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Gram +ve
S.aureus
Gram –ve
E.coli
Step 1: Crystal Violet
Step 2: Gram’s Iodine
Step 3: Decolorization
(Aceton-Alcohol)
Step 4: Safranin Red
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Step 1: Crystal Violet
Step 2: Gram’s Iodine
Step 3: Decolorization
(Aceton-Alcohol)
Step 4: Safranin Red
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Gram’s +ve Bacteria
Gram’s -ve Bacteria
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Gram’s +ve Bacteria
Gram’s -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)
Gram’s iodine (mordant)
Acetone-alcohol (decolorizing agent)
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Safranin (counter stain)
Gram Stain
Procedure:
safranin
CV
iodine
s
30
sec
30-60
sec
10
sec
2 min
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Results:
Shape: Cocci
Arrangment: irregular clusters
Colour: Violet
Gram’s reaction: Gram’s +ve
Name of microorganism:
Staphylococci
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Results:
Shape: Oval
Arrangment: Single
Colour: Violet
Gram’s reaction: Gram’s +ve
Name of microorganism:
Candida
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Results:
Shape: Bacilli
Arrangment: Chains
Colour: Violet
Gram’s reaction: Gram’s +ve
Name of microorganism:
Bacillus
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Results:
Shape: Rods
Arrangment: Single
Colour: red
Gram’s reaction: Gram’s –ve
Name of microorganism:
Gram negative bacilli
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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.
No heat fixation or strong chemicals are
used→ the bacteria are less distorted
than in other staining procedure.
Example: Nigrosine
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Negative staining
Candida
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Negative staining
Staphylococci
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Negative staining
Bacillus
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