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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 Eeasily
determined under the microscope.

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 Principle of staining
Stains → combine chemically with the
bacterial protoplasm.

Commonly used stains are salts:

v Basic dyes: colored cation + colorless
anion

chloride)
MB+ + Cl-
f
e.g. methylene blue (methylene blue

O
v Acidic dyes: colored anion + colorless
cation e
e.g. eosin ( Na+ + eosin-).
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Bacterial cells are slightly negatively

charged ( rich in nucleic acids bearing
g
negative charges as phosphate groups)
→ combine with positively charged
basic dyes
as 5
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 Identification Visualization
shape & arrangement. of structure
Gram Acid fast
stain stain Spore Capsule

00
stain stain
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 Smear Preparation:
Preparation and Fixation of Bacteria for
Staining.

Objective:
To kill the microorganism & fix them to the
I
slide to prevent them from being washed out
during the process of staining.
I
7

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,
I
safranin. a
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 Tetrads

I
Chains or Pairs

Staphylococci Micrococci 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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most get
Simple Staining
ISMAY
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:-
a
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
TIM
Name of m.o:-
Staphylococci
É
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Gram Stain:
It is the most important
differential stain used in

E
bacteriology because
it classified bacteria
into two major groups:

a)Gram positive: b) Gram negative:

Appears violet after
Gram’s stain T
Appears red after Gram’s
stain

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Crystal violet
↓
Iodine
↓
Alcohol
↓

Safranin 20
 Gram +ve Gram –ve
S.aureus E.coli

Step 1: Crystal Violet

Step 2: Gram’s Iodine

Step 3: Decolorization
(Aceton-Alcohol)
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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-positive bacteria
Have a thick peptidoglycan layer surrounds the cell.
The stain gets trapped into this layer and the
bacteria turned purple.

F
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, fangs
E.coli
Gram stain:
Crystal violet (primary stain)
Gram’s iodine (mordant)
Acetone-alcohol (decolorizing agent)
Safranin (counter stain) 25

Gram Stain

Procedure:
CV
safranin
iodine

30 sec
sec
30-60
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 candida
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 Results:
Shape: Bacilli
Arrangment: Chains

Colour: Violet
Gram’s reaction: Gram’s +ve

Name of microorganism:

Bacillus Bacillus
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Results:
Shape: Rods Poods
Arrangment: Single

Colour: red
Gram’s reaction: Gram’s –ve

Name of microorganism: E Colli

Gram negative bacilli 30
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