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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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Dyes
• make internal and external structures of cell more
visible by increasing contrast with background
• Dyes used to stain bacterial cells are organic
compounds, which have affinity for specific cellular
components.
• The many types of dyes have two features in
common: 1) they have chromophore groups, groups
with conjugated double bonds that give the dye its
color and
• 2) they can bind with cells by ionic, covalent, or
hydrophobic bonding. Dyes also contain an
auxochrome group, which in itself does not produce
color but gives the dye its acidic or basic properties.

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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 Identificatio Visualization
shape & arrangement. n of structure
Gram Acid fast
stain 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.

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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 Tetrads Chains or Pairs

Staphylococci Micrococci Streptococci

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 Simple Staining
Type of staining:- Simple
Stain
Name of dye:- Methylene
blue

Shape of cells:- bacilli

Arrangement of cells:-
chains
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
it classifies bacteria
into two major groups:

a)Gram positive: b) Gram negative:

Appears violet after Appears red after Gram’s
Gram’s stain stain

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Basic classification of bacteria is based on the cell wall
structure.

The staining technique, developed in the late 1700’s by

Christian Gram classifies the rigid cell walled bacteria into one
of two groups based on whether they are able to resist the
decolorizing action of an alcoholic solution.

Those that resist decolorization by 95% ethanol are arbitrarily

termed Gram positive and those that do not are Gram negative
(the terms positive and negative have nothing to do with
charges of the cell but based on differences in the cell wall
structure of these two groups of bacteria).
 Principle of Gram Staining
Crystal Violet (purple)
– Primary stain; positive stain
– Stains cell wall purple
Iodine
– Mordant
– Combines with CV to form an insoluble complex that gets trapped in
thicker peptidoglycan layers
Ethanol
– Decolorizer
– CV-I complex washed out of Gram negative organisms because it cannot
be trapped by peptidoglycan layer; flows right through outer membrane
Safranin (pink)
– Counterstain
– Simple positive stain that provides contrasting dye for decolorized cells
(Gram negative)
– Stains all cells, but only the negative ones actually appear pink.
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 decolorization with Aceton-Alcohol.
Therefore, cells will be counterstained with safranin
and turned red.
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There are 4 conditions to be followed for a valid Gram staining
procedure: 

Young cultures - must be young within 18-24hrs old

(older cultures lose their Gram staining properties
due to changes in the CWs as the cells get older)
Thin smear
thicker or uneven smears will result in uneven
staining
and decolorization
Fresh reagents - of proper strength
Control cultures - for a known GP bacterium and GN
culture (S.aureus & E.coli)
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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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