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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 Identification Visualization
shape & arrangement. 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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 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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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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 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: b) Gram negative:

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

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Crystal violet
↓
Iodine
↓
Alcohol
↓

Safranin 18
 Gram +ve Gram –ve
S.aureus 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)
Safranin (counter stain) 24
 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 28

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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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 ACID-FAST STAINING:
(Ziehl-Neelsen stain)

• To stain Mycobacterium species especially

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 green-
counterstain.
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.

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Result:

• Pink bacilli – Acid fast bacteria/bacilli

Eg., M.tuberculosis – long slender
bacilli.
• M. leprae – short thick bacilli.

• Blue colored bacteria – Non-acid fast

Eg., Epithelial cells, pus cells, other
bacteria.
Acid fast stain:
SPECIAL STAIN:

Used to stain special structures of

bacteria– capsule, spores, flagella,
metachromatic granules.
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.
CAPSULE STAIN BY NIGROSIN INK (BLACK)
CAPSULE STAIN- INDIAN INK(BLUE)
.
 .
Capsule stain of Streptococcus lactis
Capsule stain of Enterobacter aerogenes
 The shape of the spore is an
identifying characteristic
Swelled vs. Not swelled

spore

Bacterial cell spore

Bacterial cell
The location of the spore is also an
identifying characteristic
Central, Sub-Terminal, and Terminal
spores
Some spore forming bacteria are
capable of causing disease
Clostridium botulinum – botulism
Clostridium perfingens – gas gangrene
Clostridium tetani – tetanus
Bacillus anthracis wound infections
The Schaeffer-Fulton Stain Procedure is used to
differentiate between endospores and vegetative
cells
 Schaeffer-Fulton Stain Procedure
1. Make a smear. Air Dry. Heat fix
2. Flood the smear with Malachite Green
stain
3. Cover the flooded smear with a square of
filter paper
4. Steam slide for 10 minutes (every minute,
add a few more drops of Malachite Green
stain)
5. Allow slide to cool (after the 10 min.
steam process)
 Schaeffer-Fulton Stain Procedure
(continued)
6. Drain slide and rinse for 30 seconds with
DI water (discard filter paper)
7. Put slide on steam rack
8. Flood smear with Safranin (counter stain).
This stains the vegetative cell.
(Leave for 1 minute)
9. Drain the slide and rinse with DI water
10. Blot Dry
11. Use oil immersion objective to view
 Endospore Stain Example
Spores: Green
Cell: Red or Pink

Each Person will make a smear and Endospore

stain of: Bacillus pumilus or Bacillus subtilis
Spore stain
 FLAGELLAR STAIN – SILVER
STAIN:
This stain increases the thickness of
flagella – thus easy to see under light
microscope.
 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 don’t use heat fixation
treatment .
After air drying the slide is flooded with Leifson’s 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.
Metachromatic granule staining:

To demonstrate polar granules of

Corynebacterium diphtheriae.
Take up the stain of methylene blue – but
appears bluish black – hence granules
called metachromatic granules.
Bacilli stains blue not bluish black.
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