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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.

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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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.

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.

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.

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

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 1: Crystal Violet

Step 2: Grams Iodine

Step 3: Decolorization
(Aceton-Alcohol)

Step 4: Safranin Red

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Step 1: Crystal Violet

Step 2: Grams Iodine

Step 3: Decolorization
(Aceton-Alcohol)

Step 4: Safranin Red

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Grams +ve
Bacteria

Grams -ve Bacteria

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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.

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 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

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 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.

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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