Lab – 5

Bacterial Smear Preparation &

Staining
 Bacterial Smear Preparation
• It is a preparation that Materials
includes a small amount • Bacterial culture (whether
of biological sample or on plate or broth)
culture spread in a very • Glass slide
thin film on the surface
of the slide. • Bunsen burner.
• Smear preparation is a • Inoculating loop
step before staining. • Permanent marker or
• A good bacterial smear pencil
preparation is the key for
good stain.
Method
Step I: Properly label a new microscope slide
Step II: Sterilize an inoculating loop by burning it and
let it cool
Step III: Remove bacteria from plate or broth sample
A: From plate (solid media)
1. Place a drop of water on the slide
2. Using a sterilized and cooled inoculation loop to obtain a very small
sample of a bacterial colony from a culture plate
3. Gently mix the bacteria with the water drop.
4. Spread the mixture in a circular motion, this will avoid cell clumping.
B. From broth (liquid media)
1. Gently tapping the broth tube by the finger to re- suspense
the bacteria
2. Using a sterilized and cooled inoculation loop to obtain broth
(one or two loopfuls taken according to the size of the loop)
3. Apply the broth on the center of the slide and spread it
evenly.
Step IV: Allow the smear to dry completely by air at room
temperature
Step V: Heat fixation
i. After smear is dried completely by air, rapidly pass it 3- 4
times through flame of Bunsen burner
ii. Avoid too much heating

iii. Allow smear to cool before staining

Summery of ideal bacterial smear preparation
An ideal bacterial smear should be thin, semi-
transparent, whitish layer or film, circular or
oval with 2 cm diameter .
The importance of heat fixation is:

1) Kill the M.O.

2) Make the M.O. stuck to the surface of the slide.

3) Make the M.O. more permeable to the stain.

4) Prevent the M.O. from going autolytic changes

 Bacterial Staining
• Bacteria are colorless Types of stains:
(transparent) MO

• Staining is a process by which

1. Basic (+ve stains)
we use different dyes to
visualize the MO 2. Acidic (-ve stains)
• By staining we can study size,
shape (cocci, bacilli, cocco- 3. Neutral stains
bacilli) and arrangment (single,
pairs, clusters or chains ) of MO
• A- Basic stains:
• Also called +ve stains. They posses (+ve) charge after ionization.
• Basic stains are attracted to negatively charged molecules in the cell
including nucleic acids (DNA and RNA) and some proteins.
• Examples: methylene blue, crystal violet, malachite green, basic
fuchsin, carbolfuchsin, and safranin.

• B- Acidic stains:
• Also called –ve stain. these stains posses (-ve) charge after
ionization.
• These stains will be repelled from the bacterial cell & only the
background is stained i.e. bacteria cells remain unstained and a
clear zone around the cell will be seen
• Examples: india ink , acid fuchsin and nigrosin.

• C. Neutral stains
• Both +ve and –ve molecules in the smear are stained.
• Neutral stain are actually a salt of acidic and basic stain.
• Examples: Giemsa, Leishman, Wright stain.
 Basic stain (crystal violet stain)

Neutral stain (Giemsa stain) usually used

Negative stain (India ink) reveals cocci to stain blood smears for parasitic
bacteria single, paired and in clusters infection
Table illustrate Basic, Acidic, and Neutral stains :

Basic (+Ve Charge) Acidic (-Ve Charge) Neutral Stains

Stains Stains

 Nigrosin  Giemsa
 Acid fuchsin  Leishman
 India ink  Wright

 Neutral red
 Stains classified according to staining techniques
or their function to

1. Simple stains
2. Differential stains
3. Special stains
1. Simple stain:
It is the using of ONE STAIN (Basic stain) only to stain
microorganisms, so MO that present in the slide appears in ONE
COLOR.
It used to get information about bacterial size, shape (cocci, bacilli,
cocco-bacilli) and arrangment (single, pairs, clusters or chains )
Procedure
This procedure is simple,easy,time saving and economic. It is
applied by using only a single stain and all bacteria in the
specimen will be stained by one color.
1-Use a clean slide, mark it and fellow the steps of smear
preparation

2-Flood smear with simple stain,basic stains,such as;crystal

violet,methyl violet…

3-Wash with tap water, drain excess water & dry in air.

4-Examine under microscope using the low , high and oil

immersion lens.
Picture of simple stain. Using single basic stain (crystal
violet) and all bacteria in slide will be stained violet.
2. Differential stain
• This type of stain used to differentiate between two
organism
• The mainly used differential stain is GRAM STAIN
• This stain first discovered by HANS CHRISTIAN GRAM 1884
• By gram stain procedure the bacteria are classified into two
main groups,

Gram positive(+ ve):retain the dark blue to violet color of

primary stain (crystal violet stain) after using alcohol
(decolorizing agent)

Gram negative(-ve): those that can’t retain primary stain

when the slide washed by alcohol(decolorizing agent)but
then take the redcolor of counter stain (safranine)
Procedure for Gram staining :
1. Prepare a fixed smear after marking our slide.
2. Flood the smear with crystal violet stain for 1 minute.
3. Wash with tap water .
4. Cover the smear with Gram iodin (mordant) for 1 minute:
The stain now is combined with iodin forming what is called Crystal
Violet Iodin complex (CVI complex).This complex will be fixed in bacteria
in its cell wall more than the stain alone. At this step all types of bacteria
present in the sample smear are colored by one color which is the dark
violet color.
5. Wash with tap water and get rid of the excess water on the slide.
6. Decolorize with (which is absolute alcohol 95% or absolute alcohol +
acetone) for 5 sec.
7. Wash with running water immediately. Cover the smear with the
counter stain which is safranine in order to color the bacteria that have
lost the stain during the use of alcohol. Leave for 1 minute and wash
with tap water.
8. Leave to dry at room temperature & Examine by oile immersion lens.
9. Examine under microscope using the low , high and oil immersion lens.
In summary, the steps of gram stain
procedure include:
Principle
1. Crystal violet and iodine combined to form a dye-iodine
complex
2. Dye-iodine complex is dissociated by alcohol (decolorizing
agent)
3. The cell wall of Gram-negative organisms have relatively
little peptidoglycan and mainly consist of lipoproteins and
polysaccharides. While in Gram positive organisms the
peptidoglycan comprises the major part of the cell wall so
Gram +ve more rigid than Gram negative cells and less
permeable for the dye-iodine complex to diffuse freely out
of the cell during the process of decolorization.
4. The more acid charater of the protoplasm of Gram positive
bacteria which is also enhanced by treatment with iodine
may partly explain their strong retention of the basic dye.
3. Special stain:
This procedure is used when a certain bacteria or some
of their structures could not be stained
Special stain include:
A. Acid fast stain (Zeihl Neelsen stain)
B. Capsular stain
C. Spore stain
D. Flaggellar stain
A. Acid fast stain (Zeihl Neelsen stain)
• This technique is used for staining of M. tuberculosis
& M. leprae (the causative agents of tuberculosis and
leprosy respectively) and using for staining of Nocardia
or Actinomyces.
• The above bacteria possess a complex lipid in their cell
wall which is called wax D
• Wax D composed of two molecules of mycolic acid and
one molecule of the sugar trehalose, which mainly
gives the bacteria resistance to be stained by ordinary
stains and also lead to the formation of what is called
serpentine cords when a smear is taken from a colony
on Lownstein Jensen medium.
Procedure for Acid Fast staining :
1. Specimen:taken from either a suspected tuberculosis
patients Or formalin killed culture.
2. Prepare a heat fixed smear after marking our slide.
3. Flood the smear with Strong carbol fuchin stain and
steam for 5 minutes.
4. Leave to cool down, hold the slide with forceps in an
angle position and add 20% H2SO4 or acid alcohol
until no more color runs off.
5. Wash with tap water and add the counter stain which
is Methylene blue. .
6. Wash with tap water,Leave to dray in an angel
position .
7. Examine by oil immersion lens for Red bacilli.
B. Capsule stain :
• Capsule is a gelatinous ,non ionic structure, spherical
or oval shape surrounding certain of bacteria as a
protective extracellular structure, protect the cell from
phagocytosis by phagocytic cell,
• It is considered as virulence factor & Antigenic
structure .
• The capsule is consisting of 97-98% water and only 2-
3% of dissolved solid (polysaccharides,glycoproteins ,
• polysaccharides with certain lipids)
• Since the capsule consisting of large amount of water,
it requires a special procedure for staining . One of the
procedures is called Hiss method :
Procedure for Capsule staining (Hiss method ) :
1. Prepare a smear for bacteria that possess a capsule, such as; Klebsiella
or Pneumococci and heat fix very gently on the flame to avoid damaging
of the capsule by excess heat.
2. Flood the smear with 1% aqueous solution of crystal violet stain.
3. Steam very gently for 2-3 minutes.
4. Leave to cool and rinse with 20% copper sulfate ( CuSo4 ).
5. Leave to dray and examine under the oil immersion lens. capsule appear
faint surrounding dark blue cell.

Other methods to view capsule include :

A. Capsule-swelling test ( Quelling reaction ).
B. Negative stain : India ink , Nigrosin (-ve stain).

•
Negative stain Negative stain procedure
India ink, Nigrosin stain
• Used to visualize MO that
not easily stained e.g.
cryptococcus neoformans
or visualize capsule of
encapsulated bacteria
(eg streptococcal
pneumonia).
• Negative stain repelled
by negatively charged
bacteria cell wall so only
background will be
stained
 Negative stain

Negative stain (india ink) Negative stain (India Ink)

Capsule of bacteria Cryptococcus neoformans
C. Spore stain :
During sporulation process,the cell forms several layers :
• Core: which contains its DNA and some ribosomes and
dipicolinic acid and some other most important factors that
keep the cell resistant to most adverse conditions.
• Cortex: consist of double layer of peptidoglycan, differs from
that of the cell wall.
• Exosporium: consist of keratinized protein.
The above layers give the spore protection against several hard
environmental conditions, at the same time, become difficult to
stain the spore with the ordinary staining techniques; therefore
its staining requires a special procedure such as steaming in
order to allow the stain to penetrate into the spore.
Procedure for Spore staining ( Schaeffer-fulton method) :
1. Prepare a heat fixed smear of a spore forming bacteria
such as Bacillus subtilis or any species of Clostridium.
2. Flood the smear with Malachite green stain and steam for
5 minutes.
3. Leave to cool down and wash with tap water.
4. Add Safranine as a counter stain ,for 1 minutes.
5. Wash with tap water, Leave to dray in upright position .
6. Examine by oil immersion lens .
7. Location and the size of endospores vary with species,
thus they are of value in identify bacteria.
8. Endospores position within the cell is characteristic. It can
be either :
1.Central. 2. Subterminal. 3. Terminal.
D. Flagella stain :
• Flagella is tiny hair like structure of locomotion . Their
numbers and patterns of arrangement provide clues to
identify species.
• Flagella can be viewed by:
1. E. M.
2. D.F.M ( Dark field ).
3. Flagellar staining
• Flagellar stain contains Rosaniline dyes (basic fuchsin), and a
mordant, tannic acid, applied to bacterial suspension fixed
in formalin and spread across a glass slide .
• The dye and mordant precipitate around the formalin fixed
flagella surface, enlarging their diameter and making then
visible.
Flagella arrangement

Flagella