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MICROSCOPIC EXAMINATION
OF BACTERIA (UNSTAINED &
STAINED SMEARS)
By
Dr. Emad AbdElhameed Morad
Lecturer of Medical Microbiology and Immunology
There are two principal ways of
preparing a microbial specimen for
observation with light microscope:
1. Unstained smears (wet preparation): to
examine the motility of the bacteria.
2. Stained smears: to study the size, shape,
arrangement and staining affinity of the
bacteria.
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Stained
smears
• Study size, shape, arrangement and
staining affinity of the bacteria.
size
• Bacteria are measured in microns.
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Shap
e
o Bacteria may have several shapes:
Cocci: spherical shape
Bacilli: straight rods
Vibrios: curved rods
Spiral: spiral filaments
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Arrange
ment
Cocci: may occur in clusters (staphylococci),
in pairs (pneumococci), in chains (streptococci).
Bacilli: may be separately arranged
(salmonella), in pairs (klebsiella), in chains
(Bacillus anthracis), in Chinese letter
arrangement and club shaped ends
(Corynebacterium diphtheria).
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Cocci arranged
in clusters
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Cocci arranged
in chains
6
Stains may be:
Staining
properties
1. Simple stains: using one stain only such as
methylene blue, carbol fuchsin.
2. Differential stains: using two stains primary and
secondary separated by a step of decolorization.
Gram
stain
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ZiehlNeelsen
stain
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Gram
stain
 With Gram stain, bacteria could be divided
into:
Gram positive bacteria: bacteria that retain
crystal violet iodine dye complex and so appear
purple.
Gram negative bacteria: bacteria that destain
with 95% alcohol and appear pink due to
counterstaining with carbol fuchsin.
 This difference in staining affinity is due to
difference in the permeability of cell wall.
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Gram
staining
Preparation and fixation of
smears
Staining
Examination
Interpretation
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Preparation of
smears
Put a drop of water on the middle of the slide
using the inoculating needle.
With the sterile needle, collect bacteria from
agar surface by touching the bacterial
growth.
Rub the tip of the needle on the glass slide
in the drop of water in a circular motion till
you get homogenous smear.
Allow the smear to dry.
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Fixation of
smears
o Pass the slide with the smear side
uppermost over the flame for 2-3 times.
o Do not overheat the smear. The slide should
only be warm when you touch it with hands.
o Passage of the smear through heat has two
benefits:
1. Fixation of the smear to the slide.
2. Killing of the bacteria in the smear so it
becomes non infectious.
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Staining of
smears
o
o
o
o
o
o
o
o
o
Cover the smear with crystal violet for 1 minute.
Pour it off then wash with water.
Add iodine solution to the smear for 1 minute.
Gently wash with water.
Decolorize by 95% alcohol and rock the slide from side to
side and pour it off. Reapply alcohol till no violet color
comes off.
Wash with water
Counterstain with diluted carbol fuchsin for 1 min.
Wash with water.
Place the film at angle to air dry or blot dry with filter
paper.
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Gram
staining
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Examination of
smears
o Rack the condenser high and open the iris
diaphragm.
o Place a drop of immersion oil on the smear.
o Put the slide with the smear side up on the stage.
o Use the oil immersion lens.
o Lower the oil lens till the lens contacts the oil and
almost touches the smear.
o Look through the eye piece of the microscope.
o Focus on the object using the coarse adjustment
screw then the fine one.
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Interpretation of
smears
o Comment on the bacterial morphology as regards:
Size
Staini
ng
Shap
e
Arrangeme
nt
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Ziehl
Neelsen
stain
This stain is used for detection of bacteria which
are described as acid fast.
These bacteria are not stained with ordinary stains
but they need exposure to strong stains with
application of heat.
Once stained, they will resist decolorization with
mineral acids such as H2SO4 or HCL.
This property is due to large amount of lipids and
fatty acids especially mycolic acid wax in cell wall
of these bacteria.
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Examples of acid fast bacteria or bacterial
structures:
A. Tubercle bacilli: retain red carbol fuchsin when
decolorized with 20% H2SO4 or 3% HCL in
alcohol.
B. Lepra bacilli and saprophytic acid fast bacilli:
retain red dye when decolorized with 5% H2SO4
or 1% HCL in alcohol.
C. Actinomyces clubs and nocardia: retain red
dye when decolorized with 0.5% to 1% H2SO4.
D. Spores: tolerates only 0.25% to 0.5% H2SO4.
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Ziehl
Neelsen
staining
Preparation and fixation of
smears
Staining
Examination
Interpretation
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Preparation and
fixation of smears
 Tubercle bacilli cause tuberculosis. Pulmonary
tuberculosis is the commonest form of tuberculous
infection in which tubercle bacilli are found in the
sputum of the patients.
 Smears could be prepared from sputum
samples as follows:
o Three morning sputum samples are preferable since they
represent overnight accumulation.
o Choose a purulent portion of sputum and spread it evenly in
the middle of a new clean glass slide.
o Leave the smear to dry.
o Then fix the smear byDr.passing
through the flame.
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Staining of
smears
o Flood the smear with strong carbol fuchsin. Allow the stain
to act for 5-10 minutes.
o Heat intermittently until the vapor begins to rise. Do not
allow the stain to boil or dry.
o Pour it off then wash with water.
o Flood the smear with 20% H2SO4 or 3% HCL in 95%
alcohol. Allow to act for 1 min. then wash with water and
reapply fresh acid. Repeat this process several times till the
smear becomes colorless or pale pink.
o Wash thoroughly with water.
o Add methylene blue or malachite green for 2 min.
o Wash with water. Dry then examine.
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Kinyoun
technique
o It is called cold Z.N. because no heating is applied.
o Penetration of the dye is achieved by increasing
concentration of carbol fuchsin and incorporation of
a wetting chemical agent.
o However, acid fast bacilli stain less well by this
method than hot Z.N.
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Examination
of smears
o Put immersion oil on a dry slide.
o Examine under oil immersion lens.
o Tubercle bacilli appear pink rods that may be
single or bundles.
o The background appears blue in color.
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Positive Z.N. smear for acid
fast bacilli (AFB)
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Interpretation of
smears
o One or more bacilli / oil field
(+++)
o 10 bacilli / slide
(++)
o 3-9 bacilli / slide
(+)
o 1-2 bacilli / slide
(+/-)
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Thank you
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