Beveled Slide Style

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MICROSCOPIC EXAMINATION OF BACTERIA (UNSTAINED
STAINED SMEARS)
By
Dr. Emad AbdElhameed Morad
Lecturer of Medical Microbiology and Immunology
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• There are two principal ways of preparing a
  microbial specimen for observation with light
  microscope
• Unstained smears (wet preparation) to examine
  the motility of the bacteria.
• Stained smears to study the size, shape,
  arrangement and staining affinity of the
  bacteria.

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• Study size, shape, arrangement and staining
  affinity of the bacteria.
• Bacteria are measured in microns.

Stained smears
size
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• Bacteria may have several shapes
• Cocci spherical shape
• Bacilli straight rods
• Vibrios curved rods
• Spiral spiral filaments

Shape
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• 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).

Arrangement
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Cocci arranged in clusters
Cocci arranged in chains
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• Stains may be
• Simple stains using one stain only such as
  methylene blue, carbol fuchsin.
• Differential stains using two stains primary
  and secondary separated by a step of
  decolorization.

Staining properties
Ziehl-Neelsen stain
Gram stain
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• 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.

Gram stain
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Gram staining
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• 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.

Preparation of smears
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• Pass the slide with the smear side uppermost over
  the flame for 2-3 times.
• Do not overheat the smear. The slide should only
  be warm when you touch it with hands.
• Passage of the smear through heat has two
  benefits
• Fixation of the smear to the slide.
• Killing of the bacteria in the smear so it
  becomes non infectious.

Fixation of smears
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• 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.

Staining of smears
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Gram staining
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• Rack the condenser high and open the iris
  diaphragm.
• Place a drop of immersion oil on the smear.
• Put the slide with the smear side up on the
  stage.
• Use the oil immersion lens.
• Lower the oil lens till the lens contacts the oil
  and almost touches the smear.
• Look through the eye piece of the microscope.
• Focus on the object using the coarse adjustment
  screw then the fine one.

Examination of smears
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• Comment on the bacterial morphology as regards

Interpretation of smears
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(No Transcript)
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• 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.

Ziehl Neelsen stain
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• Examples of acid fast bacteria or bacterial
  structures
• Tubercle bacilli retain red carbol fuchsin when
  decolorized with 20 H2SO4 or 3 HCL in alcohol.
• Lepra bacilli and saprophytic acid fast bacilli
  retain red dye when decolorized with 5 H2SO4 or
  1 HCL in alcohol.
• Actinomyces clubs and nocardia retain red dye
  when decolorized with 0.5 to 1 H2SO4.
• Spores tolerates only 0.25 to 0.5 H2SO4.

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Ziehl Neelsen staining
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• 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
• Three morning sputum samples are preferable since
  they represent overnight accumulation.
• Choose a purulent portion of sputum and spread it
  evenly in the middle of a new clean glass slide.
• Leave the smear to dry.
• Then fix the smear by passing through the flame.

Preparation and fixation of smears
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• Flood the smear with strong carbol fuchsin. Allow
  the stain to act for 5-10 minutes.
• Heat intermittently until the vapor begins to
  rise. Do not allow the stain to boil or dry.
• Pour it off then wash with water.
• 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.
• Wash thoroughly with water.
• Add methylene blue or malachite green for 2 min.
• Wash with water. Dry then examine.

Staining of smears
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• It is called cold Z.N. because no heating is
  applied.
• Penetration of the dye is achieved by increasing
  concentration of carbol fuchsin and incorporation
  of a wetting chemical agent.
• However, acid fast bacilli stain less well by
  this method than hot Z.N.

Kinyoun technique
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• Put immersion oil on a dry slide.
• Examine under oil immersion lens.
• Tubercle bacilli appear pink rods that may be
  single or bundles.
• The background appears blue in color.

Examination of smears
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Positive Z.N. smear for acid fast bacilli (AFB)
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• One or more bacilli / oil field
  ()
• 10 bacilli / slide
  ()
• 3-9 bacilli / slide
  ()
• 1-2 bacilli / slide
  (/-)

Interpretation of smears
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• Thank you