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Bacterial smear preparation and Types of bacterial stains


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Title: Bacterial smear preparation and Types of bacterial stains

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A---Preparing a smear from a solid media
  • Take glass slide and dip them momentarily into
    the acid alcohol ( or only a lcohol) jar.
  • 2- Dry it with paper towel and do not touch the
    surfaces with your fingers. Acid alcohol removes
    oil and dirt particles from the slide.
  • 3- Using a glass-marking pencil, draw large
    circle in middle of slide.
  • 4- Place a drop of water in the middle of circle
    with a dropper.
  • 5. Sterilize the loop in the Bunsen burner flame
    until it glows. and let it cool.

6. Remove a very small amount of inoculum by
gently touching a single colony on the plate with
loop. 7. Spread the inoculum in the circle,
filling the circle and mixing with the water. 8.
Flame the loop again until the wire glows. Cool
the loop and put in its place. 9. Let the slides
dry in the air. 10. Fix the bacteria onto the
slide by passing the slide, smear side up,
quickly through the flame of the burner three
times. Avoid getting the slide too hot.
B- Preparing a smear from a broth
culture Repeat the 3rd steps as mention above ,
with out using water drops. 1- Sterilize the
loop using the Bunsen burner flame until it
glows. Remove the loop and air cool. 2-
Holding the culture tube in one hand at an angle
and remove the cap with other hand and flame the
mouth of the slanted broth tube. 3- Insert the
sterile, cooled loop into the culture and remove
a loopful from the tube carefully to avoid
touching the sides of the tube. 4- Flame the
mouth of the tube, and quickly return the cap to
the tube and Replace the tube in the rack.
5- Spread the inoculum in the circle, filling the
circle. Do not spread out the inoculum too
thinly. 6- Flame the loop by placing it in the
flame (by gradually moving the wire through the
flame, until the wire glows. then the loop itself
is flamed until it is glowing). In this way the
broth dries out gradually before incineration. If
the wet loop is put immediately in the flame, it
may splash out and contaminate the area. 7- Let
the slide air dry or by placing the slide in a
warm spot near a lamp or a Bunsen burner to speed
up the . 8- Fix the bacteria onto the slide by
passing the slide quickly through the flame of
the burner two or three times.
Why is it necessary to heat fix bacterial smears
prior to staining?
  • Heat fixing bacterial smears kills the bacterial
    cells so that they are fixed in place and ready
    for staining.
  • Heat fixing adheres the bacterial cells to the
    slide and allows the sample to take up the stain
    more easily .
  • fixation processcoagulates the proteins and fixes
    the bacteria onto the slide so they will not wash
    off during staining

Types of Different Staining Techniques of
There are three kinds of staining techniques
Simple stains
Differential stains
Special stains
Simple stain
  • Use asingle stain ,which is usuelly sufficient to
    reveal the basic morphological features of most
    microbial cells including relative size,shape and
    characteristic arrengments for groups of cells.
  • principles Simple staining depends on the
    fact that bacteria differ chemically from their
    surroundings and thus can be
  • stained to contrast with their environment, these
    stain contains chromophore that react chemically
    with specific cell structures making it possible
    to visualize several unque microstructures,suchas
    flagella,endospores,cytoplasmic inclusion.

1- prepare the Bacterial smear 2. Stain the
slide with (alkaline methylene blue for1 to1 1/2
minutes carbol fuchsin for 5 to 10 seconds or
with crystal violet for 20 to 30 seconds) 3.
Wash stain off slide with water for a few
seconds 4. Blot slide dry with bibulous paper 5.
Examine under the oil immersion lens
Differential staining
Principle Bacteria differ from one another
chemically and physically and may react
differently to a given staining procedure.
Differential staining can distinguish between
two types of bacteria gram positive and gram
  • Gram staining method was developed by Danish
    physician Hans Christian Gram in 1884.

Mechanism of Differential stain (Gram Stain)
At this time the exact mechanism of the Gram
stain reaction is not yet known. The most
prominent current theories are as follows (1)
The cell wall of a Gram positive bacterium is
composed of a heteropolymer of amino acids and
sugars called peptidoglycan. This wall provides a
barrier through which the crystal violet-iodine
complex cannot pass during decolorization.
Gram negative bacterium contains less
peptidoglycan and more lipid than a Gram positive
organism. These chemical characteristics cause
more effective and rapid removal of dye complex
when decolorizer is applied.
2) There may exist a specific Gram positive
substrate within the cell. All major cellular
macromolecules have been implicated, including
lipids, carbohydrates, proteins, and nucleic
acids. This theory is hard to prove because
removal of any of these components from the cell
vastly alters the chemistry of the cell wall.
However, there is some evidence for a crystal
violetribonucleic acid-iodine complex in Gram
positive cells. (3) The real situation may be
the combination of above two theories suggesting
that a Gram positive bacterium is not only less
permeable but also contains a compound that
reacts with and holds the stain complex tightly.
1-Place the slides on a staining rack. 2. Add a
few drops of crystal violet stain on the smears.
Stain for 1 min. 3. Rinse the slide with a
gentle jet of water from a wash bottle. At this
stage all bacteria will be stained purple by the
crystal violet. 4. Drain off the rinse water,
and add a few drops of Gram's iodine solution and
let the slide stand for 1 min. 5- Rinse the
slide with a gentle jet of water as described
above. 6- Decolorize the stain by ethyl
alcohol(95) or acetone run down over the until
the stain no longer is being removed fromthe
slide (20 -30 sec) .
7. Quickly rinse the slide with water. 8. Add a
few drops of Safranin and let the smears stain
for 30 seconds or carbol fuchsin 1 min. 9. Rinse
the slides with water. 10. Let the slides
air-dry. The slides should be completely dry
before adding oil for microscopic examination.
description on staining as gram stain 1- G-ve
or Gve 2- shapecocci,rod,cocobacelli 3-
arrangement chains,chains like letters 4- size
larage,small -------1micrometeres (µm) 1micron
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Procedure for Three-Step Gram Stain Difco
Laboratories has introduced reagents for a three
step Gram stain. The advantages include 1- less
reagent 2-usage versus conventional stains,
reduced chance of overdecolorization, 3- and
saved time. The procedure recommended by the
company is as follows 1. Flood smear with gram
crystal violet primary for 1 minute. 2. Wash off
the crystal violet with cold water. 3. Flood the
slide with Grams iodine mordant and let sit for
1 minute. 4. Wash off the mordant with safranin
decolorizer/counterstain solution. Then add
more decolorizer/counterstain solution to the
slide and stain for 20 to 50 seconds. 5. Wash
off the decolorizer/counterstain with cold water.
air dry and exam.
  • Several factors may affect the results of Gram
  • If the smear is too thick, proper decolorizing
    will not be possible.
  • If the smear is overheated during heat fixing,
    the cell walls will rupture.
  • Concentration and freshness of reagents may
    affect the quality of the stain.
  • Washing and drying of the smear between steps
    should be consistent.
  • Excess water.left on the slide will dilute
    reagents, particularly Gram's iodine.

  • Gram stain is reliable only on cells from
    cultures that are in the exponential phase of
  • Older cultures contain more ruptured and dead
    cells. Cells from old cultures may stain Gram
    negative even if the bacteria are Gram positive

  • Use of KOH as an Indicator of Gram Stain
  • 1. Place two drops of 3 potassium hydroxide
    (KOH) solution on a clean slide.
  • 2. Transfer bacterial cells from the culture
    medium with a wooden toothpick. get a rather good
    amount of inoculum Mix bacteria into the solution
    on the slide rapidly and in a circular motion.
  • 3. After 5-8 seconds, raise and lower the
    toothpick just off the slide surface to determine
    the stringing effect.
  • If there is stringing (i.e. increased viscosity)
    within 15 seconds the bacteria are considered to
    be Gram negative.
  • If no stringiness is observed, the bacteria are
    considered to be Gram positive.

Negative, indirect, or background staining
  • Principles
  • Sometimes it is convenient to determine overall
    bacterial morphology without the use of harsh
    staining or heat-fixing techniques that change
    the shape of cells.
  • or in case when the bacterium does not stain well
    (e.g., some of the spirochetes).
  • or when it is desirable to confirm observations
    made on the shape and size of bacteria observed
    in either a wet-mount or hanging drop slide.
  • Negative staining is also good for viewing

Procedure 1-apply a small amoun tof bacteria
to one end of a clean microscope slide 2. Add 1
to 2 loops of nigrosin, India ink, or eosin
solution to the bacteria and mixe thoroughly. 3.
Spread the mixture over the slide using a second
slide. held at a 45angle so that the
bacteria-nigrosin solution spreads across its
edge The slide is then pushed across the surface
of the first slide in order to form a smear that
is thick at one end and thin at the other This is
known as thin smear. 4-air dry Do not
heat-fix! 5. With the low-power objective, find
an area of thesmear that is of the optimal
thickness for observation. 6. Use the oil
immersion lens
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Acid-Fast Staining (Ziehl-Neelsen and Kinyoun)
(Ziehl-Neelsen) developed by Franz Ziehl, a
German bacteriologist, and Friedrich Neelsen, a
German pathologist.
Principles A few species of bacteria in the
genera Mycobacterium and Nocardia, and the
parasite Cryptosporidium do not readily stain
with simple stains. these microorganismscan be
stained by heating them with carbolfuchsin. The
heat drives the stain into the cells. Once the
microorganisms have taken up the carbol fuchsin,
they are not easily decolorized by acid-alcohol,
and hence are termed acid-fast. This
acid-fastness is due to the high lipid content
(mycolic acid) in the cell wall of these
microorganisms. Acid-fast microorganisms will
retain this dye and appear red,and blue
  • Procedure
  • Ziehl-Neelsen (Hot Stain) Procedure
  • 1- Prepare a smear
  • 2- Cover the smear with strong carbol fuchsin
    and place a small piece of filter paper on smear
    and soak with CF stain place the slide on
    astanding rack and steam over aboiling water bath
    for 5 min. adding addition stain to prevent the
    smear from drying.

3- Remove the slide, let it cool, and rinse with
waterfor 30 seconds 4. Decolorize by adding (3
v/v) acid-alcohol drop by drop until the slide
remains only slightly pink. (10 to 30 seconds)
with Carefully. 5. Rinse with water for 5
seconds 6. Counter stain with alkaline methylene
blue for about 2 minutes 7. Rinse with water
for 30 seconds. 8. Blot dry with bibulous or
filiter paper. Examine under oil immersion
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Kinyoun (Cold Stain) This is Amodification of
Ziehl-Neelsen stain that employs a wettingagent
(Tergitol No. 7) rather than heat to ensure stain
(developed by Joseph Kinyoun, German
bacteriologist, in the early 1900s). Procedure
1. Heat-fix the slide as previously directed. 2.
Flood the slide for 5 minutes with
carbolfuchsinprepared with Tergitol No. 7
(without heat). 3. Decolorize with acid-alcohol
and wash with tapwater. Repeat this step until no
more color runs off the slide. 4. Counterstain
with alkaline methylene blue for 2minutes. Wash
and dry. 5. Examine under oil. Acid-fast
organisms stain red the background and other
organisms stain blue.
Spore stain
  • Bacteria in genera such as Bacillus and
    Clostridium produce quite a resistant structure
    capable of surviving for long periods in an
    unfavorable environment and then giving rise to a
    new bacterial cell This structure is called an
    endospore sinceIt develops within the bacterial
  • Endospores are spherical to elliptical in shape
    and may be eithersmaller or larger than the
    parent bacterial cell. Endosporeposition within
    the cell is characteristic and may be central,
    subterminal, or terminal

Principle Endospores are dense,multilayerd
structure so do not stain easily, but, once
stained, they strongly resist decolorization.
This property is the basis of the
Schaeffer-Fulton (Wirtz-Conklin method ) of
staining endospores. Procedure 1- Cover the
smear with malachite green and place asmall piece
of filter paper on smear and soak with stain
place the slide on astanding rack and steam over
aboiling water bath for 5 -6min (Heat is used to
provide stain penetration). adding addition stain
to prevent the smear from drying. 2- . Remove
the paper using forceps, allow the slide to cool,
and rinse the slide with water for 30 seconds 3-
Counterstain with safranin or carbol fuchsin for
60 to 90 seconds 4- Rinse the slide with water
for 30 seconds and examin . endospores and free
spores, stain green vegetative cells stain red.

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Dorner method for staining endospores1.   Air
dry or heat fix the organism on a glass slide and
cover with a square of blotting paper or toweling
cut to fit the slide. 2.   Saturate the
blotting paper with carbolfuchsin and steam for 5
to 10 minutes, keeping the paper moist and adding
more dye as required.  Alternatively, the slides
may be steamed over a container of boiling
water.3.    Remove the blotting paper and
decolorize the film with acid-alcohol for 1
minute rinse with tap water and blot dry.4.   
Dry a thin even film of saturated aqueous
nigrosin on the slide.5.    Examine the slide
under the oil immersion lens for the presence of
endospores.  Vegetative cells are colorless,
endospores are red, and the background
Capsule Staining
  • Many bacteria have a slimy layer surrounding
    them, which is usually referred to as a capsule .
  • The capsules composition, as well as its
    thickness, varies with individual bacterial
    species. Polysaccharides, polypeptides, and
  • Often, a pathogenic bacterium with a thick
    capsule will be more virulent than a strain with
    little or no capsule since the capsule protects
    the bacterium against the phagocytic activityof
    the hosts phagocytic cells.
  • Principlescapsule dont have a charge,so use
    negative stain(india ink).
  • Note Cannot always determine if a capsule is
    present by simple staining procedures, such as
    using negative staining and India ink.

An unstained area around a bacterial cell may be
due to the separation of the cell from the
surrounding stain upon there is Two
convenient procedures for determining the
presence of a capsule are
  • Anthonys capsule staining method
  • Graham and Evans procedure

Modified Capsule Stain (Graham and Evans) 1.
Mix two loopfuls of culture with a small amount(
1 to 2 drops) of India ink at one end of the
slide.(thin smear ) 2. Dry the smear.(dont
heat) 3. Rinse with distilled water Gently so
that the bacteria do not wash off the slide. 4.
Stain for 1 minute with Grams crystal violet. 5.
Rinse again with water. 6. Stain for 1 1/2
minutes with safranin stain or carbol fuchsin. 7.
Rinse with water and blot dry. 8 . If a capsule
is present, the pink to red bacteria are
surrounded by a clear zone. The background is
Capsule Staining (Anthonys) Procedure 1-
aseptically transfer a loopful of culture with an
inoculating loop to the slide. Allow the slide to
air dry. Do not heat-fix! Heat-fixing can cause
the bacterial cells to shrink and give a false
appearance to the capsule 2. Place the slide on
a staining rack. Flood the slidewith crystal
violet and let stand for 4 to 7 minutes 3. Rinse
the slide thoroughly with 20 copper sulfate 4.
Blot dry with bibulous paper 6. Examine under oil
immersion necessary). Capsules appear as faint
halos around dark cells.
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Description of bacteria as follow
  • description on staining as gram stain
  • 1- G-ve or Gve
  • 2- shapecocci,rod,cocobacelli
  • 3- arrangement chains,chains like letters
  • 4- size larage,small -------1micrometeres (µm)
    1micron 1/1000mm.
  • Description of bacterial colonies
  • A- on solid media
  • 1- size 0.5-1mm(small), 3-5mm(larage)
  • 2- form granular,circular,irregular,filmentous
  • 3- surface smooth ,rough,glistening
  • 4- elevation raised,convex,flat,umbonate
  • 5- opacity translucent,opaque
  • 6- color white , yellow ,buff
  • 7- margin entire,undulant,erose,curled
  • 8- Odors burnt,chocolate,butter
  • B- on liquid media
  • 1-turbid growth
  • 2-deposit growth deposit at the bottom of tube
  • 3- surface growth related to aerobic nature

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