Gram Staining in Diagnostic Microbiology by doctorrao

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  Dr.T.V.Rao MD

      Dr.T.V.Rao MD   1
Hans Christian Joachim Gram
     Danish Bacteriologist

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         Dyes become Stains
    With interest in the effects of dyes on
    living tissue. In 1884 the Danish
    microbiologist Hans Christian Gram
    discovered that crystal violet irreversibly
    stains certain bacteria but can be washed
    from others. The dye has been widely
    used ever since for the Gram stain
    technique, which identifies bacteria as
    gram-positive (the stain is retained) or
    gram-negative (the stain is washed).
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     Gram staining a Important
   A staining technique used to classify
    bacteria; bacteria are stained with gentian
    violet and then treated with Gram's
    solution; after being decolorized with
    alcohol and treated with safranine and
    washed in water, those that retain the
    gentian violet are Gram-positive and
    those that do not retain it are Gram-
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        Gram positive Bacteria
   Gram positive bacteria
    have a thick cell wall of
    peptidoglycan and other
    polymers. Peptidoglycan
    consists of interleaving
    filaments made up of
    alternating acetylmuramic
    acid and
    In Gram positive bacteria,
    there are "wall teichoic
    acids". As well, between
    the cell wall and cell
    membrane, there is a
    "membrane teichoic
    acid".                    Dr.T.V.Rao MD   5
Gram Negative Bacteria
                        Gram negative bacteria
                         have an outer membrane
                         of phospholipids and
                         outside of their thin
                         peptidoglycan layer. The
                         space between the outer
                         membrane and the
                         peptidoglycan layer is
                         called the periplasmic
                         space. The outer
                         membrane protects Gram
                         negative bacteria against
                         penicillin and lysozymes.

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The Cell walls differ…

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Constituents of the Cell wall
      make difference

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        Definition of Gram stain

   A method of staining bacteria using a
    violet stain. The gram staining
    characteristics (denoted as positive or
    negative). A heat fixed bacterial smear is
    stained with crystal violet (methyl violet),
    treated with 3% iodine/potassium iodide
    solution, washed with alcohol and
    counterstained. The method differentiates
    bacteria into two main classes, gram-
    positive and gram-negative.
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Organizing the Staining Bottles

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Method of Picking material from
         Agar plates
   Wrong                     Right

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Prefer to pick up colonies with loop and
       smear on Clean glass slide

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               Making a Smear
   First prepare your slide.
    You do this by placing
    bacteria on a slide in a
    drop of water, allowing
    them to dry and then
    heat fixing them. Heating
    the slide kills the bacteria
    and makes sure that the
    bacteria a stuck to the
    slide and wont wash
    away during the staining

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    Choosing a Right Smear

 Before choosing a
  field for microscopic
  examination, it is
  important to look at
  the smear
 Note that the smear
  is easily visible in
  ordinary light
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        Requirements for Gram
          staining technique
   Glass slides (25 by 75 mm), frosted ends
   b. 0.85% Nacl, sterile
   c. Pasteur pipettes and wood applicator sticks,
   d. Microbiological loops, inoculating needles
   e. Supplies for disposal of biological waste,
    including “sharps”
   f. Bunsen burner
   g. Immersion oil
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    Making Multiple smears in
     same slide – conserve
 Making multiple
  smears make the
  optimal use of the
 Reduces the
  economic costs and
  saves the technical

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        Correct preparation
   Smear preparation: Proper smear
    preparation should produce a monolayer
    of organisms sufficiently dense for easy
    visualization but thin enough to reveal
    characteristic morphological
    characteristics. Use clean, new glass
            NOTE: When using the same pipette
      or swab, always inoculate culture media
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      Steps in Gram Staining
    Procedure- Follow the Clock
   1 On a rack, flood with filtered crystal violet   10
  2 Wash briefly in water to remove excess crystal
 3. Flood with Gram’s iodine 10 sec
 4. Wash briefly in water, do not let the section
  dry out.
 5. Decolourise with acetone until the moving dye
  front has passed the lower edge of the section
 6. Wash immediately in tap water
 7. Note : If the section appears too blue repeat
  steps 6 and 7
 8. Counterstain with safranin 15 seconds

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Proceed in organized Fashion

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Step 1

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Step 2

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Step 3

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Step 4

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Step 5

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Step 6

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Step 7

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    Observe the Following Under
        Oil Immersion lens
   i. Relative amounts of PMN’s, mononuclear
    cells, and RBC's
   ii. Relative amounts of squamous epithelial cells,
   consistent with normal micro flora, which may
    indicate an improperly collected specimen
   iii. Location and arrangements of
   Note any special character sticks

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Microscopy: The Instruments
                    In a compound
                     microscope the image
                     from the objective
                     lens is magnified
                     again by the ocular
                    Total magnification =
                     objective lens 
                     ocular lens

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    Microscopy: The Instruments
 Resolution is the
  ability of the lenses to
  distinguish two
 A microscope with a
  resolving power of 0.4
  nm can distinguish
  between two points ≥
  0.4 nm.
 Shorter wavelengths
  of light provide
  greater resolution.
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Optimal use of Microscopy
                       Gram stained
                        preparations have to be
                        observed with bright-field
                        optics. Phase-contrast
                        microscopy does not
                        allow the recognition
                        of true colours. Gram-
                        positive bacteria may be
                        seen under phase-
                        contrast as red cells.
                        Using bright-field optics,
                        Gram-positive cells are
                        purple or blue and Gram-
                        negative pink due to
          Dr.T.V.Rao MD
                        counter stain with         31
       Use Bright field optics
   With bright-field
    optics colours can
    be discriminated
    best if the
    condenser iris is
    opened as far as
    possible without
    discomfort to the

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Colors makes the Difference in
        Gram staining
   Bacteria that manage to
    keep the original purple
    dye have only got a cell
    wall - they are called
    Gram positive.
   Bacteria that lose the
    original purple dye and
    can therefore take up the
    second red dye have got
    both a cell wall and a cell
    membrane - they are
    called Gram negative.

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Observe with Caution to

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        Report as follows
 If no microorganisms are seen in a smear
  of a clinical
 specimen, report “No microorganisms
 ii. If microorganisms are seen, report
  relative numbers and
 Describe morphology.
 Observe predominant shapes of
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      Gram Stain Differentiates
      Gram positive from Gram
   Differential Stains: Gram Stain

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Nature of Morphology in guides
 early Diagnosis in uncommon

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    Creating Library of Gram
  Drain or gently blot
         excess oil
For slide libraries and
teaching collections that
will be stored for longer
periods, immersion oil
can be removed with
xylene solution and the
slides can be cover
slipped using Per mount
to prevent fading.

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    Value of Direct Smears
 Guide the physician on initial choice of
  antibiotic, pending results of culture and
 Judge specimen quality.
 Contribute to selection of culture media,
  especially with mixed flora.
 Provide internal quality control when
  direct smear results are compared to
  culture results.

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Mixed Flora of Staphylococcus and
    differentiates morphology

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Stains Several Fungi

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 Gram-negative Pseudomonas
aeruginosa bacteria (pink-rods).

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(Bacillus anthracis),

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Streptococcus pneumonia

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Streptococcus salivarius

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Gram stain of Neisseria

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Streptococcus pneumoniae in

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Clostridium spp as seen in Gram Stain

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Moraxella as seen in Gram Staining

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Clostridium difficile as seen by
         Gram staining

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Corynebacterium diptheria as seen
        in Gram staining

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Gram stain of Neisseria

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Neisseria meningitides as seen in
          Gram staining

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Nocardia spp seen in Gram

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Gram Stained Actinomyctes

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Gram stained Listeria

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Gram Stained Cryptococcus

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    Artifacts in Gram Staining
 Gram stain reagents (Crystal Violet, Iodine,
  Safranin and Neutral Red
 Dirty glass slides
 Contaminated water used to rinse slides
 When investigating non-viable organisms seen in
  Gram stains it is always wise to eliminate every
  potential source.

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Gram‟s method stains Artificacts

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     Gram stain results may not
    correlated with culture results
 Gram stain-positive, culture-negative
  specimens may be the result of
  contamination of reagents and other
  supplies, presence of
 Antimicrobial agents, or failure of
  organisms to grow under usual Culture
  conditions (media, atmosphere, etc.)
 Presence of anaerobic microorganism

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      Common errors in Staining
   Excessive heat during
   Low concentration of
    crystal violet
    Excessive washing
    between steps
   Insufficient iodine
   Prolonged
   Excessive counterstaining
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Correlation of Gram stain with
    Culture results are not correlated with
           direct gram stain results.
              Select true or false

                 1 True ( Correct )
                 2 False

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Gram staining not a fool proof
                            Gram‟s staining method is
                             not without its
                             problems. It is ,
                             complicated, and prone
                             to operator error. The
                             method also requires a
                             large number of cells
                             However, it is also
                             central to phenotypic
                             microbial identification

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Gram variable observations in
       Gram staining
   The Gram staining procedure does not
    always give clear-cut results. Some
    organisms are Gram-variable and may
    appear either Gram-negative or Gram-
    positive according to the conditions. With
    these types of organisms, Gram-positive
    and Gram-negative cells may be present
    within the same preparation

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    Overcoming in Gram Variable
   it is necessary that it is stained at two or
    three different ages (very young cultures
    should be used). If an organism changes
    from positive to negative or vice versa
    during its growth cycle, this change should
    be recorded with a statement as to the
    age of the culture when the change was
    first observed. In case a Gram-variable
    reaction is observed it is also good to
    check the purity of the culture.
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       Why Gram Variability?
   Some Gram-positive bacteria appear
    Gram-negative when they have reached a
    certain age, varying from a few hours to a
    few days. On the other hand, some Gram-
    negative bacteria may become Gram-
    positive in older cultures. For this reason it
    is strongly recommended to use very
    young cultures for the staining procedure,
    after growth has become just visible.
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       Trouble shooting in Gram
           Staining method
   It is important to note that gram-positivity (the ability to
    retain the purple crystal violet-iodine complex) is not an
    all-or-nothing phenomenon but a matter of degree.
    There are several factors which could result in a
    gram-positive organism staining gram-negatively

   1. The method and techniques used. Overheating
    during heat fixation, over decolourization with alcohol,
    and even too much washing with water between steps
    may result in gram-positive bacteria losing the crystal
    violet-iodine complex.

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     Trouble shooting in Gram
         Staining method
 2. The age of the culture. Cultures more than
  24 hours old may lose their ability to retain the
  crystal violet-iodine complex.
 3. The organism itself. Some gram-positive
  bacteria are more able to retain the crystal
  violet-iodine complex than others.
  One must use very precise techniques in
     gram staining and interpret the results
                    with discretion

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          Poor quality of slides
            Can be corrected
   Use of glass slides
    that have not been
    pre cleaned or
    degreased NOTE:
    Storing slides in a jar
    with 95% ethanol will
    ensure clean slides.
    Drain excess alcohol
    or flame slide before

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    Modification in Gram staining
             methods ?
 Since the original procedure of Gram, many
  variations of the Gram staining technique have
  been published. Some of them have improved
  the method, others include some minor technical
  variants of no value. Bartholomew (1962) has
  pointed out that each variation in the Gram
  staining procedure has a definite limit to its
  acceptability. Any final result is the outcome of
  the interaction of all of the possible variables.
 All modified methods to be practised with
  caution should suit to the laboratory, and quality
  control checks.

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    Gram staining continues to be
          Most Rapid test.
   Even new molecular methodologies typically take hours
    rather than minutes. Fortunately, Gram's stain, devised
    by a Danish pathologist in 1884, exists (see "The man
    behind Gram's stain, and "A revolution in staining
    begins," This simple staining procedure remains the
    most useful test performed in the microbiology lab.
    Results from a Gram's stain can tell volumes about an
    infection within 15 minutes of a specimen's arrival in the
    lab, while most other microbiology results require 24
    hours or more. Nevertheless, Gram's stain findings can
    be equivocal and, therefore, must be assessed carefully
    in light of the clinical picture.

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