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Gram Staining in Diagnostic Microbiology

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					GRAM STAINING
  Dr.T.V.Rao MD




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




             Dr.T.V.Rao MD    2
         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).
                      Dr.T.V.Rao MD               3
     Gram staining a Important
            Technique
   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-
    negative
                      Dr.T.V.Rao MD               4
        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
    actylglucosamine
    monomers.
    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
                         bacterial
                         Lipopolysaccharides
                         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.

         Dr.T.V.Rao MD                           6
The Cell walls differ…




         Dr.T.V.Rao MD   7
Constituents of the Cell wall
      make difference




            Dr.T.V.Rao MD       8
        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.
                      Dr.T.V.Rao MD                9
Organizing the Staining Bottles




             Dr.T.V.Rao MD    10
Method of Picking material from
         Agar plates
   Wrong                     Right




             Dr.T.V.Rao MD           11
Prefer to pick up colonies with loop and
       smear on Clean glass slide




                 Dr.T.V.Rao MD         12
               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
    procedure

                              Dr.T.V.Rao MD   13
    Choosing a Right Smear

 Before choosing a
  field for microscopic
  examination, it is
  important to look at
  the smear
  macroscopically
 Note that the smear
  is easily visible in
  ordinary light
                          Dr.T.V.Rao MD   14
        Requirements for Gram
          staining technique
   Glass slides (25 by 75 mm), frosted ends
    desirable
   b. 0.85% Nacl, sterile
   c. Pasteur pipettes and wood applicator sticks,
    sterile
   d. Microbiological loops, inoculating needles
   e. Supplies for disposal of biological waste,
    including “sharps”
   f. Bunsen burner
   g. Immersion oil
                        Dr.T.V.Rao MD                 15
Dr.T.V.Rao MD   16
    Making Multiple smears in
     same slide – conserve
           resources
 Making multiple
  smears make the
  optimal use of the
  slide.
 Reduces the
  economic costs and
  saves the technical
  time.



                        Dr.T.V.Rao MD   17
        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
    slides.
            NOTE: When using the same pipette
      or swab, always inoculate culture media
                        first
                     Dr.T.V.Rao MD          18
      Steps in Gram Staining
    Procedure- Follow the Clock
   1 On a rack, flood with filtered crystal violet   10
  sec
  2 Wash briefly in water to remove excess crystal
  violet
 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

                          Dr.T.V.Rao MD                    19
Proceed in organized Fashion




            Dr.T.V.Rao MD      20
Step 1




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Step 2




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Step 3




 Dr.T.V.Rao MD   23
Step 4




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Step 5




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Step 6




 Dr.T.V.Rao MD   26
Step 7




 Dr.T.V.Rao MD   27
    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,
    bacteria
   consistent with normal micro flora, which may
    indicate an improperly collected specimen
   iii. Location and arrangements of
    microorganisms
   Note any special character sticks


                         Dr.T.V.Rao MD               28
Microscopy: The Instruments
                    In a compound
                     microscope the image
                     from the objective
                     lens is magnified
                     again by the ocular
                     lens.
                    Total magnification =
                     objective lens 
                     ocular lens


           Dr.T.V.Rao MD                 29
    Microscopy: The Instruments
 Resolution is the
  ability of the lenses to
  distinguish two
  points.
 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.
                        Dr.T.V.Rao MD   30
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
                        safranin..
       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
    eyes


                         Dr.T.V.Rao MD   32
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.


                             Dr.T.V.Rao MD   33
Observe with Caution to
     differentiate




         Dr.T.V.Rao MD    34
        Report as follows
 If no microorganisms are seen in a smear
  of a clinical
 specimen, report “No microorganisms
  seen.”
 ii. If microorganisms are seen, report
  relative numbers and
 Describe morphology.
 Observe predominant shapes of
  microorganisms
                   Dr.T.V.Rao MD             35
      Gram Stain Differentiates
      Gram positive from Gram
             negative
   Differential Stains: Gram Stain




                      Dr.T.V.Rao MD   36
Nature of Morphology in guides
 early Diagnosis in uncommon
           diseases




             Dr.T.V.Rao MD       37
    Creating Library of Gram
             Stains
  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.


                        Dr.T.V.Rao MD   38
    Value of Direct Smears
 Guide the physician on initial choice of
  antibiotic, pending results of culture and
  sensitivity.
 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.

                    Dr.T.V.Rao MD              39
Mixed Flora of Staphylococcus and
          Streptococcus
    differentiates morphology




              Dr.T.V.Rao MD     40
Stains Several Fungi




        Dr.T.V.Rao MD   41
 Gram-negative Pseudomonas
aeruginosa bacteria (pink-rods).




             Dr.T.V.Rao MD         42
(Bacillus anthracis),




        Dr.T.V.Rao MD   43
Streptococcus pneumonia




          Dr.T.V.Rao MD   44
Streptococcus salivarius




          Dr.T.V.Rao MD    45
Gram stain of Neisseria
    gonorrhoea,




         Dr.T.V.Rao MD    46
Streptococcus pneumoniae in
         Sputum




           Dr.T.V.Rao MD      47
Clostridium spp as seen in Gram Stain




                Dr.T.V.Rao MD           48
Moraxella as seen in Gram Staining




              Dr.T.V.Rao MD      49
Clostridium difficile as seen by
         Gram staining




             Dr.T.V.Rao MD         50
Corynebacterium diptheria as seen
        in Gram staining




              Dr.T.V.Rao MD     51
Gram stain of Neisseria
    gonorrhoea,




         Dr.T.V.Rao MD    52
Neisseria meningitides as seen in
          Gram staining




              Dr.T.V.Rao MD         53
Nocardia spp seen in Gram
         Staining




          Dr.T.V.Rao MD     54
Gram Stained Actinomyctes
           spp




          Dr.T.V.Rao MD     55
Gram stained Listeria
  Monocytogenes




        Dr.T.V.Rao MD   56
Gram Stained Cryptococcus
       neoformans




          Dr.T.V.Rao MD     57
    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.




                      Dr.T.V.Rao MD              58
Gram‟s method stains Artificacts
            too




             Dr.T.V.Rao MD         59
     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

                 Dr.T.V.Rao MD        60
      Common errors in Staining
            procedure
   Excessive heat during
    fixation
   Low concentration of
    crystal violet
    Excessive washing
    between steps
   Insufficient iodine
    exposure
   Prolonged
    decolourization
   Excessive counterstaining
                            Dr.T.V.Rao MD   61
Correlation of Gram stain with
           cultures
    Culture results are not correlated with
           direct gram stain results.
              Select true or false



                 1 True ( Correct )
                 2 False


                    Dr.T.V.Rao MD              62
Gram staining not a fool proof
         procedure
                            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
                             techniques.


             Dr.T.V.Rao MD                          63
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

                      Dr.T.V.Rao MD              64
    Overcoming in Gram Variable
           Observations
   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.
                      Dr.T.V.Rao MD            65
       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.
                       Dr.T.V.Rao MD             66
       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.




                             Dr.T.V.Rao MD                     67
     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

                      Dr.T.V.Rao MD               68
          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
    use.

                         Dr.T.V.Rao MD   69
    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.

                       Dr.T.V.Rao MD               70
    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.


                            Dr.T.V.Rao MD                    71
Created for „e‟ learning to
Medical and Paramedical
 students in developing
        countreis
         Dr.T.V.Rao MD
              Email
     doctortvrao@gmail.com

             Dr.T.V.Rao MD    72

				
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Description: Gram Staining in Diagnostic Microbiology