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Simple Stains

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Simple Stains ... Different types of staining methods are used to make the cells and their ... Blot the with bibulous paper. Differential Staining ... – PowerPoint PPT presentation

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Title: Simple Stains


1
Simple Stains
  • Bacteria have nearly the same refractive index as
    water, therefore, when they are observed under a
    microscope they are opaque or nearly invisible to
    the naked eye.
  • Different types of staining methods are used to
    make the cells and their internal structures more
    visible under the light microscope.
  • Simple stains use one dye that stains the cell
    wall.
  • The cells are then visible against a light
    background.
  • Steps
  • Place the slide on the staining rack.
  • Flood the slide with a basic stain either
    crystal violet (1 min.), Safranin (2 min.), or
    Methylene blue (2 min.).
  • Wash the stain off the slide with deionized
    water.
  • Blot the slide with bibulous paper.

2
Differential Staining
  • Differential Stains use two or more stains and
    allow the cells to be categorized into various
    groups or types.
  • Both techniques allow the observation of cell
    morphology, or shape, but differential staining
    usually provides more information about the
    characteristics of the cell wall (Thickness).
  • The most common differential stain used in
    microbiology is the Gram Stain.
  • Basic stains, due to their positive () charge
    will bind electrostatically to negatively charged
    molecules such as many polysaccharides, proteins
    and nucleic acids.
  • Acid stains ( - ) bind to positively charged
    molecules which are much less common, meaning
    acidic stains are used only for special purposes.
  • Some commonly encountered basic stains are
    crystal violet, safranin (a red dye) and
    methylene blue.
  • Basic stains may be used alone (a simple stain)
    or in combination (differential stain) depending
    on the experiment involved.

3
Gram Staining
  • The Gram Stain is a differential stain.
  • Four different reagents are used and the results
    are based on differences in the bacterial cell
    wall.
  • Gram Positive bacteria have a relatively thick
    cell wall composed of a special carbohydrate
    called Peptidoglycan.
  • Gram Negative bacteria have a much thinner cell
    wall composed of the same carbohydrate,
    Peptidoglycan, but with certain chemical
    differences, such as the presence of
    Lipopolysaccharides (LPS).
  • Notice that both Gram Positive and Gram Negative
    bacteria have a cell wall composed primarily of
    Peptidoglycan.
  • Gram Staining Steps
  • Crystal violet acts as the primary stain. Crystal
    violet may also be used as a simple stain because
    it dyes the cell wall of any bacteria.
  • Grams iodine acts as a mordant (Helps to fix the
    primary dye to the cell wall).
  • Decolorizer is used next to remove the primary
    stain (crystal violet) from Gram Negative
    bacteria (those with LPS imbedded in their cell
    walls). Decolorizer is composed of an organic
    solvent, such as, acetone or ethanol or a
    combination of both.)
  • Finally, a counter stain (Safranin), is applied
    to stain those cells (Gram Negative) that have
    lost the primary stain as a result of
    decolorization.

4
Gram Staining Procedure
5
Gram Staining Results
  • When reporting the results of the Gram stain you
    indicate the type of stain used, the reaction,
    and the morphology of the cells observed.
  • Round (spherical), purple (or dark blue) cells
    are reported as Gram positive cocci (GPC).
  • Rod-shaped, purple (or dark blue) cells are
    reported as Gram positive bacilli (GPB).
  • The standard abbreviations for the four types of
    Gram stain and morphology are
  • Gram Positive Cocci (GPC)
  • Gram Positive Bacilli (GPB)
  • Gram Negative Cocci (GNC)
  • Gram Negative Bacilli (GNB)
  • Notice that both Gram Positive and Gram Negative
    bacteria have a cell wall composed primarily of
    Peptidoglycan.
  • Spiral-shaped bacterial cells do not Gram stain
    well and are usually observed using dark-field
    microscopy. There are no standard abbreviations
    for Gram stain reactions of the spirilla of
    medical importance.
  • Certain other types of bacteria may not Gram
    stain well, such as, Acid-fast Mycobacteria
    (Mycobacterium tuberculosis).

6
Bacterial Cell Shapes
  • Bacteria can have several different shapes, but
    the primary shapes we will be observing are
  • Spherical or round cells cocci (plural) or
    coccus (singular)
  • Rod shaped bacilli (plural) or bacillus
    (singular)
  • Spiral shaped spirilla
  • Some bacteria have characteristic clustering or
    arrangements, usually due to how the cells divide
    and whether they remain attached together when
    they divide.
  • Diplococci divide in one plane and remain
    attached together after cell division.
  • Streptococci divide in one plane and form long
    chains of attached cells.
  • Staphylococci divide in many planes and remain
    attached together forming a grape-like cluster
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