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    The Gram stain separates bacteria into Gram positive and Gram negative based on differences in their cell walls. Gram positive bacteria have a very thick peptidoglycan cell wall with teichoic acid, absorbing crystal violet and appearing blue. Gram negative bacteria have a thinner cell wall with lipopolysaccharide in the outer membrane, absorbing safranin and appearing red after the crystal violet is washed away with alcohol. The acid-fast stain uses carbolfuchsin to stain acid-fast bacteria red that retain the stain after decolorization with acid-alcohol, while other bacteria take on the counterstain color.

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    The Gram stain separates bacteria into Gram positive and Gram negative based on differences in their cell walls. Gram positive bacteria have a very thick peptidoglycan cell wall with teichoic acid, absorbing crystal violet and appearing blue. Gram negative bacteria have a thinner cell wall with lipopolysaccharide in the outer membrane, absorbing safranin and appearing red after the crystal violet is washed away with alcohol. The acid-fast stain uses carbolfuchsin to stain acid-fast bacteria red that retain the stain after decolorization with acid-alcohol, while other bacteria take on the counterstain color.

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    Gram Stain-FINALS

    Uploaded by

    Dexcel concepcion
    The Gram stain separates bacteria into Gram positive and Gram negative based on differences in their cell walls. Gram positive bacteria have a very thick peptidoglycan cell wall with teichoic acid, absorbing crystal violet and appearing blue. Gram negative bacteria have a thinner cell wall with lipopolysaccharide in the outer membrane, absorbing safranin and appearing red after the crystal violet is washed away with alcohol. The acid-fast stain uses carbolfuchsin to stain acid-fast bacteria red that retain the stain after decolorization with acid-alcohol, while other bacteria take on the counterstain color.

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    of 2

    Gram Stain The different stains are the result of

    differences in the cell walls of g+ and g-


     Bacteriuria colorless and usually bacteria
    invisible to light microscopy
     Gram stain separates organisms into Both bacterias have more than 1
    Gram positive and Gram negative protecting their cytoplasm and nucleus
     Microorganism morphology can also be
    The layer outside the bacterial
    seen
    cytoplasm is the peptidoglycan layer (g+
    1. Pour on crystal violet stain and wait 60 and g-)
    seconds
    The peptidoglycan layer or cell wall is
    2. Wash off with water and flood with composed of repeating disaccharides
    iodine solution. Wait 60 seconds. with 4 amino acids in a side chain
    extending from each disaccharide
    3. Wash off with water and then
    “decolorize” with 95% alcohol The gram positive cell wall - very think
    with extensive cross linking of amino
    4. Finally, counter stain with safranin. acid side chains, cytoplasmic membrane
    Wait 30 seconds and wash off with contains proteins that span the lipid
    water bilayer

     Cells that absorb the crystal violet - blue Teichoic acid - polysaccharide that is
    (g+) present in g+ cell wall. Antigenic
     Crystal violet washed off by alcohol but determinant which is important for
    absorbing safranin - red (g-) serologic identification of g+ organisms

    The different stains are the result of Gram negative cell wall - thin with fairly
    differences in the cell walls of g+ and g- simple cross linking pattern. It has
    bacteria periplasmic space between the
    cytoplasmic membrane and extremely
    Both bacterias have more than 1
    thin peptidoglycan layer.
    protecting their cytoplasm and nucleus
    Murein lipoprotein - present in
    The layer outside the bacterial
    peptidoglycan layer of g- bacteria.
    cytoplasm is the peptidoglycan layer (g+
    and g-) Murein lipoprotein is important
    because it originates from the
    The peptidoglycan layer or cell wall is
    peptidoglycan layer and extends
    composed of repeating disaccharides
    outward to bind the unique third outer
    with 4 amino acids in a side chain
    membrane
    extending from each disaccharide
    The last membrane is similar to other
    cell membranes in that it is composed
    of 2 layers of phospholipid with
    hydrophobic tails in the center

    Outermost portion of the bilayer


    contains lipopolysaccharide (LPS)

    LPS is composed of 3 layers:

    1. Outer carbohydrate chains of 1-50


    oligosaccharide units ( O-specific side
    chain or the O-antigen

    2. The center part is a water soluble


    core polysaccharide

    3. Interior to the core polysaccharide is The Acid-Fast Stain


    Lipid A ( disaccharide with multiple fatty
    Acid-fast bacteria are those that retain
    acid tails reaching into the membrane
    carbolfuchsin (basic fuchsin dissolved in
    a phenol-alcohol-water mixture) even
    when decolorized with hydrochloric
    acid in alcohol

    A smear of cells on a slide is flooded


    with carbolfuchsin and heated on a
    steam bath

    After this, the decolorization step with


    acid-alcohol is carried out, and finally a
    contrasting (blue or green) counterstain
    is applied Acid-fast bacteria appear red;
    others take on the color of the
    counterstain.

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