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    Gram Staining

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    dhanya
    The document discusses different techniques for staining bacteria, including simple staining, Gram staining, and negative staining. Simple staining involves using a single basic dye like methylene blue to color bacterial cells and reveal their morphology and arrangement. Gram staining differentiates between Gram-positive and Gram-negative bacteria based on differences in their cell wall structure and ability to retain or lose crystal violet dye after decolorization. Negative staining stains the background instead of the bacteria to view colorless microorganisms against a dark background. Together, these staining methods allow visualization and classification of bacteria under the microscope.

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    Gram Staining

    Uploaded by

    dhanya
    89 views37 pages
    The document discusses different techniques for staining bacteria, including simple staining, Gram staining, and negative staining. Simple staining involves using a single basic dye like methylene blue to color bacterial cells and reveal their morphology and arrangement. Gram staining differentiates between Gram-positive and Gram-negative bacteria based on differences in their cell wall structure and ability to retain or lose crystal violet dye after decolorization. Negative staining stains the background instead of the bacteria to view colorless microorganisms against a dark background. Together, these staining methods allow visualization and classification of bacteria under the microscope.

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    this talks majorly about the gram staining techniques

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    The document discusses different techniques for staining bacteria, including simple staining, Gram staining, and negative staining. Simple staining involves using a single basic dye like methylene blue to color bacterial cells and reveal their morphology and arrangement. Gram staining differentiates between Gram-positive and Gram-negative bacteria based on differences in their cell wall structure and ability to retain or lose crystal violet dye after decolorization. Negative staining stains the background instead of the bacteria to view colorless microorganisms against a dark background. Together, these staining methods allow visualization and classification of bacteria under the microscope.

    Copyright:

    © All Rights Reserved
    Download as PPT, PDF, TXT or read online from Scribd
    Download as ppt, pdf, or txt
    89 views37 pages

    Gram Staining

    Uploaded by

    dhanya
    The document discusses different techniques for staining bacteria, including simple staining, Gram staining, and negative staining. Simple staining involves using a single basic dye like methylene blue to color bacterial cells and reveal their morphology and arrangement. Gram staining differentiates between Gram-positive and Gram-negative bacteria based on differences in their cell wall structure and ability to retain or lose crystal violet dye after decolorization. Negative staining stains the background instead of the bacteria to view colorless microorganisms against a dark background. Together, these staining methods allow visualization and classification of bacteria under the microscope.

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    1

    Staining of Bacteria
    Bacteria cells are almost colorless and
    transparent

    A staining technique is often applied to the


    cells to color them →
    Their shape and size can be easily
    determined under the microscope.

    2
    Dyes
    • make internal and external structures of cell more
    visible by increasing contrast with background
    • Dyes used to stain bacterial cells are organic
    compounds, which have affinity for specific cellular
    components.
    • The many types of dyes have two features in
    common: 1) they have chromophore groups, groups
    with conjugated double bonds that give the dye its
    color and
    • 2) they can bind with cells by ionic, covalent, or
    hydrophobic bonding. Dyes also contain an
    auxochrome group, which in itself does not produce
    color but gives the dye its acidic or basic properties.

    3
    Principle of staining
    Stains → combine chemically with the
    bacterial protoplasm.

    Commonly used stains are salts:


     Basic dyes: colored cation + colorless
    anion
    e.g. methylene blue (methylene blue
    chloride)
    MB+ + Cl-
     Acidic dyes: colored anion + colorless
    cation
    e.g. eosin ( Na+ + eosin-).
    4
    Bacterial cells are slightly negatively
    charged ( rich in nucleic acids bearing
    negative charges as phosphate groups)
    → combine with positively charged
    basic dyes

    Acidic dyes do not stain the bacterial


    cell → can stain the background
    material with a contrasting color.

    5
    Types of staining techniques

    Simple staining Differential staining


    (use of a single stain) (use of two contrasting stains
    separated by a decolorizing agent)

    For visualization of
    morphological Identificatio Visualization
    shape & arrangement. n of structure
    Gram Acid fast
    stain stain Spore Capsule
    stain stain
    6
    7
    Smear Preparation:
    Preparation and Fixation of Bacteria for
    Staining.

    Objective:
    To kill the microorganism & fix them to the
    slide to prevent them from being washed out
    during the process of staining.

    8
    Smear preparation

    S Fixation

    9
    10
    Definition:
    It is the use of single basic dye to
    color the bacterial organism.
    e.g. methylene blue,
    crystal violet,
    safranin.
    All bacteria take the color of the dye.

    Objective:-
    To show the morphological shapes and
    arrangement of bacterial cells.
    11
    Simple Staining
    Procedure:-

    MB

    1-2 min

    12
    Basic Shapes of Bacteria

    Cocci Bacilli
    13
    Arrangements
    Cocci

    Irregular Clusters Tetrads Chains or Pairs

    Staphylococci Micrococci Streptococci


    14
    Simple Staining
    Type of staining:- Simple
    Stain
    Name of dye:- Methylene
    blue

    Shape of cells:- bacilli


    Arrangement of cells:-
    chains
    Color:- Blue

    Name of m.o:- Bacillus

    15
    Simple Staining
    Type of staining:- Simple
    Stain
    Name of dye:- Methylene blue

    Shape of cells:- cocci


    Arrangement of cells:clusters
    Color:- Blue

    Name of m.o:-
    Staphylococci

    16
    Simple Staining
    Type of staining:- Simple
    Stain
    Name of dye:- Crystal violet.

    Shape of cells:- cocci


    Arrangement of cells:clusters
    Color:- Purple

    Name of m.o:-
    Staphylococci

    17
    18
    Gram Stain:
    It is the most important
    differential stain used in
    bacteriology
    it classifies bacteria
    into two major groups:

    a)Gram positive: b) Gram negative:


    Appears violet after Appears red after Gram’s
    Gram’s stain stain

    19
    Basic classification of bacteria is based on the cell wall
    structure.

    The staining technique, developed in the late 1700’s by


    Christian Gram classifies the rigid cell walled bacteria into one
    of two groups based on whether they are able to resist the
    decolorizing action of an alcoholic solution.

    Those that resist decolorization by 95% ethanol are arbitrarily


    termed Gram positive and those that do not are Gram negative
    (the terms positive and negative have nothing to do with
    charges of the cell but based on differences in the cell wall
    structure of these two groups of bacteria).
    Principle of Gram Staining
    Crystal Violet (purple)
    – Primary stain; positive stain
    – Stains cell wall purple
    Iodine
    – Mordant
    – Combines with CV to form an insoluble complex that gets trapped in
    thicker peptidoglycan layers
    Ethanol
    – Decolorizer
    – CV-I complex washed out of Gram negative organisms because it cannot
    be trapped by peptidoglycan layer; flows right through outer membrane
    Safranin (pink)
    – Counterstain
    – Simple positive stain that provides contrasting dye for decolorized cells
    (Gram negative)
    – Stains all cells, but only the negative ones actually appear pink.
    Gram’s +ve Bacteria Gram’s -ve Bacteria
    25
    Gram’s +ve Bacteria Gram’s -ve Bacteria

    26
    Gram-positive bacteria
    Have a thick peptidoglycan layer surrounds the cell.
    The stain gets trapped into this layer and the bacteria
    turned purple.
    Retain the color of the primary stain (crystal violet)
    after decolorization with alcohol

    Gram-negative bacteria
    have a thin peptidoglycan layer that does not retain
    crystal violet stain.
    Instead, it has a thick lipid layer which dissolved
    easily upon decolorization with Aceton-Alcohol.
    Therefore, cells will be counterstained with safranin
    and turned red.
    27
    There are 4 conditions to be followed for a valid Gram staining
    procedure: 

    Young cultures - must be young within 18-24hrs old


    (older cultures lose their Gram staining properties
    due to changes in the CWs as the cells get older)
    Thin smear
    thicker or uneven smears will result in uneven
    staining
    and decolorization
    Fresh reagents - of proper strength
    Control cultures - for a known GP bacterium and GN
    culture (S.aureus & E.coli)
    29
    Negative staining
    (Indirect staining with acidic
    dye)
    The negative staining technique does not
    stain the bacteria due ionic repulsion.
    but stain the background.
    The bacteria will appear colorless against
    a dark background.
    No heat fixation or strong chemicals are
    used→ the bacteria are less distorted
    than in other staining procedure.
    Example: Nigrosine
    30
    Negative staining

    Candida

    31
    Negative staining

    Staphylococci

    32
    Negative staining

    Bacillus

    33
    34
    35
    36
    37

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