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    381 PHT Lab #2

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    Bo Naing
    The document describes various bacterial staining techniques. Simple staining involves using a single basic dye to color bacterial cells and show their morphology and arrangement. Gram staining differentiates bacteria into Gram-positive or Gram-negative based on differences in their cell wall structure. Gram-positive bacteria appear violet while Gram-negative appear red. Negative staining does not stain bacteria but instead stains the background, allowing bacteria to appear colorless against a dark background. Proper smear preparation and fixation is required before staining bacteria on a microscope slide.

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    Download as PPT, PDF, TXT or read online from Scribd

    381 PHT Lab #2

    Uploaded by

    Bo Naing
    13 views42 pages
    The document describes various bacterial staining techniques. Simple staining involves using a single basic dye to color bacterial cells and show their morphology and arrangement. Gram staining differentiates bacteria into Gram-positive or Gram-negative based on differences in their cell wall structure. Gram-positive bacteria appear violet while Gram-negative appear red. Negative staining does not stain bacteria but instead stains the background, allowing bacteria to appear colorless against a dark background. Proper smear preparation and fixation is required before staining bacteria on a microscope slide.

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    Lab 2

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    The document describes various bacterial staining techniques. Simple staining involves using a single basic dye to color bacterial cells and show their morphology and arrangement. Gram staining differentiates bacteria into Gram-positive or Gram-negative based on differences in their cell wall structure. Gram-positive bacteria appear violet while Gram-negative appear red. Negative staining does not stain bacteria but instead stains the background, allowing bacteria to appear colorless against a dark background. Proper smear preparation and fixation is required before staining bacteria on a microscope slide.

    Copyright:

    Attribution Non-Commercial (BY-NC)
    Download as PPT, PDF, TXT or read online from Scribd
    Download as ppt, pdf, or txt
    13 views42 pages

    381 PHT Lab #2

    Uploaded by

    Bo Naing
    The document describes various bacterial staining techniques. Simple staining involves using a single basic dye to color bacterial cells and show their morphology and arrangement. Gram staining differentiates bacteria into Gram-positive or Gram-negative based on differences in their cell wall structure. Gram-positive bacteria appear violet while Gram-negative appear red. Negative staining does not stain bacteria but instead stains the background, allowing bacteria to appear colorless against a dark background. Proper smear preparation and fixation is required before staining bacteria on a microscope slide.

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    381 PHT

    Lab #2
    1
    2
    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.

    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
    Results

    Type of staining:
    Name of stain:

    Shape of cells:
    Arrangement of cells:
    Color:

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

    Shape of cells:- bacilli


    Arrangement of cells:-
    strain
    Color:- Blue

    Name of m.o:- Bacillus

    16
    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

    17
    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

    18
    19
    Gram Stain:
    It is the most important
    differential stain used in
    bacteriology because
    it classified bacteria
    into two major groups:

    a)Gram positive: b) Gram negative:


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

    20
    Crystal violet
    ↓
    Iodine
    ↓
    Alcohol
    ↓

    Safranin 21
    Gram +ve Gram –ve
    S.aureus E.coli

    Step 1: Crystal Violet

    Step 2: Gram’s Iodine

    Step 3: Decolorization
    (Aceton-Alcohol)

    Step 4: Safranin Red

    22
    Step 1: Crystal Violet

    Step 2: Gram’s Iodine

    Step 3: Decolorization
    (Aceton-Alcohol)

    Step 4: Safranin Red

    23
    Gram’s +ve Bacteria Gram’s -ve Bacteria
    24
    Gram’s +ve Bacteria Gram’s -ve Bacteria

    25
    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 decoulorization with Aceton-Alcohol.
    Therefore, cells will be counterstained with safranin
    and turned red.
    26
    Gram Stain
    Materials:-
    Cultures of Staphylococcus aureus,
    Candida albican,
    Bacillus subtilis,
    E.coli
    Gram stain:
    Crystal violet (primary stain)
    Gram’s iodine (mordant)
    Acetone-alcohol (decolorizing agent)
    Safranin (counter stain) 27
    Gram Stain

    Procedure:
    CV
    safranin
    iodine

    30
    10 sec
    30-60 sec
    sec
    2 min

    28
    Results:
    Shape: Cocci
    Arrangment: irregular clusters

    Colour: Violet
    Gram’s reaction: Gram’s +ve

    Name of microorganism:
    Staphylococci 29
    Results:
    Shape: Oval
    Arrangment: Single

    Colour: Violet
    Gram’s reaction: Gram’s +ve

    Name of microorganism:

    Candida
    30
    Results:
    Shape: Bacilli
    Arrangment: Chains

    Colour: Violet
    Gram’s reaction: Gram’s +ve

    Name of microorganism:

    Bacillus
    31
    Results:
    Shape: Rods
    Arrangment: Single

    Colour: red
    Gram’s reaction: Gram’s –ve

    Name of microorganism:

    Gram negative bacilli 32


    33
    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
    34
    Negative staining

    Candida

    35
    Negative staining

    Staphylococci

    36
    Negative staining

    Bacillus

    37
    38
    39
    40
    41
    42

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