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    Prokaryotic Cell and Staining

    Uploaded by

    Kriziaoumo P. Orpia
    This document summarizes the key structures and characteristics of prokaryotic cells. It discusses their cytoplasmic structures including DNA and ribosomes. The cell envelope contains a cell membrane, cell wall, and surface polymers like capsules and slime layers. Some prokaryotes also have appendages like flagella and pili. Various staining techniques are described like Gram staining and acid fast staining to distinguish cell types. Gram positive cells have a thick peptidoglycan layer and stain purple, while Gram negatives have a thin layer and stain red. Acid fast cells contain mycolic acid and resist decolorization by acid-alcohol.

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    Prokaryotic Cell and Staining

    Uploaded by

    Kriziaoumo P. Orpia
    668 views6 pages
    This document summarizes the key structures and characteristics of prokaryotic cells. It discusses their cytoplasmic structures including DNA and ribosomes. The cell envelope contains a cell membrane, cell wall, and surface polymers like capsules and slime layers. Some prokaryotes also have appendages like flagella and pili. Various staining techniques are described like Gram staining and acid fast staining to distinguish cell types. Gram positive cells have a thick peptidoglycan layer and stain purple, while Gram negatives have a thin layer and stain red. Acid fast cells contain mycolic acid and resist decolorization by acid-alcohol.

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    This document summarizes the key structures and characteristics of prokaryotic cells. It discusses their cytoplasmic structures including DNA and ribosomes. The cell envelope contains a cell membrane, cell wall, and surface polymers like capsules and slime layers. Some prokaryotes also have appendages like flagella and pili. Various staining techniques are described like Gram staining and acid fast staining to distinguish cell types. Gram positive cells have a thick peptidoglycan layer and stain purple, while Gram negatives have a thin layer and stain red. Acid fast cells contain mycolic acid and resist decolorization by acid-alcohol.

    Copyright:

    Attribution Non-Commercial (BY-NC)
    Download as DOCX, PDF, TXT or read online from Scribd
    Download as docx, pdf, or txt
    668 views6 pages

    Prokaryotic Cell and Staining

    Uploaded by

    Kriziaoumo P. Orpia
    This document summarizes the key structures and characteristics of prokaryotic cells. It discusses their cytoplasmic structures including DNA and ribosomes. The cell envelope contains a cell membrane, cell wall, and surface polymers like capsules and slime layers. Some prokaryotes also have appendages like flagella and pili. Various staining techniques are described like Gram staining and acid fast staining to distinguish cell types. Gram positive cells have a thick peptidoglycan layer and stain purple, while Gram negatives have a thin layer and stain red. Acid fast cells contain mycolic acid and resist decolorization by acid-alcohol.

    Copyright:

    Attribution Non-Commercial (BY-NC)
    Download as DOCX, PDF, TXT or read online from Scribd
    Download as docx, pdf, or txt
    of 6

    Prokaryotic Cell

    Cytoplasmic structures
    a. No membrane bound structures
    b. DNA consist of singular circular chromosomes
    c. Attach to a mesosome which is a saclike structure
    Bacterial Ribosomes are found free to attached to the cytoplasmic
    granules
    Consist of polysaccharide such a poly - typing
    1. Cell Envelope structure
    I. Cell Membrane
    a) Lipoprotein membrane that surrounds the cell wall
    b) Location of the electron transport Chain
    c) Made of phospholipids and proteins but no sterols
    Functions:
    Regulates transport across membrane acts as osmotic
    barrier
    2. Cell Wall
    a) Rigid structures that maintains the shape of the cell
    b) Prevents bursting of the cell (cause by high osmotic
    pressure)
    Gram Positive Cell Wall – Peptidoglycan (antibiotics)
    Glycan Chains- NAG and NAM
    Techoic Acid(peptidoglycan); lipotechoic acid (Plasma
    membrane)
    Gram Negative Cell Wall- inner peptidoglycan (thinner than
    gram positive cell walls)
    Outer Membranes
    Proteins, lipids and LPS
    LPS -antigenic O-specific polysaccharide
    -core polysaccharide
    -inner lipid A (endotoxin) –fever and shock
    conditions

    Functions:
    a. Barrier to hydrophobic compounds
    b. Acts a sieve (through porins)
    c. Attachment sites

    I. Periplasmic Space
    Gel- like matrix
    Nutrient binding proteins
    Degradative and detoxifying enzymes
    II. Acid Fast Cell Wall
    Waxy layer and fatty acid (Mycolic Acid- mycobacterium spp.)
    ABSENCE OF CELL WALL
    Mycoplasma and Ureaplasma
    - Lacks Cell wall but they have sterols

    L-Forms
    -Media supplemented with sugar or serum to prevent
    osmotic rupture
    - capable of reverting to its normal form (residual
    peptidoglycan)

    Using Physical and Chemical Agents to lose Cell wall


    a. Gr (+) turns to a Protoplast
    b. Gr (-) turns to a Spheroplast- Major portion of the cell
    wall will remain

    3. Surface Polymer
    Capsules- discrete organized covering Polysaccharide, polypeptides;
    acts as virulence factors to evading phagocytes
    Slime layer- more diffuse polysaccharide inhibits phagocytosis,
    aid in the adherence to host tissue or synthetic implants.
    Glycocalyx-tangled mass of thin polysacharride fibers

    4. Cell Appendages
    FLAGELLUM- organ of locomotion

    BACTERIAL SPECIES VARY IN THEIR POSSESION OF FLAGELLA


    None - atrichous
    Extend to one end - polar

    AXIAL FILAMENTS
    Singly - monotrichous
    At one or both ends - amphitrichous
    Multiply in tuft at one - Lophotrichous
    Occurs on sides of the bacterium -peritrichous

    PILI (FIMBRIAE)
    Hair-like protein structures
    Aid in attachment to surfaces
    a. Sex pili-bacterial conjugation and gene exchange
    b. Adherence pili- allowing them to attach to specific
    eukaryotic host cells

    STAINING
    Unstained/Unfixed Preparation
    Wet Mount
    Hanging Drop
    -Natural conditions suspended in fluids
    -Bright Field, Dark Field and Phase Contrast Microscope
    -Motility
    Brownian Movement- along with the fluid
    True motility- with locomotor
    -Morphology
    Dye (Salt)
    Chromophore
    Color of the basic dye- Positive ion
    Color of the Acidic dye-Negative ion
    Bacteria are slightly negatively charged at pH 7
    The colored positive in a basic dye is attracted to the negatively
    charged bacterial cells.
    Fixed/Stained Preparation

    Smear---- Heat Fixed---------- Cell’s molecules change shape


    Chemical Fixation--------No destruction of structures
    Staining
    Increase visibility
    Reveal additional information about the bacteria
    TYPES OF STAINING
    A. Simple Staining
    -single dye
    -cells and structures stain the same color
    Characteristic of size, shape and cell arrangement

    B. Differential Staining
    -distinguish structures within the cell, or by different types of cells
    - By staining them with different colors

    React with specific microbial structures


    C. Gram Staining
    Gram Positive and Gram negative
    Most important staining
    Determine the source of microbes as contaminants
    Fundamental Step in the diagnosis and treatment of diseases
    Determine the most effective antibiotics for critical ill patients

    THEORIES
    1. Membrane Theory of Knaysi
    Gram negative bacteria have a high lipid content; alcohol added removes the
    lipid the gram negative cells, thereby increasing cell permeability resulting in the
    loss of the dye complex

    2. Isoelectric Theory
    Gram positive have lower isoelectric point or the pH at which the cells have
    no change. This makes the cells more susceptible to the entrance of the dye and
    slow to decolorization

    3. Outer Surface Theory


    The out surface of the cell wall of Gram Positive bacteria contains Mg+RNA
    which forms a complex crystal violet iodine. This complex is insoluble with alcohol

    D. ACID FAST STAIN


    Identification of genus Mycobacterium
    Mycobacterium contains high levels of lipid material (mycolic acid)
    that repel water-soluble dyes that are difficult to stain by standard procedures

    All mycobacteria are acid fast

    E. STRUCTURAL STAINS
    Selective Stain specific structures
    Endospores
    Capsules
    Flagella
    Granules
    Cell wall
    Nucleus

    Culture Media
     Right nutrients in their concentration
     Properly adjusted pH
     Sufficient Moisture
     Desired consistency
     Free inhibitory substances
     Initially sterile
    Comparison of Gram Negative from Gram Positive

    Gram Positive Gram Negative


    Gram Reaction Purple Red
    Peptidoglycan layer Thick Thin
    Techoic acid Present Absent
    LPS content Absent Present
    Periplasmic Space Absent Present
    Outer Membrane Absent Present
    Lipid content and Low High
    lipoprotein
    Resistance to Physical High Low
    Disruption
    Lyzosyme Cell wall High Low
    disruption
    Susceptibility to High Low
    penicillin
    Susceptibilty to Low High
    streptomycin,
    chlorampenicol and
    tetracycline
    Inhibition by basic High Low
    dyes
    Susceptibility to High Low
    Anionic detergents
    Resistance to Sodium High Low
    Azide

    Steps in Gram Staining

    Steps Reagent Remarks


    Primary or initial Crystal Violet Colors cytoplasm purple
    Stains regardless of cell type
    Mordant Iodine Combines with crystal
    violet to form insoluble
    complex
    Decolorizer Alcohol or Acetone Purple dye is retained
    Alcohol by gram positive cells
    but is readily removed
    from gram negative
    cells
    Secondary or Counter Safranin Stains the colorless
    Stain gram negative cell to
    red while Gram positive
    cells remain purple
    Gram Positive- Purple
    Gram Negative- Red

    Steps in Acid Fast Staining

    Steps Reagent Remarks


    Primary or initial Stains Carbolfuchsin All cells are color Red
    Mordant Fixes the stain and
    Physical Steam Heat facilitates entrance of
    Chemical Phenol the dye

    Decolorizer Acid- Alcohol Non-Acid Fast are


    decolorized; acid fast
    remain color red
    Secondary or Counter Methylene Blue Stains the colorless and
    Stain non acid fast blue while
    acid fast remains
    colored red
    Non- Acid fast bacilli- Blue
    Acid fast bacilli- Red

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