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    Microscopy and Staining

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    Denmark Manlusoc
    1) Anton van Leeuwenhoek was the first to observe microorganisms using a simple microscope and described bacteria from his own mouth in 1683. 2) The compound light microscope uses lenses, light, and sometimes stains to make small objects visible. Total magnification is calculated by multiplying the objective and ocular magnifications. 3) Staining microorganisms allows for better visibility of cells and structures under the microscope and differentiation between species. The Gram stain distinguishes between gram-positive and gram-negative bacteria based on cell wall composition.

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    Microscopy and Staining

    Uploaded by

    Denmark Manlusoc
    46 views7 pages
    1) Anton van Leeuwenhoek was the first to observe microorganisms using a simple microscope and described bacteria from his own mouth in 1683. 2) The compound light microscope uses lenses, light, and sometimes stains to make small objects visible. Total magnification is calculated by multiplying the objective and ocular magnifications. 3) Staining microorganisms allows for better visibility of cells and structures under the microscope and differentiation between species. The Gram stain distinguishes between gram-positive and gram-negative bacteria based on cell wall composition.

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    MICROSCOPY AND STAINING

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    1) Anton van Leeuwenhoek was the first to observe microorganisms using a simple microscope and described bacteria from his own mouth in 1683. 2) The compound light microscope uses lenses, light, and sometimes stains to make small objects visible. Total magnification is calculated by multiplying the objective and ocular magnifications. 3) Staining microorganisms allows for better visibility of cells and structures under the microscope and differentiation between species. The Gram stain distinguishes between gram-positive and gram-negative bacteria based on cell wall composition.

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    © All Rights Reserved
    Download as DOCX, PDF, TXT or read online from Scribd
    Download as docx, pdf, or txt
    46 views7 pages

    Microscopy and Staining

    Uploaded by

    Denmark Manlusoc
    1) Anton van Leeuwenhoek was the first to observe microorganisms using a simple microscope and described bacteria from his own mouth in 1683. 2) The compound light microscope uses lenses, light, and sometimes stains to make small objects visible. Total magnification is calculated by multiplying the objective and ocular magnifications. 3) Staining microorganisms allows for better visibility of cells and structures under the microscope and differentiation between species. The Gram stain distinguishes between gram-positive and gram-negative bacteria based on cell wall composition.

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    © All Rights Reserved
    Download as DOCX, PDF, TXT or read online from Scribd
    Download as docx, pdf, or txt
    of 7

    MICROSCOPY AND

    STAINING
    NOTES AND SUMMARY
    Historical Microscopy
     Anton van Leeuwenhoek (1632-1723)
     1st person to see individual microorganisms
     Constructed simple microscope capable of magnifying objects
    (100 to 300 times.
     1683, he described bacteria taken from his own mouth.

    Principles of Microscopy
     Microscopy is the technology of making very small things visible to
    the human eye.
     Micrometer (um) = 0.000001m
     Nanometer (nm) = 0.000000001 m
     Angstrom (A) = 0.0000000001 m
    The Compound Light Microscope
     Monocular – single eyepiece
     Binocular – with two eyepieces
     Base
     Condenser
     Iris diaphragm
     Objective lens
     Body tube
     Ocular lens
     mechanical stage
     coarse adjustment
     fine adjustment

    Total Magnification
     Scanning (3X) x (10X) = 30X magnification
     Low power (10X) x (10X) = 100X magnification
     High power (40X) x (10X) = 400X magnification
     Oil immersion (100x) x (10X) = 1000x magnification

    Other types of microscopes


     Dark-field microscope
     Phase-contrast microscope
     Fluorescence microscope
     Electron microscope
     Transmission electron microscope
     Scanning electron microscope

    Microscopic examination of Microorganisms


    Examination of unstained bacteria
     Wet mounts, in which a drop of medium containing the organisms is
    placed on a microscope slide to view the live microorganisms.
     Hanging drop, a drop of suspension is placed on a cover slip, which
    is then inverted over a concave area of hollow ground or concavity
    slide.

    Conditions where these preparations are


    preferred:
     Morphology of spiral bacteria
     Motility of bacteria
     Cell inclusions that are easily destroyed
     Reaction to chemicals or specific sera
     Cytologic changes during cell divisions

    Examination of Fixed, Stained Preparations


     Preparing a smear, thin layer of material spread across a slide for
    microscopic study.
     The thin layer of material on the slide is air dried or fixed by
    heating
     Staining, the process of artificially coloring microorganisms with dyes
    in order to facilitate their study under the microscope.

    Advantages over wet preparations


     Cells and cell structures are more clearly visible.
     Differences between cells of the same or different species can be
    demonstrated using a special staining solutions.
     Dye – an organic compound consisting of benzene rings with
    chromophore (chemicals that give color) and auxochrome (salt-
    forming properties that transfer color of a dye) group.
     Mordant – chemical added to a dye to make it stain more intensely.
     Chemical mordant: iodine, tannic acid
     Physical mordant: heat and cold.

    Types of Staining
    Direct or Positive
     Simple
     use of ordinary dye for general study of organisms with no
    need of comparisons of physiological characteristics.
     Selective or special
     Certain cell structures are selectively colored by special stains
    of dyes.
     Example: green for spores
     Differential
     Used to contrast two or more organisms in a field of study
    which maybe the same or different species.
    GRAM STAIN
    STEPS REAGENTS REACTIONS
    Initial staining CRYTAL VIOLET All cells will take up the
    dye. All appear purple
    Mordanting Gram’s iodine Forms a complex &
    fixes on the cell call. All
    takes up the violet
    color
    Mordanting Acetone-alcohol Gram – bacteria will be
    95% ethanol decolorized; gram +
    retain the violet color.

    Counter- Safranin Gram – bacteria will be


    staining decolorized; gram +
    retain the violet color.

    Rule:
     All COCCI are gram positive except:
     Neisseria
     Branhammella
     Veillonella
     Acidaminococcus
     Megasphaera
     All COCCI are gram positive except:
     Neisseria
     Branhammella
     Veillonella
     Acidaminococcus
     Megasphaera
    Theories explaining the gram reaction
     Membrane theory of Knaysi
     High lipid content of gram – can be removed by alcohol
     Isoelectric theory
     Gram + have lower isoelectric pt. or pH at which the cells have
    no strong change.
     Outer surface theory
     Gram + bacteria contain magnesium ribonucleic acids which
    forms complex with crystal violet and iodine which is insoluble
    to alcohol.

    Acid Fast Stain

    Steps Reagents Reactions

    Initial staining Carbolfuchsin All cells are colored


    red

    Mordanting Steam phenol in Fixes the stain


    - Physical carbolfuchsin
    - Chemical

    Decolorizing Acid-alcohol Non-acid fast bacteria


    are decolorized; acid
    fast remains red

    Counter- Methylene blue Acid fast remains red;


    staining Non-acid fast blue in
    color

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