Microscopy and Staining PDF

Microbiology: Principles and Explorations Microscopy and Staining  Principles of Microscopy Microscopy is the technology of making very small things visible to the human eye Table 3.1 1. Milli = one thousandth 10-3 m 2. Micro = one millionth 10-6 m 3. Nano = one billi...

Microbiology: Principles and Explorations Microscopy and Staining  Principles of Microscopy Microscopy is the technology of making very small things visible to the human eye Table 3.1 1. Milli = one thousandth 10-3 m 2. Micro = one millionth 10-6 m 3. Nano = one billionth 10-9 m Relative Sizes of Objects bacteria on the tip of a needle  Properties of Light Wavelength: Length of a light ray; represented by the Greek letter lambda () Equal to the distance between two adjacent crests or troughs of a wave Resolution: Refers to the ability to see two items as separate and discrete units Wavelength  The Electromagnetic Spectrum Only a narrow range of wavelengths, those of visible and ultraviolet light are used in light microscopy The shorter the wavelength used, the greater the resolution that can be attained  The Electromagnetic Spectrum  Resolving Power (RP) of a lens in a numerical measure of the resolution that can be obtained with that lens The smaller the distance between objects that can be distinguished, the greater the resolving power of the lens Numerical aperture (NA): A mathematical expression relating to the extent that light is concentrated by the condenser lens and collected by the objective RP= /2(NA) Resolution  Effect of Wavelength on Resolution Properties of Light:Light & Objects Reflection: If the light strikes an object and bounces back (giving the object color) Transmission: The passage of light through an object Absorption: The light rays neither pass through nor bounce off an object but are taken up by the object Various Interactions of Light  Refraction The bending of light as it passes from one medium to another of different density The bending of the light ray gives rise to an angle of refraction, the degree of bending Index of refraction: A measure of the speed at which light passes through the material Refraction Immersion Oil Diffraction  Light Microscopy Refers to the use of any kind of microscope that uses visible light to make specimens observable The compound light microscope has more than one lens 1. Objective lens 2. Ocular lens Monocular vs. Binocular Cutaway View of a Modern Microscope Objective The Compound Light Microscope  Total Magnification: calculated by multiplying the magnifying power of the objective lens by the magnifying power of the ocular lens Parfocal: The specimen will remain very nearly in focus as microscopist increases or decreases the magnification Ocular micrometer: for measuring objects viewed  Different Types of Microscopy 1. Bright-Field Microscopy 2. Dark-Field Microscopy 3. Phase-Contrast Microscopy 4. Nomarski (Differential Interference Contrast) Microscopy 5. Fluorescence Microscopy 6. Confocal Microscopy 7. Digital Microscopy Bright Field vs. Dark Field Illumination Saccharomyces cerevisiae (975X) Under Bright Field Illumination Saccharomyces cerevisiae (975X) Under Dark Field Illumination Fluorescent Antibody Staining  Confocal Microscope System  Cell With Microtubular Fragments Under Standard Fluorescent Microscopy Cell With Microtubular Fragments Under Confocal Microscopy Digital Microscope System Cyanobacterium—Chroococcus Using Digital Microscopy  Electron Microscopy Uses a beam of electrons instead of a beam of light electromagnets rather than glass lenses to focus the beam Produce electron micrographs with great detail Two most common types of electron microscopy: 1. Transmission electron microscopy (TEM) 2. Scanning electron microscopy (SEM) Electron Microscope Cross-section Modern Scanning Electron Microscope Light Microscope Image of Paramecium (160X) Electron Microscope Image of Paramecium (425X) Freeze-Fracturing and Freeze-Etching Freeze-Etch Preparation Escherichia coli-- Transmission Electron Microscopy Escherichia coli-- Scanning Electron Microscopy Aspergillus-- Scanning Electron Microscopy Actinomyces (5670X) Radiolarian (1761X)  Cyclotella meneghiniana (1584X)  Techniques of Light Microscopy Wet mounts: A drop of medium containing organisms is placed on slide and used to view living microorganisms Smears: Microorganisms are spread onto the surface of a glass slide and used to view destroyed organisms Heat fixation: destroys the organisms, causes organism to adhere to slide, and alters organism to accept stains (dyes) The Hanging Drop Technique  Treponema pallidum Using Dark Field Microscopy and hanging-drop technique  Principles of Staining Stain or dye: A molecule that can bind to a cellular structure and give it color (contrast) 1. Cationic or Basic Dyes 2. Anionic or Acidic Dyes Simple stain: makes use of a single dye and reveals basic cell shapes and arrangements Differential stain: makes use of two or more dyes and distinguishes between organisms based on structural differences The Gram Stain Gram Positive and Gram Negative Cells The Ziehl-Neesen Acid-Fast Stain  Negative Staining Specimen or part of it resists taking up a stain The Schaeffer-Fulton Spore Stain