MICROSCOPY

• Gram negative bacteria have only a thin outer membrane composed of lipopolysaccharde (LPS).
• Scanning Electron Microscopy involves subjecting the specimen to a narrow electron beam which rapidly moves over the surface of the specimen, causing the release of a shower of secondary electrons and other types of radiation from the specimen surface.
• The maximum useful magnification in Scanning Electron Microscopy is 10,000 to 1,000,000x.
• Scanning Tunneling Electron Microscope is an atomic force microscopy source.
• The crystal violet - iodine complex is easily lost through the LPS and thin peptidogly can layer when the cells are treated with a solvent.
• Gram-positive bacteria have a thick layer of peptidoglycan in their cell walls.
• Gram-negative bacteria have a thin layer of peptidoglycan in their cell walls.
• Teichoic acids and lipoteichoic acids are present in the cell walls of Gram-positive bacteria, but absent in Gram-negative bacteria.
• Lipopolysaccharide is present in the cell walls of Gram-negative bacteria, but absent in Gram-positive bacteria.
• Microscopy involves the use of either the light microscope or the electron microscope.
• All microscopes employ the principle that specific lenses magnify the image of a cell such that details of its structure are most apparent.
• Resolution, the ability to distinguish two points as separate, is dictated by the physical properties of light.
• A microscope is an optical instrument used to observe tiny objects, often objects that cannot be seen at all with the unaided eye.
• The resolving power or resolution of a microscope is the ability of the lenses to distinguish fine detail and structure; refers to the ability of the lenses to distinguish between two points as distinct and separate.
• The shorter the wavelength of light used in the instrument, the greater the resolution.
• The compound light microscope has been of crucial importance in the development of microbiology as a science and remains a basic tool of routine microbiological research.
• Total magnification is the product of the magnification of its objective and ocular lens, and is a property set by resolution.
• Rundina - dela Cruz 37 specimens Transmission Electron Microscope (TEM) 0.0002 mm (0.2 nm) X 200,000 Specimen is viewed on screen Excellent resolution Allows examination of cellular and viral ultrastructure Specimen is non living Reveals internal features of thin specimens Scanning Electron Microscope (SEM) 0.0200 mm (20 nm) X 10,000 Specimen is viewed on screen Gives the illusion of depth (three - dimensions) Useful for examining surface features of cells and viruses Specimen is nonliving Resolution is less than that of TEM Components of the Compound Light Microscope (Source: Engelkirk and D
• Resolving power is a function of the wavelength of light used and an innate property of the lens known as its numerical aperture.
• In general, magnification of lens is proportional to its numerical aperture therefore, lenses with higher magnification usually have a higher numerical aperture.
• Bright-Field Microscopy is a technique where the microscopic field is brightly lighted and the microorganisms appear dark because they absorb some of the light.
• Specimens must be stained for Bright-Field Microscopy.
• Dark-Field Microscopy is a light microscope in which the lighting system has been modified to reach the specimen from the sides only, causing the specimen to appear light on a dark background.
• The resolution in Dark-Field Microscopy is higher, allowing objects to be resolved that are not resolvable by Bright-Field or Phase-Contrast Microscopy.
• Dark-Field Microscopy is utilized in the examination of living unstained microorganisms such as in wet mount or hanging drop preparations.
• Fluorescence Microscopy involves staining microorganisms with a fluorescent dye and illuminating them with blue light, where the blue light is absorbed and green light is emitted by the dye.
• Fluorescence occurs either because of the presence within cells of naturally fluorescent substances such as chlorophyll (autofluorescence) or because cells have been treated with a fluorescent dye.
• Phase Contrast Microscopy is used in the study of living unstained cells, making use of a conventional light microscope with a phase contrast objective and a phase contrast condenser.
• Nomarski Differential Interference Contrast Microscope produces a 3D image of living specimens due to prisms and two beams of light used.
• Atomic Force Microscopy gives a 3D image, using a stylus positioned extremely close to the specimen and with repulsive atomic forces from the specimen and stylus the interaction is recorded by a computer.
• Confocal Scanning Laser Microscopy couples a laser light source to a microscope and makes use of a fluorescent dyes.
• The Electron Microscope gives a more detailed view of microorganisms wherein electromagnets act as lenses.
• Transmission Electron Microscopy involves examining the specimen prepared in an extremely thin dry film on screens.
• Shadow-casting in Transmission Electron Microscopy involves depositing an extremely thin layer of metal at an oblique angle on the organism so that the organism produces shadow on the uncoated side.
• Negative staining in Transmission Electron Microscopy is used to view fine details of objects such as viruses or bacterial flagella using an electron-dense material such as phosphotungstic acid as “stain” to outline the object.
• Ultrathin sectioning in Transmission Electron Microscopy involves embedding bacterial cells in a plastic material and then cutting blocks into ultrathin slices (60 nm) and preparing them for microscopic examination.
• Freeze-etching in Transmission Electron Microscopy is developed to prepare sections of the specimen without resorting to the chemical treatment of the fixation process, which can produce artifacts.
• Autoradiography is a cytochemical method in which the location of a particular chemical constituent in a specimen is determined by observing the site at which the radioactive material becomes positioned.
• The maximum useful magnification in Transmission Electron Microscopy is 500,000 to 1,000,000x.