뒤로Microscopy and Staining Techniques in Microbiology
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Microscopy is the use of instruments to magnify and observe microorganisms that are too small to be seen with the naked eye.
A simple microscope has one lens, while a compound microscope uses multiple lenses (objective and ocular) to magnify the image.
Total magnification = objective lens magnification × ocular lens magnification.
Resolution is the ability of lenses to distinguish two points as separate; higher resolution means clearer detail.
Shorter wavelengths of light provide greater resolution in microscopy.
The refractive index measures how much a medium bends light; it affects how light passes through specimens and lenses.
Brightfield illumination shows dark objects against a bright background; light reflected off the specimen does not enter the objective lens.
Darkfield microscopy makes light objects visible against a dark background by blocking direct light and only allowing light reflected from the specimen to enter the lens.
Phase-contrast microscopy allows observation of living organisms and internal structures by enhancing contrast between light rays.
DIC microscopy uses two light beams and prisms to split light, producing higher contrast and color in specimens.
Fluorescence microscopy uses UV light to excite fluorescent dyes or naturally fluorescent substances, emitting visible light to visualize specimens.
Confocal microscopy uses fluorochrome dyes and blue light to illuminate single planes of a specimen, creating 3D images by computer reconstruction.
Electron microscopy uses electrons with shorter wavelengths than light, providing much higher resolution to see very small structures like viruses.
TEM passes electrons through ultrathin specimen sections to produce highly magnified 2D images of internal structures.
SEM scans a specimen's surface with electrons to produce detailed 3D images of surface structures.
A smear is a thin film of microorganisms spread on a slide, fixed to attach and kill them before staining.
Basic dyes have positively charged chromophores; acidic dyes have negatively charged chromophores.
Negative staining stains the background instead of the cells, enhancing contrast without staining the microorganism itself.
A simple stain uses a single basic dye to highlight the entire microorganism, showing cell shape and structure.
A mordant helps hold the stain on the specimen or enlarges the structure for better visualization.
Differential stains distinguish between different types of bacteria, such as Gram stain and acid-fast stain.
The Gram stain classifies bacteria as Gram-positive (thick peptidoglycan walls) or Gram-negative (thin walls with lipopolysaccharides).
The acid-fast stain identifies bacteria with waxy cell walls, such as Mycobacterium and Nocardia.
Acid-fast bacteria stain red; non-acid-fast bacteria stain blue after counterstaining with methylene blue.