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Microscopy and Staining Techniques in Microbiology

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  • Units used to measure microorganisms
    Microorganisms are measured in micrometers (µm) and nanometers (nm).
  • Total magnification in a compound microscope
    Total magnification = ocular lens magnification × objective lens magnification.
  • Resolution (resolving power) of a microscope
    Resolution is the ability to distinguish two points as separate and see fine detail.
  • Limit of resolution for compound light microscopes
    Approximately 0.2 micrometers (µm), limiting magnification to about 1500x.
  • Purpose of immersion oil in microscopy
    Immersion oil reduces light refraction between the slide and objective lens, improving resolution and clarity.
  • Brightfield illumination
    Dark objects are visible against a bright background; light reflected off the specimen does not enter the objective lens.
  • Darkfield microscopy
    Light objects are visible against a dark background; uses an opaque disk to block direct light, capturing only light reflected by the specimen.
  • Phase-contrast microscopy
    Enhances contrast in unstained cells by combining direct and diffracted light rays, allowing detailed examination of living organisms.
  • Differential Interference Contrast (DIC) microscopy
    Uses two light beams and prisms to produce brightly colored, three-dimensional images with enhanced contrast.
  • Fluorescence microscopy
    Uses UV light to excite fluorescent dyes; cells emit visible light and appear bright against a dark background.
  • Fluorescent-antibody (immunofluorescence) technique
    Uses antibodies tagged with fluorochromes to specifically detect pathogens by causing them to fluoresce.
  • Confocal microscopy
    Uses fluorochrome dyes and blue light to obtain clear 2D images of a single plane; computer constructs 3D images.
  • Two-photon microscopy
    Uses two photons of long-wavelength light to excite dyes, allowing study of living cells up to 1 millimeter deep.
  • Electron microscopy advantage
    Uses electrons with shorter wavelengths than light, providing much greater resolution to see viruses and internal cell structures.
  • Transmission Electron Microscopy (TEM)
    Electrons pass through ultrathin specimen sections; magnification 10,000–10,000,000x; resolution 0.2 nm.
  • Scanning Electron Microscopy (SEM)
    Electron beam scans specimen surface; produces 3D images; magnification 1,000–500,000x; resolution 0.5 nm.
  • Scanning Tunneling Microscopy (STM)
    Uses a tungsten probe to scan surfaces at atomic resolution without special specimen preparation.
  • Atomic Force Microscopy (AFM)
    Uses a metal-and-diamond probe to produce 3D images at near atomic detail by recording probe movements.
  • Purpose of fixing a smear
    Attaches microorganisms to the slide, kills them, and preserves structures with minimal distortion.
  • Difference between basic and acidic dyes
    Basic dyes have a cationic chromophore and stain cells (which are negatively charged); acidic dyes have an anionic chromophore and stain the background.
  • Simple stain
    Use of a single basic dye to highlight the entire microorganism and visualize cell shapes and structures.
  • Gram stain purpose
    Differentiates bacteria into gram-positive (thick peptidoglycan, purple) and gram-negative (thin peptidoglycan, pink/red) groups.
  • Gram stain steps and colors
    1. Crystal violet (purple), 2. Iodine mordant (purple), 3. Alcohol decolorizer (gram-negative colorless), 4. Safranin counterstain (gram-negative pink/red).
  • Acid-fast stain use
    Identifies bacteria with waxy cell walls like Mycobacterium and Nocardia that retain carbolfuchsin dye after acid-alcohol decolorization.
  • Appearance of acid-fast and non–acid-fast bacteria
    Acid-fast bacteria stain red; non–acid-fast bacteria stain blue after counterstaining with methylene blue.
  • Capsule stain
    Negative staining technique using India ink or nigrosin to stain background, revealing capsules as clear halos around cells.
  • Endospore stain
    Schaeffer-Fulton method uses malachite green with heat to stain endospores green; counterstain with safranin colors cells red or pink.
  • Flagella stain
    Uses mordant and carbolfuchsin to thicken flagella, making them visible under light microscopy to determine number and arrangement.