L6b- Method of microbiology - Microscopic techniques

The Microscope: Window on an Invisible Realm

  • Microorganism sizes range from:

    • Smallest viruses: measured in nanometers (nm)

    • Largest protists and bacteria: up to 200 micrometers (µm)

The Compound Microscope

  • Commonly used in laboratories for teaching and research.

The Microscope: Lenses and the Bending of Light

Refraction of Light

  • Light is refracted when passing from one medium to another.

  • Refractive index: a measure of how much a substance slows the velocity of light.

  • Bending direction and magnitude depend on the refractive indices of both media.

Focal Length and Strength of Lens

  • Focal point (F): where light rays converge.

  • Focal length (f): distance from the lens center to the focal point; shorter focal length indicates higher magnification.

Working Distance

  • Distance between the lens front and the specimen surface in sharp focus.

The Microscope: Key Characteristics

Magnification

  • Magnification results from visible light passing through curved lenses.

  • Light is refracted to form an enlarged image based on object distance and illumination.

  • Magnification pathway involves:

    • Light passing through the objective lens

    • Light passing through the ocular lens.

Total Magnification Calculation

  • Total magnification = Power of objective lens x Power of eyepiece.

  • Example: 40X (objective) x 10X (eyepiece) = 400X.

The Microscope: Resolution

  • Resolution: ability of a lens to distinguish small, close-together objects.

  • Affected by:

    • Wavelength of light: shorter wavelengths yield better resolution.

    • Numerical aperture: describes lens efficiency in bending light.

    • Oil immersion lenses increase numerical aperture and resolution.

Wavelength Effect on Resolution

  • Longer wavelengths may produce fuzzy images.

  • Shorter wavelengths penetrate better and yield more detailed images.

The Microscope: The Light Microscope

The Bright-Field Microscope

  • Most commonly used in laboratories

  • Produce a dark image against a brighter background

  • Observe live or preserved stained specimens

The Dark-Field Microscope

  • Image is formed by light reflected or refracted by specimen

  • Produces a bright image of the object against a dark background

  • Used to observe living, unstained preparations

The Phase-Contrast Microscope

  • View internal cellular detail

  • Converts differences in refractive index/cell density into detected variations in light intensity

  • Some light rays from hollow cone of light passing through unstained cell slowed/out of phase (dark against bright background)

The Fluorescence Microscope

  • Exposes specimen to ultraviolet, violet, or blue light

  • Specimens usually stained with fluorochromes

  • Shows a bright image of the object resulting from the fluorescent light emitted by the specimen

  • assential tool in microbiology – fluorochrome-labeled probes, such as antibodies, or fluorochrome dyes tag specific cell constituents for identification of unknown pathogens

Confocal Microscopy

  • Confocal scanning laser microscopy (CLSM) creates sharp, composite 3D image of specimens by using laser beam, aperture to eliminate stray light, and computer interface

  • Numerous applications including study of biofilms

Electron Microscopy

  • Replaces light with electrons as the illuminating beam.

  • Shorter wavelength of electrons results in higher resolution.

  • Enables detailed study of microbial morphology.

Transmission Electron Microscope (TEM)

  • Electrons pass through thin sections, producing a clear image.

  • Denser regions scatter more electrons, appearing darker.

Preparation for Electron Microscopy

  • Specimens must be thin and treated with electron-dense materials.

  • Techniques include:

    • Negative stain

      -heavy metals do not penetrate the specimen but render dark background

      – used for study of viruses, bacterial gas vacuoles

    • Shadowing

      – coating specimen with a thin film of a heavy metal on only one side

      – useful for viral morphology, flagella, DNA

    • Freeze-etching for 3D observation.

      - freeze specimen then fracture along lines of greatest weakness (e.g., membranes)

Scanning Electron Microscopy (SEM)

  • Uses excited electrons to create detailed 3D images of surfaces.

  • Allows for the examination of microorganism locations in ecological niches.

Electron Cryotomography

  • Rapid freezing technique provides way to preserve native state of structures examined in vacuum

  • Images are recorded from many different directions to create 3-D structures

Scanning Probe Microscopy

  • Magnification 100 million times, can view atoms on surface of a solid

  • steady current (tunneling current) maintained between microscope probe and specimen

  • Up/down movement of probe as it maintains current is detected, used to create image of surface of specimen.

Fixation

  • Preserves structures; organisms are usually killed and attached to slides.

    • Heat fixation: routine for bacteria/archaea.

    • Chemical fixation: used for larger, delicate organisms.

Dyes

  • Dyes enhance visibility by increasing contrast against backgrounds.

Simple Staining

  • Uses one dye to determine size, shape, and arrangement of bacteria.

Differential Staining

  • Divides microorganisms based on staining properties:

    • Gram stain: illustrates differences in cell wall structure.

    • Acid-fast stain: targets Mycobacterium species (e.g., tuberculosis).

Gram Staining

  • Most widely used differential staining procedure

  • Divides bacteria into two groups, Gram-positive and Gram-negative, based on differences in cell wall structure

Acid-Fast Staining

  • Particularly useful for staining members of the genus Mycobacterium

Staining Specific Structures

• Endospore staining

– heated, double-staining technique

– bacterial endospore is one color and vegetative

cell is a different color

• Capsule stain used to visualize polysaccharide

capsules surrounding bacteria

– negative stain - capsules may be colorless against

a stained background

• Flagella staining

– mordant applied to increase thickness of flagella

Simple Vs Differential stains

• Simple staining

– One dye

• Differential

– Two-different colored dyes

– Example: Gram stain, acid-fast stain

• Special

– Emphasize certain cell parts

– Example: capsule stain