3.1 Units of Measurement

• A micrometer equals 0.000001 m.

  • The prefix micro indicates the unit following it should be divided by 1 million.

• A nanometer equals 0.000000001 m.

3.2 Microscopy: The Instruments

• Light microscopy refers to the use of any kind of microscope that uses visible light to observe specimens.

• A compound light microscope has a series of lenses and uses visible light as its scourge of illumination.

• An illuminator is the light source.

• A condenser is the lens that directs the light rays through the specimen.

• The objective lenses are the lenses closest to the specimen.

• The ocular lens is the eyepiece.

• The total magnification of a specimen is calculated by multiplying the objective lens magnification by the ocular lens magnification.

• Resolution is the ability of the lenses to distinguish fine detail and structure.

• The refractive index is a measure of the light-bending ability of a medium.

• We change the refractive index of specimens by staining them.

  • After the specimen is stained, the specimen and its medium have different refractive index.

  • When light rays pass through the two materials, the rays change direction from a straight path by bending or changing angle at the boundary between the materials.

  • It would increase the image’s contrast between the specimen and the medium.

  • As the light rays travel away from the specimen, they spread out and enter the objective lens, and the image is thereby magnified.

• To achieve high magnification with good resolution, the objective lens must be small.

• By focusing the light, the condenser produces a bright field illumination.

• A dark-field microscope is used to examine live microorganisms that either are invisible in the ordinary light microscope, cannot be stained by standard methods, or are so distorted by staining that their characteristics are obscured.

• Phase-contrast microscopy is useful because the internal structures of a cell become more sharply defined, permitting detailed examination of living microorganisms.

• Differential interference contrast microscopy uses differences in refractive indexes.

  • Differential interference contrast microscopy uses two beams of light.

  • Differential interference contrast microscopy has prisms that split each light beam, adding contrasting colors to the specimen.

  • Differential interference contrast microscopy image is brightly colored and appears nearly three dimensional.

• Antibodies are natural defense molecules that are produced by humans and many animals in reaction to a foreign substance, or antigen.

• Confocal microscopy is a technique in light microscopy used to reconstruct three-dimensional images.

  • Using a confocal microscopy, specimens are stained with fluorochromes so they will emit, or return, light.

  • Most confocal microscopes are used in conjunction with computers to construct three-dimensional images.

• Two-photon microscopy uses long-wavelength (red) light, and therefore two photons, instead of one, are needed to excite the fluorochrome to emit light.

• In super-resolution light microscopy, one wavelength stimulates fluorescent molecules to glow, and another wavelength cancels out all fluorescence except for that in one nanometer.

• Scanning acoustic microscopy basically consists of interpreting the action of a sound wave sent through a specimen.

• In electron microscopy, a beam of electrons is used.

  • Electron microscopes are used to examine structures too small to be resolved with light microscopes.

  • Electron microscopes use electromagnetic lenses to focus a beam of electrons onto a specimen.

• In the transmission electron microscope, a finely focused beam of electrons from an electron gun passes through a specially prepared, ultra him section of specimen.

  • In the transmission electron microscope, the specimen is placed on a copper mesh grid.

  • The beam of electrons passes through the specimen and then through an electromagnetic objective lens, high magnifying the image.

  • The electrons are focused by an electromagnetic projector lens onto a viewing screen and saved as a digital image.

• The scanning electron microscope overcomes the sectioning problems associated with a transmission electron microscope.

• The scanning-probe microscopes use various kinds of probes to examine the surface of a specimen using electric current, which does not modify the specimen or expose it to damaging, high-energy radiation.

• The scanning tunneling microscopy uses a thin tungsten probe that scans a specimen and produces an image that reveals the bumps and depressions of the atoms on the surface of the specimen.

• In atomic force microscopy, a metal-and-diamond probe is gently forced down onto a specimen.

3.3 Preparation of Specimens for Light Microscopy

• The color of basic dyes is in cation.

• The color of acidic dyes is the anion.

• Preparing colorless bacteria against a colored background is called negative staining.

• A simple stain is an aqueous or alcohol solution of a single basic dye.

• A mordant would increase the affinity of a stain for a biological specimen.

  • A mordant would coat a structure to make it thicker and easier to see after it is rained with dye.

• Differential stains react differently with different kinds of bacteria and can be used to distinguish them.

• The Gram stain classifies bacteria into two large groups: gram-positive and gram-negative.

• The gram-positive bacteria is the bacteria that lose the dark violet of purple color after decolonization are classified as gram-negative bacteria.

• Stains such as safranin that have a contrasting color to primary stain are called counterstains.

• Acid-fast stain binds strongly only to bacteria that have a waxy material in their cell walls.

• Special stains are used to color parts of microorganisms.

• Many microorganisms contain a gelatinous covering called a capsule.

• An endoscope is a special resistant, dormant structure formed within a cell that protects a bacterium from adverse environmental conditions.