Microscopy Basics in Microbiology

Microscopy

• Microscopy is the use of light or electrons to magnify objects.
• Metric units used in microbiology include: Meter (m), Decimeter (dm), Centimeter (cm), Millimeter (mm), Micrometer (µm), Nanometer (nm). The meter is the standard unit of length, while decimeters are not commonly used in microbiology.
• Microscopy depends on factors such as wavelength of radiation, magnification, resolution, and contrast. Wavelengths in different parts of the electromagnetic spectrum (e.g., gamma rays, X rays, ultraviolet, visible light, infrared, microwaves, radio waves) have different resolving powers.
• Magnification of images depends on lens thickness, curvature, and the speed of light.
• Resolution is the ability to distinguish two points that are close together.
• Contrast is the difference in intensity between two objects or objects in their background. Staining increases contrast.
• There are several types of microscopy: bright field, dark field, phase contrast, differential interference contrast, fluorescence, confocal, transmission electron microscopy (TEM), scanning electron microscopy (SEM), and probe microscopy (scanning tunneling microscopy (STM) and atomic force microscopy (AFM)). Each type has different magnifications and uses. For example, TEM has a higher magnification than the compound light microscope with resolutions capable of viewing viruses and subcellular components;
• Figure 4.3 shows a representation of various object sizes under different types of microscopes.
• Figure 4.6 and 4.7 showcase different light microscopy techniques, including bright-field, dark-field, phase-contrast, and differential interference contrast (Nomarski), and fluorescence microscopy. Various other image types are seen.
• Figure 4.8 shows immunofluorescence microscopy. Immunofluorescence is based on using a dye linked to antibodies.
• Figure 4.11 and 4.12 illustrate scanning electron microscopy (SEM) and probe microscopy, respectively. Scanning electron microscopy images detailed surface morphologies; Probe microscopy views images at the atomic and molecular level.
• Table 4.2 compares diverse microscopy types, providing information about magnification, typical image appearance, procedures, and representative applications. Microscopes can be categorized as either light microscopes (simple and differential) or electron microscopes (transmission and scanning) or probe microscopes (scanning tunneling microscopy and atomic force microscopy).

Identifying Microorganisms

• Microorganisms (MO) are identified using staining and classification methods.
• Staining methods include simple stains, differential stains, and special stains. Examples of differential stains include Gram stain (used to differentiate between Gram-positive and Gram-negative bacteria) and acid-fast stain (used for staining bacteria with waxy cell walls). Table 4.3 shows the different types of stains and their typical uses.
• Classification methods include physical characteristics, biochemical tests, serological tests, phage typing, and analysis of nucleic acids (such as G + C content).
• Physical characteristics refer to shape, size, and appearance.
• Biochemical tests identify microorganisms based on their ability to utilize certain chemicals or produce specific substances.
• Serological tests determine the presence or absence of antibodies against an antigen to assist in identification.
• Phage typing uses bacteriophages (viruses that infect bacteria) to classify bacteria to identify specific bacterial strains.
• Analysis of nucleic acids (DNA or RNA) is used to classify microbes and determine their genes.

Staining

• Dyes used in staining are usually salts. The colored portion of the dye is called the chromophore. Types of dyes include acidic and basic dyes. Acidic dyes bind to positively charged molecules, while basic dyes associate with negatively charged materials.
• Simple stains use one dye; differential stains use multiple.
• Preparing specimens for staining includes smearing and fixing the sample.
• For instance, figure 4.13 outlines the procedure when preparing a specimen for staining.
• Figures 4.14 to 4.15, display examples of simple stain techniques.
• Table 4.3 shows important stains used for light microscopy.

Classification/Identification

• Physical characteristics are used to identify microorganisms based on their shape, size, and appearance.
• Biochemical tests determine the ability of microorganisms to utilize or produce specific chemicals.
• Serological tests employ antigen-antibody reactions to identify microorganisms.
• Phage typing uses bacteriophages to classify bacteria.
• Analysis of nucleic acids determines the sequence and content of an organism's DNA (or RNA) to identify them.