Microscopy is an important component of diagnostic microbiology. Bacteria being very small cannot be visualized by the naked eye, because the limit of resolution with the unaided eye is about 200 microns. So, the study of bacteria requires the use of microscopes. A microscope is an instrument that uses one or more lenses to produce a magnified image of an object that is invisible to the unaided eye.
Types of Microscop
Light microscopy, as the name suggests, uses natural or artificial transmitted light as the source of light. Resolving power of microscope is an important component of light microscopy. It is the ability of the lens system to distinguish two closely placed objects as distinct and separate entities.
It is dependent on the wavelength of light used to illuminate the object and on the numerical aperture of the microscope. It is about half of the wavelength of light being used. For example, the smallest particle which can be resolved by yellow light with a wavelength of 0.4 µm is about 0.2 µm.
1. Bright-field microscopy: Bright-field microscopy (always referred to as ordinary light microscopy) is the most common form of light microscopy that uses a compound light microscope. A compound light microscope primarily consists of a compound lens system that contains a number of objective lenses, such as lenses of low power (*10), high power (*40), and oil immersion (*100).
It also contains a fixed ocular (eye piece) lens, usually of *10 or *5. Final magnification of an object is the multiplication of lens power of the objective with that of the eye piece
2. Dark-ground microscopy: The dark-ground microscopy makes use of dark-ground microscope, a special type of compound light microscope. The dark-field condenser with a central circular stop, which illuminates the object with a cone of light, is the most essential part of the dark-ground microscope.
This microscope uses reflected light instead of transmitted light used in the ordinary light microscope. It prevents light from falling directly on the objective lens. Light rays falling on the object are reflected or scattered onto the objective lens with the result that the microorganisms appear brightly stained against a dark background
Fluorescence microscopy is based on the principle that the specimens stained with fluorescent dye when exposed to ultraviolet light result in emission of longer wavelength of light (i.e., visible light). The bacteria stained with fluorescent dye appear as a brightly glowing object against a dark background.
Fluorescence microscopy needs a fluorescence microscope fitted with an ultraviolet light source. Auramine O, acridine orange, and rhodamine are fluorescent dyes used to visualize bacteria. The resolving power of a fluorescence microscope is increased due to the short wavelength of ultraviolet light. Auramine O, acridine orange, and rhodamine are fluorescent dyes used to visualize bacteria.
Electron microscopy utilizes a beam of electrons instead of a beam of light used in the light microscopy. The electron beam is focused by electromagnets, analogous to the lenses used in the light microscopy. The object to be examined is kept on the path of the beam that scatters the electrons and produces an image which is focused on a screen.
The resolving power of the electron microscope is extremely high, theoretically 100,000 times than that of a light microscope. This is because the electron microscope uses electrons whose wavelength is approximately 0.005 nm as compared to 5000 nm wavelength of the visible light. As mentioned earlier, the resolving power is half of the wavelength.
In practice, the resolving power of the electron microscope, however, is about 0.1 nm. There have been many developments in electron microscopy that include (a) shadow casting, (b) scanning electron microscopy, (c) immunoelectron microscopy, and (d) freeze-etching, etc.