A microscope is an instrument used for the visualization of small objects like cells and microorganisms. Microscopes can be broadly classified into types; one that uses visible light as the source of illumination (light microscope) and the other uses electron beams (electron microscope).
Light microscopes use glass lenses to bend and focus light rays and produce enlarged images of small objects. The maximum resolution of a light microscope is about 0.2 μm. The resolution is determined by the wavelength of the light and the numerical aperture of its lens system. The most common types of light microscopes are bright-field, dark-field, phase-contrast, and fluorescence microscopes. Each of these microscopes yields a distinct image of the objects and is used to observe different aspects of microbial morphology.
Bright-field microscope uses visible light as a source of illumination and the image appears dark in the brighter background. Commonly known as an ordinary microscope, this type of microscope produces a useful magnification of about 1000 times but cannot resolve structures smaller than about 0.2 µm. Stained specimens are often required to increase contrast and color differentiation. Bright field microscopes are used for routine microscopic works in diagnostic and teaching laboratories.
Dark-field microscope is used to examine living microorganisms that are invisible in bright-field microscopy, do not stain easily, or are distorted by staining. For example, in suspected cases of syphilis, chancre fluid is examined by dark-field microscopes to detect Treponema pallidum.
A dark-field microscope uses a darkfield condenser instead of the normal condenser. An opaque disk of this condenser blocks the light that would enter the objective lens directly. The only light that is reflected or refracted by the specimens enters the objective lens and forms an image. The field surrounding a specimen appears dark and the specimen appears brightly illuminated so the name dark-field microscope.
A fluorescence microscope is much the same as a conventional light microscope but it uses light of higher intensity as a light source instead of visible light.
A specimen is stained with a fluorescent dye (fluorochrome) and then exposed to the light of a shorter wavelength (ultraviolet or blue light). The light is absorbed by the specimen stained with fluorochrome and releases fluorescent (or green) light of a longer wavelength. This produces a bright image on a dark background.
- To identify different bacterial pathogens after staining them with fluorochromes. Eg: Auramine-Rhodamine staining technique for the detection of Mycobacterium tuberculosis.
- To do ecological studies. Fluorochromes like acridine orange stain the microorganisms. These stained organisms will fluoresce orange or green even in the midst of other particulate material.
- To distinguish live bacteria from dead bacteria based on the color of their fluorescence when they are treated with a special mixture of stains.
Phase-contrast microscope is a bright field-light microscope with the addition of a special phase-contrast objective (phase plate) and a phase contrast condenser (annular stop). When the light passes from one substance to another substance having a slightly different refractive index or thickness, it will change the phase. The difference in phase is translated into variation in the brightness of the structure and hence are detectable by the eye.
- To study living cells without staining. The ongoing different biological processes in the live cells can be studied.
- To study microbial motility.
- To observe endospores and inclusion bodies that contain poly- hydroxybutyrate, polymetaphosphate, sulfur, or other substances.
It is also called a dissecting microscope. It is an optical microscope designed for low magnification observation of a sample, typically using light reflected from the surface of an object rather than transmitted through it. It is also called a dissecting microscope and is used in microsurgery, watchmaking, and building and inspecting circuit boards. Students can use a stereomicroscope to observe plant photosynthesis in action.
Electron microscope uses the electron beam as a source of illumination and is used to examine structures too small to be resolved with light microscopes. The resolving power of the electron microscope is far greater than that of the light microscopes. Due to the use of a shorter wavelength of electrons, better resolution is obtained. The wavelengths of electrons are about 100,000 times smaller than the wavelengths of visible light.
The electron travels in a vacuum and the magnet focuses the beam on the sample. On the monitor, an image is created which is always black and white and can be colored artificially.
- To study smaller objects such as viruses or objects or molecules having sizes smaller than 0.2 µm.
- To study the details of the internal structure of the cells.
- To observe the ultrastructure of microorganisms, large molecules, biopsy samples, metals, and crystals.
Transmission electron microscope and scanning electron microscope are two main types of electron microscopes.
Find out the major differences between scanning electron microscopy (SEM) and transmission electron microscopy HERE
Transmission Electron Microscope (TEM)
The transmission electron microscope is used to examine cells and cell structure (even individual protein and nucleic acid molecules can be visualized) at very high magnification and resolution. The resolving power of a high-quality TEM is about 0.2 nanometers.
A special thin sectioning technique is needed to observe a bacterial cell by transmission electron microscope. A bacterial cell is cut into very thin (20-60 nm) slices and is treated with heavy metal stains (such as osmic acid, permanganate, and uranium) to obtain sufficient contrast.
Scanning Electron Microscope (SEM)
A scanning electron microscope (SEM) is used to observe the external features of an organism. The specimen is coated with a thin film of a heavy metal such as gold. An electron beam then scans back and forth across the specimen. Electrons scattered from the metal coating are collected and activate a viewing screen to produce an image. SEM can obtain magnification of as low as 15X to as high as 100,000X.
A scanning electron microscope can produce a three-dimensional image of the microorganism’s surface. SEM can be used to examine the actual in situ location of microorganisms in ecological niches such as human skin and the lining of the gut.
References and further readings
- Madigan, M. T., Martinko, J. M., Stahl, D. A., & Clark, D. P. (2011). BROCK Biology of Microorganisms (13thedition). Benjamin Cumming.
- Prescott, L. M. (2002). Microbiology (5th edition). The McGraw-Hill Companies.
Sushmita BaniyaHello, I am Sushmita Baniya from Nepal. I am a postgraduate student of M.Sc Medical Microbiology. I am interested in Genetics and Molecular Biology.
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