Various types of microscopes are available for use in the microbiology laboratory. The microscopes have varied applications and modifications that contribute to their usefulness.
The light microscope. The common light microscope used in the laboratory is called a compound microscope because it contains two types of lenses that function to magnify an object. The lens closest to the eye is called the ocular, while the lens closest to the object is called the objective. Most microscopes have on their base an apparatus called a condenser, which condenses light rays to a strong beam. A diaphragm located on the condenser controls the amount of light coming through it. Both coarse and fine adjustments are found on the light microscope (Figure ).
To magnify an object, light is projected through an opening in the stage, where it hits the object and then enters the objective. An image is created, and this image becomes an object for the ocular lens, which remagnifies the image. Thus, the total magnification possible with the microscope is the magnification achieved by the objective multiplied by the magnification achieved by the ocular lens.
A compound light microscope often contains four objective lenses: the scanning lens (4X), the low‐power lens (10X), the high‐power lens (40 X), and the oil‐immersion lens (100 X). With an ocular lens that magnifies 10 times, the total magnifications possible will be 40 X with the scanning lens, 100 X with the low‐power lens, 400 X with the high‐power lens, and 1000 X with the oil‐immersion lens. Most microscopes are parfocal. This term means that the microscope remains in focus when one switches from one objective to the next objective.
The ability to see clearly two items as separate objects under the microscope is called the resolution of the microscope. The resolution is determined in part by the wavelength of the light used for observing. Visible light has a wavelength of about 550 nm, while ultraviolet light has a wavelength of about 400 nm or less. The resolution of a microscope increases as the wavelength decreases, so ultraviolet light allows one to detect objects not seen with visible light. The resolving power of a lens refers to the size of the smallest object that can be seen with that lens. The resolving power is based on the wavelength of the light used and the numerical aperture of the lens. The numerical aperture (NA) refers to the widest cone of light that can enter the lens; the NA is engraved on the side of the objective lens.
If the user is to see objects clearly, sufficient light must enter the objective lens. With modern microscopes, entry to the objective is not a problem for scanning, low‐power, and high‐power lenses. However, the oil‐immersion lens is exceedingly narrow, and most light misses it. Therefore, the object is seen poorly and without resolution. To increase the resolution with the oil‐immersion lens, a drop of immersion oil is placed between the lens and the glass slide (Figure ). Immersion oil has the same light‐bending ability (index of refraction) as the glass slide, so it keeps light in a straight line as it passes through the glass slide to the oil and on to the glass of the objective, the oil‐immersion lens. With the increased amount of light entering the objective, the resolution of the object increases, and one can observe objects as small as bacteria. Resolution is important in other types of microscopy as well.
Other light microscopes. In addition to the familiar compound microscope, microbiologists use other types of microscopes for specific purposes. These microscopes permit viewing of objects not otherwise seen with the light microscope.
An alternative microscope is the dark‐field microscope, which is used to observe live spirochetes, such as those that cause syphilis. This microscope contains a special condenser that scatters light and causes it to reflect off the specimen at an angle. A light object is seen on a dark background.
A second alternative microscope is the phase‐contrast microscope. This microscope also contains special condensers that throw light “out of phase” and cause it to pass through the object at different speeds. Live, unstained organisms are seen clearly with this microscope, and internal cell parts such as mitochondria, lysosomes, and the Golgi body can be seen with this instrument.
The fluorescent microscope uses ultraviolet light as its light source. When ultraviolet light hits an object, it excites the electrons of the object, and they give off light in various shades of color. Since ultraviolet light is used, the resolution of the object increases. A laboratory technique called the fluorescent‐antibody technique employs fluorescent dyes and antibodies to help identify unknown bacteria.
Electron microscopy. The energy source used in the electron microscope is a beam of electrons. Since the beam has an exceptionally short wavelength, it strikes most objects in its path and increases the resolution of the microscope significantly. Viruses and some large molecules can be seen with this instrument. The electrons travel in a vacuum to avoid contact with deflecting air molecules, and magnets focus the beam on the object to be viewed. An image is created on a monitor and viewed by the technologist.
The more traditional form of electron microscope is the transmission electron microscope (TEM). To use this instrument, one places ultrathin slices of microorganisms or viruses on a wire grid and then stains them with gold or palladium before viewing. The densely coated parts of the specimen deflect the electron beam, and both dark and light areas show up on the image.
The scanning electron microscope (SEM) is the more contemporary form electron microscope. Although this microscope gives lower magnifications than the TEM, the SEM permits three‐dimensional views of microorganisms and other objects. Whole objects are used, and gold or palladium staining is employed.
Light microscopy. (a) The important parts of a common light microscope. (b) How immersion oil gathers more light for use in the microscope.
To magnify an object, light is projected through an opening in the stage, where it hits the object and then enters the objective. An image is created, and this image becomes an object for the ocular lens, which remagnifies the image. Thus, the total magnification possible with the microscope is the magnification achieved by the objective multiplied by the magnification achieved by the ocular lens.
A compound light microscope often contains four objective lenses: the scanning lens (4X), the low-power lens (10X), the high-power lens (40 X), and the oil-immersion lens (100 X). With an ocular lens that magnifies 10 times, the total magnifications possible will be 40 X with the scanning lens, 100 X with the low-power lens, 400 X with the high-power lens, and 1000 X with the oil-immersion lens. Most microscopes are parfocal.This term means that the microscope remains in focus when one switches from one objective to the next objective.
The ability to see clearly two items as separate objects under the microscope is called the resolution of the microscope. The resolution is determined in part by the wavelength of the light used for observing. Visible light has a wavelength of about 550 nm, while ultraviolet light has a wavelength of about 400 nm or less. The resolution of a microscope increases as the wavelength decreases, so ultraviolet light allows one to detect objects not seen with visible light. The resolving power of a lens refers to the size of the smallest object that can be seen with that lens. The resolving power is based on the wavelength of the light used and the numerical aperture of the lens. Thenumerical aperture (NA) refers to the widest cone of light that can enter the lens; the NA is engraved on the side of the objective lens.
If the user is to see objects clearly, sufficient light must enter the objective lens. With modern microscopes, entry to the objective is not a problem for scanning, low-power, and high-power lenses. However, the oil-immersion lens is exceedingly narrow, and most light misses it. Therefore, the object is seen poorly and without resolution. To increase the resolution with the oil-immersion lens, a drop of immersion oil is placed between the lens and the glass slide (Figure 1 ). Immersion oil has the same light-bending ability (index of refraction) as the glass slide, so it keeps light in a straight line as it passes through the glass slide to the oil and on to the glass of the objective, the oil-immersion lens. With the increased amount of light entering the objective, the resolution of the object increases, and one can observe objects as small as bacteria. Resolution is important in other types of microscopy as well.
Other light microscopes. In addition to the familiar compound microscope, microbiologists use other types of microscopes for specific purposes. These microscopes permit viewing of objects not otherwise seen with the light microscope.
An alternative microscope is the dark-field microscope, which is used to observe live spirochetes, such as those that cause syphilis. This microscope contains a special condenser that scatters light and causes it to reflect off the specimen at an angle. A light object is seen on a dark background.
A second alternative microscope is the phase-contrast microscope. This microscope also contains special condensers that throw light “out of phase” and cause it to pass through the object at different speeds. Live, unstained organisms are seen clearly with this microscope, and internal cell parts such as mitochondria, lysosomes, and the Golgi body can be seen with this instrument.
The fluorescent microscope uses ultraviolet light as its light source. When ultraviolet light hits an object, it excites the electrons of the object, and they give off light in various shades of color. Since ultraviolet light is used, the resolution of the object increases. A laboratory technique called the fluorescent-antibody technique employs fluorescent dyes and antibodies to help identify unknown bacteria.
Electron microscopy. The energy source used in the electron microscope is a beam of electrons. Since the beam has an exceptionally short wavelength, it strikes most objects in its path and increases the resolution of the microscope significantly. Viruses and some large molecules can be seen with this instrument. The electrons travel in a vacuum to avoid contact with deflecting air molecules, and magnets focus the beam on the object to be viewed. An image is created on a monitor and viewed by the technologist.
The more traditional form of electron microscope is the transmission electron microscope (TEM). To use this instrument, one places ultrathin slices of microorganisms or viruses on a wire grid and then stains them with gold or palladium before viewing. The densely coated parts of the specimen deflect the electron beam, and both dark and light areas show up on the image.
The scanning electron microscope (SEM) is the more contemporary form electron microscope. Although this microscope gives lower magnifications than the TEM, the SEM permits three-dimensional views of microorganisms and other objects. Whole objects are used, and gold or palladium staining is employed.