3-1 Use of the microscope

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The microscope, as shown in Figure 3-1, is one of the most important instruments utilized by the microbiologist. In order to study the morphological and staining characteristics of microorganisms such as bacteria, yeasts, molds, algae and protozoa, you must be able to use a microscope correctly.

Figure 3-1 The light microscope

A modern light microscope. This is an example of the kind used in the teaching labs at the University of Wisconsin-Madison. The various parts of the microscope are labeled. Please take the time to become familiar with their names.

The compound microscope used in microbiology is a precision instrument; its mechanical parts, such as the calibrated mechanical stage and the adjustment knobs, are easily damaged, and all lenses, particularly the oil immersion objective, are delicate and expensive. Handle the instrument with care and keep it clean.

The microscope is basically an optical system (for magnification) and an illumination system (to make the specimen visible). To help understand the function of the various parts of the microscope, we will follow a ray of light as it works its way through a microscope from the light source, through the lenses, up to the eye. Figure 3-8 traces the path of light through the parts of the microscope

Figure 3-8 The path of light through a microscope

Modern microscopes are complex precision instruments. Light, originating in the light source (1), is focused by the condensor (2) onto the specimin (3). The light then enters the objective lens (4) and the image is magnified. Light then passes through a series of glass prisms and mirrors, eventually entering the eyepiece (5) where is it further magnified, finally reacing the eye.

First let us consider a primary feature of all microscopes, the light source. Proper illumination is essential for effective use of a microscope. A tungsten filament lamp usually serves as the source of illumination. If reflected illumination is used, a separate lamp provides a focused beam of light which is reflected upward through the condenser lenses by a mirror.

The light from the illuminating source is passed through the substage condenser. The condenser serves two purposes; it regulates the amount of light reaching the specimen and it focuses the light coming from the light source. As the magnification of the objective lens increases, more light is needed. The iris diaphragm (located in the condenser), regulates the amount of light reaching the specimen. The condenser also collects the broad bundle of light produced by the light source and focuses it on the small area of the specimen that is under observation.

Light then passes up through the slide and into the objective lens where the first magnification of the image takes place. Magnification increases the apparent size of an object. In the compound light microscope two lenses, one near the stage called the objective lens and another in the eyepiece, enlarge the sample. The magnifying power of an objective lens is engraved in the lens mount. Microscopes in most microbiology laboratories have three objective lenses: the low power objective lens (10X), the high-dry objective lens (40X) and the oil-immersion objective lens (100X). The desired objective lens is rotated into working position by means of a revolving nosepiece.

On both sides of the base of the microscope are the course and fine adjustment knobs, used to bring the image into focus. Rotation of these knobs will either move the specimen and the objectives closer or farther apart. The coarse adjustment moves the nosepiece in large increments and brings the specimen into approximate focus. The fine adjustment moves the nosepiece more slowly for precise final focusing. In some microscopes, rotation of the fine and course adjustment knobs will move the stage instead of the nosepiece.

Magnification alone is not the only aim of a microscope. A given picture may be faithfully enlarged without showing any increase in detail. The true measure of a microscope is its resolving power. The resolving power of the lens is its ability to reveal fine detail and to make small objects clearly visible. It is measured in terms of the smallest distance between two points or lines where they are visible as separate entities instead of one blurred image. The resolving power of the objective lens, engraved on the lens, allows us to predict which objective lens should be used for observing a given specimen. However, having good resolution in the microscope does not guarantee a visible image, the resolving power of the human eye is quite limited. Often further magnification is needed to obtain a good image.

When the oil-immersion objective lens is in use, the difference between the light-bending ability (or refractive index of the medium holding the sample) and the objective lens becomes important. Because the refractive index of air is less than that of glass, light rays are bent or refracted as they pass from the microscope slide into the air, as shown in Figure 3-9. Many of these light rays are refracted at so great an angle that they completely miss the objective lens. This loss of light is so severe that images are significantly degraded. Placing a drop of immersion oil, which has a refractive index similar to glass, between the slide and the objective lens decreases this refraction, and increases the amount of light passing from the specimen into the objective lens. This results in greater resolution and a clearer image.

Figure 3-9 Refraction of light at 100X

Light passing out of the slide, into the air, toward the objective lens is refracted, due to the different in refractive index between air and glass. While the bending cause by this difference is not important at 100X and 400X, at 1000X this refraction is problematic, causing blurring of the image and significant loss of light. Immersion oil has a refractive index very similar to that of glass. Placement of a drop of oil between the objective lens and the slide prevents the bending of light rays and clarifies the image. The blue dashed line represents a potential light ray if immersion oil is not present. The red dashed line represents a light ray if immersion oil is present.

The image of the specimen continues on through a series of mirrors and/or prisms that bend it toward the eyepiece. A further magnification takes place at the eyepiece producing what is called a virtual image. Total magnification is equal to the product of the eyepiece magnification and the objective magnification. Most often eyepiece lenses magnify 10-fold resulting in total magnifications of 100, 400, or 1000X, depending upon which objective is in place. Many modern microscopes will also have focusable eyepieces to compensate for differences between individuals and even between individual's eyes. The adjustment of these is important and is described below.

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