Introduction
Microorganisms, as their name implies, cannot be seen with the naked eye. Although they were observed as early as 1674 by Anton Van Leeuwenhoek using a simple, single-lens microscope, it was not until the development of the modern compound microscope that the real diversity of microorganisms became apparent.
There are two basic categories of microscopes:
light
microscopes and electron microscopes. Light, or optical, microscopes require light waves to
provide the
illumination while electron
microscopes use electrons to provide the
illumination. Light microscopes are used for most general
laboratory work, while electron microscopes are used to view extremely
small objects such as sub-cellular components or viruses.
In each basic category of microscope, there are a
variety of sub-types. Light microscopes may be bright field, dark
field, phase contrast, or fluorescence, while electron microscopes can
be either transmission or scanning. The most commonly used
laboratory microscope is the bright-field microscope, so this lab will
be concerned exclusively with bright-field microscopes.
For this lab, you will need to use a microscope,
which should be available at most wastewater treatment plants.
Read all of the introductory information carefully before
beginning the exercises. The first exercise is meant to
familiarize
you with the parts of the microscope. Then you will learn to
focus the
microscope while viewing a sample of pond water.
Parts of a Microscope
The main parts of a bright-field microscope are shown by the diagram and table below.
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Basically, the microscope consists of a support system, a light system, a lens system, and a focusing system. Each of these systems works together to produce a magnified image of the specimen.
The support system consists of the base, arm, and stage. The base and arm are structural elements which hold the other parts of the microscope in place while the stage holds the slide. Depending on the microscope, the slide can be positioned under two spring clips and moved by the fingers, or it can be held in place by a mechanical stage and moved by means of two control knobs.
Light System
The light system passes light through the specimen using the light source, the condenser, and the iris diaphragm. In a bright-field microscope, an incandescent bulb is usually used as the source of illumination. Light from the light source then passes through the condenser which focuses the light on the specimen. An iris diaphragm is used to control the intensity, or brightness, of light which passes through the specimen, thus allowing the operator to adjust the intensity and achieve an optimum viewing contrast.
Lens System
The lens system forms the actual image which you will see when you look through a microscope. A typical compound microscope has two lenses - an objective lens near the specimen and an ocular lens at the top - each of which magnifies the image of the specimen by a certain amount.
The ocular lens on most microscopes magnifies 10x (meaning that the image produced by the ocular lens is ten times as large as the specimen). In contrast, the typical microscope has at least three objective lenses mounted on a revolving nosepiece to allow for different magnifications. The low-power objective is the shortest and generally magnifies 10x; the middle-sized lens is the high-dry objective which usually magnifies between 40x and 45x; and the longest lens is the oil-immersion lens which usually magnifies between 97x and 100x.
To determine the total magnification of the image, simply multiply the magnification power of the ocular lens by the magnification power of the objective lens which is being used. For example, if you are using a high-dry objective with a magnification power of 40x, then the total magnification will be 10 × 40 = 400x. Alternatively, using the 10x low-power objective, the total magnification would be simply 10 × 10 = 100x. As you can see, you would use the higher power objective lenses to magnify smaller objects and the low-power objective lens to magnify large objects.
There is a limit to the amount of useful magnification one can achieve with a light microscope. The highest magnification which can be achieved without producing a poorly resolved image is known as the resolving power of the lens. The resolving power is the shortest distance between two closely adjacent points which can be seen and is based on the wavelength of light used for illumination and on the nature of the lens. Because of these constraints, the maximum resolving power normally possible with a light microscope is about 0.2 micrometers (um), or 1/100,000 inch. Smaller objects can be viewed using an electron microscope.
Focusing System
The final system at
work in the microscope is the focusing system. So far, we have
learned how all of the components of the microscope are held together
by the support system, how the light system sends light through the
specimen, and how the lens system uses that light to magnify the
specimen's image and transmit it to our eyes. The focusing system
adjusts the distance between the slide and the objective lens so that
the image comes into focus.
The focusing system
consists of two knobs - the coarse adjustment knob and the fine
adjustment knob. When focusing, the operator first turns the
coarse adjustment knob (which is the larger focus knob) in order to
move the stage a large distance and bring the image into the focal
plane of the objective lens. At this stage, the image will be
visible but fuzzy. Then the operator turns the smaller knob,
known as the fine focus knob, to fine tune the focus and to make the
image sharply focussed.
Care of the Microscope
Microscopes are delicate pieces of
equipment, so you should follow a few basic rules to prevent damage to
the microscope. These rules are meant to prevent you from
dropping the microscope, from damaging the lenses, or from storing the
microscope improperly.
Dropping a microscope can break the
lenses or can alter the alignment of the lenses. To prevent this
damage, you should always carry the microscope with two hands - one
hand under the base and the other hand on the arm of the
microscope. When using the microscope, keep the instrument at
least six inches from the edge of the lab table and keep any excess
electrical cord on the table top to keep the microscope from being
pushed or pulled off the table.
The microscope's lenses are very delicate and can easily be scratched or damaged by oils. Lenses should be cleaned before and after each use with special lens paper. (Cleaning with paper towels or cloth can damage the lenses.) In addition, you should refrain from touching the glass lens with your finger to avoid depositing oils or scratching the glass.
When using the microscope to view a
specimen, you should follow common sense rules of behavior. Do
not tamper with any part of the microscope unless you understand its
purpose. A common mistake is to focus quickly while looking
through the eyepiece of the microscope so that the objective lens bumps
into the slide. To prevent damage to the lens, you should always
make large focus changes slowly while observing the movement of the
objective lens from the side of the microscope.
Finally, the microscope should be stored carefully. Unplug the electrical cord by pulling on the plug instead of the cord. Remove oil from the oil-immersion objective using lens paper, then turn the nosepiece so that the low-power objective is in place. Carefully lower the objective to its lowest position by turning the coarse adjustment knob. Then store the microscope under a dust cloth.
