Although there are many kinds of microscopes available to the microbiologist today, only four types will be described here for our use: the brightfield, darkfield, phase-contrast, and fluorescence microscopes.
If you have had extensive exposure to microscopy in previous courses, this unit may not be of great value to you; however, if the study of microorganisms is a new field of study for you, there is a great deal of information that you need to acquire about the proper use of these instruments.
Microscopes in a college laboratory represent a considerable investment and require special care to prevent damage to the lenses and mechanicals.
The fact that a laboratory microscope may be used by several different individuals during the day and moved around from one place to another results in a much greater chance for damage and wear to occur than if the instrument were used by only one individual.
The complexity of some of the more expensive microscopes also requires that certain adjustments be made periodically.
Knowing how to make these adjustments to get the equipment to perform properly is very important. An attempt is made in the five exercises of this unit to provide the necessary assistance in getting the most out of the equipment.
Microscopy should be as fascinating to the beginner as it is to the professional of long standing; however, only with intelligent understanding can the beginner approach the achievement that occurs with years of experience.
A microscope that allows light rays to pass directly through to the eye without being deflected by an intervening opaque plate in the condenser is called a brightfield microscope.
This is the conventional type of instrument encountered by students in beginning courses in biology; it is also the first type to be used in this laboratory.
All brightfield microscopes have certain things in common, yet they differ somewhat in mechanical operation. An attempt will be made in this exercise to point out the similarities and differences of various makes so that you will know how to use the instrument that is available to you.
Before attending the first laboratory session in which the microscope will be used, read over this exercise and answer all the questions on the Laboratory Report. Your instructor may require mat the Laboratory Report be handed in prior to doing any laboratory work.
Microscopes represent considerable investment and can be damaged rather easily if certain precautions are not observed. The following suggestions cover most hazards.
When carrying your microscope from one part of the room to another, use both hands when holding the instrument, as illustrated in figure 1.1.
If it is carried with only one hand and allowed to dangle at your side, there is always the danger of collision with furniture or some other object. And, incidentally, under no circumstances should one attempt to carry two microscopes at one time.
Keep your workstation uncluttered while doing microscopy. Keep unnecessary books, lunches, and other unneeded objects away from your work area. A clear work area promotes efficiency and results in fewer accidents.
Microscopes have been known to tumble off of tabletops when students have entangled a foot in a dangling electric cord. Don't let the light cord on your microscope dangle in such a way as to hazard foot entanglement.
At the beginning of each laboratory period check the lenses to make sure they are clean. At the end of each lab session be sure to wipe any immersion oil off the immersion lens if it has been used.
In most laboratories dustcovers are used to protect the instruments during storage. If one is available, place it over the microscope at the end of the period.
Before we discuss the procedures for using a microscope, let's identify the principal parts of the instrument as illustrated in figure 1.2 (above).
All microscopes have a basic frame structure, which includes the arm and base. To this framework all other parts are attached. On many of the older microscopes the base is not rigidly attached to the arm as is the case in figure 1.2 (above); instead, a pivot point is present that enables one to tilt the arm backward to adjust the eyepoint height.
The horizontal platform that supports the microscope slide is called the stage. Note that it has a clamping device, the mechanical stage, which is used for holding and moving the slide around on the stage. Note, also, the location of the mechanical stage control in figure 1.2. (above).
In the base of most microscopes is positioned some kind of light source. Ideally, the lamp should have a voltage control to vary the intensity of light.
The microscope in figure 1.2 (above) has a knurled wheel on the right side of its base to regulate the voltage supplied to the light bulb. The microscope base has a knob (the left one) that controls voltage. Most microscopes have some provision for reducing light intensity with a neutral density filter.
Such a filter is often needed to reduce the intensity of light below the lower limit allowed by the voltage control.
On microscopes such as the Olympus CH-2, one can simply place a neutral density filter over the light source in the base. On some microscopes a filter is built into the base.
All microscopes have three lens systems: the oculars, the objectives, and the condenser.
The ocular, or eyepiece, is a complex piece, located at the top of the instrument, that consists of two or more internal lenses and usually has a magnification of 10X. Although the microscope in figure 1.2 (above) has two oculars (binocular), a microscope often has only one.
Three or more objectives are usually present. Note that they are attached to a rotatable nosepiece, which makes it possible to move them into position over a slide.
Objectives on most laboratory microscopes have magnifications of 10X, 45X, and 100X, designated as low power, high-dry, and oil immersion, respectively.
Some microscopes will have a fourth objective for rapid scanning of microscopic fields that is only 4X.
The third lens system is the condenser, which is located under the stage. It collects and directs the light from the lamp to the slide being studied.
The condenser can be moved up and down by a knob under the stage. A diaphrag m within the condenser regulates the amount of light that reaches the slide.
Microscopes that lack a voltage control on the light source rely entirely on the diaphragm for controlling light intensity.
On the Olympus microscope in figure 1.2 (above) the diaphragm is controlled by turning a knurled ring. On some microscopes a diaphragm lever is present.
The concentrically arranged coars e adjustment and fine adjustment knobs on the side of the microscope are used for bringing objects into focus when studying an object on a slide. On some microscopes these knobs are not positioned concentrically as shown here.
On binocular microscopes one must be able to change the distance between the oculars and to make diopter changes for eye differences.
On most microscopes the interocular distance is changed by simply pulling apart or pushing together the oculars. To make diopter adjustments, one focuses first with the right eye only.
Without touching the focusing knobs, diopter adjustments are then made on the left eye by turning the knurled diopter adjustment ring (figure 1.2 - above) on the left ocular until a sharp image is seen. One should now be able to see sharp images with both eyes.
The resolution limit, or resolving power, of a microscope lens system is a function of its numerical aperture, the wavelength of light, and the design of the condenser. The optimum resolution of the best microscopes with oil immersion lenses is around 0.2 p.m.
This means that two small objects that are 0.2 p.m apart will be seen as separate entities; objects closer than that will be seen as a single object.
To get the maximum amount of resolution from a lens system, the following factors must be taken into consideration:
A blue filter should be in place over the light source because the short wavelength of blue light provides maximum resolution.
The condenser should be kept at its highest position where it allows a maximum amount of light to enter the objective.
The diaphragm should not be stopped down too much. Although stopping down improves contrast, it reduces the numerical aperture.
Immersion oil should be used between the slide and the 100X objective.
Of significance is the fact that, as magnification is increased, the resolution must also increase. Simply increasing magnification by using a 20X ocular won't increase the resolution.
Keeping the lenses of your microscope clean is a constant concern. Unless all lenses are kept free of dust, oil, and other contaminants, they are unable to achieve the degree of resolution that is intended.
Consider the following suggestions for cleaning the various lens components:
Only lint-free, optically safe tissues should be used to clean lenses. Tissues free of abrasive grit fall in this category. Booklets of lens tissue are most widely used for this purpose.
Although several types of boxed tissues are also safe, use only the type of tissue that is recommended by your instructor.
Various liquids can be used for cleaning microscope lenses. Green soap with warm water works very well.
Xylene is universally acceptable. Alcohol and acetone are also recommended, but often with some reservations.
Acetone is a powerful solvent that could possibly dissolve the lens mounting cement in some objective lenses if it were used too liberally.
When it is used it should be used sparingly. Your instructor will inform you as to what solvents can be used on the lenses of your microscope.
The best way to determine if your eyepiece is clean is to rotate it between the thumb and forefinger as you look through the microscope. A rotating pattern will be evidence of dirt.
If cleaning the top lens of the ocular with lens tissue fails to remove the debris, one should try cleaning the lower lens with lens tissue and blowing off any excess lint with an air syringe or gas cannister.
Whenever the ocular is removed from the microscope, it is imperative that a piece of lens tissue be placed over the open end of the microscope.
Objective lenses often become soiled by materials from slides or fingers.
A piece of lens tissue moistened with green soap and water, or one of the acceptable solvents mentioned above, will usually remove whatever is on the lens.
Sometimes a cotton swab with a solvent will work better than lens tissue. At any time that the image on the slide is unclear or cloudy, assume at once that the objective you are using is soiled.
Dust often accumulates on the top surface of the condenser: thus, wiping it off occasionally with lens tissue is desirable.
If your microscope has three objectives you have three magnification options: (1) low-power, or 100X total magnification, (2) high-dry magnification, which is 450 X total with a 45 X objective, and (3) 1000X total magnification with a 100X oil immersion objective.
Note that the total magnification seen through an objective is calculated by simply multiplying the power of the ocular by the power of the objective. Whether you use the low-power objective or the oil immersion objective will depend on how much magnification is necessary.
Generally speaking, however, it is best to start with the low-power objective and progress to the higher magnifications as your study progresses. Consider the following suggestions for setting up your microscope and making microscopic observations.