Riley, M. 2003. Basic Microscopy - An Important Skill for Plant Pathologists.The Plant Health Instructor. DOI: 10.1094/PHI-I-2003-0130-02
Melissa B. Riley, Department of Plant Pathology and Physiology, Clemson University
You are about to proceed on the adventure of a lifetime. A microscope is one of the most useful tools a plant pathologist has when trying to identify the causal agents of plant diseases. Sometimes they will provide you with information that will allow you to know exactly what is causing a plant disease. They may be important in other cases in determining that certain pathogens are not causing a problem.
Proper microscope use is one of the most important skills that a beginning plant pathology student can learn. Microscopes were developed during the late 17th century and continue to be important in identifying fungi and other causal agents of plant diseases. Although microscopes often are introduced to students early in their science classes, students frequently do not learn how to use and care for microscopes. In fact, in many classes microscopes are set up by someone else and students are told to not change anything other than possibly the focus. This teaching aid provides basic information and instructions on how to set up, use, and care for microscopes, essential skills for plant pathologists.
Two types of light microscopes commonly are used in introductory plant pathology courses. These microscopes are the compound microscope (Figure 1) and the dissecting or stereo-microscope (Figure 2). Dissecting microscopes are commonly used for the observation of larger objects and generally have magnifications of less than 100x. The light source used with the dissecting microscope can be located above the microscope stage, or the light may be transmitted through the base of the stage. Compound microscopes are used for the observation of smaller specimens which are placed on microscope slides and topped with a cover slip. Such specimens should be transparent or translucent because light must be transmitted through the specimen to reach the lens of the microscope. Magnifications commonly found on compound microscopes are between 10x and 1000x.
Components of this exercise include:
CLICK HERE FOR INSTRUCTOR'S NOTES.
Parts of MicroscopesYou should familiarize yourself with the different parts of the microscopes before using them because you will need to know where the different parts can be found on your microscopes. Some of these components may be located in slightly different positions on different models of microscopes. Take a few minutes to study Figures 1 and 2 before proceeding. The major components of the compound and dissecting microscopes and their purposes are outlined in Table 1. Note the differences between the two types of microscopes before continuing.
Q. What components are present on the compound microscope that are not present on the dissecting microscope based on the Figures 1 and 2?
Q. What components are present on the dissecting microscope that are not present on the compound microscope?
How to Move and Transport Microscopes ProperlyMicroscopes are often stored in cabinets when they are not in use and must be removed in order to use them. To remove microscopes from the storage area, place one hand completely around the arm, and then place your other hand underneath the microscope base. Always carry the microscope in an upright position. Carrying in any other way may allow parts to fall from the microscope. Use care to ensure that electrical cords are not entangled with those of other microscopes. Place microscope on a clean area of the desk or laboratory bench.
Light sources are required for most microscopic observations. The cord for the light should be plugged into a suitable outlet. Dissecting microscope lights are often a unit separate from the microscope. Such lights may be attached to the arm to illuminate the stage from above the sample. Alternatively, light sources may be placed next to the stage at the level of the sample, or below the stage.
Q. Note the differences between your compound microscope and the compound microscope illustrated in Figure 1. If components are in different locations, these altered locations should be noted below.
Q. Is your dissecting microscope different from the one illustrated in Figure 2? If so, please note differences below for your dissecting microscope.
Determination of MagnificationMagnification is a measure of the ability of the microscope to enlarge an image. Resolution is a measure of the ability of the microscope to separate different points of the image. Determining magnification is vital when comparing the sizes of different objects being viewed with a microscope. Note the magnification of the eyepieces and the different objectives on your microscope. The magnification is printed or etched on the side of the objective and on the side or top of the eyepiece. This magnification is shown as a number followed by an x (e.g. 15x). Dissecting microscopes may have an additional lens (referred to as an auxiliary or supplemental lens) on the base of the objective cover in order to increase magnification. Note if your dissecting microscope has such an auxiliary lens. Dissecting microscopes also may have a magnification knob which changes the magnification when turned. Note the markings on or near this knob that are used to determine the magnification.
Compound microscopes often have objectives which are designed strictly for use with immersion oil. These objectives are identified by having the word "oil" engraved on the side, near the number stating the magnification of the objective. Oil objectives cannot be used without immersion oil. Other objectives cannot be used with oil and can be damaged if inadvertently immersed in oil. The use of an oil immersion lens is essential when viewing structures less than 10 µm in size. For example, magnification requiring an oil immersion lens is necessary to determine the shape of an individual bacterium. Oil immersion does not increase the magnification of the lens, but it improves the resolution or sharpness of the image produced by the objective. When light passes through any material to another, such as from glass to air, the light is refracted or bent. Light of different wavelengths bend at different angles. Such refraction results in distortion which can be significant at higher magnifications. By putting a drop of immersion oil, which has the same refractive index as glass, between the 100x objective and your slide you significantly reduce the light scattering that would otherwise occur, thereby increasing the resolution of the image.
To determine the magnification of any image, you will need to multiply the eyepiece magnification by the objective magnification. If an auxiliary lens is present on a dissecting microscope, its magnification must be multiplied times the objective and eyepiece magnifications. It is important to note the magnification on all drawings so that you can compare the relative sizes of images that you observe at various times. In many cases you will need to use two or more different magnifications to view all the details of a sample.
Q. What is the magnification of the eyepieces on your dissecting and compound microscopes?Dissecting _______________Compound _______________
Q. What are the magnifications of objectives of your compound microscope? ____________
Q. Does your microscope have an oil immersion lens?______
Q. What is its magnification? ___________
Q. What is the maximum magnification which you can obtain with your compound microscope?With use of oil? ____________________________
Q. Without use of oil? ___________________________
Q. Does your dissecting microscope have an auxiliary or supplemental magnification lens at the bottom of the objective cover (Figure 3)? _____________________________
Q. If so what is its magnification factor? _______________________________
Q. What is the minimum and maximum magnification that you can obtain when using the adjustment associated with your magnification knob located on the side of your dissecting microscope (Figure 4)? __________________________
Q. What is the maximum magnification available when using your dissecting microscope?
Q. Remember to include the auxiliary or supplemental lens factor if present on your microscope and the maximum for the adjustable magnification knob and eyepieces. ______________________
Adjustment of EyepiecesSeveral adjustments are necessary before looking at specimens. On binocular microscopes (microscopes with two eyepieces) the distance between eyepieces should be adjusted so that you can look through both eyepieces easily at the same time and see a single image. There are several different methods of adjusting this distance depending on the microscope being used. In some microscopes there is a knob at the top of the microscope between the eyepieces that adjusts the eyepiece distance (Figure 5A). Other microscopes are adjusted by pushing on the eyepieces (Figure 5B). This adjustment is specific to each person so minor adjustments may be required when one person looks through a microscope set up by another person. Make this adjustment before continuing. Adjusting for differences between a person's eyes using the focusing sleeve on left or both eyepieces is another important adjustment to prevent discomfort and fatigue of the microscope user. The procedure for making this adjustment follows the initial focusing since a specimen has to be observable for making the adjustments.
Placement and Focusing of Specimen SlideSpecimen slide placement and focusing are the first techniques that need to be considered. To proceed, obtain an "e" provided by the instructor. After focusing the "e" will be used to illustrate the movement and orientation of materials on the slide.
Orientation of Materials on a SlideMaking accurate drawings of your observations can be extremely important. These drawings will be especially important when you are making comparisons of drawings made at different times. It may be advantageous to draw a circle representing your field of view and then add a grid. The circle and grid will assist you in drawing details in the correct place and size. Make sure to note the magnification at the bottom of your circle.
Q. Draw what you observe. Note the top of the "e" as you look through the microscope. How does the position of the top of the "e" compare with what you observe when you are not looking through the microscope? Are they different? Is this important?
Q. If you need to look at the material on the left side of your image which way do you need to move the slide? _______________________
Q. If you need to look at the material at the top of your image which way do you need to move the slide? ___________________________
Q. Why is this important?
Q. Note the differences that you can see within the "e" when you switch to higher magnification below.
Use of Dissecting Microscope There are several differences between using a compound microscope and using a dissecting microscope (sometimes referred to as a stereo-microscope because it is like two microscopes set to focus on one point providing a 3-dimensional or stereoscopic view of the specimen). Generally most dissecting microscopes do not have a fine adjustment knob. The light source (illuminator) may also be separate and it may be mounted on the arm, below the stage plate, or at the level of the stage. Your instructor will give you specific instructions related to your microscope. Many dissecting microscopes do not have separate objectives to increase the magnification but they have a magnification knob that can be turned to increase or decrease magnification. Note: Some dissecting microscopes also have an auxiliary or supplemental lens on the bottom of the objective cover or body. If this lens is present, it must be included in the determination of magnification.
Samples are placed on the stage plate and are moved by hand. Light can be transmitted through the slide by adjusting the mirror below the sample or the sample may be viewed with the light coming from above. When viewing the sample with the light coming from below you may need to adjust the mirror so that the light is properly adjusted for viewing. Use the steps as outlined below, ignoring those that do not apply to your microscope.
To view the "e" it is best to have the light coming from below the sample (Figure 12). Follow Steps 1-4 for the compound microscope as appropriate.
Q. Are there any differences from what you observed earlier with the compound microscope? If so, what are they?
Q. Eyepiece X Auxiliary Lens X Magnification Knob Setting = Total Magnification________ X ________ X ________ = __________
Q. Is this different or the same as what you observed with the compound microscope?
Q. When would you want to use a light source that is shining directly on the sample (light positioned above the sample)?
Q. What is the advantage of this?
After you have finished using your microscope for the day, care should be taken in properly storing it. Make sure that objectives are clean and slides are removed. Turn off all lights. Unplug electrical cords and loosely wrap around the base. Remove lights that are associated with dissecting microscopes. Place all microscopes and lights carefully back into their proper storage area. Instructors may provide further instructions as appropriate.
If you have any problems, ask your instructor for assistance. Do not disassemble microscopes.
http://www.microscopyu.com - Web site for Nikon Microscopy - contains feature articles and tutorials as well as microscopic images. It also includes technical developments related to new techniques and microscopes.