Chapter 2. Laboratory Exercises: Microscopy
It is standard in botany courses, at the high-school level and above if not before, to learn the proper use of a microscope, a tool or instrument of considerable importance to most if not all biological disciplines. If you have not done so already, you should now follow the link to the Wikipedia article on microscopes:
- Read: Optical microscope
If you are not reading this text as part of a formal course, or for whatever reason do not have access to a microscope, you can make good use of a hand-lens or magnifying lens. If you have intentions of doing field botany, you should certainly invest in a hand-lens such as the one shown on the previous page. Although a lupe will work as well (and some lupes are designed to fit in the eye socket), this device is not really intended for field use, and (like the hand lens) is more appropriate for close-up work at the desk or the laboratory bench.
Types of Microscopes
There are two basic kinds of optical microscopes used in class work in botany (there are many other kinds of microscopes used by botanists in research work) widely called compound and stereo. These terms may be a bit confusing, because a stereo microscope is technically a compound microscope (the image is magnified by more than one lens) and you will find that all higher quality optical microscopes have binocular eyepieces, although all do not provide a 3-dimensional (stereo) view of the specimen or target. Preferred are the terms high power and low power; these better specify the use of the two types in the laboratory and not the quality of the instrument.
- High power microscope – Provides (depending upon lens system) magnifications between about 300X and 1000X. A very small or thin specimen is placed on the stage (usually on a glass slide under a cover slip) and viewed close to the objective lens, typically illuminated by light passing through from below. Used to view cells, tissues, very small organisms such as single-celled algae and bacteria. Attention to sample preparation is necessary to achieve good results.
- Low power microscope – Provides magnifications typically in the range of 60 to 500X. Specimen is placed on the stage and lighted from above or the side. There is ample room to access and manipulate the specimen while viewing it and for this property, this type is also called a dissecting microscope for its use in plant and small animal dissections. Although most low power microscopes have binocular eyepieces and give a stereo view, some student models do not. Sample preparation is not especially important; what you have is what you will see.
Using the compound (high power) microscope
Assuming you have access to a compound microscope, it is important to familiarize yourself with the instrument and the tools used to prepare objects for viewing, BEFORE YOU JUMP IN AND ATTEMPT TO VIEW SOMETHING. The reason being that you can damage the optics or mechanical systems on a compound microscope if you are not familiar with their proper use. Although the microscope is a simple instrument to operate and you will learn proper use quickly, proceed initially with caution. You should first review the diagram of a typical microscope as shown at Optical microscope and understand where these parts are located on your microscope (which may differ somewhat from the picture shown):
- eyepiece, objective lens, coarse focus, fine focus, and stage
You will need to become familiar with other features of the microscope as we proceed, but locating each of the above on your microscope is an essential first step.
If you are new to using a microscope, an onion tissue mount is a good first exercise for a botany student, and you should follow all of the instructions provided in the laboratory module, preparing the mount as described. Realize that the high power microscope requires light to pass through the specimen in order for you to view it through the eyepiece. The onion tissue both lacks pigments (such as chlorophyll) and is inherently thin. Thus, it provides a readily available source of plant tissue material requiring minimal preparation for viewing of plant cells. This exercise will introduce you to some of the techniques and accessories used with the high power microscope.
- Read Glass microscope slide and understand glass slide, cover slip, stage clips, and wet mount.
- For more advanced information: Read Microscopy.
- Read Cell staining techniques, introduction and sections on iodine and methylene blue.
If you use an iodine solution, you should understand the basics of why it works as a vital stain. Iodine binds to starch, and starch is a storage product made by plant cells from the glucose produced by photosynthesis. The living cells can break the starch polymer down into glucose sugar for use in energy reactions as needed. Onion epidermis cells do not contain a lot of starch, so they do not stain strongly with iodine. Take a small piece of potato and put a drop of iodine solution on it (not under the microscope) and observe the much darker staining reaction. A potato is a food storage organ for the potato plant.