Bio5LAManual12f

With the right eye closed and the left eye open focus

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Unformatted text preview: ing ray path forming ray path vision of each eye. Making this adjustment: With the inin a CompoundLight Microscope a Compound Light Microscope left eye closed and the right eye open, focus the microscope with the lower focus controls until you see a sharp image. With the right eye closed and the left eye open focus with the diopter ring until a sharp the image is obtained. The microscope is now adjusted to compensate for the differences between eyes. Some people have difficulty using binocular microscopes that is not related to either of the above adjustments. One explanation for this problem is that the microscope has been damaged by a negligent previous user thus making binocular viewing impossible. Another common problem here is that the viewer is holding his/her eyes too close to the oculars. The solution is to back your eyes off slightly from the oculars and redo the two adjustments described above. If you suffer from near- or far-sightedness, you should use the microscope without your glasses; it corrects for faulted vision. The microscope cannot correct for astigmatism. *** [next page please] Biology 05LA – Fall Quarter 2012 Lab 1 – page 7 PRACTICAL MICROSCOPE USE AND THE CONCEPT OF IMAGE CONTRAST In this section, we will look at some plant and animal cells with the goals of practicing and refining your microscope skills as well as introducing another important microscopy concept; contrast. Contrast. You have learned that what can be seen in the light microscope is limited by the size of the object. Another limiting factor in microscopy concerns image contrast. Contrast relates to the ability of the viewer to distinguish an object from its background. For example, it is easier to see dark spots on a white suit than it is to see dark spots on a dark suit. Problems of inadequate contrast are common in microscopy. In Biology 5 we will use two methods to increase contrast: partial closure of the condenser diaphragm and differential staining. 1. Closure of the condenser diaphragm. For each of the specimens that will be provided, your first observation will be of fresh, untreated tissue. As you will discover, it is difficult to see much detail when the condenser diaphragm is wide open. However, if you slowly close the diaphragm while observing the specimen, you should see that more detail becomes visible as the diaphragm is closed. This improvement is realized only up to a certain point, after which contrast is not improved and resolving power is lost. Examine the effect of manipulating the condenser diaphragm for each of the available tissues and then make a sketch of the best images in your lab notebook. NOTE: The condenser must be properly focused to optimize the use of the condenser diaphragm to increase contrast. 2. Differential staining. Objects seen in the compound microscope are discerned as a result of their interaction with light. Biological material frequently absorbs only a small amount of light and thus has poor contrast. Differential staining involves the use of stains (dyes) which bind to various components of the specimen to increase contrast. Stains usually bind specifically to particular substances in cells or tissues with a unique chemical make-up. Today, we will use three different stains with three different binding affinities: Acetocarmine. This is a natural stain that comes from an insect. This stain binds to chromatin and stains it red; thus it is useful for identifying nuclei. I2KI. This stain is a mix of iodine (I2) and potassium iodide (KI). I2KI works through a chemical reaction between the iodine and the starch granules of green plants. The resulting starch-iodine complex has a deep purple/blue color. Toluidine Blue. Unlike the preceding two stains, toluidine blue is a “metachromatic” stain when applied to biological material at an acid pH. This means that when the stain binds to different cellular components, the color produced may be different and dependent upon the chemical composition of that component. The above stains will be used in the following exercise. For each of the specified tissues, you will be drawing images of the specimens reacted with these stains in your lab notebooks and comparing these images with those drawn from the unstained material. While doing this, you should be thinking about what you think is uniquely colored with the different stains. Biology 05LA – Fall Quarter 2012 Lab 1 – page 8 The Tissues. – Each lab table will do one of the following tissues and report their findings to the rest of the class. Onion epidermis. Your TA will show you how to obtain a small strip of onion epidermis that is one cell in thickness. Once you can do this with ease, proceed as follows: 1. Make a wet mount of a small epidermal strip as described in the section below entitled the “Making of wet mounts.” Here you should use tap water as the mounting liquid and float the strip on the drop with the torn surface against the water. Don’t forget to add a cover slip...
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This note was uploaded on 08/27/2013 for the course BIO BIOL05LA taught by Professor Abbottl during the Fall '12 term at UC Riverside.

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