Laboratory Introduction

Laboratory Introduction

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. Since a large majority of marine algal species are members of the phytoplankton and therefore microscopic, the competent and careful use of the microscope is essential. In this lab, you will need to use two different types of microscopes, a dissecting or stereomicroscope and a compound microscope. Read all of the introductory information carefully before beginning the exercises. There is a series of short exercised that you should accomplish during today s lab: 1) familiarize you with the parts of the compound microscope and achieve Koehler illumination, 2) learn how to use a dissecting microscope, 3) familiarize yourself with the art of producing wet mount, 4) compare animal, plant and bacterial cell types and finally, there are some microscope care and cleaning instructions. Exercise I - Parts of a Microscope The main parts of a bright-field microscope are shown listed below. 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. Support System - 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 that focuses the light on the specimen. An iris diaphragm is used to control the intensity, or brightness, of light passing 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 that 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 4x; the middle-sized lenses are the high-dry objective that usually magnify between 10x and 40x; and the longest lens is the oilimmersion lens, which usually magnifies at 100x but will not produce a clear picture unless immersion oil is used.

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 that 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 focused.

Koehler illumination is proper alignment of the incident or illuminating light for microscopy. Every time you use the microscope you should align the condenser lens to assure Koehler illumination is optimal or you will end up with poor resolution, contrast artifacts, and unevenly lit pictures. Köhler illumination is simple to achieve with any microscope equipped with a field diaphragm, which in your microscopes will be located in the base just beyond the lamp With the new microscopes Köhler Illumination is quickly and easily achieved in five easy steps 1. Locate the specimen and bring it into focus (usually done with the condenser iris closed to yield maximum contrast.) 2. With the 10X or 40X objective, close the field diaphragm in the base sufficiently to be able to see its edges. This is done by rotating the control ring on top of the lens where the light leaves the base. Notice that the edges are NOT in sharp focus. It should look like this: 3. Using the condenser focus knob on the left side beneath the stage, raise or lower the condenser to bring the edges of the field diaphragm into sharp focus. When the edges are in sharp, clear focus it should look like this: 4. Open the field diaphragm. 5. Now you may use the condenser iris control to achieve best balance between contrast and resolution. Incidentally, another use for the field diaphragm is when viewing one small object in a particularly densely packed field. So many small objects can be confusing. Closing the field diaphragm down to include only area surrounding the object of interest can be a great help. A very important point!!! The condenser iris is NOT intended to control brightness!! That should be done by the rheostat ( dimmer control ) or with filters. When changing objectives use the knurled ring on the turret, don t grab the objectives. The manufacturer has extended this ring to make it easier to grasp than are the objectives.

Exercise II The use of the dissecting microscope 1. Adjust the magnification to its lowest power with the magnification knob on the top or side of the microscope body. 2. Adjust the interpupillary distance of the ocular lenses. Look through the ocular lenses. If you see one image, no adjustment is necessary. If you see two images, or a lot of black, adjust the distance between the ocular tubes until you see one image. You also may need to move your eyes closer to or farther away from the ocular lenses so that the specimen's image fills the lenses. You may need to move the ocular lenses far apart or close together. 3. Focus on the specimen. This is a two-step process. In the first step, you will roughly focus on the specimen with the objective lens. In the second step, you will compensate for any differences in strength between your eyes to obtain the sharpest image possible. 4. Rough focus. Lower the microscope body to its lowest point with the focusing knob on the sides of the microscope arm. Use the focus knob to raise the microscope body until the specimen image is the sharpest. Compensate for any differences in strength between your eyes. (The following directions are written for microscopes with diopter adjustment rings on the right ocular tube. Obviously, if you have a scope with the diopter adjustment on the left ocular tube, you will start with your right eye closed.) a. Close your left eye. Adjust the diopter adjustment ring until the image is in focus for your right eye. You may want to adjust the ring back and forth (i.e., in and out of focus) a few times until you are sure you have the best focus for yourself. b. The first time through the diopter adjustment you may want to repeat steps a through d-sometimes our eyes automatically compensate for out-offocus images seen in the microscope, and eyestrain results. Who wants a headache in marine bio lab?? 5. If you change the magnification, you may need to adjust the focus again. 6. When you are finished with the microscope, unplug it and lightly wind the cord around the arm, clean the stage (this is pertinent when working with marine organisms and cultures because seawater is incredibly corrosive) and lower the body all the way down before storing it away. Exercise III Preparing Wet Mounts The purpose of this exercise is to prepare usable wet mounts that may be viewed under the microscope using both microscopy cultures and macroscopic preparations. Procedure A Preparing a wet mount using microscopic cultures 1. Using the dropper, place a few drops of pond water onto the center of a clean, dry slide. 2. Hold the side edges of the coverslip and place the bottom edge on the slide near the drop of pond water. 3. Slowly lower the coverslip into place. The water should spread out beneath the

coverslip without leaving any air bubbles. If air bubbles are present, you can press gently on the coverslip to move the air bubbles to the sides. Procedure B Preparing a wet mount using oral epithelial cells The overall steps are the same as in procedure A with the exception that you will need to remove some epithelial cells out of your mouth. Since these cells are not easily visible unless stained, use the methylene blue provided as a stain. 1. Using the dropper, place a drop of methylene blue onto a clean slide 2. Take a coverslip and gently scrape the side of your tongue with one of the straight sides (do NOT use the pointed tips of the coverslip) 3. Slide the side of the coverslip inoculated with you epithelial cells onto the slide so that it touché the drop of methylene blue and carefully lower it. (You might want to gently move it around in order to disperse cells slightly. Procedure C Preparing a wet mount using an Elodea leaf Repeat the procedure discussed in A but place a light green (younger and therefore thinner) Elodea leaf into the water before lowering the coverslip. Since Elodea leaves will always be rather thick compared to other microscopic materials, the coverslip may not fit snugly. Exercise IV - Viewing specimens Using the wet mounts that you prepared in exercise III as well as a prepared bacterial slide, you should also be able to detect the subtle differences between eukaryotic animal and plant cells as well as prokaryotic bacterial cells. This course will cover the marine autotrophs of different varieties, from prokaryotic cyanobacteria (Kingdom Monera), to eugenics, diatoms, dinoflagellates, red and brown macroalgae (Kingdom Protista) and even plants including the green algae, seagrasses etc. (Kingdom Plantae). Since these organisms may either have bacterial characteristics, both animal and plant characteristics and typical plant characteristics you will need to be able to recognize the difference between the different cell types. 1. View the specimen with the low-power objective: a. Turn the nosepiece until the low-power objective locks into place. b. Place the slide on the stage and center it over the condenser. c. Looking from the side, turn the coarse adjustment knob until the low-power objective is in its lowest possible position. d. Looking through the ocular lens, slowly turn the coarse adjustment knob in the other direction. This raises the low-power objective away from the slide. Continue until a clear image appears. e. Slowly turn the fine adjustment knob until the object comes into sharp focus. f. Adjust the light for maximum contrast using the iris diaphragm. g. Move the slide around on the stage using your fingers or the control knobs until

you find a microorganism. h. Open the iris diaphragm until optimum contrast is achieved. 2. If the microscope has an oil immersion objective, then view the microorganism using this lens. a. Turn the nosepiece so that no objective lens is directly over the slide. Then place a small drop of immersion oil on top of the coverslip. b. Turn the nosepiece so that the oil immersion objective is locked into place over the specimen, making sure that the lens is immersed into the oil. c. Slowly turn the fine adjustment knob until the image comes into focus. d. Adjust the iris diaphragm to achieve maximum contrast. e. When you are finished, remove the oil from the oil immersion lens using lens paper. f. Remove the oil from the slide by wiping it gently with a paper towel. 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 tabletop 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.