BIO 121 LAB INSTRUCTIONS

Lab 3 - Introduction to Microscopy

I. Overview of Microscopy

Biologists spend a lot of time looking at organisms. Many of these are clearly visible to the naked eye, but many others are not and to see them their image must be magnified. In some cases a magnifying glass will do, but frequently a microscope is needed.

There are many different kinds of microscopes. You will become most familiar with compound microscopes and dissecting or stereo-microscopes. These microscopes differ in three respects: 1) the compound microscope that you will use in BIO 121 can magnify objects up to 1000-fold, whereas your dissecting microscope's potential is only 30-fold. 2) the dissecting microscope gives you a three-dimensional view of the outer surface of the object, whereas the compound microscope's image is two-dimensional. 3) to observe an object under a compound microscope, it must be extremely thin and placed on a glass slide. Thus, a thick structure must be sliced into thin sections using a sharp instrument of some kind. Sectioning and slide preparation is not necessary for the dissecting microscope.

Compound and dissecting microscopes are similar in that they use visible light (radiation of wavelengths 400-700 nm) to illuminate the object. The problem with using visible light is that it has a limit of resolution (the smallest structure visible) of 200 nm, regardless of the quality of the microscope's lenses. Unfortunately, many sub-cellular features are smaller than 200 nm and are therefore too small to be seen by light microscopy. To see such minute features they must be illuminated with another type of radiation.

One solution to this problem is to illuminate the samples with electrons. These have a much smaller wavelength than visible radiation, and therefore have a much lower limit of resolution. One can therefore see much smaller structures with an electron microscope than with a light microscope.

Although electron microscopes are more powerful in their ability to resolve tiny structures, they are much more expensive and complex than light microscopes. Therefore, BIO 121 will be limited to light microscopy.

II. Compound Microscope

Your compound microscope is located in the cabinet at the back of the room. Go to one of the cabinets, as directed by the instructor, and carefully remove your microscope from the cabinet, place it on the lab bench in front of you and remove the dust cover. Notice that there are a variety of controls and other parts. These will be discussed in turn. A generalized description of a typical compound microscope is provided on p. 17 of your laboratory manual, and you should each ahve a copy of the instruction manual for your Leica ATC 2000 microscope at your pod. It was manufactured by Leica in Germany, and has a replacement value of approximately $1800. Therefore, please treat it with respect, and remember that you are financially responsible for any damage you do to it!

Starting at the top, you see the eyepiece or ocular lens. Carefully remove it from the body tube and then reinsert it. Notice that the rim has "10x" engraved into it. That means that the lens magnifies all images 10-fold. In looking through the lens, you might notice a dark line. That is a hairline, and is useful for pointing to objects viewed under the microscope. Do not remove it!

Next, look at the nosepiece and objective lens assembly. Your microscope has three objective lenses, and the whole assembly can be rotated. Carefully rotate the assembly and notice that each lens clicks into place. The lens in front is the one that is engaged. Note that each lens has a different colored band: the red one has 4x magnification, the yellow lens has 10x, and the blue lens has 40x. The total magnification is the ocular lens (10x) times the objective lens. Please calculate the magnifications for these lenses and enter the answers into Table I on page 1 of your datasheet.

TO PREVENT DAMAGE TO THE LONGER LENSES, ALWAYS ENGAGE THE 4X (RED) LENS WHEN PLACING OR REMOVING A SLIDE FROM THE MICROSCOPE.

Now observe the stage, which is the black, flat surface beneath the objective lenses. Slides will be positioned on the stage, secured by the stage clip and bracket assembly. Move the stage clip to the left, and notice that it will return to its original position by spring action. The horizontal and vertical adjustment knobs are located to the lower-right of the stage. Turning the upper knob will cause the stage to move toward and away from you. Turning the lower knob will cause the clip and bracket assembly to move to the left and right.

Beneath the stage you find the condenser assembly, which controls and focuses the light passing to the slide. The single round knob under the left side of the stage controls the height of the condenser. Lowering the condenser decreases the amount of light reaching the slide. Notice the diaphragm lever at the front of the condenser. Moving the lever to the left and right adjusts the size of the iris diaphragm, which further regulates the amount of light passing to the slide.

The two large knobs at both sides of the base of the arm are the focus adjustment knobs. The inner (larger) knob on either side is the coarse focus. Turn that knob and notice the stage moves up and down slightly. The outer knob is the fine focus, which allows for more precise focusing.

Finally, the light source is located beneath the condenser. The on-off switch is located on the left side of the microscope. At the front of the microscope is a knob,the rheostat knob, which controls the brightness of the light. Always start with it at "0", to avoid blinding yourself! Behind the rheostat on the base is a the field diaphragm lever, which controls the width of the field diaphragm.

The only maintenance that you will need to perform on your microscope is periodically cleaning the objective and ocular lenses, with the lens paper provided in the dispenser on the wall. Should the light-bulb burn out, or any other malfunction occur, please notify the instructor or T.A. at once. Remember that you are financially responsible for this expensive piece of equipment, and will pay for any damage that you inflict on it!

III. Viewing a Prepared Slide

Obtain a slide of the letter "e." Use the coarse focus adjustment knob to raise the nosepiece as far as possible, then click the 4x (red) lens into position. Place the slide on the stage, then follow the instructions on page 4 of your Leica instruction manual to adjust your microscope.

Once adjusted, move the stage until the letter e is over the hole in the stage. Move the slide until the e comes into view in the oculars, then get it properly focused. Please note the following:

  1. the image that you see is inverted relative to its position on the slide.
  2. what happens to the image when you move the slide from left to right, and towards you?
  3. when an object is in focus under 4x, it is safe to switch to 10x. After refocusing, it is safe to then switch to 40x. Do not switch to 40x until you have focused the object under 10x. Failure to do so may cause damage to the 40x lens because it rides very close to the slide. Do not hesitate to ask for help if needed.

IV. Estimating the Field of View

The field of view is the size of the diameter observed under a given objective lens. The field of view decreases as you switch from the low power lens to the high power lens. You can observe the field of view directly when the 4x lens is in place by obtaining a slide with graph paper divided into 1 mm x 1 mm squares from the side table and placing it on your stage.

Examine the graph paper with the 4x lens in place and enter the diameter of the field of view into table II on page 1 of your datasheet. It is impossible to directly measure the field of view under 10x, 40 x and 100x. Instead, please estimate them using the following formula
Field 2 = (field 1 X magnification 1) / magnification 2.
Please enter your estimates into table II.

V. Scaling

Today you will be making diagrams of objects viewed under the microscope. It is important to know how large the various structures really are. To do so, you will scale your diagrams.

The idea behind scaling should be familiar to anyone who has used a map. You can estimate the distance between two cities by measuring the distance between them on the map, then consulting the scale to determine the distance that represents in real life. For example, if two cities are 5 cm apart on the map, and 1 cm represents 50 km, you can estimate that the two cities are 250 km apart.

For each diagram that you make, you will provide a scale. In doing so you must decide: 1) how long the scale be should (in µm), and 2) how long the line that you draw on your datasheet to represent that scale should be (in mm). To decide how long the scale should be you need to know: 1) which objective lens you have in place, and 2) the field of view (in µm). The scale should be a round number between 1/10 to 1/3 of the field of view. For example, assume that you were observing something under low power and that the field of view is 4000 µm. The range of possible scale lengths would be 400 µm (1/10 x 4000) to 1333 µm (1/3 x 4000). Within that range, round numbers such as 500µm, 750µm, and 1000µm would all be acceptable. Let's assume that you select 1000µm. To decide how long the line you draw on your paper to represent 1000 µm should be you use the equation:

a = (b x c) / d

where: a is the length of the line (in mm) that you want to draw,

b is the diameter of the circle (in mm) that you drew on your datasheet (representing the circle that you observe under the microscope),

c is the length of the desired scale (in µm) (in this case it is 1000 µm)

d is the field of view (in µm) (in this case it is 4000 µm)

VI. MAKING A WET MOUNT

Not everything that you want to observe under a microscope will be prepared for you. You will often need to make your own slides from fresh material. In today's lab, you will be making five wet mounts. The first is of onion epidermis in order to clearly see cell structure. The second will be of a potato tuber, to allow you to see how stains make certain features more evident. The third will be some of your own cheek cells, so that you can compare animal cells with plant cells. The fourth will be of yogurt, in order to compare prokaryotic and eukaryotic cells. The final will be of some lovely pond water, in order to gain some appreciation for the diversity of microorganisms.

A. Onion Epidermis

Obtain a small piece from a single layer of onion flesh. Be sure to isolate it from one of the fleshy inner leaves of the onion, not from one of the outer papery leaves. Snap the piece in half, then use forceps to peel off a piece of the transparent epidermis from the inner layer. It should look like saran wrap.Place the epidermal section on a clean slide, add a drop of water and a coverslip. Make sure that the bottom of the slide is dry.

Place it on the stage and focus under the 4x objective. Notice the brick-shaped cells. Then switch to 10x and refocus. If you desire, switch to 40x. However, if the cells are so large that a single cell cannot fit within the field, switch back to 10x.

Using a pencil, draw the cells of the onion epidermis so that they are in the same shape and proportion as the cells appearing within the microscope. Draw just a few cells, but draw them carefully to show that you were really looking at them. We would much rather see 3-4 well-drawn cells than a circle full of ball bearings.

Have the instructor or T.A. comment on your diagram before proceeding. Next, scale your diagram as outlined above, and label the visible structures by drawing a line to them with a ruler, then labeling them outside the circle. Be sure to give a title and caption for your figure. Provide a discussion (in ink) in which you describe the shape, size and color of the cells.

B. Potato Tuber

Using a razor blade obtain an extremely thin section (ca. 5 mm x 5 mm) of the white part of a potato tuber. Add a drop of water and a coverslip. Examine under your microscope and notice the whitish spherical structures. Note, though, that the structures are not as distinct as they were for the onion epidermis. To make the structures in the potato tuber more visible you will stain them with iodine. Since potatoes contain lots of starch, a noticeable blue color should develop. Add Lugol's iodine to your original potato slide by slowly inserting a drop under one edge of the coverslip. You might want to place a bit of paper towel along the opposite edge of the cover slip to blot up the water already there and to draw the dye under the cover slip. Wait about 2 minutes for the color to develop and observe under your microscope. Notice the spherical, dark blue structures (these are starch granules or amyloplasts) and the ghost-like cell walls.

Draw the cellular portion (not the loose amyloplasts) on your datasheet, in the same manner as you did for the onion epidermis. Provide a title, caption. scaling and a discussion. In your discussion, again comment on the size and shape of the cells. Describe the difference in appearance caused by adding the iodine.

C. Cheek Cells

To observe a sample of your own cells, obtain a toothpick and gently scrape the inner surface of one of your cheeks. Smear the residue on a slide, add a drop of water, and a drop of methylene blue stain. Add a cover slip and place on your microscope. BE SURE TO DISCARD THE TOOTHPICK INTO THE "BOOGER-BAG."

Examine using the 4x, 10x, then 40 x objectives. Notice the blue ovoid cells, which often occur in clumps.

Make a diagram, again being sure to provide a title, caption, labeling, scaling and discussion. In your discussion, comment on the shape and size of the cells, especially in comparison with the plant cells.

D. Prokaryotic cells

Prokaryotic cells are structurally much simpler than eukaryotic cells. They completely lack subcellular compartmentalization, although some may show invagination of the plasma membrane. In today's lab, you will examine prokaryotic cells from two sources: yogurt and prepared slides. You will note their size and shape. Examination of their internal contents will be exceedingly difficult, due to the small size of the cells.

A container of yogurt containing an active bacterial culture is available on the side table. Put a drop of water on a slide, then dip a toothpick in the yogurt and then stir it in the drop of water to evenly mix it. You only need a little bit of yogurt! Put on a cover slip, then e xamine under your microscope using 4x, 10x then 40x power. You may find that reducing the light will make the bacteria more visible. The bacteria should mostly appear as chains of minute circular cells, but you should also see small, rod-shaped bacteria.

Diagram a few of the yogurt bacteria in one of the sectors of the 10 cm circle on the page labeled "prokaryotic cells,", being sure to draw a few of each type. Take care to show the proper shape and size of the cells. Can you see any sub-cellular features? Next, observe the prepared slides of bacterial cells. Make a separate diagram of each slide in one of the remaining sectors. Be sure that the cells are drawn in correct proportion relative to the width of field. Label and scale each diagram.

Write a discussion in which you compare the cell structure of the prokaryotes with those of the eukaryotic cells. Indicate the chief similarities and differences between the two.

E. Pond Life

Prepare a wet mount of the pond water available on the side table. Examine the slide using the 4x, 10x, then 40 x objectives. Divide the circle into thirds, and in each third draw a different type of organism. Provide a title, caption, scaling and discussion. (Note that labeling is optional). In your discussion comment on the number of different kinds of organisms which you observed, their relative sizes, and whether the majority appeared to be unicellular or multicellular.

F. Clean-up

When you are finished, wash and dry the slides and coverslips, and replace in their boxes. Discard all unwanted pieces of plant material, paper towels, and tissue. Unplug your microscope, wrap the cord around the base, and carefully return to the cabinet. Wash off your table-top.