LAB #4

Using Microscopes

Focused Reading: "Microscopes:..." pp 69-71 Stop @ "Electron microscopy..."
Read Table 4.1 (pg 70) and figure 4.1 (pg 65)

Goals for This Lab

During this lab, you will learn how to use a compound microscope that has the ability to view specimens in bright field, dark field, and phase-contrast illumination. You will also learn about a model research organism, Chlamydomonas. Chlamydomonas is a unicellular green alga that has two flagella and can reproduce asexually by mitosis, or sexually after undergoing gametogenesis.

I. Care and Use of the Compound Microscopes

A compound microscope is illustrated in Fig. 1 and can magnify from 40 to 2000 times (40 ­ 2000 X). Microscope quality depends, however, on resolving power and not so much on magnification. Resolving power is the ability to distinguish between two points in the field of view. Thus, if you can magnify 1000-fold yet cannot resolve detail, your microscope would be of little value. Even more important may be the abilities of the microscopist to learn the capabilities of his or her microscope and to gain proficiency in the use of the instrument.

Do's and Don'ts

1. Always carry a microscope with both hands, one grasping the handhold in the back and one grasping the bottom.
2. Do not swing the microscope at arms length and do not bang it onto the bench top.
3. Never place the microscope near the bench's edge and keep electric cords out of the way, towards the center of the bench.
4. All of our compound microscopes are parfocal, meaning that the objects remain in focus as you change from one objective lens to another. Examine your material first using the lower power objective (i.e. 10X); then use a higher power objective (i.e. 20X or 40X). Because the objectives are parfocal, you need to use only the fine focus knob to fine tune your image. Never use the coarse adjustment to focus downward. Replace and remove a slide only after the lowest power objective has been rotated into viewing position.
5. Never attempt to repair a microscope or force an adjustment knob. You may severely damage the instrument.

II. Parts of a Microscope (see tutorial with images and movies):

Ocular: The piece you look through. Sometimes called an ocular lens or eyepiece, this unit is really a series of lenses. Our microscopes are binocular, having two oculars. Learn to use both eyes; focus your eyes as if you were looking at an object about five to ten meters in front of you. You should adjust the width of the oculars to match the width of your eyes.

Objective lens: Sometimes called the objective; a set of self-contained lenses. The objective gathers light from the specimen and directs it through the tube to the oculars. These scopes have three phase contrast objectives (10X, 20X and 40X with red lines on them) and one bright field objective (20X with no red line).

Nosepiece: The rotating turret to which objectives are mounted. There are preset positions for each objective, detected by slight pressure changes while turning the nosepiece and usually a clicking noise. You should not grab the objectives to turn the nosepiece ­ use the black ring instead.

Stage: The flat surface upon which slides are placed. On your microscopes, the stage moves up and down and the slide is manipulated by a geared device. A moveable stage is sometimes called a mechanical stage. The slide is moved left/right and front/back by two knobs projecting downward from the stage.

Condenser: A lens system under the stage that gathers light from the light source and focuses it on the specimen. There is a diaphragm in one part of the condenser that can be adjusted to allow the viewer to see different parts of the cell when using bright field illumination. You should experiment with this control. These condensers also have phase rings but you should not have to make any adjustments to them.

Condenser Adjustment Control: Under the stage on the left side is a small knob that is used to adjust the height of the condenser. For the most part, this will always be all the way up.

Light Switch Control: The light switch and intensity controls are on the right side of the microscope base, about half way up the side. There is an on/off switch as well as a brightness control. Use only as much light as necessary to illuminate the specimen.

Light Source: On our microscope the light source is built into the base and is directly under the condenser.

Adjustment (Focus) Knobs: Both coarse (large) and fine (small, inner) adjustment knobs are found on both sides of our microscopes. Remember that the coarse adjustment is used only with the low-power objective. These control a gear mechanism that raises and lowers the stage.

Different Types of Microscopy: Bright Field, Dark Field, and Phase-Contrast

There are three different ways that we can view specimens with these microscopes. The type of illumination that you are most familiar is called Bright Field. Think of the light source as producing a solid tube of light that travels up to and through the condenser. When you view specimens with all of this light, you are using bright field illumination.

Dark Field: Dark field illumination seems like an oxymoron, but in this case it describes an unusual way of viewing specimens in some compound microscopes. The light that passes directly through the condenser does not enter the objective lens. Only light that has been scattered or reflected by the specimen enters the objective. As a result, you wind up seeing bright objects on a dark background.

Phase-Contrast: Phase-contrast microscopy allows us to see otherwise transparent organelles and structures. We will make extensive use of this for viewing flagella. In a phase-contrast scope, the light hits the specimen and some of the light continues in a direct path. Other portions of the light pass through membranes which redirects the light. This redirected light is slowed down by 1/4 a wavelength (a phase shift of 1/4) by passing through a special filter. This special filter is shaped like a doughnut and is called a phase ring. The redirected and out of phase light eventually reaches your eyes but not at the same time as the unaltered light that passed straight through. The end result is that you can see transparent structures because they altered the pathway of light as it went through the structures. This allows us to view subcellular structures within living cells.

III. Viewing A Specimen:

The entire lab will follow the procedure for view a specimen as a group. Your instructor will demonstrate how to make a wet mount (see below) and show you the differences between bright-field, dark-field, and phase-contrast microscopy using a microscope that is equipped with a camera and projector. In this lab, we will be looking primarily at wet mounts. A "wet mount" is a specimen mounted in aqueous solution but you do not expect to keep the slide for very long. If your preparation begins to dry out while you're working with it, make a new one.

Every time you work with a microscope:

1) Position the scope so it is directly in front of you and your chair is adjusted so that you do not have to strain to view a specimen.
2) Make sure the light intensity control is turned all the way off before turning on the microscope.
3) Make sure the 10X objective is in place over the specimen.
4) If you are making a wet mount, clean the microscope slide by fogging it with your breath and then wiping it with a Kimwipe.

Bright Field

1. Switch on the light source and then dial the adjustment knob to about 3.5. Start with the oculars set so they are at equal heights.
2. Turn the condenser so that the "O" is facing you. This is the bright field slot on the condenser.
3. Position the low-power (10X) objective over the specimen and, looking from the side, raise the stage as high as possible. How close to the objective is the stage?
4. Use the coarse adjustment to lower the stage away from the glass slide while looking through the oculars until the specimen comes into focus. Adjust the focus to its sharpest with the fine adjustment knob.
5. Now it is time to make sure both oculars are focused. Use the fine focus while looking through the right ocular and close your left eye. Pick one object to focus on. Then close your right eye and focus the left ocular by turning it up and down with the focusing ring for the left eye but do not touch the fine focus control during this time.
6. Readjust the light intensity to reduce glare and center the specimen in the field of view by moving the stage.
7. Use the knob on the left side of the condenser to move the condenser up as high as possible. You might also want to adjust the condenser's diaphragm to maximize the resolution but minimize the "graininess" of the image.
8. Place the 20X objective over the specimen and sharpen the focus with the fine adjustment knob (only!) as necessary.
9. Adjust the condenser's diaphragm to maximize the resolution of the structure you are trying to see. The actual setting will depend on what you are trying to see. Small translucent objects will be seen more easily with the diaphragm closed substantially while large pigmented structures are easier to see with the diaphragm wide open.
10. Repeat steps 8 and 9 but use the 40X objective instead of the 20X.

Dark Field

11. Turn the condenser ring so that the "D" is facing you. This will permit you to see objects in dark field illumination. You must also adjust the condenser so that it is as high as it can go - use the knob on the left side of the scope. You can use dark field illumination with any of the 4 objective lenses.

What structures can you see now that you could not see in bright field?
What is difficult to see in dark field that was easy to see in bright field?


12. When you use phase-contrast, you must match the objective lens with the phase ring in the condenser. Therefore, you must follow this table:

 Objective Lens Phase Ring
10X, 20X, (red lines) 10
40X (red line) 40

A Reminder: only the objectives with red lines can be used for phase. The 20X objective that does not have a red line on it is not equipped with phase rings.

13. Select the appropriate objective lens and phase ring pair. You might need to increase the amount of light since images do not appear as bright in phase. Once you have done this, you should adjust the condenser vertically with the knob on the left side of the condenser. Once these adjustments are made, using a phase-contrast microscope is similar to using a bright field scope.

What structures can you see now that you could not see in bright field? Dark field?
What is difficult to see in phase-contrast that was easier to see in bright field?
Do you see the same colors in phase that you saw in bright field? Dark field?

A Series of Experiments on Microscopic Mating

Focused Reading: p 538-541 "Chlorophyta"
p 540 Figure 26.24 26.15


Over the next three weeks you will become comfortable with a fundamental tool in biology - the compound microscope. We will conduct a series of experiments on a unicellular green alga, Chlamydomonas reinhardtii, or Chlamy for short. Chlamy is a biflagellated green plant that reproduces asexually (by mitosis) and sexually (via meiosis, mating, and zygote formation).

A Word About Cooperative Learning

The laboratory is a place where scientists (that includes you) come together to work as teams and talk about methods, results, and conclusions. It is also a place to assert yourself, take responsibility for your own education, and trust your common sense. For example, you will view cells that are immobilized which requires you to kill the cells by chemically cross-linking the all proteins, euphemistically called fixing the cells. The other three people in your group suggest that you pour the Lugol's fixative solution into your one and only supply of cells even though you also are supposed to mate LIVING cells later in the lab. Unfortunately, you have been cursed with short-sighted lab mates. To you, it seems obvious that you cannot kill all the cells in step 3 if you need live ones for step 7. So, you assert yourself and persuade your lab mates that they have made a miscalculation (this works better than calling them idiots, even if they are). This is not confrontation but cooperative learning. Each of you can be both student and teacher if you think while you are in lab; don't just hurry through in order to finish. What you learn from each other in the lab is just as important as what you learn in the class. That's why lab material is tested in the "lecture" exams.

Background Information on Chlamy

There are several reasons why Chlamydomonas is such a useful model organism. It is a haploid organism which means there is only one copy of each chromosome. Therefore, the genotype is always expressed in the phenotype (unlike diploids that may have a recessive mutation that is not revealed in the phenotype). It has a generation time of 2 weeks (from mating of one generation to when the next generation can mate). Finally, there are hundreds of mutant strains (stored at Duke University) that have been generated over the years and can be used for research. For example: ac-17 cannot fix carbon during photosynthesis, arg-7 requires the amino acid arginine to be added to the medium since it cannot synthesize its own; act-1 is resistant to the translational inhibiting drug cycloheximide; and pf14 has straight and paralyzed flagella so it cannot swim.

Why would anyone want to know how efficient Chlamy sex is? Well, if you are trying to study the process of gametogenesis at the molecular level, you would need to be able to compare wild-type mating to abnormal, or mutant, mating. To compare these two, you might use mating efficiency as an indicator of the ability of a gamete to mate. Since human subjects are reluctant to submit to experiments such as these, especially experiments on mating efficiency, you would be forced to find an alternative organism to study and one that has a short generation time. For example, imagine you are trying to learn how gametes fuse and you decide to generate a mutant strain of Chlamy that cannot fuse. (There are several strains like this and some Davidson students are conducting their honors research on them.) Once the mutant is generated, you can try to clone the gene that has been altered which would allow you to identify the gene that encodes the "fusing gene". Maybe a new contraceptive will result from your research?!

Chlamy cells come in 2 sexes called mating-type plus (mt+) and mating-type minus (mt-). When a mitotically dividing cell is deprived of nitrogen, it differentiates into gametes; mt- cells differentiate into minus gametes and mt+ cells differentiate into plus gametes. These two gametes of opposite sexes will fuse to form a diploid zygote that becomes a metabolically inactive spore. When conditions are favorable for mitotic growth (i.e. there is enough nitrogen), the zygote spore undergoes meiosis and germination to produce a tetrad of four haploid progeny: two mt+ cells and two mt- cells. We will be working with plus and minus gametes in today's lab.


A reminder

 Objective Lens Phase Ring
10X, 20X (red lines) 10
20X (no red lines) cannot be used with phase
40X (red line) 40

Each person should:

A) Place 25 µl of minus gametes on a clean (use a kimwipe) glass microscope slide and cover this with a coverslip. Do not press down on the coverslip or else you will crush the cells. Place the slide on the stage of the microscope and use the 10X objective lens to observe the cells swimming around. Start with bright field, then try dark field and phase-contrast.
1. Can you see the flagella?
2. Which form of illumination allows you to see them the best?
3. What is the total magnification you are using with a 10X objective lens and the 10X oculars?

B) Increase the magnification by using the 20X objective lens. Again, view the cells in bright field, dark field, and phase-contrast. Remember to use lenses with the red ring for phase and the 20X without the red ring for bright-field.
1. What is your total magnification now?
2. Can you see the flagella? Which form of microscopy is the best for seeing flagella?
3. Can you see any other organelles in these cells?
4. Do you see any other colors besides green? If so, where and what organelle could this be?
(Hint: "The better to see you with, my dear.")

C) On the same slide but separate from the previous sample, place 12.5 µl of mt- cells into 12.5 µl Lugol's fixative [this is a dye that stains the sugars which are covalently bound to the proteins (sugar coated proteins are called glycoproteins) on the surface of the flagella]. Examine this preparation of stained cells under the microscope. View the cells at all three magnifications with each form of illumination.
1. What structure(s) can you see better with fixed cells than when you observed live, unstained cells? Give 2 reasons why.

As a group:

D) In a microfuge tube, mix 150 µl of plus and 150 µl minus gametes and record the time.

Each person should:

1. Take out a 25 µl subset, or aliquot, of the mating cells shortly after you have mixed them together and observe these mating cells (still alive) under the microscope using the 10X and 40X objectives. Choose the form of illumination that will allow you to see the flagella the best.
2. What is going on? Describe how Chlamy cells mate. Pay special attention to the tips of the flagella.
3. Using phase-contrast and the 40X objective, look carefully for some round gray objects floating around. What are these?
4. After the cells have mated for at least 15 minutes in the original tube, take an aliquot of mating cells and fix them in Lugol's stain. Record the amount of time the cells have been mating. Observe these stained cells using the 40X objective lens and phase-contrast.
5. Do you see any cells that look like diploid zygotes instead of haploid gametes?
6. What 2 or 3 features are noticeably different in zygotes?
(Look carefully for a cell that is different from the haploids you have looked at until now.)
7. Each person should count the first 25 cells with flagella that you see and "score" them as either gametes or zygotes. Record the number of each type of cell. Try to be random in your selection of cells to count; do not hunt for one kind of cell over the other.
8. Compare your data with the data from the other 3 people in your group. Did you all get the same numbers?
9. Determine the % mating efficiency using the formula below. Write your results on the board.

% mating efficiency = 2(no. of zygotes) ÷ [2(no. of zygotes) + (no. of gametes)]

or written in words:

% mating efficiency = no. gametes fused ÷ [total of all gametes in original mixture]

10. Do your numbers agree well with your colleagues in other groups?
11. Can you imagine any reason why your results would vary significantly from another person's?
12. What modification to your technique would you make to avoid this problem next time (like next

Turning off the scope

1) Turn the light down to zero.
2) Turn off the power.
3) Rotate the 10X objective in place and turn the condenser to bright-field.

Before you leave the lab, you should know the following:

- How can you see flagella better without staining them?
- What colors do you see in a Chlamy cell and what structures are responsible for the colors?
- How do you calculate the total magnification you are using on a microscope?
- With what appendage(s) do Chlamy cells mate? How efficient was this mating process?
- How can you standardize your methodology so that 2 people can get similar results when counting cells?

Before next week, your lab group should meet so that you can be prepared for next week's lab. Timing will be very important - you snooze, you lose. You can meet at the end of lab today or later.

 Lugol's Fixative (protect from light and made fresh each semester)-
1 g iodine
2 g K+ Iodide
12 ml H20
dissolve the KI first, then add the iodine
filter undissolved crystals and store in a dark container

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