6: Plankton and Microscopes

Microscopic Terms

To get the most out of your microscope, it is worthwhile to take a moment to review the three principles that govern its use as an investigative tool. These are magnification, resolution, and contrast:

Magnification is the factor by which the image of an object is increased in size relative to the actual size of the object (when held from a specified distance). While most people believe that the purpose of the microscope is to magnify, in fact, this is only partly true. Magnification is a necessary step that enables us to see details that are normally lost to our eyes. But the real purpose of the microscope is to increase resolution, which is accomplished by making the object appear larger and clearer.

Resolution can be defined as the smallest distance between two points when they can still be seen as being separate. While increasing the virtual size of the object does increase our ability to resolve some details, recognize that this process has its limitations, just as infinitely zooming in on a photograph will not enable you to see infinite amounts of detail. The limit of resolution is set by, among other things, the nature of the imaging source, which in this case is visible light. The third parameter that influences your ability to study a specimen with the microscope is contrast.

Contrast is the degree to which the image of an object stands out relative to the background. This can be how a cell is revealed relative to the surrounding cells, or how some transparent creature is shown against a background. To enhance contrast, microscopists can treat the specimens they are observing with chemical dyes or stains. These are pigments that, due to their chemical properties, preferentially bind to certain parts of the specimen, making them appear more prominent relative to those areas that don’t take up the stain. In marine biology, we are often working with living organisms, so adding chemicals to increase contrast often is not possible, but with different lighting, we can change the contrast as well.

Dissection Microscope

Although of much lower magnification than the compound microscopes, dissection scopes are incredibly useful because of the large working distance between the subject and the microscope head and the fact that we can look at thick, opaque objects. This allows us to look at large, still living organisms, and even allows us to do things like dissect and manipulate the specimens with probes, etc. The new Leica EZ4 dissection microscopes have a 3-way incident light feature using three different sets of LEDs, which can be switched individually, dimmed, and combined with transmitted light from underneath the subject. The switch for the scope is on the back, and the lights are controlled by switches on the top of the stage. Dissecting microscopes always have two objective lenses and two oculars. This arrangement provides a three-dimensional image with a large depth of field, two features that are essential to the kinds of activities performed with a stereoscope.

Activity

1. Take a coin (or some other object) you have in your pocket and place it on the stage of the microscope. Adjust the ocular lens distance so that when you look into the scope, you see only one circle of light when viewed with both eyes at the same time. Make sure the zoom is on its lowest setting and then, while looking in the scope, focus on the object with the large knob.
2. Play around with the different lighting controls and see what a difference the direction of light plays in how the object looks and on the contrast.
3. Get comfortable with the scope and practice zooming in and out of the object. Do you need to re-focus the scope after zooming?
4. Draw the object under the scope in the first plankton “box” (see last page). Using the ruler, measure the size of the object under the scope to the nearest millimeter (mm).
5. After your work is done (at the end of the lab), carefully wipe down any drops of water from the scope! Seawater is very harmful to the microscopes and will do a lot of damage if not cleaned up. If you had a lot of seawater on the stage, wipe it down with freshwater, then dry it with a towel.

Dissection Microscope. Image by Kevin Raskoff. CC BY-SA 4.0.

The Compound Microscope

Biologists of all types use microscopes as a principal investigative tool. From genetics, molecular, and cell biology all the way to evolution and ecology, this instrument provides useful and often critical information relating to the action of genes, the structure of cells, the presence of particular tiny adaptations that are key to an organism’s survival. The microscope consists of a lens system, a controllable light source, and a geared mechanism for focusing the specimen by adjusting the distance between the lens system and the specimen or object being observed. The light on compound scopes always comes from behind (transmitted) the objects, so they must be either very small, transparent, or very thin to see through.

Lens and magnification: The magnification achieved by a compound microscope is the result of two systems of lenses: the objective lens, nearest the specimen, and the ocular, or eyepiece lens, at the upper end of the instrument. To achieve different degrees of magnification, three or four objective lenses are provided. These are attached to the nosepiece, which revolves around an axis above the specimen, enabling you to gradually increase the magnification by simply rotating from the shortest lens in the active position, in sequence, to the longest objective. The magnification of each objective is indicated on its side and can be specified from the shortest to the longest, as follows: Scanning objective (4X), low power (10X), high dry objective (40X), and the optional oil immersion lens (100X). To determine the total magnification of your image, it is necessary to multiply the magnification of the objective lens you are using by the magnification of the ocular, which is fixed at 10X. So, for example, if the specimen is being viewed with the 40X objective, multiply 40 by 10 to get a total magnification of 400X.

Compound Microscope. Image by Kevin Raskoff. CC BY-SA 4.0.

Activity

Making a Wet Mount: Wet mounts are used to study fresh, living material. They can be used only for a little while because they will soon dry out. They are useful for observing qualities such as natural color, movement, or behavior that cannot be observed on dead, fixed, and stained material.

1. Obtain a clean slide and coverslip from the supply counter. If the slides and/or coverslip are not clean, wipe them gently with a Kimwipe or get a new one.
2. Place a drop of the living material (plankton) on your slide with the bulb pipette.
3. Touch the edge of the coverslip to the drop of water on the slide and gently lower the coverslip onto the slide. If you have been careful, the slide will not have any bubbles. If not, you will see the air bubbles as circles of various sizes with very dark edges when you look at your slide with the microscope.
4. Click the scanning objective lens into position. The scanning lens is the shortest lens and has a magnification, by itself, of 4X.
5. Using the coarse focus knob, lower the stage as far as possible.
6. Place the slide on the stage and position the cover slip directly over the hole in the center of the stage.
7. Now, looking from the side and using the coarse focus knob, bring the scanning lens as close to the slide as it will go.
8. Look into the microscope and slowly turn the coarse focus knob so that the scanning lens is moving away from the slide. Continue looking and turning until the plankton comes into focus. Fine-tune the image with the fine focus.
9. Using the travel knobs on the mechanical stage, move the slide away from you and note what happens to the image. Does the image move in the same direction? Move the slide to your left, then to your right. What does the image do when you make these movements?
10. Note that the inverted image always moves in the direction opposite to the direction in which the slide is moved.
11. Now, switch the objective lens to low power. The low-power lens is a bit longer than the scanning lens and has a magnification by itself of 10X. Since these lenses are “parfocal”, you will only have to slightly adjust the fine focus. If you do not see the plankton any longer, it is probably because you did not center the image in the scanning field before switching to the 10X lens.
12. Once again, center the image in the middle of the low-power field of view.
13. Move the high-power objective lens (40X) into position, making certain that it clicks in correctly. You may have to fine-tune the image with the fine focus knob. DO NOT USE THE COARSE FOCUS KNOB WHILE UNDER HIGH POWER, it can easily break the lens and the slide!
14. Observe the slide and wonder at the magnificent things to be seen in this microscopic world! Imagine what it must have been like for the first microscopists to look into a drop of pond water and see all these amazing creatures!
15. After your work with all the slides has been done, carefully wipe down any and all drops of water from the scope! Seawater is very harmful to the microscopes and will do much damage if not cleaned up.

Dissection Scope Reticle Scale

Equation is: 1000/Mag=Unit

Compound Scope Scale

Using the reticle, you can see this object is 10 units long (pay attention to the littlest lines, not the bigger ones). If this were viewed under 400x magnification, then the size would be: 10 x 2.5 μm = 25 μm.

Activity

Drawing Plankton: Using several different samples of the plankton in both the dissection and compound microscopes, you will draw and try to identify several groups of plankton. Using the techniques outlined above and the books and guides provided, carefully examine the specimens and draw their details on the plankton observation pages provided. Use the magnification and microscope best suited to the organism under study, and use the entire circle of the drawing area to represent the field of view of the microscope.

For each drawing of a plankton, try to list the following items:

1. Common Name:
2. Scientific Name (Genus and species):
3. Phylum:
4. Class:
5. Which Microscope?
6. Size of creature in micrometers (μm)
7. Other notes on its behavior or look