All living things are composed of cells. This is one of the tenets of the Cell Theory, a basic theory of biology. This remarkable fact was first discovered some 300 years ago and continues to be a source of wonder and research today. Cell biology is an extremely active area of study and helps us answer such fundamental questions as how organisms function. Through an understanding of how cells function we can discover how human ailments, such as cancer and AIDS, can be possibly treated.
The Cell Theory
- All life is composed of cells
- Cells are the fundamental units which possess all the characteristics of living things
- New cells can only come into existence by the division of previously existing cells
Notice that this scientific concept about life is called a theory. In science, unlike the layman’s definition, the word theory is used for a hypothesis about which there is a large body of convincing evidence. Under experimental conditions all observations have thus far confirmed the theory. The evidence that helped formulate the theory was obtained using the microscope. The microscope is of enormous importance to biology and has extended our ability to see beyond the scope of the naked eye.
When we look at cells under the microscope, our usual measurements fail to work. In science, the metric system is used to measure objects and, as you will see, is vastly superior to our antiquated English system of measurement. Here are the basic units:
| Length | Volume | Weight |
|---|---|---|
| 1 meter (m) | 1 liter (L) | 1 gram (g) |
| 1 millimeter (mm) = 0.001 m or 10−3 m or 1/1,000 m | 1 milliliter (ml) = 0.001 L or 10−3 L | 1 milligram (mg) = 0.001 g or 10−3 g |
| 1 micrometer (mm)= 0.000001 m or 10−6 m or 1/1,000,000 m | 1 microliter (ml) = 0.000001 L or 10−6 L | 1 microgram (mg) = 0.000001 g or 10−6 g |
| 1 nanometer (nm)= 0.000000001 m or 10−9 m or 1/1,000,000,000 m |
There is also a different scale for temperature: Celcius.
- 100˚ Celcius (C) = water boiling (equivalent to 212˚ F)
- 0˚ C = water freezing (equivalent to 32˚ F)
Converting between units can be confusing. The most effective way to do this is by using conversion factors and canceling units. For example, if you want to know how many liters are in 425 milliliters, you can set up a simple equation that looks like this.
[latex]\displaystyle{425}\text{ ml}\times\frac{1\text{ liter}}{1000\text{ ml}}=\frac{425\text{ ml}}{1000\text{ ml}}=0.425\text{ L}[/latex]
Practice
| 1.2 mm = ________ mm | 0.224 m = ________ mm | 225 nm =___________mm |
| 0.023 L = ________ ml | 750 ml = _________L | 50 ml =___________ L |
Part 1: Microscope Parts
The compound microscope is a precision instrument. Treat it with respect. When carrying it, always use two hands, one on the base and one on the neck.
The microscope consists of a stand (base + neck), on which is mounted the stage (for holding microscope slides) and lenses. The lens that you look through is the ocular (paired in binocular scopes); the lens that focuses on the specimen is the objective.
Your microscope has four objectives of varying magnifications (4x, 10x, 40x, and 100x) mounted on a revolving nosepiece. The 100x objective is a special oil immersion objective that needs to be used with oil—we won’t use the oil immersion objective for this course.
Positioning the specimen requires that you turn the mechanical stage controls, which operate the slide bracket on the surface of the stage. One control moves the specimen in the x-direction, and the other moves the specimen in the y-direction.
Focusing on the specimen is achieved by knobs that move the stage up and down, so that it is closer or farther from the objective. There are two knobs, an outer coarse focus and an inner fine focus.
The substage condenser directs light through the slide into the objective. An iris diaphragm on the substage condenser controls the amount of light reaching the objective, and also affects the contrast of the specimen.
Part 2: Magnification
The compound microscope has two sets of lenses; the ocular lens (or eye piece) which magnifies an object 10 times its normal size, and the objective lenses located on a revolving nosepiece. Rotate the nosepiece and notice how each objective lens clicks into place. Each objective lens has a different magnification of power written on it (such as 4, 10, 40, or 100). This number is the power of magnification for each of the objective lenses. For total magnification multiply the ocular power (10x) times the objective lens that is in place. For example, if you have a 10x ocular and a 10x objective, the total magnification is: 10x × 10x = 100x.
Use this information to fill in the following table:
| Ocular Lense | Objective Lense | Total Magnification | ||
|---|---|---|---|---|
| 10 | × | ________ (scanning) | = | ________ |
| 10 | × | ________ (low power) | = | ________ |
| 10 | × | ________ (high power) | = | ________ |
| 10 | × | ________ (oil immersion) | = | ________ |
Part 3: Using the Compound Light Microscope
After the instructor explains the proper carrying procedures, each student should get out a compound microscope and place it before them on the bench. The instructor will then go over the procedures for using your scope. You will not need to memorize its parts.
Complete the following procedure EVERY TIME you get your microscope out and EVERY TIME you put it away.
Getting Started
- Get your microscope out of the cabinet in the lab. Carry it with TWO HANDS to your table.
- Before plugging in your scope, always make sure that the voltage control is at its lowest level and the light switch is off.
- Plug in the microscope and turn on the light source.
- Raise the substage condenser to its top position and open the iris diaphragm all the way.
- Turn the nosepiece so that the 10x objective is lined up with the light source.
- Place a slide on the stage and use the mechanical stage controls to move it into place.
- Turn up the light to a comfortable level.
Getting a Focused Image
- Adjust the interocular distance (distance between the oculars) by gently pressing the oculars together or pulling them apart until you see a single circular field of view.
- Look through both oculars (i.e., keep both eyes open), but think right eye and adjust focus until the specimen is clear in your right eye.
- Now think left eye and turn the diopter adjustment (the moveable ring) on the left eyepiece to adjust the focus for your left eye. You should have a sense of the image suddenly “popping out” at you, sharp and clear.
Optimizing Resolution and Contrast
Resolution is the ability to distinguish two closely spaced points on your specimen, and it is always best with the iris diaphragm wide open. Contrast is the magnitude of difference between light and dark objects, and it increases as you close the aperture of the iris diaphragm. Getting the best image, then, requires that you find the right balance. Slowly open and close the iris diaphragm to get a feeling for the effect this has on your image.
Changing Magnification
Always start with the lowest power objective (4x) to get oriented and locate an area of interest, and then switch to higher power to examine interesting regions more closely. To change magnification, simply rotate the nosepiece to bring one of the other objectives into the light path.
Finishing Up
In this order: Turn down the illumination; turn off the power; switch back to the 4X objective; remove your slide; unplug the power cord and wrap it around the base of the scope; lower the stage to hold the cord in place; return your scope to the cabinet.
Part 4: The Letter e
Materials
- Light microscope
- Letter “e” slides
Procedure
- Note the position of the letter “e” on the slide (using your eyes only). Now center the slide of the letter “e” on the stage with the “e” in its normal upright position. Bring the letter into focus under low power using the procedures described above.
- Draw what you see through the eyepiece.
- Compare what you see through the eyepiece with what you saw using your eyes only.
- What do you notice about the position of the “e”?
- While looking through the microscope, move the slide to the left, notice which way the letter “e” moved. Now move the slide to the right. Notice which way the letter “e” moved. Do the same with moving the slide away and towards you.
- When you move the slide to the left on the stage, what direction does the image appear to move?
- When you move the slide away from you on the stage, what direction does the image appear to move?
- Why is it important to explore this?
Part 5: Colored Threads
Materials
- Light microscope
- Colored thread slides
Procedure
- Obtain a slide of colored threads and view them under the scanning power.
- Which thread is on top? Which is on bottom?
- View the threads under high power (not oil immersion). Use the fine focus to figure out the order of the threads from top to bottom. As you rotate the fine focus, different strands will go out of focus while others will become more sharply focused.
- Are all of the threads in focus at the same time?
- What is the order (from top to bottom)?
- “Depth of field” refers to the thickness of the plane of focus. With a large depth of field, all of the threads can be in focused at the same time. With a narrower depth of field, only one thread or a part of one thread can be focused at a time. In order to view the other threads, you must focus downward to view the ones underneath and upward to view the ones that are above.
- What happens to the depth of field when you increase to a higher magnification (increases, decreases, or remains the same)?
- Explain how the slide with threads could be used to answer the question above.
Part 6: Plant Cells
Preparing a Wet Mount
If you want to look at something small under the microscope, you must know how to prepare a wet mount of the specimen.
- Place a drop of water on the center of a microscope slide.
- Pull off a single Elodea leaf (also called Anacharis in the aquarium trade) and place it within the drop of water.
- Carefully place a coverslip at an angle against the water droplet. Then drop the coverslip onto the water and the leaf. This will reduce the number of air bubbles caught under the coverslip.
- Make sure the scanning power objective is selected. [Always begin on scanning power!]
- Place your slide onto the stage and secure with the clip.
- Do not look through the ocular lens. Use the mechanical stage knobs to center the specimen under the scanning objective. Crank the coarse adjustment so that the scanning lens is close to the slide (look directly at the slide).
- Now look through the ocular lens and slowly crank the coarse adjustment back until something comes into focus. Use the mechanical stage knobs to search for your specimen. Once the specimen is positioned in the center of the field of view, use the fine adjustment knob to resolve in more detail.
- Search for any cellular organelles, such as chloroplasts, that you can find.
- Remember, the leaf is alive! Can you spot cytoplasmic streaming?
Estimating the Size of Objects
To determine the size of the object you are viewing, you must know the distance across the field of view (the diameter of the total circular area you see when looking through the microscope). Millimeters (mm) are used to measure distances across the field of view on scanning power, whereas micrometers (mm) are used for greater magnification. The fields of view and approximate distances across for scanning, low, and high power are as follows:
- Carefully draw your Elodea at all three magnifications. Determine the length of your specimen at each magnification and place this number under the measurement bar that you draw under the specimen. Include any organelles you see.
- Draw a measurement bar on each field of view and indicate the length of the bar.
- There are three structures that distinguish plant cells from animal cells. Label these structures in your high power drawing.
Part 7: Animal Cells
Materials
- 1 toothpick/ person
- Tap water
- Methylene blue
- Slide
- Coverslip
Procedure
- Take the flat end of a toothpick and gently scrape the lining of your cheek inside your mouth.
- Spread the sample on a drop of water you have already placed on a microscope slide.
- Place a coverslip on top and carefully add one or two drops of methylene blue dye to the edge of your coverslip.
- Allow the dye to diffuse across the slide as you examine your cells under the microscope.
- Draw a typical cheek cell that has been stained with dye and LABEL all visible parts. Include a scale bar in your drawing.