4.2: Lab 4 Procedures

PROCEDURE 1: PARTS OF THE COMPOUND MICROSCOPE

Equipment we’ll need:

• Compound microscope (one per person)

1. On the Answer Sheet, label the diagram of the microscope. Use the list of keywords as a guide.

Figure 2. Labomed CxL, Compound microscope.

2. Use the formula above to complete Table 1 in the Answer Sheet.

PROCEDURE 2: FOCUSING THE COMPOUND MICROSCOPE

Equipment we’ll need:

• Compound microscope
• Prepared slide of the letter “e”

Examining specimens with the microscope can take some practice. Follow the procedures below to become familiar with how to properly focus on specimens under the microscope.

Low Power

1. Obtain a “letter e” slide. Observe the orientation of the “e” on the slide before putting it on the microscope stage and record it in Answer Sheet.
2. Obtain the microscope. Use lens paper to gently wipe clean the ocular lens and the objective lenses.
3. If necessary, rotate the nosepiece until the scanning objective is in position. Use the coarse focus knob to lower the stage all the way.
4. Pull the stage clip lever handle to open the bow shaped stage clip, place the prepared slide of the letter “e” on the stage, move the slide toward the back of the stage until it sits snug, and release the stage clip.
   • The slide should be flat on the stage and the stage clip should be resting against the corner of the slide (not on top of it).
5. Use the stage control knobs to center the letter “e” over the circular opening above the condenser.
6. Use the coarse focus knob to raise the stage all the way up.
7. While looking through the ocular lens, slowly turn the coarse focus knob to lower the stage. Stop turning the coarse focus knob when the specimen comes into view. Slightly adjust the focus until the specimen is clear.
8. Adjust for best illumination by adjusting the brightness control dial and/or the condenser diaphragm. See tips above.
9. Using the stage control knobs, move the stage to the left. What direction does the letter e move while looking through the oculars?
   • Move the stage away from the arm. What direction does it move while looking through the oculars?
10. Center the specimen directly in the middle of the field of view.
11. Once the specimen is in focus, centered, and the illumination is right, rotate the nosepiece until the low power objective is in position. We should hear a click.
12. Use the fine focus knob to adjust for clarity.
    • Remember - do not use the coarse focus knob with objectives more powerful than the scanning objective!
13. Answer questions on the Answer Sheet.

High Power

In order to see the specimen under higher magnification, be sure that the specimen is in the very center of the field of view and adjusted for best clarity.

1. Rotate the nosepiece to the high power objective. We should hear a click.
2. Adjust for clarity and sharp focus using the fine focus knob only.
3. Adjust for proper illumination.

Tip: If we ever lose the specimen (i.e., cannot find it) while viewing under high power magnification, rotate the nosepiece back to the scanning objective to find it again. Then, re-focus the specimen and return to high power magnification.

4. Answer questions on the Answer Sheet.

PROCEDURE 3: MAKING A WET MOUNT SLIDE

The slide we just observed was prepared for us. However, we will need to prepare many of our own slides of specimens throughout the course. Care should be taken to avoid getting any liquids on the objectives or other areas of the microscope.

Equipment we’ll need:

• Compound microscope
• Clean slide and cover slip
• Pond water
• Sterile plastic pipette

1. Obtain a clean slide and cover slip. (Cover slips are the plastic squares; disregard the packing tissues.)
2. Add just 1 drop of the pond water to the slide.
   • A little goes a long way! Adding more liquid runs the risk of having water drip off the slide and onto the microscope.
3. Place one edge of the cover slip next to the water drop and slowly lower the cover slip on top of the sample. Be sure there is no water on top of the cover slip or leaking out from around the edges. Water will damage the objective lenses. Be sure the bottom of the slide is dry to avoid making a mess on the stage.
   • If there is any water outside the coverslip (especially if it is on the bottom of the slide), use a paper towel to gently wick away the water.
4. Use the same procedure as with the “letter e” to gradually focus the specimens. Start with the scanning objective, then after the focus and illumination are right, move on to the stronger magnifications.
   • Here, it is especially important to get the illumination right. We will likely have to play with the brightness and contrast (using the brightness control dial and condenser diaphragm) until we see some organisms come into view.
   • If we don’t see anything right away, and we’re confident that we’re focusing on the right area, use the stage control knobs to move the slide to see different spots in the water.
5. Answer the questions in the Answer Sheet.
6. Rinse the glass slide in the sink and set it on the towel next to the sink to dry. Throw the coverslip away in the trash.

Common cells/organisms we may see

Here are some examples of cells or microscopic organisms we may see in the pond water.

Figure 3. Examples of organisms we may see in pond water, including A.) Amoeba (image source), B.) assorted microorganisms (image source), C.) rotifer (image source), D.)Paramecium (image source), and E.) Euglena (image source).

Things often mistaken for cells

It is common for students learning to use the microscope to see various structures and mistake them for cells or microorganisms. Below are some examples. If we see these, keep looking! These are not cells!

Figure 4. Examples of things mistaken for cells under the microscope. These include A-B.) air bubbles, C.) lint or a scratch in the slide, D.) the glass slide, and E.) dust (dry, dead skin cells). Image credit: Ernie Kwok.

PROCEDURE 4: HUMAN CHEEK EPITHELIAL CELLS

Now we get to look at some of our own human cells (and not so human cells) under the microscope! We are going to look at some cells from our cheeks, and along with them some bacteria that call our mouths their home. But our cheek cells are rather difficult to see because there is no color to them, so first we have to stain them. The stain, called methyl blue, is very strong and can easily stain clothes, so be careful not to spill!

Equipment we’ll need

• Clean glass slide and coverslip
• Toothpick
• Methylene blue

1. Place a small drop of methylene blue stain in the center of a clean glass slide.
   • Remember to be careful with the stain! Avoid spilling it, especially on any clothing. If any gets on our skin, wash with soap and water immediately.
2. Use a clean toothpick to gently scrape the inside of your cheek.
3. Carefully rub the toothpick in the stain on the slide to stain and deposit your cheek cells on the slide. Dispose of the toothpick in the trash can.
4. Slowly lower a clear plastic coverslip on top of the sample.
5. Be sure there is no stain on top of the cover slip or leaking out from around the edges. Any liquid will damage the objectives. Use a paper towel to dab away any excess stain.
6. Focus the microscope in the same way as the previous procedures. Use either the low power (10x) or high power (40x) objective.
7. Draw or take a picture of a few of the cheek cells and bacterial cells on the Answer Sheet.
8. Rinse the glass slide in the sink and set it on the towel next to the sink to dry. Throw the coverslip away in the trash.

PROCEDURE 5: PLANT CELLS

Elodea is an aquatic, multicellular, eukaryotic plant that is commonly used in freshwater aquariums. Interestingly, the leaves are just one cell layer thick, making it very easy for us to observe individual cells. We will also observe the organelles that make these cells green.

Equipment we’ll need:

• Clean glass slide and coverslip
• Elodea
• Forceps

1. Use forceps to remove a leaf from a sprig of Elodea and place it on a clean glass slide.
2. Add a single drop of water over the leaf.
3. Gently add a plastic cover slip. Be sure that the top and bottom of the slide are dry.
4. Focus the microscope in the same way as the previous procedures. Use either the low power (10x) or high power (40x) objective.
5. Draw or take a picture of a few of the plant cells on the Answer Sheet.

Clean up/Disposal

Microscope

1. Put the 4x objective lens in place over the stage.
2. Lower the microscope stage all the way down.
3. Remove slide from microscope.
4. Turn off the microscope.
5. Unplug the microscope and wrap the cord neatly around the base.
6. Cover the microscope and return to its appropriate cabinet. Then lock the cabinet and return the key.

Slides

1. Wipe down any prepared slides and return to the wooden slide holder by the front of the room.
2. Remove the coverslip from the wet mounts. Dispose of the coverslip in the trash.
3. Dispose of any large sample materials (such as the Elodea leaf) in the trash. Do not rinse this down the drain.
4. Rinse the slide clean. Scrub off any residue or stain left behind.
5. Set slides out to air dry on the towels.