Things you should be able to explain to someone else after this lab:

• Compound
• Total magnification
• Binocular
• Field of view
• Depth of focus
• Inversion phenomenon

Slideshow

http://www.slideshare.net/CandelaContent/microscope-biology-i-lab

Background

Many organisms (bacteria) and parts of organisms (cells) that biologists study are too small to be seen with the human eye. We use microscopes to enlarge specimens for our investigation. The word microscope means “to see small” and the first primitive microscope was created in 1595.

There are several types of microscopes but you will be mostly using a compound light microscope. This type of microscope uses visible light focused through two lenses, the ocular and the objective, to view a small specimen. Only cells that are thin enough for light to pass through will be visible with a light microscope in a two dimensional image.

Another microscope that you will use in lab is a stereoscopic or a dissecting microscope. This type of microscope uses visible light view thicker, larger specimens, such as an insect, in 3D. Since you are viewing larger samples, the magnification range of the dissecting microscope is lower than the compound light microscope.

Your instructor will review the parts and functions of the compound light microscopes that we will be using throughout the semester. Fill in the table on the next page to help you remember this important information. You will likely refer back to this page frequently. Here is a picture of a light microscope for you to label and take notes on.

Rules and Instructions for using the Compound Light Microscopes

Your instructor will discuss the use of the microscope with your class. The proper steps to follow for correct focusing will be reviewed. You are to follow the step-by-step sequence as your instructor indicates. Even if you are familiar with this type of microscope, you are still expected to move through the focusing review with the rest of the class.

1. Important general rules:
   1. Always carry the microscope with 2 hands—place one hand on the microscope arm and the other hand under the microscope base.
   2. Do not touch the objective lenses (i.e. the tips of the objectives).
   3. Keep the objectives in the scan position and keep the stage low when adding or removing slides.
   4. Always look at the microscope from the side when you are making large changes in the height of the stage.
   5. The objective lenses are to be cleaned only with special lens paper and lens-cleaning fluid.
   6. Do NOT play handyperson—no microscope is to be disassembled. Report malfunctions to your instructor.
2. To obtain a microscope from laboratory cabinet:
   1. First clear area on table for microscope—avoid a crowded working area.
   2. The microscopes are numbered according to where you are sitting. Find the number under your bench and use the corresponding microscope.
   3. Carry the microscope with TWO hands.
   4. Secure the electrical cord—don’t let it hang off the table!
3. When returning the microscope to the laboratory cabinet:
   1. Lower the stage.
   2. Rotate the scan objective into position over the stage.
   3. Remove your slide from the stage.
   4. Clean the slide and objective using the special lens-cleaning fluid and paper provided.
   5. Center the stage so that it does not project too far to either side.
   6. Secure the cord by wrapping it under the stage of the microscope..
   7. Replace the dust cover.
   8. Carry the microscope with TWO hands.
   9. Return the microscope to the same cubby from which you obtained it making sure to put it in arm out.
4. To focus the microscope:
   Look at the microscope from the side:
   1. Lower the stage as far as it will go.
      1. Rotate the objectives so that the scan objective is pointing down at the stage.
      2. Adjust the stage so that the aperture (the opening in the middle of the stage) is centered.
      3. Place your slide on the stage, using the stage clamp to secure it. The stage clamp moves only within the horizontal plane of the stage, and it secures the slide by just barely touching the bottom right corner of the slide.
      4. Center the specimen on your slide under the objective by moving the stage.
      5. Move the stage up almost as far as it will go, being careful not to let the slide touch the objective lens—look from the side so that you can see how high to raise the stage.
   2. Look through the oculars:
      1. Use the coarse adjustment knob to move the stage downward until your specimen comes into focus, then sharpen the focus.
      2. Center the specimen in microscope field by moving the stage.
   3. Look at microscope from the side:
      1. Slowly rotate the low power (10X) objective into position.
   4. Look through the oculars:
      1. Sharpen the focus, if necessary, with the coarse adjustment knob. Only a minimal amount of adjustment is usually necessary.
      2. Center the specimen in field, if necessary.
   5. Look at the microscope from the side:
      1. Rotate the high power objective into place very carefully!
   6. Look through the oculars:
      1. Using the fine adjustment knob only, sharpen focus. Re-center if necessary. (If your specimen has “disappeared”, immediately return to low power and re-center the specimen.)
      2. When removing a slide, always return the objectives to the scan position and lower the stage before lifting the slide off!

Part 1: Total Magnification

Magnification is the ratio of the image size with the microscope to the actual size of the object. When you say that the magnification is 10, the image you see using the microscope is ten times bigger than viewing the specimen with the naked eye. Remember with a compound light microscope you are magnifying with two lenses, so to calculate the total magnification you multiple the objective magnification by the ocular magnification. View the microscope and use the chart below to calculate total magnification for each lens:

Part 2: Inversion Phenomenon

Obtain a letter “e” slide available in the classroom. View the slide with your eyes, and then place it onto the microscope. Use the focusing sequence to view the slide under low power.

Procedure

1. Draw the letter “e” as it appears when you look at the slide without the microscope.
2. Draw the letter “e” as it appears when you look at the slide under the microscope.
3. Move your slide to the right using the mechanical stage lever while looking through the oculars. Which way does the “e” move?
4. Move your slide away from you using the mechanical stage lever while looking through the oculars. Which way does the “e” move?

State the inversion phenomenon in your own words.

Part 3: Field of View

The field of view is the amount of the specimen you see when you look through the objectives. The field of view decreases at higher magnifications. 

Procedure

Place a blue plastic ruler across the stage aperture so that the edge of the ruler is visible as a vertical line along the field diameter. Estimate the field size in millimeters for each of the objective lenses.

Scan __________                    Low (10X) __________                    High (40X) __________

Part 4: Depth of Focus

The depth of focus is the thickness of the specimen that remains in focus at a given magnification. Depth of focus decreases at higher magnifications.

Procedure

Obtain a colored threads slide, and view it under scan or low power. Then, determine which color thread is:

• On the top
• In the middle
• On the bottom

Hint: using the instructions for focusing, focus on the area where the three threads cross. Use the fine focus to discern the order of the threads.

Part 5: Making a Wet Mount

Throughout the semester, you will be expected to successfully make a number of simple slide preparations called “wet mounts.” The specimen to be observed is placed on a clean slide, a drop or two of water added, and a cover slip carefully placed over the water and specimen. Your instructor will demonstrate this technique.

Make a wet mount of pond water following the procedure below:

1. Place a drop of pond water on a clean glass slide using the plastic disposable pipette.
2. Cover the pond water on the slide with a coverslip. Try to place the coverslip on at an angle to avid air bubbles.
3. Wipe off any excess liquid on your slide.
4. Place the slide on the microscope to observe the specimen.
   1. Start on the scan objective
   2. Carefully switch to the low and high objectives as necessary.

Hopefully you will see some live organisms in your pond water. If you view green material, it is probably some sort of plant. Look to see if there is anything moving. In the space below, describe what you are observe under the microscope and draw a simple picture. 

Part 6: Stereoscopic Dissecting Microscope

Your instructor will demonstrate proper use of the dissecting scope. These microscopes generally give a lower magnification than the compound microscope you are using.

Procedure

View the specimens available at the lab table using the dissecting microscope. Notice that the microscope has two light sources, one from the base and one from above. The specimens are not always mounted on a slide. Also note that when you view objects under the dissecting microscope they are three-dimensional.

Cleaning and Caring for the Microscope

1. Describe how and with what you are to clean the lens of the microscope.
2. List four things that you are to do when you are finished using the microscope at the end of lab.