BIOLOGY LAB
Care and Use
of the Microscope


PURPOSE: To learn how to use the microscope.

You will need to record data and answer questions from each part of this lab.   All drawings must be labeled with what it is and with the magnification used.

Students will work in lab pairs on this lab.  There will be 1 microscope for 2 people.

INTRODUCTION:

Because biological objects can be very small, a microscope is often used to view them.  We have two kinds of light microscopes available in the lab.  Light microscopes use light rays that are magnified and focused by means of lenses. The binocular dissecting microscope is designed to study entire objects in three dimensions at low magnification.  The compound light microscope is used for examining small or thinly sliced sections of objects under magnification that is higher than that of the dissecting light microscope.  Illumination is from below, and Th. light passes through clear sections but does not pass through opaque sections.  To improve contrast, the microscopist uses stains or dyes that bind to cellular structures and absorb light.  The following link will take you to a site with some images from a light microscope: IMAGES

THE COMPOUND LIGHT MICROSCOPE

Identification of Parts

After the instructor has explained how to carry a microscope, obtain one from the cabinet and place it securely on the table.  Identify the following parts on your microscope, and record the answers to the questions in your lab notebook.
  1. Eyepiece (ocular lens): Topmost series of lenses through which an object is viewed.

    2. What is the magnifying power of the ocular lens on your microscope?
  2. Body tube: holds nosepiece at one end and eyepiece at the other end; conducts light rays.
  3. Arm: Supports upper parts and provides carrying handle.
  4. Objectives (objective lenses):
    • Scanning power objective: Holds 4x lens used to view the whole slide.
      Does your microscope have a scanning power objective?   If so, what is its magnifying power?
    • Low-power objective: Holds 10x lens used to view the object in greater detail.
      What is the magnifying power of this lens on your microscope?
    • High-power objective: Holds 40x lens used to view the object in even greater detail.
      What is the magnifying power of this lens on your microscope?
  1. Coarse-adjustment knob: Knob used to bring object into approximate focus; used only with low-power objective.
  2. Fine-adjustment knob: Knob used to bring object into final focus.
  3. Diaphragm or diaphragm control lever: Controls amount of illumination used to view the object.
  4. Light source: An attached lamp that directs a beam of light up through the object.
  5. Base: The flat surface of the microscope that rests on the table.
  6. Stage: Holds and supports microscope slides.
  7. Mechanical stage: A moveable stage that aids in the accurate positioning of the slide.

    8. Does your microscope have a mechanical stage?
    • Mechanical stage control knobs: Two knobs that are usually located below the stage.  One knob controls forward/reverse movement, and the other controls right/left movement.

RULES FOR MICROSCOPE USE

Always observe the following rules for microscope use:
  1. Carry the microscope with two hands from the microscope cabinet to your lab desk.
  2. The lowest power objective should be in position both at the beginning and end of microscope use.
  3. Use only lens paper for cleaning lenses.
  4. Do not tilt the microscope when viewing a wet mount.
  5. Keep the stage clean and dry to prevent rust and corrosion.
  6. Do not remove pats of the microscope.
  7. Report any malfunctioning.

Focusing the Microscope

Lowest Power (Scanning)

  1. Turn the nosepiece so that the lowest power lens is in straight alignment over the stage.
  2. Always begin focusing with the lowest power objective lens.
  3. With the coarse-adjustment knob, lower the stage until it stops.
  4. Place a slide of the letter e on the stage, and stabilize it with the clips (instructor will demonstrate if necessary).
  5. Again, be sure that the lowest power objective is in place.  Then, as you look from the side, decrease the distance between the stage and the tip of the objective lens until the lens comes to an automatic stop or is no closer than 3 mm above the slide.
  6. While looking into the eyepiece, rotate the diaphragm (or diaphragm lever) to give the maximum amount of light.
  7. Slowly increase the distance between the stage and the objective lens, using the coarse-adjustment knob, until the object - in this case the letter e - comes into view, or focus.
  8. Once the object is seen, you may need to adjust the amount of light.  To increase or decrease the contrast, rotate the diaphragm slightly.
  9. Use the fine-adjustment knob to sharpen the focus if necessary.
  10. Practice having both eyes open when looking through the eyepieces, as it greatly reduces eyestrain.

Higher Powers

Compound microscopes are parfocal; that is, once the object is in focus with lowest power, it should also be almost in focus with the higher power.
Bring the object into focus under the lowest power by following the instructions in the previous section.
Make sure that the letter e is centered in the field of the lowest objective.
Move to the next higher objective (low power, 10x) by turning the nosepiece until you hear or feel it click into place.  Do not change the focus; parfocal microscope objectives will not hit normal slides when changing the focus if the lowest objective is intially in focus.
If any adjustment is needed use only the fine-adjustment knob.  Always use only the fine-adjustment knob with higher powers.

    Lab Exercise 1 - Letter "e" Lab

    Supplies:
    Standard Equipment Needed:
    • Microscope 
    • Hand Lens 
    • Pencil 
    • Colored Pencils 
    • Colored Paper
    • Prepared letter "e"
    • Paper containing "e"s 
    • Scissors
    Lab Procedure:

    1. Using microscope, slide, cover slip, water, scissors, and newspaper make a wet-mount slide of a small-case letter "e".
    2. Cut a small-case letter "e" from a newspaper.
    3. Put the letter "e" on a microscope slide.
    4. Using a dropper bottle, put a small drop of water on the letter "e".
    5. Cover the letter "e" with a cover slip.
    6. Look at the letter "e" using the low power objective lens.
    7. What is the total magnification when using low power?
    8. Draw what you see.
    9. Rotate the nose piece to the medium power objective lens and observe the letter "e".
    10. What is the total magnification when using medium power?
    11. Draw what you see.
    12. Rotate the nose piece to the high power objective lens and observe the letter "e".
    13. What is the total magnification when using high power?
    14. Draw what you see.

    The letter"e" is a great way to learn how a microscope works. In this lab you will set up your microscope and view what a simple letter e cut out from a piece of paper looks like. First let's examine the letter "e" with your naked eye and through a hand lens. Draw what you see in the circles below:
    Carefully carry your microscope and place it on your lab desk. Identify the following items on your microscope:
    1. Eye piece  
    2. Objectives (low, medium, and high) 
    3. Light source - illuminator 
    4. Stage and stage clips 
    5. Focus control 
    Follow these steps to use the microscope with a prepared slide:
    • Place your letter "e" slide, coverslip side up, on the stage. Use the low power objective.
    • Secure the slide with the stage clips.
    • Turn on the light.
    • Focus on the letter "e" using the coarse focusing knob. 
    • Draw what you see in the circle below. Try using the high power objective. After focusing (fine focus only), draw what you see. 
    Analysis Questions :

    What are some of the ways the e you see with the microscope is different from the e you see with the hand lens?

    If you are looking at the "e" through the microscope and you push your slide to the left, which way does the e in the microscope move? (Try this!)

    If you push the slide away from you, which way does the e in the microscope move? (Try this!)

This phenomenon is known as inversion.

Total Magnification

Total magnification is calculated by multiplying the magnification of the ocular lens (eyepiece) by the magnification of the objective lens.

Exercise 2:

Calculate the total magnification for each objective, and record your figures in a table.  To calculate the total magnification, multiply the power of the ocular lens by the power of the objective lens.  Your table should include the powers of both lenses and the total magnification.

Microscope
Total Magnification
Value for each ocular unit at 4X (Scanning) .
Value for each ocular unit at 10X (Low Power) .
Value for each ocular unit at 40X (High Power) .

 

Diameter of Field

The diameter of the field (the circle visible through the lens) is the length of the field across the center.

Exercise 3:

SCANNING (4x)
Place a clear plastic ruler across the stage so that the edge of the ruler is visible as a horizontal lilne along the diameter of the low-power field.  Be sure that you are looking at the millimeter side of the ruler.  You will use the ruler to measure the diameter of the field of view under various magnifications.
Estimate and record the number of millimeters (mm), to tenths, that you see along the field: (Hint: Start with one of the millimeter markers at the edge of the field.)

Convert the figure to micrometers (mm) and record.  This is your scanning diameter of field .  To convert from millimeters to micrometers, multiply the millimeter value by 1,000.

Microscope
Field of View
Value for each ocular unit at 4X (Scanning) .
Value for each ocular unit at 10X (Low Power) .
Value for each ocular unit at 40X (High Power) .


LOW POWER (10x) Repeat the steps above to determine(LPD)
HIGH POWER (40x) Repeat the steps above to determine(HPD)

Does low power or high power have a larger field of view and allow you to see more of the object?
Which has a smaller field but magnifies to a greater extent?

Reminder:
To locate small objects that are seen on low power, place these objects in the center of the field before rotating to high power.

Depth of Focus

The vertical distance that remains in focus at one time is called the depth of focus.

Exercise 4:

Prepare a slide with three or four colored threads or hairs mounted together. See Sample
With low power, find a point where the threads or hairs cross.
Slowly focus up and down.  Notice that when one thread or hair is in focus, the others seem blurred.
Determine the order of the threads or hairs, and record the order.  Remember, as the lens moves down (or the stage moves up) with focusing, objects on top come into focus first.
Switch to high-power, and notice that the depth of focus is more shallow with high power than with low power.

Constant use of the fine-adjustment knob when viewing a slide with high power will give you an idea of the specimen's three dimensional form.

Conclusion Questions:

  1. What did you learn about the care and use of the microscope?
  2. Why is it helpful for a microscope to be parfocal?
  3. What is inversion?
  4. How is the image affected by the microscope?
  5. Explain how to determine the total magnification and field of view for you microscope.
  6. Why is locating an object more difficult if you start with the higher power objectives than with the scanning objective?