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NS 1011: Introduction to Biology: Cells and Organisms

Microscope and Cell Observation Lab

PURPOSE: To learn how to use the microscope, and to observe some cells.

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

Students will work individually on this lab.  There will be enough microscopes for everyone.

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 the 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

RULES FOR MICROSCOPE USE

Always observe the following rules for microscope use:

  1. The lowest power objective should be in position both at the beginning and end of microscope use.
  2. Use only lens paper for cleaning lenses.
  3. Do not tilt the microscope when viewing a wet mount.
  4. Keep the stage clean and dry to prevent rust and corrosion.
  5. Do not remove parts of the microscope.
  6. Keep the microscope dust-free by covering it after use.
  7. Report any malfunctioning.

Identification of Parts

The instructor will explain how to set up and move a microscope.  Identify the following parts on your microscope, and record the answers to the questions in the table below.
  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?
  3. Body tube: holds nosepiece at one end and eyepiece at the other end; conducts light rays.
  4. Arm: Supports upper parts and provides carrying handle.
  5. Objectives (objective lenses):
    1. Scanning power objective: Holds scanning lens used to view the whole slide.
    2. What is its magnifying power?
    3. Low-power objective: Holds low power lens used to view the object in greater detail.
    4. What is the magnifying power of this lens on your microscope?
    5. High-power objective: Holds high power lens used to view the object in even greater detail.
    6. What is the magnifying power of this lens on your microscope?
    7. Oil immersion objective: Holds oil immersion lens and is used in conjunction with immersion oil to view objects with the greatest magnification.
    8. Does your microscope have an oil immersion objective?  What is its power?
      This lens will be used rarely, if at all, in this course.
  6. Coarse-adjustment knob: Knob used to bring object into approximate focus; used only with low-power objective.
  7. Fine-adjustment knob: Knob used to bring object into final focus.
  8. Diaphragm or diaphragm control lever: Controls amount of illumination used to view the object.
  9. Light source: An attached lamp that directs a beam of light up through the object.
  10. Base: The flat surface of the microscope that rests on the table.
  11. Stage: Holds and supports microscope slides.
  12. Mechanical stage: A moveable stage that aids in the accurate positioning of the slide.
  13. Does your microscope have a mechanical stage?
  14. 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.

Total Magnification

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

Microscope Parts data table

Eyepiece magnifying power:  
Scanning objective power: Total magnification:
Low-power objective power: Total magnification:
High-power objective power: Total magnification:
Oil immersion objective power: Total magnification:

Focusing the Microscope

Lowest Power

  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.

Exercise 1

  1. Draw the letter e as it appears on the slide (look from the side, not through the eyepieces).
  2. Draw the letter as it appears when you look through the eyepieces.
  3. Move the slide to the right.  Which way does the image appear to move?
This phenomenon is known as inversion.

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.
  1. Bring the object into focus under the lowest power by following the instructions in the previous section.
  2. Make sure that the letter e is centered in the field of the lowest objective.
  3. 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.
  4. If any adjustment is needed use only the fine-adjustment knob.  Always use only the fine-adjustment knob with higher powers.

  5. On your drawing of the letter e  that you made for exercise 1, draw a circle around the portion of the letter that you are now seeing with the higher-power magnification.
  6. Repeat steps 2-4 with the 40x power objective lens.
  7. When you have finished your observations of this slide (or any slide), rotate the nosepiece until the lowest power objective clicks into place, and then remove the slide.

Diameter of Field

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

Exercise 2:

    LOW POWER (10x)
  1. 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 (not scanning) 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.
  2. 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.)
  3. Convert the figure to micrometers (mm) and record.  This is your low-power diameter of field (LPD).  To convert from millimeters to micrometers, multiply the millimeter value by 1,000.

          HIGH POWER (40x)

  1. Record the following data for your microscope:

    1. LPD=low-power diameter of field (in micrometers).
    2. LPM=low-power total magnification (not scanning).
    3. HPM=high-power total magnification.
  2. Compute the high-power diameter of field (HPD) by substituting the preceeding data into the following formula:

                               HPD = LPD x (LPM / HPM)

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

LPD: LPM:
HPM: HPD:

Questions:

  1. Does low power or high power have a larger field of view and allow you to see more of the object?

    __________________________________________________
     
  2. Which has a smaller field but magnifies to a greater extent?

    __________________________________________________

Microscopic Observations

When a specimen must be prepared for observation, the object should always be viewed as a wet mount.  A wet mount is prepared by placing a drop of liquid on a slide or, if the material is dry, by placing it directly on the slide and adding a drop of water or stain.  The mount is then covered with a coverslip.  The instructor will demonstrate how to make a wet mount.

Exercise 3:

HUMAN EPITHELIAL CELLS
Epithelial cells cover the body's surface and line its cavities.
  1. Obtain toothpick and sanitize it with alcohol.
  2. Gently scrape the inside of yourcheek with the toothpick.
  3. Place the scrapings on a clean, dry slide.
  4. Add a drop of very weak methylene blue stain and cover with a coverslip.
  5. Observe under the microscope, using the directions for focusing given above.  Start with the scanning power objective to find some cells, then observe under both low and high power.
  6. Locate the cell membrane, the cytoplasm, and the nucleus.
  7. Make a drawing of what you see.  Be sure to label the drawing with what it is, and what power you were using when you made your drawing.

Exercise 4:

PLANT CELLS
  1. With a scalpel or your fingers, strip a thin, transparent layer of cells from a piece of onion.
  2. Place it gently on a clean, dry slide.
  3. Add a drop of methylene blue stain and cover with a coverslip.
  4. Observe under the microscope and draw what you see.  Be sure to label your drawing.
  5. Locate the cell wall.  Is a nucleus visible?
  6. Count and record the number of cells across the diameter of the high power field, both lengthwise and side to side.
  7. Using the number you calculated in exercise 3, calculate and record the length and width of an onion cell in micrometers.
  8. Record some obvious differences between the human cheek cells and the onion cells.
  9. Record any similarities between the two types of cells.
  10. If there is time, obtain another plant sample, make a slide (without methylene blue, use water) and observe under the microscope.
  11. Draw what you see.  Be sure to label your drawing.
  12. Record some differences and similarities between the different plant cells you observed.

Exercise 5:

PROTISTS (optional, if there is time)
  1. Obtain a sample of a protist from one of the containers at the front of the room.  The instructor will help you obtain this sample.
  2. Place a drop containing one or more protists on a slide and cover with a cover slip.
  3. Scan the slide for organisms: Start at the upper left-hand corner, and move the slide forward and back as you work across the slide from left to right.
  4. Experiment by using all available lenses (NOT the oil immersion!), by focusing up and down with the fine-adjustment knob, and by adjusting the light so that it is not too bright.
  5. Draw any organisms you see and label your drawings.

Conclusion Questions:

  1. Why is it helpful for a microscope to be parfocal?
  2. What is inversion?
  3. Why is locating an object more difficult if you start with the higher power objectives than with the scanning objective?

Lab Report:



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