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Unit Labs The Matter Energy of Living Systems

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                                LAB – Using the Microscope
Introduction
The compound microscope contains two sets of lenses: an objective
lens that produces a magnified image, and an eyepiece, or ocular lens
that further magnifies the image. The total magnification available is
the product of the magnifications produced by each of the two lenses.
Our microscopes generally have a 10X ocular, and three objective
lenses: low power (4X), medium power (10X), and high power (40X).
Therefore, the total magnifications available are 40X, 100X, and
400X. Other important parts of the compound microscope are labeled
in Figure 1 on the second page of this lab.
A stereo microscope has double oculars and objectives, providing 3-D vision. It can be used to view
objects with reflected as well as transmitted light. It is useful for viewing dissection specimens or the
observation of objects too large to be seen as a whole under the monocular compound microscope. It is
also useful in working with opaque objects. Examine the labeled drawing of the stereo microscope in
Figure 2 on the second page of this lab.
The microscope may be used not only to study small objects, but also to measure their dimensions.
The unit of measurement used in microscopy is the micrometer (µm). The micrometer is equivalent to
1 x 10-6 meters, or 0.000001 m.

Materials
     compound microscope            newspaper scraps                  50 mL beaker
     stereo microscope              microscope slides                 transparent ruler
     lens paper                     cover slips                       prepared slides:
     scissors                       eye dropper                         colored threads, paramecium

The Compound Microscope
Skill: Identify Parts of the Compound Microscope
    1. First, it is important to know the name and function of each of the parts of the microscope.
         Use Figure 1 to help you locate each part on your compound microscope. Predict what each
         part does, based on its name and location.
     2. Next, find where the magnification power is engraved on each lens (the eyepiece AND all three
        objectives). It will be a whole number, like 4, or 10, not a decimal.

Skill: Calculate Total Magnification
    3. In your lab notebook, calculate the total magnification for the low, medium and high power
        objectives by copying the chart below. (Use information in the Introduction to help you!)

                      Objective Magnification   Eyepiece Magnification    TOTAL MAGNIFICATION
          low
        medium
          high
Skill: Making a Wet Mount Slide
    4. Cut out a small, lowercase “e” from the newspaper. To make a wet mount slide of this letter,
        place the “e” on a clean slide, and with an eyedropper place 1-2 drops of water on the letter.
        Then hold the coverslip by its edges at an angle of 45 to the slide; one edge of the coverslip
        should be touching the slide. Then lower the coverslip until it is parallel to the surface of the
        slide. Lowering the coverslip at a 45° angle should prevent air bubbles under the coverslip.
   5. Use the revolving nosepiece to turn the low power objective (4X) into place, if it is not already.
      Plug in the microscope and turn on the light below the stage. Place the wet mount slide you
      made on the stage, and clip it down. Move the slide so that the letter is in the middle of the
      stage opening. When focusing a specimen under the microscope, always begin with a LOW
      power scan using the coarse adjustment (the larger focus knob) to focus. The coarse adjustment
      knob raises the stage and slide closer to the objective lens.

   6. Looking through the eyepiece, compare the position of the image of the letter as viewed
      through the microscope with the actual position of the letter on the slide. Make a sketch of how
      the “e” looks on the slide and how the “e” looks through the eyepiece in your lab notebook.

   7. Now move the slide slightly to the right. Notice the direction in which the image appears to
      move. Move the slide left, and again notice the image’s movement. Move the slide toward you
      and away from you and watch the resulting movement of the image. Write a general statement
      about your observations in your lab notebook.
   8. Adjust the light with the disc diaphragm. Some materials are best viewed in dim light; others
      are more easily viewed in bright light. Try all of the different diaphragm openings until you
      find the one that gives you the most light and that you can still see your specimen.
Skill: Focusing the Microscope
    9. Again, when focusing a specimen under the microscope, always begin with a LOW power
        scan using the coarse adjustment (the larger focus knob) to sharpen the image.

   10. When you want to move to MEDIUM power for a closer look:
             a. Move the slide so that the detail of interest is in the center of the field of view.
             b. Use the revolving nosepiece to turn the medium power objective (10X) into place.
             c. Look through the eyepiece and if needed, sharpen the image using ONLY the fine
                adjustment (the smaller focus knob).
       How does the change from low to medium power affect the brightness and the size of the field
       of view? Record your observations in your lab notebook. Remember - some specimens are best
       viewed under medium power; it may not always be necessary to move to high. However…

   11. When you want to move to HIGH power to get a really close look:
             a. Use the revolving nosepiece to turn the high power objective (40X) into place.
             b. Look through the eyepiece and if needed, sharpen the image using ONLY the fine
                adjustment. Check out how close the high power lens is to the slide! Why should
                you NOT use the coarse adjustment under high power??

   12. Repeat steps #9-11 until you are comfortable focusing a specimen under the microscope and
       can explain how to do it. You will be asked to focus a slide on a lab practical test this unit!
       13. Then clean your slide and coverslip and return them to your tray. Use the revolving nosepiece
           to change the lens back to low power (4X).


      eyepiece
  (ocular lens)
                                                                                            revolving
    low power                                                                               nosepiece
 objective lens


         stage                                                                              high power
                                                                                            objective lens

                                                                                            arm

                                                                                            coarse adjustment
disc diaphragm
                                                                                            fine adjustment

 stage opening

    stage clips                                                                             lamp



                                     Figure 1 – Compound microscope




             oculars




                                                                                       adjustment knob
           objectives


    working distance

                                                                                       specimen plate




                                        Figure 2 – Stereo microscope
Skill: Sketching Specimens under the Microscope
    14. Next, obtain a prepared slide of different colored threads crossed over one
        another. Observe the threads under low, medium, and high power,
        adjusting the focus as you do so (remember steps #9-11!). It is
        important to remember that by using the adjustments, you are bringing
        the microscope into focus at many different levels. At each setting, you
        can see only one plane of the object clearly. It is impossible to see all
        parts of the thick object clearly.

   15. Sketch the threads under HIGH power in your lab notebook. All sketches
       should be drawn to scale (as they appear under the microscope).
       Accurate drawings include texture and color. The label includes the
       object or specimen and the magnification as well. Your lab notebook
       sketch should be set up like the example on the right.

   16. Now obtain a prepared slide of a paramecium, a microorganism. Position one paramecium in
       the center of the field of view. Then observe the slide at all three magnifications (beginning
       with the lowest), making a sketch at each magnification in your lab notebook. Be sure to write
       the magnification below each drawing!

Skill: Measurements under the Microscope
    17. Obtain a plastic ruler. With the low power objective in position, place the
        millimeter side of the ruler on the center of the stage so that you can clearly
        see the scale as you look through the eyepiece. Carefully move the ruler
        until one of its vertical lines is lined up with the left boundary of the
        circular field of view. The distance from the center of one line to the center
        of the next is one millimeter (or one thousand micrometers); see Figure 3.
   18. Determine the diameter of the field of view in millimeters (mm) for                        Figure 3 –
       both the low and medium power objectives. Record the measurements in               Microscopic View of a Ruler
       your lab notebook, in mm.
   19. Then convert the diameter into micrometers using dimensional analysis (or unit conversions,
       from Freshman Physics!). Use the conversion 1 mm = 1,000 µm. Show all of your work, like in
       the example below.
               14 mm x 1000 µm         = 14,000 µm
                 1      1 mm

   20. Carefully rotate the high power objective into position. Note that now the diameter of the field
       of view is less than one millimeter! Instead of attempting to measure the diameter of the high
       power field directly, we may derive it mathematically:
       Step One: divide the magnification of the high power objective by the magnification of the low
       power objective
       Step Two: then divide the diameter of the low power field by the number you got in Step One
                                                                           high 50
       Example: 1) If high power were 50X and low power were 10X, then               5
                                                                           low 10
                 2) If we measured the low power field to be 3,000 µm then the high power field
                           3000
                 would be        600 µm
                            5

   21. Knowing how to measure under the microscope, can you find the length of one paramecium??
       Yes, you can! Using the prepared slide of the paramecium, compare the length of the organism
       on the slide with the diameter of the field of view. For example, if the length of the organism
       was one-fourth the distance across the field, and the diameter of the field was 6,000 µm, then
                                                  6000
       the organism would have dimensions of             1,500 µm.
                                                    4
   22. Determine the length of your specimen using both low and medium powers, recording all work
       in your lab notebook.

       Hint: Try to estimate how many paramecia could fit side by side in your field of view. If you
       could fit 8 paramecia head to tail, and the diameter is 2,000 µm, then ONE paramecium is
       2,000 µm divided by 8, or 250 µm. Just try it…



The Stereo Microscope

   1. Now using the stereo microscope, learn the names of the major parts of this type of microscope.
      Use Figure 2 to help you locate each part on your stereo microscope. Re-read the introduction
      about why this microscope is different than the compound microscope.
   2. Plug in the microscope, and turn on the lights. Turn the objective lens to the low magnification.
      Place a transparent ruler on the specimen plate, with the printing on the ruler centered. While
      looking through the eyepieces (ocular lenses), turn the adjustment knob to focus the ruler.
   3. Move the ruler slightly to the right. Notice the direction in which the image appears to move.
      Move the ruler a short distance to the left. Again, watch the image. Move the ruler towards you
      and away from you, and note the corresponding direction in which the image moves. Record
      your observations in your lab notebook.
   4. Change to the high magnification by gently turning the objective, and note how the field of
      view has changed. Record the relationship between magnification and field size.
   5. Now it’s time to view what you want! Examine some material under the stereo microscope.
      This could be your hand and fingernails (which can look pretty gross!), a dollar bill, or a coin.
      What about a lead pencil tip? Check with your instructor if you’d like to view something else.



Care and Storage of the Microscopes

When you finish, follow these directions to properly store your microscope:
      Make sure that all microscopes are stored with the low power objective in place.
     Clean the objective lenses after use – only use lens paper to gently clean the lenses! Other
      materials will scratch them.
     Make sure that the stage is clean and dry. If they’re dirty, you can simply wipe them off with a
      damp (not wet!) paper towel.
     Be sure that you turn off the light and gently wind up the cord after using the microscope.



Analysis Questions

  1. If the ocular lens magnification of a compound microscope is 15X, and the objective lenses are
     4X, 10X and 97X, what are the possible TOTAL magnifications?

  2. When using the compound microscope, what appears to happen to the image as you move the
     slide in different directions? How does this differ with the stereo microscope?

  3. In what way does the change from low to high power affect the brightness and size of the field
     of view in the compound microscope?

  4. When handed a slide, describe the 3 specific steps one should follow when bringing a
     specimen into focus under high power:
            First, find the specimen under _________ power; use the ____________ adjustment.
            Then, switch the objective to ___________ power; use ONLY the _______ adjustment.
            Lastly, switch the objective to __________ power; use ONLY the _______ adjustment.


  5. The organism pictured is in your field of view at 10X.
     Will it be in view at 100X? Why or why not?




                                                                  

  6. How is a wet-mount slide made? Either draw the process or list the steps needed to make a wet
     mount slide of a specimen of interest.

  7. Why do objects have to be cut very thin to be viewed through a compound microscope?

  8. Why should the cover slip be lowered over the specimen at a 45° angle?


  9. Given a cell with the shape shown to the right, which microscopic power would
     probably allow the greatest depth to be viewed at one time?
   10. What type of objects can a stereo microscope view that a compound microscope cannot?




Dimensional Analysis Practice

In Freshman Physics, you learned about “dimensional analysis,” or a way to convert the units of
something into units of something else. For example, if I want to know how old I am in seconds, I can
convert my age in YEARS to my age in SECONDS using a series of unit conversion factors:

              30 yrs x 365 days x 24 hrs x 3600 sec = 946,080,000 seconds!
                1         1 yr     1 day    1 hr

Instructions: Complete the following problems using dimensional analysis. Show ALL work. You
may need to use some of the unit conversion factors below to solve the problems.

       1 m = 100 cm
       1 m = 1,000 mm
       1 m = 1,000,000 μm
       1 cm = 10 mm
       1 mm = 1,000 μm


   1. When viewing an object through a compound microscope using LOW power, the diameter of
      the field of view is only 0.0015 meters (m).

              a) What is the diameter of the field of view in millimeters (mm)? Show your work!



              b) What is the diameter of the field of view in micrometers (μm)? Show your work!




   2. On the other hand, when viewing an object through a microscope using HIGH power, the
      diameter of the field of view is 0.0175 centimeters (cm).

              a) What is the diameter of the field of view in millimeters (mm)? Show your work!



              b) What is the diameter of the field of view in micrometers (μm)? Show your work!
3. How many human red blood cells could be placed side by side along the diameter of a field of
   view measuring 1.25 mm? The average human red blood cell has a diameter of 7.5 μm. Show
   your work!

				
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