LAB 4

THE USE OF THE MICROSCOPE

 

Because biological objects can be very small, it is often necessary to magnify them in order to observe and study them. Cells, the basic organizational unit of life, are almost always viewed microscopically. This is the overall function of the microscope. The microscope is then a major tool of the biologist.  Without it, many advances in the scientific investigation of biology would not have been made and we would lack most of our present knowledge of living things too small to be seen with the unaided eye.  For instance, the cell theory would not have been discovered without the aid of the microscope.

 

Such a device composed of a system of lenses was first constructed by the Jansen Brothers of Holland, circa 1590.  The best of the early "microscopes" had only a single lens and was built and used by Anton van Leeuwenhoek, often referred to as the Father of Microscopy.  Observe figure 1: Leeuwenhoek's microscope, circa 1673.

 

 

Question 1:             What is microscopy?

 

 

 

A number of techniques have become available for obtaining greater magnification and for visualizing objects in different ways.  Such techniques led to the invention

of several types of microscopes. 

 

1.      Light microscope: uses light rays that are magnified and concentrated by means of lenses. 

a.      dissecting microscope: designed to study thick objects at low magnification. 

      A 2.     Compound light microscope has three lenses (hence the name compound) and is used for the observation of small and/or thin objects under higher magnification.  Objects must be thin enough for light to pass through them from below. You will be using a compound light microscope in this lab.

 

 

3.     Electron microscope: uses a beam of electrons magnified and focused on a photographic plate by means of electromagnets. 

4.     Dark-field microscope: the illuminating light enters from the side and is reflected off the object being observed.  The object appears bright against a black background.

5.     Phase-contrast microscope:  This microscope operates on a principle whereby differences in thickness and composition of transparent objects are converted to visible differences.  Phase-contrast microscopy is useful in observing living objects.

 

            Other types of microscopes include dissecting, reflecting, polarizing, x-ray, fluorescence and ultra-violet.

 

Question 2:            What is the overall function of the microscope?  

 

LAB OBJECTIVES: 

       

            To identify the parts of the microscope, describe their function, requirements for and practice of proper usage and storage.

            To gain through practice and repetition skill in locating, focusing, and observing a prepared and fresh specimens.  

            To prepare and observe a temporary wet mount..

            To calculate the total magnification and field size. 

 

I.          Handling the microscope.

 

The microscope is an expensive, delicate and valuable tool which must be used correctly to avoid damage and/or inefficiency of operation.  Be careful!

 

            Expectations for care and use of the microscope are explained below.   Listen and observe carefully as your instructor identifies each part and describes its function.  You are expected to comply with these practices.  Failure to do so may affect your lab grade. Figure 2 will help you in  locating the parts of the microscope.

 

A.     When removing the microscope from its cabinet space, use TWO hands.  One grasps the arm of the microscope and the other provides support beneath the base. 

            B.      Slide the microscope straight from the shelf.  Raising or tilting may damage the ocular to striking the top or sides of the shelf. 

            C.      Before operating the microscope, check for slides which may have been left on the stage.  Please turn in slides left on the stage to your instructor.

            D.      Begin by cleaning all lens surfaces only with special lens paper.  (A Kimwipe is NOT lens paper!) 

            E.      You may adjust the height of your chair so that you can look into the oculars easily.  

            F.      Keep both eyes open. Adjust the distance between the ocular while looking through them until you see a single circle.

            G.      When you have finished using the microscope:

1.       Remove slide from stage and return to proper slide tray.    

2.       Wipe all lens surfaces with lens paper. 

3.       Rotate the nosepiece until the 4X objective clicks into place 

4.       Turn light source off 

5.       Set stage and objectives at their closest distance

6.       Unplug the microscope by the base of the plug  and secure cord around the base of the microscope 

7.       Carefully return it to the microscope cabinet, matching the number marked on the shelf with the number located on the backside of the arm of the microscope.

                               

 

II.          Parts of the microscope. 

Your instructor will assign you a microscope.  RECORD your microscope number.  You will use the same microscope for the entire semester.

 

 

            The kind of microscope you will be using is a compound‑light microscope.  Using figure 2, locate and learn the following parts of your microscope and their functions.

 

 Figure 2: Your instructor will help you identify and label the major parts.

 

              A.      Eyepiece or ocular:  Top most series of lenses; magnifies by a power of 10.  A microscope with one ocular is a     

                        monocular microscope.  Your microscope is binocular.  The oculars may is adjusted to fit the distance between your

                        eyes. 

              B.      Body tube:  Holds the nosepiece at one end and the eye piece at the other end; conducts light rays. 

              C.      Arm:  Supports upper parts and provides a carrying handle.

              D.      Nosepiece:  Revolving disc that holds objectives. 

              E.      Objectives:  

                            4X objective: scanning objective; holds 4X lens used locate object on the slide. 

2.           10X objective: low power objective. 

3.           40X objective: High 'dry'  objective; used to view objects in greater detail. 

            100X objective: Oil immersion objective holds 100X lens and used with a drop of immersion oil; for viewing             

           extremely small objects in greater detail such as bacteria, gametes, etc.  This objective will not be used in this course. 

            F.        Stage:  Holds and supports the microscope slides.  Slides are placed flat on this surface. 

            G.       Stage clips:  Clips on the stage are used to hold the slide in place while viewing and should always be 

                        used.

                        Coarse adjustment knob:  Knob used to bring object into focus, and for making large adjustments in focus. 

                        Never use this knob with the 40X objective.

             I.         Fine adjustment knob:  Knob used to bring object to a clear final focus and for making very small adjustments in

                        focus.  Never use this knob with 4X or 10X objectives unless the image is not sharp - then turn it only slightly. 

            J.         In-stage condenser:  Lens may be seen through the hole in the center of the stage; this produces a cone of light 

                        which illuminates the object. 

            K.         Iris diaphragm:  The opening which controls the amount of light entering the condenser.  A lever extends forward 

                         beneath the stage; moving the lever changes the size of  the opening of the iris which either increases or  decreases

                         the amount of light entering the in-stage condenser.   

   Light switch:  For turning illuminator on and off.

                        Illuminator:  Located in base of microscope, and provides a light source for viewing objects.  

                                                                                                             Base:  The bottom of the microscope which supports all parts above.

 

 

PROCEDURE:

 

III.         Getting started.  (Your instructor will assign you a microscope number.  You will use this scope throughout the semester.)

             A.  Setting up and using the microscope:   

                       1.      Wrap the cord around the gas jet, plug in your microscope and make the following observations: 

         

           2.       Rotate the nosepiece until it clicks and note how each objective may be aligned with the hole in the center of the stage, consequently aligning it with the eyepiece.  When an objective is thus aligned, it is in proper position for use. 

 

Question 3:            How can you tell when an objective is in the proper position?  

 

          3.    Spread the oculars as far apart as possible.  While looking into one ocular with both eyes open, slowly move the ocular together until you see a single circular bright field of light.  (Do this every time to get out your microscope.  This allows a 'personal fit' to reduce eye strain and increase your success!) The bright circle seen when an objective has clicked into position and the light is on is called the field of vision or the visual field.  When observing an object, the image will appear in this field.  When increasing magnification by rotating to a higher objective, center the specimen in the field of vision.                           

 

          4.       Locate the lever that operate the iris diaphragm.  Now, while looking through the oculars,  move the lever and note how the light intensity changes. (This is one of several ways you have to regulate the light.)

 

B.      Microscope slides:

 

The specimen is placed on a glass slide and covered with a cover slip.  You will encounter two types of slides:  prepared slides which are commercially prepared and temporary wet mounts which you will prepare.

Microscopists and tissue culturists prepare slides that are sold commercially. The specimen is stained and embedded in wax.  Thin slices are cut, placed on a slide and sealed beneath a protective cover slip.  The slide is labeled with the name of the specimen and the type of section, identified as w.m., c.s., or l.s. mount (whole mount, cross section, and longitudinal section).  Wet mounts are freshly prepared slides assembled for "on the spot" use. 

 

Question 4:            What is meant by the type of section?  

 

C.      How to Prepare a Wet Mount: 

At this point you should be prepared to place an object on the stage, locate the image and observe other features of the microscope.  Before proceeding, read the following section on the preparation of a wet mount.  Prepare a wet mount following this procedure and referring to figure 3.

 

1.       Obtain a clean slide and cover slip. 

2.       Place a drop of liquid material on the slide and cover with a cover slip. If the material is dry, place the dry material directly on the slide and add a drop of water, 

           then cover with a cover slip. (Remove excess liquid by holding absorbent paper to the edge of the cover slip; this will soak up excess water.)

                      3.       Discard wet mounts as directed by the instructor. 

4.       Prepare a wet mount of a lower case letter e cut from the newspaper.  Return to your microscope and continue with your microscopy. 

          Your instructor may choose for you to observe a prepared slide of the "Letter e".

 

5.       D.  Focusing the microscope:

 

          1.  ALWAYS begin with the 4X SCANNING objective in place and the light on. 

1        2.  Place the slide of the 'Letter e' on the stage securely between the stage clips.

tops.  3.  Center the e in the opening of the stage so it is illuminate by the light. 

(3)      4.  While looking through the oculars, turn the coarse adjustment knob to focus the

                 letter e.

           5.   Looking through the oculars, adjust the iris diaphragm to maximize the image. 

(5)       6.   If the object appears to be blurred, make the image sharp by using the fine

                 adjustment knob.

 

  7.  Now, carefully rotate the nosepiece until the 10X objective snaps into place. 

        adjust only the fine focus.

c.         

                   Inversion:  Look at the letter e as it appears on the slide (not through the    

                   microscope). 

(                  In the space below, draw the letter e as it appears on the slide. 

 

   

 

 

 

       

            In the space above, draw the letter e as it appears when you look  through

            the microscope. 

  

Question 5:            8.  What differences do you notice in the appearance of the letter e through the         

                                     microscope and with the unaided eye?  

 

Question 6:           

 

                               

 Question 7:           While looking through the ocular, move the slide to the right.           Which direction does the image move?

 

Question 8:      Move the slide toward you.  Which way does the image move? 

 

 Question 9:            What is the difference between the object and the image? 

 

 

Compound light microscopes are  are said to be parfocal, i.e., once the object is in focus with 10X, it should also be nearly in focus with 40X.  You will be able to see only a portion of the letter e using the 40X objective. 

 

 

9.      Rotate the 40X objective into place by turning the nosepiece. 

10.    If any adjustment is needed (image not sharp - blurred), use the fine adjustment knob.  With 40X use only the fine adjustment knob. 

11.   On your drawing of the letter e as it was observed with 10X, draw a circle around  the portion of the letter that you are now seeing with 40X objective.

12.   When you have finished your observations of this slide or any slide, rotate the nosepiece until the 4X objective is in place and then remove the slide from the stage.  Return the slide to the proper tray.                                     

 

Question 10:         Why are you able to see only a portion of the letter e and not the entire letter? 

 

E.      Total Magnification

 

Obviously, the image is many times larger than the actual size of the object being observed.  How much larger depends on the magnification power of the ocular lens and the magnification power of the objective lens being used.

 

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

The magnification of your ocular lens is 10X.

Question 11:         Determine the total magnification of the following:

 

 

 

F.      Depth of Focus    

 

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

 

1.       Obtain a slide of 'Colored Threads'.  Begin with the 4X objective and work up to the 10X.  Find a point where the threads cross.  Slowly focus up and down.  Notice that when one thread is in focus, the others seem blurred.  Determine the order of threads and record the colors in the space below. 

 

 

 

2.       Switch to 40X and notice that the depth of focus is more shallow.

 

Question 12:         What is the advantage of the depth of focus aspect of the microscope? 

 

 

IV.        Observing Biological Objects or Specimens

 

            The pioneer biologist, Robert Hooke, was using Leeuwenhoek's microscope when he discovered the cell.  Observing a piece of cork with the microscope, Hooke noticed that the cork was not one piece of material.  Instead it was composed of tiny units which reminded him of individual rooms in a monastery called cells.  Hooke cleverly called these tiny units of cork cells.  The cell is the basic unit of life.

 

            Some living organisms are composed of only one cell and are termed unicellular.  Multicellular organisms are composed of several to millions of cells.

 

            Now you are ready to practice using your microscope. Be patient.  Use of the microscope is an art!  The more your practice the more skillful you will become. 

    

A.      Human Epithelial Cells  (Refer to figure 3 on how to prepare a wet mount.)

 

          Gently scrape the inside of your cheek with a clean, flat toothpick and rotate the toothpick in a drop of iodine.   Observe under the microscope. You are examining your own squamous epithelial cells.   Locate the nucleus, a central round body in each cell.  The area surrounding the nucleus is the cytoplasm of the cell.  Refer to figure 4. 

 

Question 13:  In the space provided, sketch a few cheek cells and label the nucleus, the cytoplasm, and the cell  membrane.    

 

 

 

            B.      Elodea Plant Cells

 

                      Prepare a wet mount of an Elodea leaf.  Elodea is a common aquarium plant.  Beginning on 4X, work your way up to the 40X objective.  Focus.  Compare your Elodea cells with figure 5.  Notice the rectangular boundaries of each cell.  This is the cellulose cell wall, typical of all plant cells.

 

Question 14:         Did you see cell walls in your cheek cells?

 

         Observe the green football-shaped structures inside of each of these cells.  These are chloroplasts containing chlorophyll.  Clan you see them moving within the cytoplasm?  This movement is a feature of all living things.

 

 

Question 15:                 What would you say is the function of these structures?  Are you able to locate a nucleus?

                               

                                    C.  Onion Plant Cells

                                    Prepare a wet mount in a drop of iodine of the skin from an onion.  Separate only one layer of onion 'skin' from the onion and 

                                    apply to the slide.  and observe with 10X objective.

 

Question 17:             How do these cells differ from the cells of the Elodea leaf? 

 

            C.          D.  Examination of Pond Water

                           Now this is the fun stuff!  Prepare a temporary wet mount using a drop of pond water.  Who know what lurks within? 

 

              Using a pipette, add a drop of pond water to the center of the slide.  Cover the drop with a cover slip but to avoid air bubbles    

              lower one end of the cover slip so that it touches one side of the drop of water.  After the water has spread across the edge of the

              cover slip, carefully lower it on the slide to eliminate air bubbles. 

 

              Under the 10X objective and with reduced light, make a survey of the drop of pond water.  Identify as many of the organisms as    

              you can.  How many different structures do you see? what methods of movement.  Figures 6a, 6b, 6c, and 6d should help you

              identify what you see.

 

 

          WHEN YOUR WORK IS COMPLETED, CLEAN AND DRY ALL SLIDES USED.  DISCARD THE COVER SLIPS.

 

Question 18:         Sketch at least 3 different organisms observed in your sample of pond water.

 

 

 

Question 19:         When scanning for an object, which objective should you use?

 

Question 20:         List some similarities among the cheek cells, Elodea cells, and organisms found in the pond water.

 

 

 

 

 

Question 21:         Using the table below, note differences between plant and animal cells.

 

 

 

 

CLEAN YOUR WORK AREA AND RETURN YOUR MICROSCOPE TO THE CABINET MATCHING THE NUMBER OF YOUR MICROSCOPE WITH THE NUMBER ON THE SHELF.  REFER TO THE EXPECTATIONS ON HANDLING THE MICROSCOPE.

 

 

                                                                              

Amoeba	Paramecium	Stentor

 

                                                       

 

 Figure 6a:  Protozoans commonly found in pond water