Lab Exercise: Microscopy I, an Introduction to the Compound Light Microscope (Revised, Spring 2012) page 1

Laboratory Exercise: MICROSCOPY I Introduction to the Compound Light Microscope The compound light microscope is a valuable tool to view biological specimens too small to be seen by the naked eye. The compound light microscope uses two sets of lenses and light to magnify the image of the specimen. Often times, the specimen is stained or dyed; these stains or dyes will bind to cellular structures and absorb light which increase contrast allowing better viewing of the specimen. In this exercise, we will demonstrate the proper use of a compound light microscope to look at slides of biological specimens.

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Activity 1: Microscope Care and Storage (The Scope Check) Successful operation of the microscope depends upon the proper maintenance and correct use. Here is a list of rules governing the use of our microscopes. Procedure: Carefully read through the following list of rules regarding microscope care and storage. Record the procedure(s) and answer questions regarding this information in your lab notebook. 1. Always use the microscope reserved for your station or seat number.

You are expected to use the microscope assigned to that seat, located in the same assigned position in the microscope cabinet.

2. Transport the microscope with two hands.

When carrying the microscope, grasp the handle (next to the power cord) and place your other hand under the base as a support. The microscope is always moved in an upright position.

3. Perform a Scope Check. You should always assess the condition of your microscope BEFORE you begin using it. If you microscope was not properly stored, report it to your lab instructor immediately. a) The covered microscope is covered.

b) The microscope should have been placed in its proper numbered location in the cabinet with

the handle visible.

c) The stage should be positioned in its lowest possible position.

d) The scanning objective lens should be in position over the stage.

e) There should never be any evidence of spills on the stage or lenses, no slides on the stage, etc.

f) The light source should be off when the microscope is plugged in to the power source.

g) The light control (rheostat) should be at its lowest position.

h) You should always clean all of the microscope ocular and objective lenses.

i) The electrical cord should be secure.

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4. Clean your microscope.

Only Lens Paper will be used to wipe clean the lenses and other glass portions of the microscope. There is a specific order that the lenses are cleaned with the Lens Paper:

(1) ocular lenses of the eyepiece (2) scanning objective lens (3) low power objective lens (4) high power objective lens (5) oil immersion objective lens.

Each time the lens paper is used on a lens, the microscopist should look at the lens paper and make sure that the lens is clean before moving to the next lens in the order listed above. You may use a drop of Windex, the blue solution, on a Q-tip to help clean the high power and oil immersion lenses as needed. One end of the Q-tip is wetted with Windex and used to clean the High power objective lens, and then the OTHER end of the same Q-tip is wetted with Windex and used to clean the Oil Immersion lens.

Note: Windex is only used to clean the high power and oil immersion objectives.

5. Never remove any item from the microscope. As you use the microscope, you may notice that the eyepieces are movable (do NOT remove these) or any other parts of the microscope are loose.

6. Never force the microscope to turn or move.

If the microscope does not move or turn as you expect it should, notify the instructor immediately.

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Activity 2: Microscope Parts and Their Function In today’s lab activities, you will learn the different parts of the microscope and then practice using the microscope. The following is a brief description of the most commonly used parts of the microscope. Procedure: Use the labeled diagram of the microscope at the front of this exercise and the following descriptions to complete your lab notebook for this activity. Descriptions and diagrams are to be done before arriving to lab. 1) Eyepiece (contains the ocular lenses). The eyepieces contain lenses that contribute to the

overall magnification of the image of the specimen. The eyepieces will move to adjust for individual interpupillary distance. You should always have these adjusted at a distance which allows you to view a perfect circle of light when using the microscope—any other shape indicates the eyepieces are too close together or too far apart for comfortable viewing.

2) Body tube: holds the eyepiece at one end and the nosepiece at the other end. The light rays are conducted from the objective lens to the eyepiece.

3) Arm: supports the upper parts and contains the carrying handle. 4) (Revolving) Nosepiece: holds and rotates the objective lenses.

a. Scanning Power Objective Lens: This is the shortest of the objective lenses attached to the revolving nosepiece. The scanning objective lens should always be in position over the stage when the microscope is not in use. Please make sure that the scanning objective lens is secured in position over the stage.

b. Low Power Objective Lens: This lens is slightly longer than the scanning objective lens and

you can view the specimens in greater detail.

c. High Power Objective Lens: This lens is slightly longer than the low power objective lens. We will use this objective lens to view many specimens in even greater detail.

d. The largest objective lens is the oil immersion lens. We may be using this lens in future lab

activities and it requires special procedures that will be described at that time. The oil immersion objective lens is commonly used to view smaller specimens like bacteria, and extremely small cellular organelles like cilia, flagella, etc.

5) The total magnification of an image of your specimen is determined by multiplying the ocular magnification by the objective magnification. You will find the magnification printed on the side of the lens beside the ‘X’. During lab, record the lens magnification, the color of the band on each objective lens and calculate total magnification when using each objective lens in your lab notebook.

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6) Stage: The stage is beneath the

objective lenses. The stage functions to hold and support the slides for viewing.

7) Stage clip: (label 3) spring-action clip

that sits gently on the corner of the slide to hold it in place on the stage. To move the stage clip, move the ‘bump’ labeled 1.

8) Mechanical stage: (label 4) the slide sits

into this ‘L’ shaped apparatus which can be moved allowing you to view various areas on a slide.

9) Mechanical stage knobs: these knobs

extend down from the mechanical stage. By turning the two knobs, one moves the mechanical stage (slide) right and left, while the other knob moves the mechanical stage (slide) forward and back.

10) Coarse-focus knob: (Label 2) The Coarse-focus knob is used with the lower power objective

lenses only. It is used to bring the objects or specimens into approximate focus. 11) Fine-focus knob: this knob extends from the center of the coarse-focus knob. It is used with the

high power objective lenses only and will bring the specimens into final focus. 12) Condenser: Beneath the stage, there is a cylinder which contains lenses to help focus the beam

of light on the object or specimen you are viewing. 13) (Iris) diaphragm lever: extending from the condenser is a flat lever that moves left and right. This

lever helps to adjust the amount of light that passes through the condenser onto the specimen. 14) Light source: an attached lamb that directs the beam of light up through the condenser and the

specimen.

a. The power switch is rocked on or off to turn on or off the light. b. The rheostat adjusts the intensity of the light ranking the intensity 1-6 (the least intense

is ranked 1 through the brightest light intensity ranked 6). Most prefer the light intensity to be adjusted at about 4-5.

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Activity 3: Focusing Materials: Compound Light Microscope, Letter ‘e’ slide In order to successfully focus on a slide, the microscopist must follow procedure. The procedure is designed to maximize efficiency so each student can view a slide easily and quickly. Procedure: Review the following protocol

for preparing to focus and focusing your microscope. In your own words, answer the questions and write a brief description of the procedure to focus in your lab notebook prior to lab. Every time you use your microscope it is important that you follow this protocol. 1) Check your microscope to make sure that it was stored properly (perform a scope check). 2) After the scope check, in order to focus your microscope, your scanning objective lens should be

snapped into position over the stage. 3) Use the coarse-focus knob to lower the stage to the lowest position possible. 4) Plug in your microscope into the nearest electrical outlet. 5) Turn the power to the ‘ON’ position. 6) Rotate the rheostat to adjust the light brightness. Most students prefer 4-5 on the rheostat dials. 7) Make sure that the iris diaphragm lever is moved, such that, light is visibly passing through the

stage. 8) NOW peer through your eyepieces. Adjust the distance between the eyepieces so that you see a

single perfect circle of white light. Move the eyepieces slowly and hold your head still. (If you see an oval or ‘8’ shape then the eyepieces are too close together or too far apart).

9) Gently clean the slide you plan to view with Kimwipes by gently wiping the surface of the slide.

10) With the scanning objective snapped into position over the stage, and the stage in the lowest

position possible, press on the stage clip and place the letter ‘e’ slide in the mechanical stage letting the stage clip gently come to rest on the corner of the slide.

11) Use the mechanical stage knobs to move the letter ‘e’ into position over the light passing through

the stage. 12) Use the coarse focus knob to raise the stage to the highest position possible.

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13) Looking through the eyepieces, using the coarse-focus knob, lower the stage slowly until the letter ‘e’ comes into focus.

14) Use the mechanical stage to center your letter in your field of view (circle of light you see as you

look through the eyepieces). 15) Rotate the revolving nosepiece so the low power objective lens is snapped into position over the

stage. 16) Looking through the eyepieces, using the coarse-focus knob, slowly focus your letter ‘e’ 17) Use your mechanical stage knobs to move some part of the ‘e’ into the center of your field of view. 18) Rotate the revolving nosepiece so that the high power objective lens is snapped into position over

the stage. The ability of your microscope to stay in relative focus as you change objective lenses, change magnifications, is described as being parfocal.

19) Looking through the eyepieces, using the fine-focus knob, focus again on the letter ‘e’.

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Activity 4: Inversion As the light travels through the microscope, the lenses bend the light of the image to magnify the image which allows us to see the image inverted or in a different orientation.

Procedure: Follow the directions below during lab and record the indicated information in your lab notebook. Upon completion, answer all remaining questions in your notebook. 1. You should already have the letter ‘e’ slide on your microscope stage. Rotate the nosepiece so

that your scanning objective is snapped into position. Draw a picture of your letter as you see it through the microscope in your lab notebook.

2. Now, lower your stage to the lowest position possible, using the coarse focus knob. 3. Remove the slide from the stage and look at the letter ‘e’ using your naked eye(s). Draw a picture

of the letter ‘e’ in the space provided above in your lab notebook. 4. Return the letter ‘e’ slide to the stage. 5. Working in pairs, one student should look through the eyepieces and view the letter’e’ using the

mechanical stage knobs to move the letter ‘e’ right/left and forward/back. The other student should observe the direction the slide moves while the microscopist is moving the mechanical stage. Record your obaservations.

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Metric Ruler: inches below and centimeters above divided into smaller increments of millimeters

Ocular micrometer is viewed through one eyepiece

Activity 5: Estimating Cell Size Human body cells range in size from very small, red blood cells and sperm, to much larger cells like epithelial cells that line the inside of your mouth. We can use the microscope to estimate average size which is often helpful in identification of cells and/or may aid in indication of homeostatic imbalance. We will be using microscopes that have an ocular micrometer (it looks like a miniature metric ruler) in one eyepiece which can be calibrated to make microscopic measurements. Materials: letter ‘e’ slide, calculator, compound light microscope with an ocular micrometer Procedure: Using the Ocular Micrometer to Estimate Cell Size As you follow these steps during lab, be sure to record your observations and calculations in your lab notebook. 1) Look through your microscope, open and close each eye individually, to find the eyepiece that has

the ocular micrometer. 2) The compound light microscope magnifies the image of a specimen, when we try to measure the

size of the specimen it is unrealistic to use units like millimeters or inches. A realistic measuring unit is micrometers.

1000 micrometers (µµµµm) = 1 millimeter (mm)

Micrometers are very small units of length. If you take a meter stick (1 meter or about 39 inches) it can be divided into 100 equal units which are the centimeters. Check out your metric ruler—you can see about 15 cm on the metric ruler. Each centimeter is divided into 10 equal units called millimeters. Now, try to imagine, dividing each millimeter into 1000 equal units—now we’re talking

micrometers (µm)! Note: Whenever you make a measurement, the measurement does not have meaning unless the units of measurement are also reported. For example, miles, feet, meters, milliliters, grams, ounces, etc.

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Since the ocular micrometer does not change size as objective lenses change, the units represented by the ocular micrometer does change. Your ocular micrometer has been calibrated so each unit has a known length in micrometers.

3) Record the conversions used for estimating cell size for each objective lens into your lab

notebook before lab. You will be required to estimate cell size in future lab activities and these conversions are necessary for those activities.

Objective Lens Conversion

Scanning Objective 1 ocular unit = 25 µm or 25 mcm

Low Power Objective 1 ocular unit = 10 µm or 10 mcm

High Power Objective 1 ocular unit = 2.5 µm or 2.5 mcm Oil Immersion Lens 1 ocular unit = 1 µm or 1 mcm

4) Calculate (estimate) the size of the letter ‘e’ using your scanning objective lens.

a. Use your mechanical stage knobs to move the letter ‘e’ beneath the ocular micrometer.

b. Draw the letter ‘e’ you are viewing in the space provided of your lab notebook. c. Use the ocular micrometer to determine the diameter (distance across) the letter ‘e’.

Record the distance across your letter ‘e’ into your notebook in the space provided for the calculation.

d. Multiply your measured ocular units by the conversion factor from the table above. Because

you are viewing your scanning objective lens you must use 25 µm as the factor.

e. The product of your multiplication is your estimated size, in micrometers, of the letter ‘e’.

f. Describe this procedure and show the supporting work, with labeled units, in your lab notebook.

5) Calculate (estimate) the size of the letter ‘e’ using your low power objective lens. Repeat the

procedure described above, however, you must use the appropriate conversion factor for the low power objective lens. Record your calculations and measurements in your lab notebook.

6) Try to calculate (estimate) the size of the letter ‘e’ using your high power objective lens. Repeat the procedure described above, however, you must use the appropriate conversion factor for the low power objective lens.

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Activity 6: Depth of Field Materials: Compound Light Microscope, Thread slide. Depth of field refers to the vertical distance that remains in focus at one time. In this experiment we will observe the changes in depth of field at the intersection (overlap) point of three different colored threads using different objective lenses. Procedure: Determining Depth of Field As you follow through these steps, record your observations in your lab notebook.

1) Obtain a prepared slide of three different colored threads.

2) For each objective, focus on the intersection point of the three threads; this is where the three threads cross each other.

3) Record the number of threads you can clearly see in focus for each objective lens in the

appropriate cell of the table in your lab notebook. If the number of threads that you see is less than three, try adjusting the focus slightly—can you see different threads in focus now?

4) As you begin to focus on any slide, you begin with either scanning or low power objective lens

in position over the stage and the stage is in its highest position possible. Focus requires the stage to be lowered into the focal point. The first part of the specimen or thread that passes through the focal point will be the first thing that comes into focus. Use this information to answer the questions in your lab notebook and determine the order of the colored threads on the slide.

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Activity 7: Observing Human Epithelial Cells

The first human body cells we will be observing will be human epithelial cells from the inside of our oral cavity. In order to view our body cells they require stain (dye) that increases the contrast between the cellular structures. Materials: (clean) slide, Methylene blue (dye), cover slip, sterile tooth picks

Lab Safety If you get methylene blue on your skin, wash with soap and water. Use care when slides. Review the location of the broken glass container. Procedure:

1) Read through this entire procedure before Lab. All histology drawings should be complete prior to lab—as you read through this procedure before lab, be sure to draw a picture of the cheek epithelial cells you will be viewing, include labels in your drawing in your Histology Atlas.

2) Gently scrape the inside of your cheek with a flat toothpick. 3) Transfer the epithelial cells from the toothpick to the center of a clean/dry microscope slide. Try

to spread the cells evenly on the central section of the slide over an area no larger than a dime.

Place the used toothpick in the biohazard container.

4) Allow the slide to dry completely; molecules in the saliva will act as an adhesive sticking most

of the cells to the glass slide. 5) At this point, your instructor may ask you to view your unstained cells microscopically under

high power.

6) Place a few drops of methylene blue over the cells. Leave the stain on the slide for 2-3 minutes do NOT allow the stain to dry.

7) Remove the excess stain from the slide by rinsing the stain off the surface of the slide by

gently squirting water from the water bottle as your hold the slide at a 45° angle over the sink. Continue to gently rinse until the water flowing over the area with the cells runs clear.

8) Allow the slide to dry completely. 9) Do NOT use a coverslip. View your stained slide microscopically using your 40X objective

lens.

10) Compare your stained cells to the labeled photograph and your drawing in your lab notebook’s Histology Atlas.

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11) Use your ocular micrometer to estimate the average cell size of a single cheek epithelial cell.

Show your work/calculation in the space provided within your lab notebook. You may want to confirm with your instructor that your measurement and calculation are reasonable estimations of your cell size.

12) Dispose of your slide in the biohazard container when you have completed your observations and measurements.

13) Review the microscope storage procedure. Return your microscope to the microscope cabinet

upon completion of today’s activities. Note: You are responsible for the condition of your microsope, make sure that you have cleaned and stored it properly.

At the end of this exercise you should have answered all of the questions/definitions; recorded all of your measurements, observations and calculations in your lab notebook. Write a conclusion that provides a summary of to day’s activities, this must be completed before the next scheduled lab meeting.

Nucleus is an organelle which is usually centrally located and stains darker than the rest of the cell.

Cell membrane is a structure that makes up the outer boundary of the cell (separates the inside from the outside).

Cytoplasm includes the contents of the cell—it includes everything except the cell membrane and the nucleus.