Exercise 4 - The Microscope - Lake-Sumter State College | … · · 2013-11-06Exercise 4 - The...

Lake-Sumter State College, Leesburg Laboratory Manual for BSC 1010C 31

Exercise 4 - The Microscope Introduction The microscope is an essential tool in modern biology. It allows us to view structural details of organs, tissue, and cells not visible to the naked eye. This laboratory exercise is designed to demonstrate some of the potential uses of various types of light microscopes and to help you become familiar with proper microscopic techniques. Materials Equipment compound microscope dissecting microscope microscope slides coverslips droppers lens paper forceps toothpicks

Biological Specimens Allium (onion) pond water Prepared Slides newspaper print colored threads Paramecium

Reagents IKI methylene blue Detain (or Protoslo)

Part A: Care and Use of the Compound Microscope

ALWAYS CARRY THE MICROSCOPE UPRIGHT WITH TWO HANDS, ONE ON THE BASE, THE OTHER ON THE ARM

MAKE SURE YOUR WORKBENCH IS FREE OF CLUTTER BEFORE YOU PLACE THE MICROSCOPE ON THE BENCH

DO NOT DRAG OR SHOVE THE MICROSCOPE ACROSS THE LAB BENCH – ALWAYS LIFT TO MOVE OR TURN IT

The steps on the next few pages represent the correct procedure for viewing a specimen under a compound microscope. Your instructor will demonstrate the proper use of the microscope as well as describe its features. Refer to Fig. 4.1 to familiarize yourself with the parts of the microscope as you study each step in the procedure.

Exercise 4 –The Microscope


Fig. 4.1 The Compound Light Microscope



Viewing a Specimen with a Compound Light Microscope Procedure

1. Clean the slide and coverslip by rubbing them gently with lens paper 2. Use the coarse focus adjustment knob to maximize the working distance (the distance between

the stage and the objective lens) 3. Rotate the revolving nosepiece into position with the scanning power (4x) objective lens in the

viewing position 4. Center the slide holder of the mechanical stage on the microscope stage 5. Place the slide between the stage clip and push it all the way back to the bar 6. Plug in the microscope and turn on the light switch 7. Using the mechanical stage drive knobs, center the coverslip and specimen over the stage

aperture 8. While carefully watching the slide on the stage, use the coarse focus adjustment knob to move

the specimen towards the scanning objective lens until it stops. The stage will come close to the lens but will not touch it

9. Adjust the interpupillary distance until you see a single circle while looking through the microscope with both eyes open. This circle of light is called the field of view

10. While looking through the ocular lenses, turn the coarse and fine focus adjustment knobs of the microscope until you see something you believe is the specimen. Stop. Move the slide back forth using the mechanical stage drive knobs. The item you thought was specimen should likewise be moving back and forth

11. Cover of close the eye that is not looking through the ocular containing the diopter ring. Viewing with only that eye focus using the coarse and fine focus adjustment knobs. Adjust the light using the iris diaphragm adjustment lever and/or the light adjustment. Then close your other eye adjusting the diopter ring on that ocular lens to bring the object into focus

12. Adjust the condenser to the highest position 13. Using the mechanical stage drive knobs, center the specimen of choice in the viewing area 14. These microscopes are parfocal (if one lens is in focus, all other lenses are, at least, close to

focus). In order to change to the next highest magnification, simply rotate the nosepiece to the low power (10x) objective lens

15. These microscopes are also parcentral (if an object is in the center of the field of view for one lens, it will be, at least, close to the center of the field of view at other lenses)

16. Using the mechanical stage drive knobs, re-center the specimen in the viewing area 17. With the low power (10x) objective, use the coarse and fine focus adjustment knobs to focus

the view of the specimen and the iris diaphragm adjustment lever to increase the light intensity on the specimen

18. Re-center the specimen in the field of view. Rotate the nosepiece to the high power (40x) objective lens. Use the FINE FOCUS ADJUSTMENT KNOB ONLY to focus and the iris diaphragm adjustment lever to increase the light intensity on the specimen. If needed, use the light adjustment to provide additional light

19. When removing the slide, rotate the nosepiece so the scanning power (4x) objective is in the viewing position, then use the coarse focus adjustment knob to maximize the working distance

20. After you have completed the laboratory activity, turn the light switch off. Clean all microscope lenses (objective and ocular) with lens cleaner and lens paper



21. Prepare the microscope for storage using the checklist below. Be sure a. The scanning power (4x) objective is in the viewing position b. The mechanical stage has been positioned so the stage arm is flush with the right side

of the stage c. The cord is wrapped securely around the microscope arm d. The stage has been adjusted all the way down e. The condenser has been adjusted all the way up f. The light adjustment is turned all the way down and the light is turned off

Part B: Magnification There is a set of three objective lenses on your microscope. The magnification (or power) of each objective lenses is engraved on the side of the objective. The ocular lens is also normally engraved with its magnification (typically 10x). To determine the total magnification of a specimen, use the following formula:

Total Magnification = Ocular Magnification x Objective Magnification Procedure

1. Use Table 4.1 to record the magnification values for each objective lens and the ocular lens on the microscope

2. Calculate total magnification (using the formula above) for each objective lens and record n Table 4.1

Table 4.1 Total Magnification of Microscope

Objective Lens Name Magnification

Objective Lens Ocular Lens Total

Scanning

Low Power

High Power

Part C: Working Distance and Diameter of the Field of View Part C1: Working Distance Working distance is the distance between the stage and objective lens (Fig 4.2). Because objective lenses vary in lengths, the working distance will change as you switch from one objective lens to the next. In a microscope, as magnification increases, working distance ______________________.



Fig. 4.2 Working Distances with Various Objective Lenses

Part C2: Diameter of Field of View The approximate size of a specimen can be estimated if the diameter of the field of view (DFV) is known. In parfocal microscopes, if we know the magnification and DFV for one objective lens, we can calculate the DFV for a second objective on the same parfocal microscope using the following formula:

M1 x DFV1 = M2 x DFV2

where M1 and DFV1 = magnification and diameter of the field of view, respectively, of objective 1, M2 and DFV2 = magnification and diameter of field of view, respectively, of objective 2. As magnification increases, the diameter of the field of view ______________________ (Fig. 4.3). Fig. 4.3 Diameter of the Field of View (DFV) with Various Objective Lenses

4x 10x 40x



Fill in Table 4.2 for your microscope using the values given for the scanning objective and the above formula. Table 4.2 Diameter of Field of View (DFV) for the Compound Microscope

Objective Lens Magnification DFV (mm) DFV (μ)

Scanning 4 4 __________

Low Power 10 __________ __________

High Power 40 __________ __________

Part C3: Depth of Focus The depth of focus for a particular objective refers to the power of the objective to produce an in-focus image from objects that are slightly different distances away from the objective lens. As magnification power increases, the depth of focus decreases. When viewing specimens under a microscope, it is beneficial to keep in mind that as magnification power increases the microscope’s field of view becomes smaller, thinner, and darker (Table 4.3). Table 4.3 Changes in a Microscope’s Field of View as a Function of Magnification Power

Scanning Low Power High Power

Diameter of Field of View (DFV)

Depth of Focus

Light

Gets Smaller

Gets Thinner

Gets Darker



Part D: Newsprint (dry mount) Procedure

1. Obtain a prepared slide of newspaper print 2. View the newsprint under the microscope using the scanning power (4x) objective

Move the slide slowly to the right as you view the image in the field of view. In which direction do the letters appear to move? Move the slide slowly away from you as you view the image in the field of view. In which direction do the letters appear to move?

Part E: Depth of Focus Procedure

1. Obtain a prepared slide of colored threads. The threads have been arranged to intersect at a single point

2. Focus on the intersection of the three threads first with the scanning power (4x) objective lens and then the low power (10x) objective lens

3. Very slowly rotate the fine focus adjustment knob while looking at the intersection of the threads

Which thread is on bottom? __________ In the middle? __________ On top? __________

Part F: Viewing specimens Specimens are often mounted in water (or other liquids) on a glass slide and then covered with a small thin glass or plastic coverslip to prepare for microscopic viewing. These wet mounts are unstained and sometimes difficult to see. Replacement staining can add color and contrast enhancing the detail of the specimen. It is important to be able to estimate the sizes of different specimens under the microscope. Already knowing the diameter of the field of view for a particular objective (Table 4.2), we can utilize the following formula to estimate size:

size of cell=DFV

# of cells across DFV

At which magnification do you think you are able to get the most accurate estimate of cell number and thus the most accurate estimation of cell size? __________. Why?

Part F1: Paramecium Procedure

1. Obtain a prepared slide of the single-celled protozoan, Paramecium 2. Use the correct focusing technique to find the Paramecium at high power 3. Estimate the # of Paramecium cells required to fill across the DFV end-to-end 4. Use the formula to calculate Paramecium length in microns



5. Estimate the # of Paramecium cells required to fill across the DFV side-by-side 6. Use the formula to calculate Paramecium width in microns

Paramecium cells arranged end-to-end Paramecium cells arranged side-to-side Paramecium Length __________ (in microns) Paramecium Width __________ (in microns) Part F2: Allium (onion) epidermis (wet mount) Procedure

1. Prepare a wet mount of Allium (onion) epidermis 2. Place one or two drops of water on a clean slide 3. Peel the epidermis (thin skin) off the inside of a piece of sliced onion using forceps 4. Place the epidermis carefully in the water on the slide 5. Place a coverslip over the epidermis 6. Observe the cells under the microscope and sketch what you see



7. Stain the onion cells with IKI using the replacement staining technique

a. Place a few drops of IKI on the slide against one edge of the coverslip b. Place the smooth edge of a single layer of paper towel up against the opposite edge of the

coverslip. The paper towel will pull the water out from underneath the coverslip. In turn, the water as it exits will drag the IKI stain underneath the coverslip

c. Continue this process, adding more IKI if necessary, until the stain covers the area under the coverslip

d. Examine under the microscope 8. Observed the cells under the microscope again and sketch what you see

9. Can you see more or less detail after staining compared to the unstained cells? _____________ 10. Estimate the length and width of an onion cell (in microns) Onion Cell Length __________ (in microns) Onion Cell Width __________ (in microns)



Part F3: Cheek Cells (wet mount) Procedure

1. Place one or two drops of water on a clean slide 2. Obtain a clean toothpick and collect cheek cells by gently scraping the inside of your cheek 3. Swirl the tip of the toothpick in the water on the slide (immediately discard your toothpick) 4. Stain your cheek cells with methylene blue stain 5. Place a coverslip over your cheek cells 6. Observe and sketch the stained cheek cells. Identify the nucleus, cytoplasm, and cell membrane

7. How do these cells differ from onion cells?

8. Estimate the diameter of one of your cheek cells (in microns)

Cheek Cell Diameter __________ (in microns) Part G: Pond Water Although staining cells makes it easier to see their detail, most staining techniques also kill any live specimens. Thus, looking at microorganisms can be a challenge. Living microorganisms are also difficult to see clearly because many of them are motile and must be chased around the slide while you are focusing. Procedure

1. Place a drop of pond water on a clean microscope slide. Try to obtain a sample that is near any floating debris and organisms tend to congregate there. Be careful not to shake the jar

2. Add a coverslip 3. Examine under the microscope 4. Try to keep motile microorganism in focus by following them around as they move on the slide.

If they move too quickly, carefully lift up the coverslip and add a drop of Detain (or Protoslo) 5. Draw a few of the critters you see in space provided



Part H: The Dissecting Microscope It is possible to have too much magnification when viewing some specimens. For example, how would you use your compound light microscope to view an entire earthworm? Larger specimens may require lower magnification. For this, biologists use dissecting microscopes (Fig. 4.4). Fill in Table 4.4 and notice the diameter of the field of view in these microscopes is substantially larger than that in compound light microscope. Your instructor will describe the use and features of this microscope.



Fig. 4.4 The Dissecting Microscope



Table 4.4 Diameter of the Field of View for the Dissecting Microscope

Objective Lens Magnification DFV (mm) DFV (μ)

Lowest Power 2 10 10,000

Highest Power 4 5 __________

Procedure

1. Obtain a dissecting microscope using two hands to carry it 2. Identify the parts as per Fig. 4.4 and their functions 3. Observe the various objects made available in lab using the dissecting microscope

Using the information in Table 4.4, complete this sentence for the dissecting microscope As magnification increases, DFV __________ Have you seen this relationship before? __________ Where? __________________________



Practice Problems and Review Questions

1. What is the total magnification of an object if the ocular lens magnification is 20x and the objective lens magnification is 45x?

2. Which objective lens is in place if the object you are viewing is magnified 1000x assuming an ocular lens magnification of 10x?

3. What is the diameter of the field of view (DFV) of a 1000x objective lens if the DFV of a 400x

objective lens is 500 μ? Express your answer in mm.

4. What is the DFV of a 40x objective lens if the DFV of a 10x objective lens is 3 mm? Express your answer in μ.

5. When viewing an organism using the 40x objective lens from question 4, you estimate 6 organisms could fit across the DFV if they were laid end-to-end and 20 could fit is stacked side-by-side. What is the length and width of this organism (in microns)?

6. What is the DFV of a 25x objective lens if the DFV of a 100x objective lens is 1.5 mm?

7. Using the 100x objective lens from question 6, you estimate 12 organisms could fit across the DFV if they were laid end-to-end and 30 could fit is stacked side-by-side. What is the length and width of this organism (in microns)?



8. What is the magnification of an objective lens with a DFV or 0.8 mm if the DFV of a 100x objective lens is 2 mm?

Exercise 4 - The Microscope - Lake-Sumter State College | … · · 2013-11-06Exercise 4 - The...

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