BIO170 General Biology Freeman/Mac Leod FMCC Spring 2014

THE COMPOUND MICROSCOPE AND MAKING WET MOUNTS Objectives: After completing this exercise, you should be able to do the following:  Identify the parts of compound microscope.  Know the correct procedure for using and caring for the compound microscope.  Understand the concept of depth of focus.  Know the correct procedure for preparing a wet mount.  Describe features of a human cheek cell. Introduction: According to cell theory, the cell is the fundamental biological unit, the smallest and simplest biological structure possessing all the characteristics of the living conditions. All living organisms are composed of one or more cells, and every activity taking place in a living organism is ultimately related to metabolic activities in cells. Thus, understanding the processes of life necessitates an understanding of the structure and function of the cell. The earliest known cells found in fossilized sediments 3.5 billion years old (called prokaryotic cells) lack membrane-bound nuclei and membrane-bound organelles. Cells with a membranebound nucleus and organelles (eukaryotic cells) do not appear in the fossil record for another 2 billion years. But the eventual evolution of the eukaryotic cell and its internal compartmentalization led to enormous biological diversity in single cells. The evolution of loose aggregates of cells and colonies of connected cells provided for specialization, so that groups of cells had specific and different functions. This early division of labor included cells whose primary function was locomotion or reproduction. The evolution of multicellularity appears to have originated more than once in eukaryotes and provided an opportunity for extensive adaptive radiation as organisms specialized and diversified, eventually giving rise to fungi, plants and animals. This general trend in increasing complexity and specialization seen in the history of life will be illustrated in this lab exercise. Given the fundamental role played by cells in the organization of life, one can readily understand why the study of cells is essential to the study of life. Cells, however, are below the limit of resolution of the human eye. We cannot study them without using a microscope. The microscope has probably contributed more than any other instrument in the development of biology as a science and continues today to be the principal tool used by biologists. There are four types of microscopes used by biologists: compound microscope, stereomicroscope, scanning electron microscope and transmission electron microscope. You will learn how to use the compound microscope in today’s laboratory. Biologists in numerous subdisciplines use microscopes: genetics, molecular biology, neurobiology, cell biology, microbiology, evolution and ecology. The knowledge and skills you develop today will be used and enhanced throughout this course and throughout your career in biology. It is important, therefore, that you take the time to master these exercises thoroughly.

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BIO170 General Biology Freeman/Mac Leod FMCC Spring 2014 The microscope is designed to make objects visible that are too difficult or too small to see with the unaided eye. There are many variations of light microscopes, including phase-contrast, dark field, polarizing, and UV. These differ primarily in the source and manner in which light is passed through the specimen to be viewed. The microscopes in biology lab are usually compound binocular or monocular light microscopes. Compound means that the scopes have a minimum of two magnifying lenses (the ocular and the objective lenses). Binocular microscopes have two eyepieces, monocular microscopes have only one eyepiece, and light refers to the type of illumination used, that is, visible light from a lamp.

A. Care and Handling of the Compound Microscope There are only a few ABSOLUTE rules to observe in caring for the microscopes you will use. Taken care of, these instruments will last many decades and continue to work well. Please report any malfunctions immediately to your instructor. 1. ALWAYS use two hands to carry the scope - one on the arm and one under the base - NO EXCEPTIONS! NEVER carry the scope upside down, for the oculars can and will fall out. 2. Use lens paper to clean all lenses before each lab session and after using the oil immersion lens. DO NOT EVER, NOT NOW, NOT EVER, USE ANYTHING BUT LENS PAPER TO CLEAN THE LENSES. Other papers are too impure and will scratch the optical coating on the lenses. Also, do not use any liquids when cleaning the lenses - LENS PAPER ONLY! 3. Always use the proper focusing technique to avoid having the objective lens come in contact with a slide - this can scratch or break the objective lens and/or ruin an expensive slide. 4. Always turn off the light when not using the scope. 5. Always carefully place the wire out of harm's way. 6. Always replace the cover on the microscope when you put it away

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BIO170 General Biology Freeman/Mac Leod FMCC Spring 2014

B. IDENTIFY PARTS AND FUNCTION OF COMPOUND MICROSCOPE Read through this information carefully. Be sure you understand each item before proceeding to the next step. 1.

Refer to Photo Atlas page 2 fig 2.a as you work through this assignment

2.

Review how to carry a microscope. Obtain a microscope.

3.

The major parts of the compound microscope are shown in Figure 1. As you read this text, refer to this diagram and label the parts that appear in bold print. (Your microscope may be slightly different from that illustrated, nevertheless, the parts functionally are the same.)

4.

The base rests on the table and contains a built-in light source and a light switch. The arm rises from the base and supports the stage, lens system, and control mechanisms. The stage is the flat surface on which microscope slides are placed for viewing. A mechanical stage holds the slide in place with a clip and can be adjusted side to side and forward and back with control knobs.

5.

The condenser is located below the stage. It concentrates the light on the object and may be raised or lowered by the condenser control knob. Usually the condenser should be raised to its highest position. Check to see that it is.

6.

An iris diaphragm is built into the base of the condenser. The iris diaphragm control lever varies the amount of light entering the condenser and lens system. Move the lever back and forth to see how this works.

7.

The body tube is supported by the arm and has a pair of ocular lenses at the upper end. Notice that the ocular lenses move apart and together to adjust for the distance between your eyes.

8.

Now find the revolving nosepiece with attached objective lenses at the lower end. The nosepiece is rotated to bring different objectives into viewing position. The objectives will click into place when they are in viewing position. Try this now.

9.

The magnification of each objective is indicated on the objective itself. An objective with a magnification of 4x is called a scanning objective. A low-power objective has a magnification of 10x. A high-power objective has a magnification of 40x. Finally, the objective with a magnification of 100x is an oil-immersion objective.

10.

Look at your microscope and list the objectives it has in your notebook.

11.

There are two focusing knobs that are used to raise and lower the stage. The coarsefocusing knob has the largest diameter and is used to bring objects into rough focus when using the 4x and 10x objectives. The fine-focusing knob has a smaller diameter and is used 3

BIO170 General Biology Freeman/Mac Leod FMCC Spring 2014 to bring objects into fine focus. It is the ONLY focusing knob used with the high-power and oil-immersion objectives. 12.

Check to see that all parts of Figure 1 are labeled. Tape or paste a copy of this in your notebook. (There is an extra copy of this diagram at the back of this handout if you need it.)

Figure 1: Controls of a typical binocular compound microscope

C. MAGNIFICATION Magnification is a function of making an object appear bigger, such as when we use a hand lens to enlarge a printed word. Merely magnifying an object without a simultaneous increase in the 4

BIO170 General Biology Freeman/Mac Leod FMCC Spring 2014 amount of detail seen will not provide the viewer with a good image. The ability of a microscope (or eye) to see detail is a function of its resolving power. Resolving power is defined as the minimum distance between two objects at which the objects can just be distinguished as separate and is a function of the wavelength of light used and the quality of the optics. In general, the shorter the wavelength of the light source, the higher the resolution of the microscope. 1. The magnification of a microscope is determined by the power of the ocular and objective lenses being used together. The ocular of your scope has a magnification of 10x. You have a list of the objectives above (in item 8). Total magnification is determined by multiplying the power of the ocular by the power of the objective. 2. Enter the objective and ocular magnifications from your microscope in the table below. Calculate the total magnification when each of the four objectives is being used. OBJECTIVE OBJECTIVE MAGNIFICATION scanning low-power high-power oil-immersion

OCULAR MAGNIFICATION

TOTAL MAGNIFICATION

3. Put a copy of this table in your lab notebook. (There is an extra copy of this table at the back of this handout if you need it.)

D. USING A MICROSCOPE The following exercise is designed to help you develop skill using the compound microscope. Follow the steps and check them off as you go. Write answers to all of the questions. You will need patience! 1. Use a piece of lens paper to clean the ocular and objectives of your microscope. Only use specially prepared lens paper on the lenses. You could damage the optics using anything other than lens paper. 2. Plug the microscope cord into the outlet at your lab station. 3. Swing the scanning lens into place – this should already be in place – it is the starting point. You will hear the nosepiece click when the objective is in position. 4. Looking from the side, NOT INTO THE SCOPE, rotate the coarse adjustment knob onehalf turn. Rotate it the other way. What happens to the stage? 5. In the same way, rotate the fine adjustment knob one-half turn. Rotate it the other way. How far did the stage move this time compared with the coarse adjustment?

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BIO170 General Biology Freeman/Mac Leod FMCC Spring 2014

6. Turn the coarse adjustment so that the stage is as near to the scanning objective as possible. Using a paper ruler, measure the distance between the tip of the objective and the stage. This is a good measure of how far the objective will be from the slide and is called the working distance. 7. Now swing the low power objective into place, making sure you hear the click. Measure the working distance of the low-power lens. Is it larger or smaller than that for the scanning objective? 8. Repeat the measuring procedure after swinging the high-power objective into place. Watch to see that it doesn’t hit the stage. What happens to the working distance between objective and slide as magnification increases? 9. Be aware of this change in working distance with increasing magnification so as to prevent damage to your specimens. 10. Why should you NEVER USE THE COARSE ADJUSTMENT WHEN USING THE HIGH POWER OBJECTIVE? THE FOLLOWING PROCEDURE IS THE EXACT SEQUENCE OF STEPS THAT YOU ALWAYS FOLLOW WHEN USING A MICROSCOPE! PAY ATTENTION!!! 1.

Put the scanning objective into place.

2.

Turn on the light of your microscope.

3.

Raise the condenser to its highest position.

4.

Looking through the eyepiece, sweep the iris diaphragm control all the way from left to right. What happens?

5.

Move the control about midway between the extreme left and right positions. You should now have even illumination over the field of view (FOV). The amount of light will have to be adjusted for different specimens and different levels of magnification.

6.

Take a slide of the letter “e”. Place the slide on the mechanical stage so that the clip holds it securely in place. Check with your instructor.

7.

Using the knobs on the mechanical stage – not your hands! - center the letter over the stage opening.

8.

Show how the letter “e” is oriented on the slide, as it appears WITHOUT LOOKING THROUGH THE MICROSCOPE Draw the letter “e” as you see it in your notebook. Be sure to label and give the total magnification.

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BIO170 General Biology Freeman/Mac Leod FMCC Spring 2014 9.

Turn the coarse adjustment knob to position the stage as close to the scanning objective as possible.

10.

Look through the ocular and adjust the light so that there is even illumination across the whole field of view.

11.

Slowly turn the coarse adjustment until you have the letter in view. If after a few turns it doesn’t appear, try centering the slide and adjusting the light again. Repeat the focusing procedure. Now use the fine adjustment for getting sharp focus.

12.

Compared to the picture that you drew without magnification, is the letter “e” right side up or upside down?

13.

Does it face the same way or is it backwards?

14.

Using the knobs on the mechanical stage, move the slide to the left slightly. Which way does the image move?

15.

Move the slide down. Which way does the image move?

16.

In your own words, summarize what happens to the orientation and movement of objects when viewed through the compound microscope. Do this in your lab notebook.

17.

Draw the letter “e” as you see it in your notebook. Be sure to label and give the total magnification.

18.

Before changing to a different objective, make sure that the object is in the center of your field of view and in sharp focus. Looking at the SIDE OF THE MICROSCOPE, swing the 10x objective into place.

19.

Now use the fine adjustment knob to bring the image into sharp focus. You should not have to adjust it more than ½ turn at any time. It may be necessary to increase the light intensity when you use this objective.

20.

How large is the letter “e” compared to what you saw at 40x total magnification (using the scanning objective)?

21.

Draw the letter “e” in in your notebook. Be sure to label and give the total magnification.

22.

Looking at the SIDE OF THE MICROSCOPE, carefully swing the high power objective into place. You should not have to touch the adjustment knobs.

23.

Remember, the coarse adjustment knob is NEVER used with high power. Focus with the fine adjustment knob only.

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BIO170 General Biology Freeman/Mac Leod FMCC Spring 2014 24.

What happened to the light when you changed to high power?

25.

Draw what you see in your notebook and label it.

26.

Swing the scanning objective back into place then remove the slide.

E. DEPTH OF FOCUS 1. Obtain a color-threads slide. 2. Using the procedures you learned in the last section, center the crossed threads on the stage. 3. Focus first using the scanning objective, then the low-power objective. 4. Center the threads so that the crossing point is in the middle of your field of view. 5. Now position the stage as close to the low power objective as possible using both the coarse and fine adjustments. 6. Looking into the oculars, slowly move the stage down using the coarse and then fine adjustment knobs until the first thread is in sharp focus. What color is the thread? 7. This is the bottom thread on the slide! How do you know this is the bottom? 8. Now continue to move the stage down using the fine adjustment until the second thread is in focus. What color is it? 9. Does this thread lie over or under the thread you saw in number 6? How do you know this? 10. Now continue to move the stage down using the fine adjustment until the third thread is in focus. (You may not be able to see all three threads in one place. If this is the case, determine which thread is on bottom and top for each pair of threads. Then, using logic, figure out the order for all three!) What color is it? 11. When one thread is in focus, what is the appearance of the other two threads? 12. The importance of depth of focus lies in the fact that all layers of a specimen on a slide are seldom in focus at the same time. When the upper layer looks sharp, the middle and lower layers are generally blurred. To examine those other layers, we must use the fine adjustment to move the object upward. The vertical distance that remains in focus at any one time is called the depth of focus. 13. In your own words, summarize how you can tell which structure lies over another structure when you are looking through a compound microscope. (You can use threads as an example.) Do this in your lab notebook.

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BIO170 General Biology Freeman/Mac Leod FMCC Spring 2014

F. PREPARING A WET MOUNT SLIDE 1. Watch carefully as your instructor demonstrates the process. 2. Please use your Goggles for this procedure! 3. Obtain a clean slide, coverslip and toothpick. 4. Using the blunt end of the toothpick, gently rub up and down against the inside surface of your cheek. A whitish material should collect on the toothpick. 5. Rub the whitish material (mucus and cells) in the center of the slide using a circular motion to create a thin layer. Allow it to dry 30-60 seconds. 6. Place the slide on a piece of paper towel. Using an eyedropper, cover the cheek material with one drop of methylene blue stain. 7. Hold the coverslip by the edges between your thumb and index finger. Place one edge of the coverslip over the specimen supporting it with a toothpick as shown in Figure 1. When the coverslip is nearly parallel to the slide, withdraw the toothpick so that the coverslip is “floating” on the drop of liquid.

Figure 2. Making a Wet Mount

Figure 3. Human cheek cells

8. Study the cells under the microscope. Be sure to start with the scanning objective to center the cell mass and adjust light before going to higher magnifications.

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BIO170 General Biology Freeman/Mac Leod FMCC Spring 2014

9. Make a drawing (in your notebook) of one cell that you can see particularly well under high power. Title it and give the magnification. Label the nucleus, cytoplasm and plasma membrane (you cannot actually see the membrane but can assume it is surrounding the cytoplasm). 10. Leave your notebook with your instructor for review.

G. PUTTING THE MICROSCOPE AWAY – at the end of lab! 1. Remove the microscope slide and return it to its proper place. 2. Turn off the light. 3. Clean all lenses using lens paper. 4. Place the scanning lens in starting position. 5. Move the mechanical stage so that there are no parts sticking out. 6. Place the cover on the microscope if it has one 7. Carefully wrap the cord electrical cord around the microscope. 8. Put the microscope in the proper cabinet with the arm facing you.

Extra table for your notebook:

OBJECTIVE OBJECTIVE MAGNIFICATION scanning low-power high-power oil-immersion

OCULAR MAGNIFICATION

TOTAL MAGNIFICATION

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BIO170 General Biology Freeman/Mac Leod FMCC Spring 2014

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