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Lab 2: Microscopy & Tests for Biological Molecules Week of September 9, 2013

Name: _________________________________ Lab section: ____________

Prelab Exercises

*Complete this page as a Pre-Lab Activity* Turn in this page when you arrive at your lab session.

1. Copy down the equation for total magnification found on page 4 of this lab. 2. Using the equation above, fill in the correct values for total magnification in the following table (ocular magnification is 10x): Scanning power 4x objective total magnification = _____x Low power 10x objective total magnification = _____x High power 40x objective total magnification = _____x 3. The large circle represents the field of view for a microscope image at 4x magnification. If the diameter of the field of view is 4 mm, how long is the little critter we’re examining?

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Lab 2: Microscopy & Tests for Biological Molecules Week of September 9, 2013

Preparation

1. Read the entire exercise before lab and mark anything you will need to ask about. This will prepare you to work more efficiently in lab because you might not then have to read through all of the background information while you could be doing the activities. We will be able to tell who is prepared and who did not read their lab material thoroughly.

2. Study the included microscope diagram so that you can locate microscope parts by name. Reference The attached pages on microscope parts/function, microscope care, and how to prepare a wet-mount slide, will be a valuable resource for this lab and future labs during the semester. Objectives

1. Know the names, functions, and correct operation of microscope parts. 2. Use good technique in preparing and studying fresh specimens. 3. Use good technique in drawing and labeling diagrams. 4. Estimate the true size of objects seen in the microscope. 5. Know the names of the basic tests for carbohydrates and proteins. 6. Be able to accurately interpret the results of the tests for biological molecules 7. Use your knowledge of the basic tests for carbohydrates and proteins to determine what is

in an unknown solution.

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Part I: Microscopy Resolution and Magnification Microscopes are magnifying tools used to produce enlarged images of tiny objects to bring them into the range of resolution of the human eye. Magnification refers to how many times bigger the image is than the object itself. It is measured in terms of the number of times the diameter is increased—10x, for example, means that the diameter of the image is ten times the diameter of the actual object. Resolution, or resolving power, refers to the clarity of the image and is measured as the smallest space that can be seen between nearby objects. The human eye cannot distinguish between objects that are closer together than about 0.1 mm; they blur together and look like a single object. The resolving power of the eye is therefore said to be about 0.1 mm (or 100 µm). This also means that the unaided eye cannot detect an object less than 0.1 mm wide, which is about the width of a human hair, because it blurs into the background. If the object is magnified so that the eye looks at a larger image, it can be seen—if it is magnified accurately. A microscope (or any other magnifying system) is useful only if its resolving power is better than that of the eye. A microscope with poor resolution but high magnification simply produces a large fuzzy image. As photographers know, if you want to blow up a picture to a very large size and still have sharp details, you need to start with a high-resolution image. Resolving power depends on (1) the quality of the lenses and (2) on the wavelength of light used. Excellent light microscopes can resolve objects that are about 0.2 µm apart. This means also that objects only 0.2 µm wide can be seen with such a microscope—provided they are magnified enough to bring the magnified image within the resolving power of the eye. This would require a magnification of at least 500 times, which is a little more than the microscopes we use in lab can produce. But our microscopes do not have lenses capable of resolving such small objects, so higher magnification would be wasted. Electron microscopes use a beam of electrons instead of a beam of light. The wavelength of a beam of electrons is many times smaller than the wavelength of any visible light, so an electron microscope can resolve much smaller objects. But an image cannot be formed by focusing a beam of electrons on the retina of the eye. Instead, the image is formed on a television screen, and then viewed and/or photographed. Contrast is another critical factor in microscopic visualization. Contrast refers to differences in color or intensity between different structures. If all of the parts of an image are the same color, or the same shade of gray, they are difficult to distinguish. At the microscopic level, there is very little natural contrast, and so material that is prepared for the microscope must often be stained with something that will make structural differences show up better. Stains can do this only if they color some structures more readily than others. Different materials, therefore, require different stains. Inevitably, though, using a stain means changing the specimen and stains must be used cautiously and studied carefully to be sure they are not introducing meaningless differences. Some advanced microscopes manipulate the light coming through an object so that small differences are enhanced, increasing contrast. On our microscopes in lab, adjusting the iris diaphragm allows you to control the amount of light passing through the specimen on the slide, thus affecting contrast.

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Using Our Microscopes During the course of the semester, we will use two kinds of microscopes in lab: Light microscope Dissecting (stereoscopic) microscope -for slides -for large, opaque, 3D objects. Both are compound microscopes because there are two stages at which the image is magnified on its way to your eye. (A magnifying glass is a simple microscope—there is only one stage of magnification.) The image is first magnified by an objective lens (that is, one close to the object), and then the lens through which you look—the ocular lens, which is mounted in an eyepiece, magnifies it again. Some microscopes are monocular (one-eyepiece) and some are binocular (two-eyepieces). When looking through a microscope, it is important to know how much the image you see has been magnified. Since we have compound microscopes, meaning two sets of lenses are working to magnify the image, we must calculate total magnification using the equation below: Total magnification = magnification of the x magnification of the objective lens ocular lens Magnification of the objective lenses is written on the lenses themselves. For most of our microscopes with 3 objective lenses the magnifications are 4x, 10x, and 40x. The magnification of the ocular lens is 10x. There may be another objective lens marked 100x which we generally will not use as it requires a special technique, called “oil immersion” to produce a focused image. For this lab and others, you will be asked to draw images of cells you are viewing under the light microscope. Part of a properly labeled drawing includes reporting the total magnification of the image, not simply the magnification of the objective lens.

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Monocular Compound Microscope:

Note: the fine focus knob of the Leica microscope is in a different location than shown in the above diagram. Your instructor will show you its location. Microscope Parts (clockwise from top) Eyepiece contains the ocular lens (i.e., the one closest to the eye). On all our

microscopes, the ocular lens magnifies the image 10x. Body tube contains mirrors that reflect the image toward the ocular lens Nosepiece rotates to move different objective lenses into place above the slide Objective lenses are the lenses closest to the object. The three lenses of our microscopes

(from shortest to longest) magnify 4x (low power, or “scanning lens”), 10x (medium power), and 40x (high power). The magnified image from the objective lens passes through the body tube to the ocular lens, which magnifies it ten times. Therefore total magnification = objective magnification times ocular magnification.

Each lens “sees” only what was in the center of the field at lower power. When the image has been focused at a lower power, the next higher lens

can be rotated into place without moving the focus knobs. Minor adjustment of focus may be necessary at the new magnification.

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Stage supports the object being studied (normally a glass slide). Stage clips hold the slide in place over the opening in the stage and help to stabilize it

when it is moved to view a new area. Lamp provides light that passes through the object; absorption and refraction by

the object alter the beam of light and produce the image that is subsequently magnified as it passes through the lenses. (Switch is also in base.)

Condenser located under the stage and above the lamp; contains the lever for the iris

diaphragm and a lens. The lens focuses the light from the lamp on the specimen so that it is evenly illuminated. For most purposes, the condenser should be raised at or near its highest level.

Lever for Iris Diaphragm operates diaphragm that opens and closes to admit more or less light from

the lamp through the object. Generally, very transparent objects and/or high magnification require less light than intensely colored objects and/or low magnification.

Base supports the optical parts of the microscope and must always be placed on

a flat, stable surface; one of the parts to hold when carrying the microscope

Coarse focus knobs (on both sides of arm) move the objective lenses significantly closer to or

further away from the object. Coarse focus knobs are never used at high power because they move the lens too fast, and at high power the lens almost touches the slide.

Fine focus knobs are used to make delicate adjustments to focus, especially at high power. Arm should be used to carry microscope, with the other hand supporting the

base.

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General Principles for Care and Use of Microscopes Care of microscope • Always carry a microscope with both hands, one holding the arm and the other supporting the

base. • Clean lenses and slides with special lens tissue only. • Always use a coverslip with wet preparations. • When finished, remove slide; rotate low-power lens into position; turn coarse-focus knob all

the way away from you so the objective is close to the stage (i.e., focus down); wrap cord around microscope and tuck plug securely in; put cover (if available) on microscope. Return microscope to cabinet.

Use of microscope • Always secure slide with stage clips. • Begin examination of slide with scanning-power lens (shortest one), and specimen (if visible

with unaided eye) in center of opening in stage. Turn coarse-focus knob all the way forward (i.e., focus down), then turn it gradually towards you until image comes into focus. Move slide until desired area is in center of field. (Microscope rotates image, making it necessary to move slide in the direction opposite to the one that seems required. Practice!)

• When image is focused at scanning power, rotate the low-power lens into place without touching focus knobs until lens is in place. Then re-focus, using either focusing knob.

• Adjust light if necessary by moving iris-diaphragm lever. • If higher magnification is desired (usually necessary for study of cells), move desired area to

center of field and carefully rotate high-power lens into place. (Some slides are too thick for this lens; don’t force the lens into place.) Use ONLY the fine-focus knob to adjust focus.

• Adjust the light using the iris diaphragm. (Do this frequently to see if it improves image.)

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Skill Building Exercises: Materials: for each pair of students

one light microscope one slide with a printed phrase or word

Becoming confident using a microscope takes time and practice. No doubt there will be times when you cannot find your image. Below are four common things to try in order to bring your image into focus. 1. Slowly move the slide around on the stage. If the object you want to view is not centered on

the stage you will not be able to see it in your field of view. It might be just out of the field of view. Remember the microscope rotates the image, so when you turn the stage knobs to move the slide, it will be moving in the direction opposite to the direction you want the image to move.

2. Go back to the lowest magnification where you last saw your object by rotating a different

objective lens into position. Always begin by finding your object in focus on low power then proceed stepwise through medium and high power, making sure the image is centered and focused before moving to the next magnification.

3. Change the focus. There are focus knobs on both sides of the microscope. The coarse-focus

knob is used only at low and medium power; the fine-focus knob is most useful at high power, though it can be used also at other magnifications.

4. Change the amount of light coming through the object by moving the lever that opens and

closes the iris diaphragm. Generally, you will need more light as you go to a higher magnification or if you use a slide with a lot of color. If your preparation is pale, too much light will wash it out. Get in the habit of trying different light levels to see their effect on a particular slide.

How to find something on the slide 1. Hold the slide so that the label is to your left. Draw the word or phrase on your slide as it appears to your naked eye (i.e. do not put it under the microscope just yet).

2. Check to make sure the scanning power objective lens (4x) is clicked into position. You should ALWAYS begin looking at a slide under scanning power or low power even if you eventually plan to view the object under high power.

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3. Now, put the slide on the stage so that the label is to your left. Pinch the projecting handles to open the “jaws” of the slide-holder, position the slide securely, and then carefully release the handles. Use the knobs to move the slide so that part of the phrase is over the hole in the stage. 4. Adjust the coarse and then fine adjustment knobs as needed until one or more of the letters in your phrase are completely in focus on low power. Draw the letter(s) as it appears through the microscope. Label your picture with a title and total magnification. Title: __________________ Total Magnification: ______________ 5. Explain what this quick demonstration has taught you about how the lenses of the microscope affect the orientation of your image (hint: it’s not just simply upside-down). If you need help figuring out what the lenses are doing, try this: turn the stage knobs so the slide moves away from you. Watch in the ocular lens at the same time. Is your image in your field of view also moving away from you? Now try moving the slide to the left on your stage. Is the image in the field of view also moving to the left? How to keep the object in view when you change magnification When you increase magnification, you look at a smaller part of the object, more highly magnified. Therefore, you need to be sure that the thing you want to study is in the center of the field before you change magnification. 6. Get the desired object in the center of the field and focused with the scanning power

objective (4x). Then without changing the focus, carefully rotate the 10x objective lens (low power) into position and re-check the focus. Adjust it carefully, using the fine-focus knobs so you do not overdo it.

7. Now move to the highest-power lens (40x). DO NOT TOUCH THE COARSE

ADJUSTMENT KNOB. I promise that the high power lens will fit, although it will look like it might hit your slide. Make sure it is clicked into place. Now look through the microscope. If you do not see anything there are 4 things to try BEFORE calling over your instructor:

1. Adjust the FINE focus knob (DO NOT TOUCH THE COARSE KNOB).

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2. Use the knobs to move your slide around slightly since you could be looking at the space between letters.

3. Return to low power (10x objective) and get your letter(s) in focus and centered and then try again to switch to high power.

4. Clean the lenses of the objectives with lens paper and lens cleaner. Estimating the actual size of an object seen with the microscope: There are devices that can very accurately measure the size of microscopic objects. For our purposes, however, an estimate is sufficient. A reasonable estimate can usually be made by using the field of view (the lighted area you see through the lens) as a crude ruler. Exercise 1: Determining the Diameter of the Field of View: 1. Using a slide with graph paper or metric ruler/grid, you will measure the diameter of the field of view with the 4X and 10X objective only. You cannot accurately measure the field of the highest power objective (40x), because the markings are too far apart.

a. Place the grid of the slide over the hole in the stage of the microscope right over the area

where the light shines. b. While looking through the ocular, align the grid to measure the diameter of the circular

field of view. c. The lines are 1mm apart. Enter the diameter of the field of view for the 4X and 10X

objectives in the table below: d. Calculate the diameter of the field of view in microns (µm) and enter it in the table

below:

Objective Lens Total Magnification (objective x ocular)

Field of View Diameter (mm)

Field of View Diameter (µm)

Scanning 4x Low power 10x High power 40x

2. Because the slide cannot be used for the highest power objective, simple math can be used to calculate the diameter of the field of view. Look back at your drawings of the word slides. What happened to the size of the field of view when you increased the magnification? ________ _____________________________________________________________________________ As magnification increases, the field of view decreases proportionately. Knowing this, you can use the following formula to calculate the field of view size for the 40X objective: Mag4x X FD4x = Mag40x X FD40x Where: Mag = magnification of that objective FD = field of view diameter of that objective 3. Fill in the last row of the table.

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Exercise 2: Measuring an actual object 1. Obtain a slide with a word or phrase. Estimate the width of a letter “e” using the following strategy:

a. Choose the best magnification at which the object (“e”) to be measured fits entirely within the field.

b. Move the slide so that one edge of the object (“e”) is at the edge of the field.

c. Estimate how many objects of this kind could fit end-to-end (or side by side) across the

center of the field of view.

d. Determine the approximate diameter (or length, or width) of the object using the following equation:

Estimated diameter (mm) = Field of View Diameter ÷ # times your object (from table) could fit across the f.o.v. Show your work below. You should include a drawing of your “e” at the edge of the field of view (to scale; label total magnification) and write out the formula used. Find the answer in mm and micrometers (µm). Ask your instructor to check your work before continuing. Formula used: __________________

Total magnification: _____________ Diameter in mm: ___________ Diameter in µm: ____________

Little swimmy critters! Pond Water Using a drop or two from the jar of pond water, prepare a wet mount according to the directions below. Make two separate drawings of two different organisms you find in your sample of pond water. Record the magnification at which you view the organisms, and estimate the length (i.e., the longer dimension) of the organisms in micrometers (µm) using the estimation technique you learned.

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Preparing a wet mount Have slide, coverslip, and pencil (with eraser) ready. • Place specimen (see previous section) in middle of slide and

cover with a drop of water or stain. If the specimen is in water, like your pond water, adding water is not necessary.

• Put one edge of coverslip against slide at one edge of water drop, and lower coverslip carefully onto drop.

• Press lightly against coverslip with pencil eraser to squeeze out air bubbles.

Pond Water Drawings:

Total Magnification: Total Magnification: Estimated length: ­ _____mm _____µm Estimated length: _____mm _____µm Using Stain to Visualize Cell Structures: Parenchyma cells of potato Parenchyma cells are large cells commonly found in many different parts of a plant. Some types of parenchyma cells may contain chloroplasts and carry out photosynthesis. Other parenchyma cells may be adapted for storage of substances, like starch, which you’ll remember as being a type of storage polysaccharide. Let’s look at how starch is stored in the parenchyma cells of a potato:

1. Using a wet, single-edged razor blade, cut an extremely thin slice from the freshly cut surface of a potato.

2. Specimen Preparation: A good specimen will occupy no more than about one-fourth of the area under the coverslip, and it must be somewhat transparent in order to be seen. For plant material, tear off a small piece or use a razor blade to shave a thin slice from a larger specimen. It sometimes works to cut a wedge-shaped piece first and put it on the slide, then cut and save only the thin edge of the wedge.

3 . Prepare a wet mount, in water, of this ultrathin slice of potato. If you sliced it thinly enough, the cover slip will lie flat and even.

4. Examine the parenchyma cells under low power, and then increase the magnification. Look for typical plant cell components.

5. Draw what you observe in the space provided. 6. Without removing the coverslip, stain the potato slice with iodine according to the

following procedure.

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Staining a specimen that has already been prepared in water: It’s generally a good idea to examine a specimen in water before staining it, in order to understand the effects of staining. You can use low or medium power without a coverslip, and simply add a drop of stain and a coverslip after the initial examination. However, if you need to use high power before staining, you must put a coverslip on the preparation. The water can be exchanged for stain (or for any other solution) without removing the coverslip, and even without removing the slide from the microscope stage:

Potato in water only Potato stained with iodine 400X (high power) 400X (high power)

Clean up: BEFORE PUTTING AWAY YOUR MICROSCOPE, HAVE YOUR INSTRUCTOR CHECK TO MAKE SURE YOU HAVE PREPARED IT PROPERLY (lenses clean, low power objective down, no slides on the stage, cord wrapped). Wash and dry the microscope slides. Return the slides to your table.

• Put a drop of the new solution at one edge of the coverslip.

• At the other edge, hold a strip of paper towel. As the paper towel draws water from under the coverslip, the solution from the other edge will flow in.

• Repeat with additional drops as necessary.

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Part 2: Tests for Biological Molecules Benedict’s test for Sugars Benedict’s solution is composed of sodium carbonate, sodium citrate, and copper sulfate. When Benedict’s solution is heated in the presence of a reducing substance, the blue cupric ions (Cu++) are reduced to red, insoluble cuprous ions (Cu+). The color varies from greenish to orange to red to brown, depending on the amount of reducing substances present. Thus, Benedict’s solution allows us to test quantitatively for the presence of reducing sugars. Procedure: 1. Half fill the beaker with water and heat to a gentle boil. 2. Using a Sharpie, number the test tubes 1-7. 3. Add 1 ml (10 drops) of the correct test solution to each tube, as indicated in the table. 4. Add 2 ml (20 drops) of Benedict’s solution to each of the 7 test tubes. 5. Place the test tubes in a boiling water bath for approximately 3 minutes. 6. Allow the tubes to cool to room temperature and record your results below:

General conclusions: Compare the Benedict’s test results obtained on the different solutions. How can you explain these results? ________________________________________________ _____________________________________________________________________________

Tube #

Test Solution

Color/appearance before adding Benedict’s

Color right after adding Benedict’s

Resulting Color after

heating

Conclusion (Does the substance in the

test tube contain a reducing sugar?)

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Water

2

Glucose

3 Sucrose

4 Fructose

5 Lactose

6 Starch solution

7 Egg Albumin

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Iodine Test for Starch Iodine potassium iodide solution enters the amylase coils of the starch molecule. When this happens, the color of the iodine potassium iodide solution changes from a yellowish orange to a bluish black. This was seen previously when the starch in the parenchyma cells of the potato was stained with iodine. Procedure: 1. Obtain 4 clean test tubes and number them 1-4. 2. Add 1 mol (10 drops) of the correct test solution to each tube as indicated in the table below. 3. Add (5 drops) of Iodine Solution to each of the 4 tubes. Mix by gently shaking the tube. *DO NOT HEAT * tubes containing Iodine solution. 4. Record any color change for each tube in the table below.

General conclusions: Compare the Iodine test results obtained on the different solutions. How can you explain these results? _______________________________________________ ____________________________________________________________________________ Biuret Test for Protein The Biuret Test is one method of detecting the presence of proteins. The name biuret is derived from the colored chemical complex formed between copper and proteins under alkaline conditions. Whole proteins will give a violet color and if polypeptides are present rather than whole proteins, a pink color results. A number of complex substances besides proteins and polypeptides can respond with a violet color to this test. Thus, a positive biuret test does not in itself conclusively prove the presence of protein but since all proteins give a positive test even in high dilution, a negative biuret test can be concluded as the near absence of protein. Procedure: 1. Obtain 4 clean test tubes and number them 1-4. 2. Add 1 mL (10 drops) of the correct test solution to each tube as indicated in the table below. 3. Add 1 mL (10 drops) of Biuret Solution to each of the 4 tubes. 4. Wait 5 minutes. Record any color change for each tube in the following table.

Tube #

Test Solution Color/appearance before adding

Iodine

Color after

adding Iodine

Conclusion (Does the substance in the test tube contain starch?)

1 Egg albumin 2 Starch 3 Glucose 4 Water

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General Conclusions: Compare the Biuret test results obtained on the different solutions. How can you explain these results? ________________________________________________ Determining the components of an unknown solution: Obtain a sample of unknown solution. This sample contains a mixture of biological molecules you have just learned to test. Using what you have learned, determine the types of biological molecules present in the solution (reducing sugar, starch, protein or mixture of 2 or more molecules). Record the process of your investigation, results, and conclusions. When you have finished, check with your TA to find out if you’re right. Then go celebrate your success (or try to figure out what might have gone wrong)! Clean up: Washing test tubes: 1. Carefully pour the contents of the test tubes down the sink drain. 2. Fill the test tubes with soapy water. Then using a test tube brush, scrub the inside of the test

tube. Be sure to remove all of the red colored residue (from the sugar tests). Also, remove your markings on the outside of the tubes.

3. Rinse well with tap water and return the tubes to your bench. 4. Invert the tubes so that they will drain. Make sure the hot plate is turned off.

Tube #

Test Solution

Color before adding Biuret

Color right after

adding Biuret

Resulting Color after 5 minutes

Conclusion (Does the substance in the test tube contain protein?)

1 Egg albumin 2 Starch 3 Glucose 4 Water

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Discussion Questions 1. List the 4 basic types of biological macromolecules: 2. What are the building blocks of each of these types of macromolecules? 3. List the functions of each of the 4 types of biological macromolecules 4. With regard to biological molecules, what does it mean to be hydrophilic? What does it mean to be hydrophobic? Can a molecule be both at the same time? Look at the food labels:

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5. What is a polyunsaturated fat? What is a monounsaturated fat? 6. Judging by the numbers on these labels, how many food calories are there in a gram of fat? Protein? Carbohydrate? 7. Which type of biological macromolecules would be best to use for energy storage?