Exercise No 1 Microscopy (1) 6l4t1g

Exercise No. 1 COMPOUND MONOCULAR MICROSCOPE The microscope is one of the principal tools of the biologist. It was invented through the efforts of Dutch scientist Anton Van Leeuwenhoek. In the laboratory, the microscope serves as a very useful tool to help you discover the fascinating secrets of the living world which the unaided eye cannot see. In this exercise, you will study the proper use and care of this delicate instrument. OBJECTIVES At the end of the period, students should be able to: 1. identify the parts of a compound microscope and learn the function of each part; 2. manipulate the different parts correctly. 3. compute for the magnification of drawing. 4. differentiate the low power objective from the high power objective in relation to the size of the field of vision, magnification, and resolving power. MATERIALS compound microscope glass slide cover slip colored threads

human hair (gray, black, and colored) medicine dropper transparent or plastic ruler small letter "e" cut out from an old newspaper

PROCEDURE: Before studying the different parts of your microscope, bear in mind the following points: 1. Other students in different classes also use your microscope. Always inspect it before using and report to the instructor any damage you can see. 2. The microscope is a delicate and expensive instrument. Exercise utmost care when using it so that other students will be able to use it too. 3. Manipulate the microscope only when you are thoroughly familiar with it. A. Identification of the Microscope Parts 1. Using Figures 1 and 2 as a guide, identify the different parts of your microscope and learn the function of each. 2. Ocular or eyepiece. It is where you look into when examining objects in the microscope. Inserted into the draw tube, it contains lenses to increase magnification. Draw tube. A cylindrical part where the eyepiece is inserted. Body tube. The barrel which holds the lenses of the eyepiece and objectives at a proper distance from each other. Coarse adjustment knob. A large wheel which moves the body tube up or down to bring the specimen into focus. It is used when the low power objective is in place. Fine adjustment knob. A smaller wheel which brings the specimen to its sharpest focus by moving the body tube up or down very slightly. Dust shield. A rounded metal directly attached to the end of the body tube which protects the objectives from dust. Revolving nosepiece. The bottom end of the body tube where the objectives are attached. It rotates to allow changing from one objective to another. Objectives. Small tubes attached to the nosepiece which contain lenses of different magnifications. The objective marked 10x is the low power objective (LPO). Sometimes a much shorter objective marked 4x or 5x may be present; this is known as the scanning objective. The high power objective (HPO) is the one marked 40x (44x or 45x in some models). More advance microscope models also have the oil immersion objective (100x). Stage. The flat area where the slide to be examined is placed. Stage aperture. The hole or opening at the center of the stage. It its light from the mirror. Stage clips. Two pieces of metal which holds the slide in place. Arm. s the body tube and is used for carrying the microscope. Mirror. Directs reflected light through the stage aperture to light the specimen in the slide. It usually has a concave and flat surface. The concave surface is used especially when the microscope does not have a condenser. (The condenser is a series of lenses below the stage that helps focus light on the specimen). If a condenser is present, the flat-surfaced mirror may be used. In some microscopes, a lamp provides the light source instead of the mirror. Pillar. The upright bar which provides and connects the base with the other part of the microscope,

Diaphragm. Regulates the amount of light ing through the specimen. The diaphragm may be in the form of a rotating disk with holes of various sizes, or it may have an adjustable iris opening. In the latter case, it is known as an iris diaphragm. Inclination t. Allows the microscope to be inclined at an angle. Base. The horseshoe-shaped bottom part of the microscope which s the whole structure. Table 1. Characteristics of the objectives: LPO

HPO

OIO

Focal length (mm) Working distance (mm) Linear magnification (X) Numerical aperture (N.A.) Focal length (mm), an optical constant of the lens system, is the distance from the center of the lens to the point where parallel rays entering the lens are brought to a focus Working distance (mm.) is the free space between the specimen surface and the objective. Numerical aperture (N.A.) is a measure of the resolving power of an objective. An objective with 0.25 N.A. allows the viewer to distinguish as separate 25000 lines per inch. B. Microscope Manipulation 1. After familiarizing yourself with the parts of the microscope and the function(s) of each, you may now proceed to manipulate it. Before you proceed, here are some reminders each time you prepare for microscopic work: a. Place the microscope in an upright position with the arm directed towards you. Make sure that it is at least two inches from the edge of the table. b. Make certain that the low power objective is in position (a slight "click" will indicate that the LPO is already in place). c. Adjust the diaphragm to its largest opening. d. Look through the eyepiece with one eye while keeping the other eye open and adjust the angle of the mirror to obtain good illumination. Note: Keeping the eyes both open while using the microscope prevents eye strain. 2. Cut out a small letter "e" from an old newspaper. Prepare a wet mount slide of the letter "e" following the procedure outlined in Figure 3. 3. Place the wet mount slide on the stage of the microscope with the letter "e" in its normal reading position. See to it that the letter "e" is right in the center of the stage aperture. Secure the slide into place with the stage clips.

4.

Slowly turn the coarse adjustment knob downward to lower the body tube until the tip of the LPO almost touches the slide. Do this while looking from the side to prevent the unlikely event of breaking the slide. While looking into the ocular, slowly turn the coarse adjustment knob upward to raise the LPO until the letter comes into focus. Turn the fine adjustment knob slightly to obtain a sharp image of the letter. a.

Draw the letter "e" in its normal size and position, and as seen under the LPO.

Fig.B.1 Letter “e” on the slide Fig.B.2. Letter “e” under LPO (mag. ____x) b. Compare the size and position of the letter "e" as viewed under the LPO with the actual position of the letter "e" on the slide. C. Orientation 1. While looking into the ocular, move the slide slightly to your right. Next move it to your left. Then upward and downward. a. In which direction does the image of the letter “e” appear to move in each case? 2. Move the slide towards you and then away from you. b. In what direction does the image appear to move in each case? 3. Rotate the nosepiece to put the high power objective into position. Make sure that as the objective moves into position, it does not hit the stage or the slide. Listen for a "click" sound which means that the objective is in place. Most microscopes today are parfocal. This means that once an image is in sharp focus under LPO, it remains in focus under HPO without further adjustment. If the image is not in sharp focus, bring it into sharp focus with a slight turn of the fine adjustment knob. If at first try you fail to see the image under HPO, refocus under LPO then repeat the procedure for focusing with the HPO until you see the image. Note: Do not use the coarse adjustment knob when the HPO is in place. The reason for this may be obvious if you look at the distance between the HPO and the top of the slide. The distance is so close that a downward movement of the objective has the danger of hitting and breaking the slide. With the fine adjustment knob, the upward and downward movements of the objective are very slight and do not normally hit the slide or the stage. D. Magnification and Reduction The magnifying power of an objective is usually indicated on its side as 5x, 10x, or 40x. If the objective is marked 40x, it means that the lens in the objective forms an image that is 40 times larger than the specimen or object. If the ocular is marked 10x, it means that the ocular magnifies the image produced by the objective an additional ten times. The total magnification (Mtotal) or the image you see in the microscope is obtained by multiplying the magnifying power of the ocular (M ocular) with the magnifying power of the objective (Mobjective), or briefly M total = (Mocular) X (Mobjective). Therefore, if you use the combination of 40x objective and I0x ocular, you obtain a total magnification of 400x. ThIs means that the image that you see in the microscope is 400 times larger than the object. E. Field of Vision The field of vision is that circular lighted field where you see the image of the object or specimen. 1. Return the letter “e” to the center of the field of view. Without moving the coarse adjustment knob, shift the objective from LPO to HPO by turning the nosepiece. Sharpen the focus using the fine adjustment knob. If necessary readjust the opening of the diaphragm (or aperture disc) and the position of the mirror to improve light and contrast. 2. Draw the resulting image of the letter “e” under HPO as Figure E.1.

a. b. c. d. e. f. g.

Fig. E.1. Letter “e” under HPO (mag. ____x) Is there a change in the level of brightness of the field of view when the objective is shifted from LPO to HPO? Describe the change. What portion of the letter “e” is magnified under HPO? Is there an increase or a decrease in the area of specimen seen? Is the orientation of the letter “e” changed by shifting from LPO to HPO? If the tail end of the letter “e” is to be viewed under HPO, where should it (tail end) be positioned under LPO before shifting into the HPO? To ensure easier focusing, what should be done first before the HPO is swung into position? Compute the theoretical magnification of the letter “e” under LPO and HPO.

F. Resolving Power of the Microscope and Depth of Focus The resolving power of the microscope is its ability to distinguish two adjacent points as distinct and separate. The depth of focus is a property of the microscope that indicates how deep the space the microscope can bring into clear view at any one time. The materials used for microscopic examination have depth, as well as length and width so that you need to focus at different levels of the specimen. 1. Mount a gray and black hair across each other on a microscope slide. Add a drop of water and cover with a cover slip. Focus under low power objective. Turn the fine adjustment knob back and forth slowly. Draw the resulting image as Figure F.1. a. Can you determine which hair is on top when looking through the microscope? 2. Move the slide to position the hair strands at the center of the field of vision. Shift to HPO and bring the hair strands into sharp focus with the fine adjustment knob. Draw the resulting image as figure F.2. b. Is the resolving power in the HPO as great as in the LPO? 3. Obtain a prepared slide with colored threads. Place the slide on the stage and focus under LPO the point where all three threads intersect. Draw the intersection of the colored threads under LPO as Figure F.3. Rack the body tube to the lowest position without hitting the slide. The threads may be out of focus. Slowly rack the body tube upward. The first thread that comes into focus is the bottom one. Focus the other threads. c. When one thread is in focus, are the other threads in focus too? d. What is the color sequence of the threads from bottom to top? 4. Shift to HPO. Slowly rotate counter clockwise the fine adjustment knob until all the threads are out of focus. Next, slowly rotate it clockwise. The first thread that comes into focus is the top one. Focus the other threads. Draw any of the colored threads as Figure F.4. e. When one thread is in focus, are the other threads in focus too? f. In which objective is it easier to determine the sequence of the colored threads? Why?

Fig. F.1. Intersection of hair strands under LPO. (mag. ____x)

Fig. F.2. Hair strands under HPO. (mag. ___x)

Questions to Answer: 1. How can each objective be identified if the identification marks are removed? 2. How does a microscope magnify an object? 3. Explain why an inverted image is seen under a compound microscope? 4. What is the purpose of the oil in the oil immersion objective? 5. Why should the ocular micrometer be calibrated for each objective? References: Duka. I. A. and M. G. Diaz. 2007. Biology 1 Laboratory Manual: An Investigative Approach. 8 th ed. UPLB. pp. 6 – 13. Fernandez, W.L. et al. 1986. General Microbiology Laboratory Manual. UPLB. pp. 1 – 15. UST General Microbiology Manual

Format of Post Lab Report Exercise No. 1 Compound Monocular Microscope Name: Date Performed Date Submitted: Instructor: I. Objectives: II. Materials: III. Methodology: (Summarized form and should be in a ive and past tense form) IV. Results and Discussion A. Identification of the Parts of a Microscope Table 1. Characteristics of the Objectives. B. Manipulation of the Microscope Fig. B.1. and Fig. B.2. Answer to question C. Orientation a. b. E. Field of Vision Fig. E.1. Table 2. Field of Vision Using Different Objectives. a. b. c. d. e. f. g. F. Resolving Power of the Microscope and Depth of Focus Fig. F.1 and F.2 a. b. c. d. e. f. V. Answers to Questions VI. Conclusion