EXERCISE 1 THE COMPOUND MICROSCOPE 1.

Introduction
The microscope is an indispensable tool in Botany as in all Biological Sciences. It enables us to see plant structures too small to be seen by the unaided eye. To date , the light and electron microscopes are in use. Since the invention of the light microscope in the 1590s, there have been numerous improvements and modifications on the performance of the instrument itself. However, part of this improvement depends on the development of new techniques for specimen preparation. Regardless of the kind of microscope being used, three elements are needed to form an image: a source of illumination, the specimen to be observed, and a system of lenses to focus the illumination on the specimen and to form the image. The compound light microscope is a microscope that uses natural or artificial light and series of lenses: the condenser, the objectives, the intermediate lenses and the ocular lens. The objective lens magnifies and projects virtual image into the body tube while the ocular lens magnifies that image further and projects the enlarged image into the eye. 3. To become familiar with measuring microscopic objects. 4. To determine magnification/reduction in size of objects viewed under the microscope. 5. To become acquainted with the preparation of wet or fresh mounts for microscopic examination.

III. Materials
Equipment/Apparatus: Compound microscopes Ocular and stage micrometers Glasswares: Slides and cover slips Plant Specimens: Leaves Rice grains Hydrilla verticillata (digman) Other materials: Small letter e (newsprint) Lens paper or cotton with alcohol Pair of scissors 1V. Procedure A. Familiarization with Parts of the Microscope The three basic parts of the microscope are: (a) mechanical parts; (b) magnifying parts; and (c) illuminating parts.

II. Objectives
At the end of the exercise, we will be able to: 1. To identify the different parts and functions of the microscope. the

2. To develop basic skills in operation of the microscope.

A. Mechanical Parts (i.e. those parts concerned with the support and adjustment of the optical parts) Parts 1. Base 2. Pillar 3. Handle or Arm 4. Inclination Screw 5. Body Tube 6. Ocular Tube or Draw Tube 7. Revolving Nosepiece 8. Dust Shield 9. Adjustment Screws a. Coarse Adjustment Screw b. Fine Adjustment Screw 10. Stage 11. Mirror Rack Functions stand that supports the microscope. a short piece of metal that attaches to one end of the base; also supports the microscope. curved metallic part arising from the pillar used for holding the microscope. found at the junction of the pillar and the handle used for tilting the microscope. cylindrical structure vertically arising from the handle; holds the dust shield and nosepiece. upper smaller end of the body tube bearing the eyepiece or ocular lens. circular structure where the objectives are attached that permits the shifting of objectives. circular structure above the nosepiece used to protect the lenses of the objectives. two pairs of wheels attached to either side of the body tube. These are: used to adjust the low power objective in focusing. used for delicate focusing in connection with the high power and oil immersion objectives. A square or round platform with an opening at the center where the slide is placed. found below the stage and attached to the pillar; holds the mirror in place.

B. The Magnifying Parts (i.e. those parts concerned with image enlargement of the specimen) Parts 1. Ocular or Eyepiece Functions found on the draw tube through which the operator peeps during actual focusing; usually carries magnification of 10X. A thin, black line that appears to cut halfway across the field of view which the student or instructor can use to point out regions of the specimen under observation is called a pointer. tube-like structures attached to the revolving nosepiece. These are: carries a magnification of 4-5X. carries a magnification of 10-12X. carries a magnification of 40-60X. carries a magnification of 97-100x;requires the use of cedar oil.

2. Objectives : a. Scanner b. Low Power Objective (LPO) c. High Power Objective (HPO) d. Oil Immersion Objective

C. The Illuminating Parts (i.e. those parts concerned with light provision and regulation to the specimen. Parts 1. Mirror 2. Diaphragm 3. Condenser Functions found below the stage near the base used to collect and direct light to the specimen. found below the stage used to regulate the entry of light onto the specimen. There are different types of diaphragm: iris, plate or fan. lens found immediately beneath the hole of the stage used to concentrate light rays on the specimen.

1. Get a microscope from the cabinet. Use both hands to carry the microscope and hold it in an upright position. With one hand, secure the microscope by its arm and support the base of the microscope with your other hand. 2. Set the microscope down in a sideview position to better locate the other parts. 3. Identify the parts of your microscope and label the drawing in worksheet 1. B. Use of the microscope After familiarizing yourself with the parts of your microscope, you are now ready to use the microscope. 1. Set the microscope down with the arm towards you and the stage away from you. 2. Rotate the nosepiece until the low power objective clicks into position. 3. Slowly raise the stage or lower the objective by rotating the coarse adjustment until the low power objective is about 1 cm from the stage. When trying to locate the specimen, the low power objective is always used first before shifting to the high power objective for a detailed study of the specimen. 4. Always relax and keep both eyes open when using the microscope to prevent eye strain. 5. While looking through the ocular, orient the mirror toward a light source so that it reflects the light up through the open diaphragm, condenser, opening in the stage and body tube. If this is done properly, a bright, evenly distributed circle of

light called the microscope field will be visible. If the field is too bright, close the diaphragm to reduce the amount of light reflected from the mirror or if it is too dim, open the diaphragm. 6. Cut a small letter e from newspaper clippings available in the laboratory. Place the letter e on a glass slide. Cover with a cover slip. Place the slide on the center of the stage. 7. When proper illumination of the field has been achieved, slowly raise the body tube (or lower the stage) by means of the coarse adjustment knob until the letter comes into focus. It is helpful to move the slide slowly back and forth while doing this in order to help locate the materials being studied. 8. Observe the letter under low power. Make a sketch of what you observe in worksheet 1. C. Measurement of microscopic objects using a micrometer eyepiece Sometimes the biologist needs to know the dimensions of the object being observed under the microscope. If you want to estimate the size of microscopic objects more accurately, a microscope equipped with an ocular micrometer can be used. An ocular micrometer is a small glass disc etched with uniformly spaced lines. When an object is examined under a microscope with an ocular micrometer, the micrometer acts as a ruler that is superimposed on the object being measured. However, the units of distance between the spaces of the ocular micrometer is arbitrary. Therefore, before an ocular micrometer

can be used for actual measurements of microscopic objects, it must first be calibrated against a standard. You need a stage micrometer for calibration. A stage micrometer is a glass slide with scribed lines that are exactly 0.01 mm (or 10 micrometers) apart. Calibration of an ocular micrometer 1. Get an ocular and a stage micrometer. These are delicate instruments and must be protected from dust, grease, fumes, and friction. These micrometers must be handled at the edges.

6. Observe another point where the lines of the two micrometers coincide again. 7. Now count the number of spaces of the stage micrometer included within a given number of spaces in the ocular micrometer. 8. Since the smallest spaces on the stage micrometer are known to be 0.01 mm apart, compute the distance of 1 ocular division or calibration constant as follows:
1 ocular division = No. of stage ms x 0.01 mm No. of ocular ms

2. How to attach an ocular micrometer

to the microscope. Detach the ocular tube from the draw tube. Unscrew the eyepiece lens (the top lens for some microscopes but for Zeiss microscopes, it is the lower part of the eyepiece) of the ocular tube. Very carefully insert the ocular micrometer. Put back the eyepiece lens and insert the ocular tube back into the draw tube. 3. Mount the stage micrometer. Center its scale and focus with the low power objective. 4. While looking through the low power objective, rotate the ocular and adjust the stage micrometer until the lines of the ocular micrometer are parallel with those of the stage micrometer. 5. Match the lines at the left edges of the two micrometers by moving the stage micrometer.

where ms = micrometer spaces 9. What is the calibration constant of the microscope you are using? Show your computation on the worksheet. 10. You may now remove the stage micrometer and replace it with the slide containing the specimen that you want to measure. 11. The calibration constant is used to determine the length or width of the microscopic specimen that you are measuring. Multiply the number of divisions of the micrometer eyepiece covered by the specimen by the calibration constant.
Length or Width = No. of ocular ms x calibration constant

12. Prepare a wet mount of a Hydrilla leaf and measure the diameter of a tooth cell. D. Preparation of a wet mount In Botany, you will be asked to make a wet or a fresh mount to facilitate viewing of some plant parts. 1. Use forceps or tweezers to pick off a young leaf from a portion of the digman stem.

2. Place the leaf on the center of a clean glass slide. 3. With a dropper, put a drop of water over the leaf. 4. Get a cover slip and put one edge of the cover slip on one side of the drop of water. Then with a dissecting needle, slowly bring the cover slip down on the water. If air bubbles form, gently tap the cover glass. Bubbles interfere with viewing of the specimen.

1. To determine the total magnification of the specimen as seen through the microscope, multiply the magnification of the ocular by the magnification of the objective.
Total Magnification = Magnification of eyepiece x Magnification of objective

2. To determine the magnification in a

drawing made, divide the size of drawing by the actual size of object or specimen.
Magnification of drawing = Size of drawing Actual size of object

5. Focus under the LPO of the microscope. Measure the diameter of a tooth cell which is located along the sides of the leaf. What is the diameter of the tooth cell? Show your computation on the worksheet.

3. Compute for the magnification of the

length of a potted plant, length of rice grain and diameter of tooth cell drawn 5 cm long.

6. If you are asked to make wet mounts

of roots, stems, or fruits, take a sharp blade and make thin sections of the specimen. Place these sections on a glass slide and proceed as above in number 4. E. Computation of magnification Magnification may be defined as the number of times an object is enlarged by the magnifying lens or the number of times a drawing is enlarged or reduced from the original size of the object.

4. Show

your worksheet.

computation

on

the

F. Reminders on the care of the microscope 1. Never touch the lens of the microscope with your hands. If the lenses are dirty, use lens paper or cotton with alcohol. If the microscope is out of order, notify your laboratory instructor.

2.

3. Report any missing parts of your microscope.

References 1. Abramoff, P. and R. Thomsom. 1982. Laboratory Outlines in Biology III. W. H. Freeman and Co. San Francisco. pp. 1-9. 2. Barbour, M. G., B. A. Bonner and G. J. Breckon. 1975. Botany: A Laboratory Manual. 5th edition. John Wiley & Sons, Inc. New York. pp. 5-10. 3. Becker, W. M., J. B. Reese and M. F. Poenie. 1996. The World of the Cell. The Benjamin/Cummings Publishing Company, Inc. California. Pp. 841-845.

Students Name ___________________________Date Performed __________ Course/Year/Section _______________________Date Submitted __________ Professors Name___________________ WORKSHEET # 1 THE COMPOUND MICROSCOPE A. Label the parts of the microscope B. Using the microscope Before viewing Viewed under the microscope

Sketch of the letter e using the LPO.

Sketch of the letter e using the HPO

1. Is the position of the letter e the same as when viewed with the naked eye or is it inverted? 2. When you move the slide to the left, in which direction does the letter image appear to move? 3. When you move the slide toward you, in which direction does the letter image appear to move? 4. Explain your observations.

5. When you shift to HPO, what happens to the letter image and field of view?

C. Measurement of microscopic objects using a micrometer eyepiece Calibration constant (computation)

Width/length of the tooth cell

E. Magnification Specimen Hydrilla tooth cell (diameter) Actual size Magnification of a 5cm drawing (Show computation)

Length of a potted plant

Rice grain (length)