PH-PHR 112 LAB AY 2020-2021: 1 - MICROSCOPY PREPARED BY: KAREN JEANET NAVARRO, RPh OUTLINE Learning Objectives Introduction/Theory Procedure PH-PHR 112 LAB AY 2020-2021: PH-PHR112 LAB AY 2020-2021 EXPERIMENT 1- MICROSCOPY LEARNING OBJECTIVES At the end of the exercise, the student should be able to: • Identify the parts and functions of a compound light microscope; • Demonstrate an understanding of the proper skill in using the compound light microscope; • Demonstrate an understanding of how to calibrate an ocular micrometer using a stage micrometer; • Apply knowledge learned in measuring microscopic specimens and make unit conversions; and • Apply knowledge learned on how to derive the magnification of PH-PHR of 112specimens. LAB AY 2020-2021: drawings PH-PHR112 LAB AY 2020-2021 EXPERIMENT 1- MICROSCOPY INTRODUCTION/THEORY Microscopy is the technique utilized to view samples and objects that cannot be seen with the unaided eye or not within the resolution range of the normal eye. Microscope is the combination of two words; “micro” meaning small and “scope” meaning view. In botany, the use of a compound microscope is very vital in studying tiny objects such as plant cells hence a thorough understanding of its parts, functions, and skill in its use and manipulation are prerequisites for successful laboratory observations. PH-PHR 112 LAB AY 2020-2021 : PH-PHR112 LAB AY 2020-2021 EXPERIMENT 1- MICROSCOPY INTRODUCTION/THEORY Types of Microscope PH-PHR 112 LAB AY 2018-2019: Simple Microscope PH-PHR112 LAB AY 2020-2021 Compound Microscope Dissecting Microscope EXPERIMENT 1- MICROSCOPY PARTS OF THE MICROSCOPE The eyepiece or ocular contains one set of lenses and rests loosely on the top of the body tube. If you move the eyepiece carefully, handling only the metal parts, you will find its printed magnification. PH-PHR 112 LAB AY 2018-2019: PH-PHR112 LAB AY 2020-2021 EXPERIMENT 1- MICROSCOPY PARTS OF THE MICROSCOPE The lenses are contained in the objective which are screwed into a revolving nosepiece at the lower end of the body tube. The lens system nearest the specimen called the objective magnifies the real image, yielding a virtual image which is seen by the eye. PH-PHR 112 LAB AY 2018-2019: PH-PHR112 LAB AY 2020-2021 EXPERIMENT 1- MICROSCOPY PARTS OF THE MICROSCOPE Microscope Objectives Objective Description Scanner Shortest objective used 4x for scanning the viewfield or searching for the structures in the specimen PH-PHR 112 LAB AY 2020-2021: Low-power PH-PHR112 LAB AY 2020-2021 Magnification objective (LPO) Short objective, but 10x with larger lens opening High-power objective (HPO) Longer, but with smaller lens 40x Oil immersion (OIO) Uses immersion oil 100x EXPERIMENT 1- MICROSCOPY PARTS OF THE MICROSCOPE The revolving nosepiece puts the desired objectives to be used. The stage is a flat platform that extends between the upper lens system (ocular and objectives) and the lower set of devices for providing light. PH-PHR 112 LAB AY 2018-2019: PH-PHR112 LAB AY 2020-2021 EXPERIMENT 1- MICROSCOPY PARTS OF THE MICROSCOPE The revolving nosepiece puts the desired objectives to be used. The stage is a flat platform that extends between the upper lens system (ocular and objectives) and the lower set of devices for providing light. Adjustment knobs are located at the side of the stage. These used to laterally move the slide on the stage. This type of stage PH-PHR 112 LAB AY 2018-2019: is referred to as mechanical stage. PH-PHR112 LAB AY 2020-2021 EXPERIMENT 1- MICROSCOPY PARTS OF THE MICROSCOPE The coarse adjustment knob moves the stage (or the body tube in some models) over a greater distance and brings the specimen into approximate focus. The fine adjustment knob moves the stage (or the body tube) more slowly for precise final focusing. The condenser lens causes the light rays to converge at the opening in the stage of PH-PHR 112 LAB AY 2018-2019: the microscope. PH-PHR112 LAB AY 2020-2021 EXPERIMENT 1- MICROSCOPY PARTS OF THE MICROSCOPE The iris diaphragm, which can be opened or closed to control the amount of light which enters the tube of the microscope. A lever (sometimes called a rotating knob) is provided on the condenser for operating the diaphragm. PH-PHR 112 LAB AY 2018-2019: PH-PHR112 LAB AY 2020-2021 EXPERIMENT 1- MICROSCOPY PARTS OF THE MICROSCOPE A build-in illuminator at the base is the source of light. Light is directed upward through the condenser. In some models without an illuminator, a mirror is found below the condenser. One side of the mirror is flat (plane) surface and the other one is curves (concave) surface. The mirror can be adjusted to direct the light rays into the stage opening or through the condenser. PH-PHR 112 LAB AY 2018-2019: PH-PHR112 LAB AY 2020-2021 EXPERIMENT 1- MICROSCOPY PROCEDURE Handling the Microscope 1. 2. 3. 4. Make sure that the electrical cord is secure. Check if the scanner or LPO is aligned with the eyepiece tube. Use the arm when picking up or moving the microscope. When carrying the microscope, place one hand under the base for support and lift it by arm with another hand. Never lift microscope by the part that holds the lenses. 5. Always keep it in an upright (vertical) position PH-PHR 112 LAB AY 2020-2021: PH-PHR112 LAB AY 2020-2021 EXPERIMENT 1- MICROSCOPY PROCEDURE Handling the Microscope 1. Place the microscope so that the arm is nearest you. It should be at least 5 cm away from the edge of the table. 2. Look at the ocular and adjust the angle of the mirror so that the maximum amount of light enters when the diaphragm is open and the condenser is as near the stage as it can move. The amount of light may then be decreased if desired by lowering the condenser or closing the diaphragm. 3. Plug in the microscope to turn on the lamp. The field of view is the entire area of light that can be seen through the microscope. The proper control of light is the most important single step in studying the microscope. PH-PHR 112 LAB AY 2020-2021 : PH-PHR112 LAB AY 2020-2021 EXPERIMENT 1- MICROSCOPY PROCEDURE Handling the Microscope 4. Place the prepared slide of algae on the stage. See to it that the LPO is in place and about ¼ inch from the slide. 5. Locate the coarse adjustment knob on your microscope and rotate it gently, noting the upward or downward movement of the stage. 6. Use the coarse adjustment knob to move the stage downward until the focus of the specimen is obtained. When adjusting the position of the stage, always look at the it from the side to avoid striking the objective lens. 7. If necessary, adjust the amount of light by turning the illumination intensity control that can be found near the light switch. PH-PHR 112 LAB AY 2020-2021 : PH-PHR112 LAB AY 2020-2021 EXPERIMENT 1- MICROSCOPY PROCEDURE Handling the Microscope 8. When the specimen appears distinct, sharpen the focus with the fine adjustment knob. The fine adjustment knob moves the stage to such a slight degree that movement cannot be observed from the side. 9. Turn the adjustment knobs slowly and gently, as you pay attention to the relative positions of the objective and specimen. ! Avoid bringing the objective extremely close to the specimen with the fine adjustment while viewing, because even this slight motion may force the lens against the slide. Bring the lens close to the stage using the coarse knob;112 then, while through the ocular, turn the fine knob to move PH-PHR LAB AYlooking 2018-2019: the stage until you have a clear view of the object. PH-PHR112 LAB AY 2020-2021 EXPERIMENT 1- MICROSCOPY PROCEDURE Handling the Microscope 10. Adjust the iris diaphragm for the best illumination. 11. Next, shift the microscope to high-power objective (HPO) by turning the revolving nosepiece until the HPO is in place. The focus only needs a slight movement of the HPO, so use only the fine adjustment knob. This will also avoid breaking the slide. 12. If necessary, re-adjust the opening of the iris diaphragm and if necessary, the illumination intensity. In most microscopes, when a new objective is brought into use, the specimen should be in focus (parfocal) and the same position in relation to the field of view as in previous objective (parcentral). PH-PHR 112 LAB AY 2020-2021 : PH-PHR112 LAB AY 2020-2021 EXPERIMENT 1- MICROSCOPY MICROSCOPE Accessories Ocular Micrometer An Ocular Micrometer is a glass disk that fits in a microscope eyepiece and that has a ruled scale; when calibrated with a slide micrometer, direct measurements of a microscopic PH-PHR 112 LAB AY 2020-2021 : object can be made. Figure 1. Ocular Micrometer PH-PHR112 LAB AY 2020-2021 EXPERIMENT 1- MICROSCOPY MICROSCOPE Accessories Ocular Micrometer (installation guide) PH-PHR 112 LAB AY 2020-2021: PH-PHR112 LAB AY 2020-2021 EXPERIMENT 1- MICROSCOPY MICROSCOPE Accessories Stage Micrometer The stage micrometer is a thick glass slide. Each big division is equivalent to 0.1 mm and each small division is equivalent to 0.01 mm. PH-PHR 112 LAB AY 2020-2021 : Figure 2. Stage Micrometer PH-PHR112 LAB AY 2020-2021 EXPERIMENT 1- MICROSCOPY MAGNIFICATION of the Specimen Linear Magnification Magnification = (MPE) (MPO) *MPE- magnifying power of eyepiece *MPO- magnifying power of objective Example: PH-PHR MPE- 10x112 (eyepiece); MPO- 10x (LPO) LAB AY 2020-2021 : Computation: Magnification= 10 X 10 = 100x PH-PHR112 LAB AY 2020-2021 EXPERIMENT 1- MICROSCOPY MAGNIFICATION of the Specimen Non-microscopic specimens Magnification of drawing (M) = (D) size of the image or drawing in mm or cm (S) actual size of the specimen in mm or cm Example: D- 6cm ; S- 1.5cm PH-PHR 112 LAB AY 2020-2021 : Computation: Magnification= 6/1.5= 4x PH-PHR112 LAB AY 2020-2021 EXPERIMENT 1- MICROSCOPY MAGNIFICATION of the Specimen Microscopic specimens PH-PHR 112 LAB AY 2020-2021 : Figure 3. Stage micrometer labelled PH-PHR112 LAB AY 2020-2021 EXPERIMENT 1- MICROSCOPY MAGNIFICATION of the Specimen Microscopic specimens PH-PHR 112 LAB AY 2020-2021 : Figure 4. Calibration PH-PHR112 LAB AY 2020-2021 EXPERIMENT 1- MICROSCOPY MAGNIFICATION of the Specimen Microscopic specimens PH-PHR 112 LAB AY 2020-2021 : Figure 5. Cell measurement PH-PHR112 LAB AY 2020-2021 EXPERIMENT 1- MICROSCOPY PH-PHR 112 LAB AY 2020-2021: 1 - MICROSCOPY PREPARED BY: KAREN JEANET NAVARRO, RPh Edited by: MARIE ESTHEL FAMILAR-SAMONTE, RPh.
