3-The Microscope.pdf

THE MICROSCOPE Karlo Antonio A. Palabrica, RMT HISTORY The first microscope was developed in 1590 by Dutch lens grinders Hans and Zacharias Janssen. In 1667, Robert Hooke described the microscopic appearance of cork and used the term cell to describe the...

THE MICROSCOPE Karlo Antonio A. Palabrica, RMT HISTORY The first microscope was developed in 1590 by Dutch lens grinders Hans and Zacharias Janssen. In 1667, Robert Hooke described the microscopic appearance of cork and used the term cell to describe the compartments he observed. Anton van Leeuwenhoek was the first person to observe living cells under the microscope in 1675—he described many types of cells, including bacteria. Since then more sophisticated and powerful scopes have been developed that allow for higher magnification and clearer images. INTRODUCTION A microscope is a laboratory instrument used to examine objects/ specimens and obtain valuable information about patients by viewing structures invisible to the naked eye. Microscopy is used by scientists and health care professionals for many purposes, including diagnosis of infectious diseases, identification of microorganisms (microscopic organisms), and determination of the effect of pathogenic (disease-causing) microbes on human cells. A microscope that allows light rays to pass directly to the eye without being deflected by an intervening opaque plate in the condenser is called a brightfield microscope. Components The ocular, or eyepiece, is a complex piece, located at the top of the instrument, that consists of two or more internal lenses and usually has a magnification of 10×. Most modern microscopes have two ocular (binocular) lenses. The oculars are correctly positioned when you see a single circle of light. If you see two circles of light, continue moving the oculars until the circles merge. Keep both eyes open while viewing your slides. Body. The body contains mirrors that bend light into the oculars. The orientation of the microscope’s optical elements causes the image to be reversed (right to left) and inverted (top to bottom), producing a virtual image of the specimen. Components Nosepiece. The objectives are attached to the nosepiece. Rotate the nosepiece to move the objectives over the stage. You should hear a clicking sound when the objectives are correctly aligned. Body. The body contains mirrors that bend light into the oculars. The orientation of the microscope’s optical elements causes the image to be reversed (right to left) and inverted (top to bottom), producing a virtual image of the specimen. Objectives. The objectives contain lenses that magnify the image. Your microscope has 4×, 10×, 40×, and 100× objectives. Components LENS COLOR MAGNIFICATION OCULAR LENS TOTAL MAGNIFICATION Scanning Red 4x 10 40 Low Power Yellow 10x 10 100 High Power Blue 40x 10 400 Oil Immersion Black 100x 10 1000 The total magnification is the eyepiece lens x objective lens!! Components Arm. The arm supports the nosepiece and holds the on/off toggle and focus adjustment knobs. Stage/mechanical stage. The stage is the flat platform on which the slide rests. The mechanical stage is the silver or black apparatus that actually holds the slide and moves the slide to position it on the stage. Stage adjustment knobs. The stage adjustment knobs located below the stage move the mechanical stage. The mechanical stage control consists of two knobs. One knob moves the mechanical stage from right to left. The other knob moves the mechanical stage from front to back. Components Substage condenser. The condenser is made of at least two lenses that focus the light passing through the specimen and improve image sharpness. Although the condenser can be moved up and down, for most applications it should be in its uppermost position, close to the slide. Iris diaphragm. The diaphragm controls the amount of light entering the substage condenser from the light source. This is probably the most important mechanism for adjusting light. Set the diaphragm half open when using the 10× and 40× objectives. Open the diaphragm completely when using the oil immersion lens. Too much light can bleach out your specimen. You need to use the appropriate light level for each magnification. Iris diaphragm lever. This diaphragm opens and closes to increase and decrease light from the light source. Components Fine focus adjustment knob. By rotating the fine focus adjustment knob, it is possible to bring the image into sharp focus. This knob raises and lowers the stage in very small increments. Coarse focus adjustment knob. The large coarse focus adjustment knob moves the stage up and down by larger increments and brings the image into focus. Light intensity control/ Rheostat. This knob increases or decreases light output from the light source. At lower magnifications and with unstained specimens, you may need to decrease the light output with this knob. When using the oil immersion lens with stained slides, one should maximize light output. Light source. The illuminator is a halogen bulb that produces light. Base. The base holds the illuminator and supports the rest of the microscope. Care and Handling Lens Care. Cleaning Tissues. Only lint-free, optically safe tissues should be used to clean lenses. Tissues free of abrasive grit fall in this category. Booklets of lens tissue are most widely used for this purpose. Although several types of boxed tissues are also safe, use only the type of tissue that is recommended by your instructor. Solvents. Cleaning fluid recommended by the manufacturer: Use as specified. Oculars. If cleaning the top lens of the ocular with lens tissue fails to remove the debris, one should try cleaning the lower lens with lens tissue and blowing off any excess lint with an air syringe or gas canister. Objectives. Objective lenses often become soiled by materials from slides or fingers. A piece of lens tissue moistened with the recommended cleaning fluid or an acceptable alternative solvent. Care and Handling The microscope is a very important tool in microbiology, and it must be used carefully and correctly. Follow these guidelines every time you use a microscope. Carry the microscope with one hand beneath the base and one hand on the arm. Do not tilt the microscope, instead, adjust your stool so can comfortably use the instrument. Observe the slide with both eyes open, to avoid eyestrain. Always focus by moving the lens away from the slide. Care and Handling Always focus slowly and carefully. When using the low power lens, the iris diaphragm should be barely open so that good contrast is achieved. More light is needed with higher magnification. Before using the oil immersion lens, have your slide in focus under high power. Always focus with your low power first. Keep the stage clean and free of oil. Keep all lenses except the oil immersion lens free of oil. Keep all lenses clean. Use only lens paper to clean them. Wipe oil off before putting your microscope away. Do not touch the lenses with your hands. Care and Handling When you take a microscope from the cabinet at the beginning of the period, you expect it to be clean and in proper working condition. The next person to use the instrument after you have used it will expect the same consideration. Check over the following list of items at the end of each period before you return the microscope to the cabinet. 1. Remove the slide from the stage. 2. If immersion oil has been used, wipe it off the lens and stage with lens tissue. Also, make sure that no immersion oil is on the 40× objective. This lens often becomes contaminated with oil as a result of mistakes made by beginning students. (Do not wipe oil off slides you wish to keep. Simply put them into a slide box and let the oil drain off.) 3. Rotate the scanning or low power objective into position. 4. Lower the stage. Adjust the mechanical stage. 5. If the microscope has a long attached electric cord, wrap it around the base. 6. Adjust light intensity to its lowest setting before turning off. 7. Cover microscope with dustcover. 8. Return the microscope to its correct place in the cabinet