Ch. 2-101-F13 PDF

Preview

Summary

Ch. 2-101-F13...

Description

Microscopic Examination of Cells Objectives • To learn how to use the microscope, an important basic laboratory skill that you will apply in Introductory Biology. Return to this chapter throughout the semester, for reference whenever necessary. 7RLQYHVWLJDWHVRPHRIWKHSURSHUWLHVLQFOXGLQJVL]HRIGLͿHUHQWFHOOW\SHVLQFOXGLQJEDFteria, protozoa, animals and plants.

Pre-lab Preparation: • Read this lab manual chapter. • Review pages 1-10 of the “Photographic Atlas for the Biology Laboratory” on microscopy and cells. • Review chapter 6 (A Tour of the Cell) in your Biology textbook, especially the section on microscopy and size (pages 94-97).

Introduction Compound Microscope Techniques Two types of microscopes are used in Introductory Biology: compound microscopes and dissecting microscopes. The compound microscope is the more complicated of the two and is the one you will probably use most often. Our compound microscopes are used to observe PDWHULDOVSODFHGRQJODVVVOLGHV7KHGLVVHFWLQJPLFURVFRSHVKDYHPXFKZHDNHUPDJQLÀFDtion and are used to observe larger objects that are not mounted on slides. The compound microscopes are high-quality, delicate instruments that cost thousands of dollars each. Please treat them with special care. It is important that you learn to use the compound microscope correctly. Incorrect use could damage the microscope, give you a headache, and prevent you from seeing the materials you are supposed to study.

2-1

Some general rules of microscope use that you must always follow include: • Always lift the microscope with both hands, with one hand grasping the arm of the microscope and the other supporting the instrument from underneath. • Never adjust anything without knowing exactly what you are doing. • When starting to use a compound microscope, always start by adjusting the ocular lenses correctly for your eyes. $OZD\VREVHUYHDQHZVOLGHXQGHUWKHZHDNHVW[REMHFWLYHOHQVÀUVW)RFXVXVLQJWKH [OHQVWKHQÁLSWRWKHQH[W[OHQVDQGIRFXVLW7KHQÁLSWRWKH[OHQVIRFXV it, etc. • Never remove any lenses from the microscope. • Be especially careful that you do not rotate the focusing knob in such a way that the objective lens comes into direct contact with–or smashes through–the slide. This will destroy the objective lens. This mistake is particularly easy to make when using the 40x and 100x objective lenses, because these lenses need to be close to the slide for proper viewing. 7XUQRͿWKHOLJKWVRXUFHXVLQJWKHWRJJOHVZLWFKEHIRUHXQSOXJJLQJWKHPLFURVFRSH Before plugging in a microscope, check that the previous user turned the light source RͿFRUUHFWO\3OXJJLQJWKHPLFURVFRSHLQZLWKWKHOLJKWWXUQHGRQWHQGVWREXUQRXW the light bulb.

2-2

Parts of the ComPound miCrosCoPe Please refer to the diagram on page 2-4. 1. Ocular Lenses. Twin 10x lenses housed within the two eyepieces. It is important to adjust the ocular lenses to match your individual eyes, as described on page 2-5. 2. Objective Lenses.$VHWRIIRXUOHQVHVRIYDU\LQJPDJQLÀFDWLRQ[[[DQG[ These are mounted on a rotating bezel called the revolving nosepiece. Always start with the 4x objective and work your way sequentially to the higher power lenses. See page IRUGHWHUPLQLQJWRWDOPDJQLÀFDWLRQ6HHSDJHIRUWKHXVHRIWKH[REMHFWLYH 3. Arm.$OZD\VOLIWWKHPLFURVFRSHE\ÀUPO\JUDVSLQJWKHDUPDQGVXSSRUWLQJWKHEDVH 4. Plain stage. Supports a slide placed in the mechanical stage. 5. Mechanical Stage. A slide clipped into the mechanical stage can be moved by turning the two stage adjustment knobs. See page 2-6 for a description of how to insert a slide into the mechanical stage. 6. Focusing Knobs.7KHRXWHUNQRELVXVHGIRUFRDUVHIRFXVLQJDQGWKHLQQHUNQREIRUÀQH focusing adjustment. Turning the focusing knobs moves the objective lenses in relation to the slide. While adjusting the focus, be careful not to ram the objective lens into the slide. 7. Eyepiece screw. Loosening this screw allows the entire eyepiece assembly to be rotated. )RU\RXUSXUSRVHVWKLVLVQRWQHFHVVDU\DQGWKHVFUHZVKRXOGEHWLJKWHQHG 8. Main light switch.7XUQVODPSRQDQGRͿ$OZD\VWXUQRͿEHIRUHVWRULQJPLFURVFRSH 9. Light intensity knob. A knob used to adjust light intensity. 10. Condenser. A lens system that concentrates the light on the slide. This is used for improving the contrast of the image. You can adjust the condenser in two ways: with the black knob on the left that raises and lowers the condenser in relation to the stage, and with the aperture iris diaphragm ring on the front of the condenser unit.

2-3

1. Ocular lenses (10X)

Revolving nosepiece 7. Eyepiece screw

3. Arm Ocular lens focusing knob (adjusts for your eyes) 2. Objective lenses (4X, 10X, 40X, and 100X)

5. Mechanical Stage (holds and moves slide) 6. Course (outer) DQGÀQHLQQHU focusing knobs

4. Plain stage

10. Condenser Unit Mechanical Stage Controls (turning these knobs moves the slide) 0DLQOLJKWRQRͿVZLWFK 9. Light intensity knob

2-4

adjusting the miCrosCoPe for Your eYes 7KHUHDUHWZRZD\V\RXFDQDGMXVWWKHFRPSRXQGPLFURVFRSHWRÀW\RXUH\HV,WLVKLJKO\ worthwhile to make these adjustments before you start to use a given microscope, as a poorly adjusted microscope will cause you all sorts of problems (probably including a bad headache). 1. Adjust for the distance between your eyes 7KHÀUVWWKLQJ\RXVKRXOGGRLVDGMXVWWKHH\HSLHFHVWRPDWFKWKHGLVWDQFHEHWZHHQ\RXUSXSLOV You can do this by simply pulling the eyepieces apart (away from one another) or pushing them together (towards one another) while simultaneously looking through them with both eyes. When set to the proper distance, your overlapping view through both eyepieces will appear as one large even circle. 2. Adjust the microscope to match the relative focusing of your two eyes. This adjustment is more complicated than the one described above, but it takes only a second RQFH\RXXQGHUVWDQGZKDWWRGR,WLVOLNHO\WKDW\RXUOHIWDQGULJKWH\HVGLͿHUVOLJKWO\IURP one another in the way that they focus upon an object. You need to adjust the ocular lenses WRWDNHWKLVGLͿHUHQFHLQWRDFFRXQW 1. Look at the eyepieces of your microscope. The left one has a thick rotating knob at the base with a scale printed in white. 2. Put a slide on the stage and roughly focus on it using the 4x objective. 3. Close your left eye. In other words, look only through the right eyepiece. Pick one SDUWLFXODUVSRWRQWKHVOLGHDQGIRFXVRQLWH[DFWO\XVLQJWKHÀQHIRFXVLQJNQRE5Hmember, while focusing look only through the right eyepiece. 4. After you have focused exactly on your chosen spot, don’t touch the master focusing knobs. Instead, look at that same spot through your left eye, keeping your right eye FORVHG7XUQWKHURWDWLQJNQREDWWKHEDVHRIWKHOHIWH\HSLHFHZLWK\RXUÀQJHUVXQWLO that same spot comes into exact focus. This time, while focusing look only through the left eyepiece. 5. You have now adjusted the microscope to match your eyes. If you leave the rotating eyepiece alone, the microscope should remain in adjustment throughout the lab period.

2-5

PlaCing and moving a slide on the stage Glass slides come in a standard size. There is a curved clip on the stage that will hold a slide in SODFHDVVKRZQLQWKHÀJXUHEHORZ7KHDVVHPEO\WKDWKROGVWKHVOLGHLVFDOOHGWKHmechanical stage. Once a slide is clipped into place in the mechanical stage, it can then be moved from right to left and forward and backwards by turning the two knobs connected to the mechaniFDOVWDJHVHHPLFURVFRSHÀJXUHV Note: If there is water or another liquid on the bottom of the slide or on the plain stage, then the VOLGHZLOODGKHUHWRWKHSODLQVWDJHYLDVXUIDFHWHQVLRQDQGEHGL΀FXOWRULPSRVVLEOHWRPRYH using the mechanical stage. To prevent this problem, dry the slide and stage before use.

Mechanical stage with slide correctly inserted, as seen from above. (Note: this image should be inverted for the new CX21 microscopes)

determining magnifiCation There are two sets of lenses on each compound microscope: the ocular lenses and the objective lenses. The ocular lenses are in the eyepieces and the objective lenses are on the revolving nosepiece just above the stage. On our compound microscopes, the ocular lenses are all 10x. That is, they magnify the image by a factor of 10. There are four objective lenses on most of the microscopes. These objective lenses vary in PDJQLÀFDWLRQWKH\XVXDOO\FRQVLVWRIRQHHDFKRID[[[DQG[OHQV :KHQDVNHGZKDWPDJQLÀFDWLRQWKHPLFURVFRSHLVVHWWRSHRSOHRIWHQORRNDWWKHREMHFWLYH OHQVDQGUHVSRQGZLWKWKDWYDOXH)RUH[DPSOHVRPHRQHXVLQJWKH[REMHFWLYHPLJKWWKLQN WKDWWKHWRWDOPDJQLÀFDWLRQLV[7KLVLVLQFRUUHFWWKHWRWDOPDJQLÀFDWLRQLVDFWXDOO\WKH product of the power of the ocular and objective lenses. Because our ocular lenses are all 10x, the math is simple. Just multiply the power of the objective lens by 10: Objective lens 4x 10x 40x 100x

7RWDO0DJQLÀFDWLRQ 40x 100x 400x 1000x 2-6

use of the Condenser The condenser is a lens system that concentrates the light on the slide. This is used for improving the contrast of the image by controling light intensity and focus. You can adjust the condenser in two ways: with the knob to the left of it that raises and lowers the condenser in relation to the stage, and with the aperture iris diaphragm ring on the front of the condenser unit. )RUWKHEHVWRSWLFVXVLQJRXUPLFURVFRSHVWKHFRQGHQVHUVKRXOGEHDGMXVWHGVRWKDWLW is raised as high as it can go in relation to the stage. Rotate the condenser height adjustment knob to move the condenser to the highest position. However if the entire observed ÀHOGRIYLHZLVQRWEULJKWHQRXJKEULJKWQHVVPD\EHLPSURYHGE\ORZHULQJWKHFRQGHQVHU slightly. 7KHDSHUWXUHLULVGLDSKUDJPULQJKDVDQREMHFWLYHPDJQLÀFDWLRQVFDOH;;; ;5RWDWHWKHULQJVRWKDWWKHPDJQLÀFDWLRQRIWKHREMHFWLYHLQXVHIDFHVIURQWZDUG

using the oil immersion lens (100x objeCtive) The 100x objective lens is used only for a technique called oil immersion. If you look through this lens without using immersion oil, the image you see will be very blurry....