Lab A:

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LAB A:
Compound Microscope Practical

Preparing For Class: It is highly recommended that you read through material twice and take notes, especially before you take the online quiz. -review and study the syllabus -rewatch and study the safety video To Be Safe on youtube http://www.youtube.com/watch?v=YdYapyzJNsE -read and study this lab manual on the microscope skills lab -Complete the online quiz in Laulima available from Sun., Jan 15 to Mon., Jan 16 -Make sure you know what time to report to class for this Microscope Skills Lab. You should be divided up into groups of ten and have just 30 minutes to complete the assignment.

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During this semester, you will be working with microscopes often. Although there are multiple types of microscopes, you will be working only with two main types, the dissection (also called stereoscope) and the compound microscope. Both are light microscopes, meaning they use a light source to illuminate the subject being studied. A dissection microscope is commonly used for dissection to provide a magnified view of the specimen being dissected. The image in the viewfinder depicts the three dimensional nature of the larger specimen. It only has a low magnification and thus is not used for viewing individual cells. The compound microscope is the most commonly used microscope in most laboratories. It is capable of a very high magnification, thus allowing the viewer to see individual cells, even living ones. The image seen is two-dimensional and does not provide a very high resolution. Specimens are usually mounted on glass slides.

Carrying and Handling the Microscope

Microscopes, like a camera, rely on the maintenance of various glass lens and are expensive pieces of equipment. -Carry the microscope with two hands. Place one hand under the base and grasp the arm with the other hand. Carry it upright and ALWAYS use two hands.

Caring for Microscope

-Protect microscope by covering when not in use -NEVER touch the lenses with your fingers -Keep the lenses clean but do not clean often or unnecessarily. Clean gently by wiping with a clean kimwipe. If absolutely necessary, use a clean kimwipe dampened with distilled water and then dry with a dry kimwipe. -The 100x lens objective is for oil-immersion viewing. Remove the oil from lens after use by gently cleaning with a clean, dry kimwipe. Make sure oil has not creeped onto other lenses. If the lamp burns out or you have other problems, inform your TA. DO NOT try to fix the problem yourself.

Storing the Microscope

-Rotate the nosepiece so that it is on the low power objective -Wind up power cord -Cover microscope with cover and place safely in cabinet.

Introduction to the Parts of the Microscope

A compound microscope has two basic lens systems at work: 1. Ocular the eye piece 2. Objective closest to the object, right above the slides

Lab A: Ocular Lens the Eye piece

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Some microscopes have only one ocular. Your microscope will most likely have two oculars and they pivot in order to adapt to the distance between your eyes. Pivot the right and left oculars inward and outward until you can see clearly with both eyes. Look for the magnification of the eyepiece. You might see a label such as WF 25X. This would mean that the magnification of the ocular lens is 25x. Fill out number 2 on your worksheet. What is the magnification is your ocular lens? The left ocular can be adjusted to compensate for your individual eye strength. You can adjust its ability to focus (diopter adjustment) if you have slightly weaker vision than your lab partner. The ocular lenses are sitting on the microscopes ocular head which rotates. You can easily rotate the ocular head so others can look through your microscope without disturbing the microscope body and specimen. Care: Do not remove or touch ocular lens. You can clean the outermost lens with kimwipes . Objective Lens rotating lens above slide stage The objective lenses are responsible for the image quality. These lenses are extremely expensive because they are made of high optical quality glass and covered with special optic films. The microscope will have multiple objective lenses (usually 3-4) of varying magnification strength. They are held together on the revolving objective head which allows you to easily switch between magnification lenses without disturbing the specimen being studied. Common magnification for objective lens 4x 10 x 40 x 100x Low power Low Power High Power, dry oil immersion

DO NOT FOCUS WITH COARSE ADJUSTMENT KNOB, only use fine DO NOT FOCUS WITH COARSE ADJUSTMENT KNOB, only use fine

Looking at each lens, you will see numbers on the sides. The magnification and numerical aperture is printed on each objective. Example 10/0.25 PHP means that the objective lens has a 10x magnification. Look at your objective lens and fill out #3 on your worksheet. You may see other numbers on the lens barrel. Those are used in more advanced labs with phase contrast filters so we wont focus on that for this semester. The total magnification of the image that you see depends on the combination of the ocular and objective lenses. Multiply the magnification of the ocular lens by the magnification of the objective lens being used to determine the total magnification. Complete # 4 of your worksheet. Body Tube The light shines through the specimen into the objective lens and then travels up through the body tube into the ocular lens, letting you view the image. In some microscopes, the body tube is straight, in

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others, the oculars are held at an angle. The body tube contains a prism that bends the light rays so they will pass through the oculars. ARM The arm of the microscope is the main body of the microscope that holds and supports both lens systems, the ocular and objective heads. This is one of the sturdiest parts of the microscope along with the base (bottom) and these two structures should be held when transporting the microscope. Stage The stage or mechanical stage is the platform that holds the microscope slide securely. The stage can be moved in two directions using the mechanical stage adjustment knobs. Locate the knob on your microscope (some might be on the right underside of the stage) and become familiar with moving the stage around. This is especially useful when examining slides under higher magnification and you wish to observe adjacent areas on the slide. Illuminator Situated on the microscope base directly beneath the opening in the stage is an illuminator, the microscopes source of light. A microscope might just have a optical mirror to reflect ambient light upwards and through the specimen being studied as its only light source. Some microscopes will have a light source that shines a cone of light upward from the base to the stage. In order to see the light, youre going to have to plug in your microscope and turn it on first. Sub-Stage Phase Contrast Condenser The sub-stage phase contrast condenser is right below the stage, between the stage and the illuminator. It focuses the light from the illuminator onto the specimen. It also has several filters but we will be using bright field microscopy. Thus, ALWAYS check that the filter wheel is set to O which means no filter. Diaphragm The diaphragm controls the amount of light which passes to the specimen and can drastically affect the focus of the image. The diaphragm can affect the focus of the image. Learn to use the diaphragm as focusing problems may be due to the incorrect adjustment of light. For now, we suggest that beginners always strive to ensure the lens diaphragm is open to the maximum extent so all the available light enters the lens system. This will ensure an evenly lighted and magnified image. If youre still having trouble, try adjusting the diaphragm. There are two types of diaphragms: - iris diaphragm- look for a lever just under the stage near the front - dial type- Just below the stage or right above the illuminator there may be a rotating dial having different size apertures (holes)

Lab A: Focusing

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The lenses are focused using the focusing knobs. There are two focusing knobs: 1. Coarse Adjustment Knob - larger knob on arm of microscope 2. Fine Adjustment Knob smaller knob To focus, always begin with the lowest power objective (shortest and smallest objective lens) like the 4x when first examining a specimen. Use the coarse adjustment knob first and then the fine adjustment focus to achieve as crisp a focus at the lowest magnification. One focus is achieved, switch to the objective at the next highest magnification (like 10x) and focus again. Always work from the lower power to the higher power. Your microscope is designed to be parfocal, which means that if an image is in focus when using a lower power objective, it should remain in focus when the head is changed to a higher power objective, ex. from 10X to 40X. Only the fine focus adjustment should be necessary to bring the image into a clear, sharp focus (focusing described later in this handout). For the high power 40x and 100x objectives, only use the fine adjustment knobs. Using the coarse knobs can cause the lens and slide to collide and break both. Thats dangerous and very expensive. Getting Started: 1. 2. 3. 4. 5. 6. Place the microscope on a table and plug in. Make sure that youre using the lowest power objective when you start. Turn the coarse adjustment knob to lower the stage until youre ready to focus. Place the Letter e slide on the stage and secure in place with clips or metal arm. Turn on the lamp using the switch on the base. Looking from the side, use the stage adjustment knobs to move the stage left/right and forward/back until the letter e on the microscope slide is over the light source. 7. Look through the ocular lens. a. Adjust the ocular lens until you can comfortably see the image through both lenses. b. Adjust the left ocular diopter focus if needed. c. Adjust the slide position if needed using the stage adjustment knobs until the e is located center in the image. 8. Look through the eyepiece, VERY SLOWLY turn the course adjustment knob until the specimen comes into focus. Then Turn the fine adjustment knob if sharper focus is needed. 9. To switch to the high power objective lens, look at the microscope from the side. CAREFULLY revolve the nosepiece until the high-power objective lens clicks into place. Make sure the lens does not hit the slide. 10. Look through the eyepiece again and this time focus by turning only the fine adjustment knob until the specimen comes into focus. Now youre ready to complete the worksheet

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LAB A WORKSHEET: Learning your microscope

Name: _________________

1. What company made the compound microscope you are using? 2. What is the magnification of the ocular lens? ___________ 3. List the number of objective lens and their magnification:

4. What is the magnification of an image if you are looking at it through the lens system of your compound microscope and you are using the lowest magnification objective lens? 5. What is the magnification of an image if you are looking at it through the lens system of your compound microscope and you are using the highest magnification objective lens?

6. Draw the "e" as it appears at the three lowest magnifications. Drawings should be drawn to
scale and you should note the orientation of the e in the viewing field (is it upside down or right side up?) and denote the relative thickness of the ink line.

Objective Magnification?

Total Magnification?

View of e image through lens

_______x

_______x

_______x

_______x

_______x

_______x

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7. Switch to your lowest magnification objective lens and use the stage adjustment knobs to move the e to the right. Which way does the e move in your view through the ocular?

8. Use the stage adjustment knobs to move the e away from you. Which way does the e move in your view?

9. Answer True/False to the following statements T F On high power magnfication, you should use the coarse adjustment knob. T F The diaphragm determines how much light shines on/through the specimen. T F The fine focus knob visibly moves the stage up and down. T F A compound microscope is a light microscope, unlike the dissection microscope.
10. Draw your compound microscope as seen from the side and label the following parts of the microscope: Ocular lens Arm Stage adjustment knobs Objective lens Base Revolving objective head Stage Illuminator

Please put away your microscope correctly and hold on to your worksheet (you will need it for quiz and lab practical)
-Rotate the nosepiece so that it is on the low power objective -Wind up power cord and cover microscope with cover.

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For your next class: There is no written assignment. This worksheet will not be collected. Instead, hold onto it and use it to study for this weekends quiz that will cover this and your reading for next weeks lab. You will also need to study this for your first lab practical where microscope skills will be a part of the exam. Dont forget to download and study next weeks lab manual this Friday and to take your next quiz this Sunday (Jan 22) to Monday (Jan 23).

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-THIS PAGE CAN BE USED FOR NOTES-

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