Introduction to Microscopes

The purpose of this laboratory is to introduce you to the microscopes that you will use to observe many biological structures and processes during this course. The familiarity that you gain with this exercise will allow you to spend your time in subsequent labs more effectively (and with less frustration).

The microscope used most frequently in the biology lab is the compound light microscope. The compound microscope is used to magnify and resolve fine detail within a transparent specimen (one through which light can pass). The compound light microscope is so called because it has two separate lens systems; an objective lens, located near the specimen, which magnifies the specimen a certain amount, and an ocular lens, or eyepiece, which further magnifies the image formed by the objective lens. The total magnification observed by the human eye is the product of the magnification of the two lenses (objective X ocular). Exercise I: getting to know your Microscope In order to use a microscope properly you must be familiar with the optical and mechanical parts of the microscope. You must also learn the appropriate care of this delicate (and expensive) instrument. The microscope gives you the opportunity to see a world of things you may never have available again; but you must know how to use it to enjoy the experience. When carrying a microscope, grasp the arm of the microscope and support the base of the microscope with your other hand. Take the microscope to your desk. The diagram on page 4 and list of terms will help you to become familiar with the microscope. As you read through the list locate each of the parts on your microscope. If you don't understand how something functions or can't find it on your microscope, check with your instructor. Basic Microscope Care: (abuse of microscopes will result in O (zero) points for the lab! Additionally, depending on the severity of the abuse you may lose all microscope-use-privileges. SIGN UP (with a lab partner) for a microscope. Always carry the microscope in an upright position using both hands; one on the arm and one on the base. Keep the microscope away from the edge of the table and place the cord out of the way where it will not catch on something. The objective lenses, ocular and top of the condenser require frequent cleaning to remove smudges. You will discover that the most common cause of not being able to focus on a specimen is a dirty lens or slide. A finger print that you may not be able to see can prevent a crisp focus. Use lens paper and lens cleaner to clean your microscope. Keep the stage dry at all times. A wet stage will prevent the slide from being accurately positioned. Some liquids may also corrode the microscope. When returning your microscope to its proper place always: Remove the slide from the mechanical stage Rotate the turret so that the scanning lens is in place. Clean all lens surfaces and the stage. Secure the cord so that it does not hang down.

I will check that microscopes are put away properly and points will be deducted if not.

Microscope Parts List: 1. Adjustment Knobs Your microscope has two adjustment knobs which are used to focus the specimen to be studied. The largest is the coarse adjustment knob. This is used for rapid (or coarse) focusing of the specimen when using the scanning objective lens. The coarse objective knob is rotated until the specimen is roughly in focus and then left alone. The fine adjustment knob controls precise focusing of the object. Only the fine adjustment knob should be used with the higher magnification objective lenses. Moving the fine adjustment knob also helps you to determine the third dimension (depth) of the specimen you are studying. 2. Stage The stage holds the slide to be observed. Moving the coarse and fine adjustment knobs moves the nosepiece with the objective lenses up or down to bring the specimen into focus. 3. Mechanical Stage Your slide is fixed into position on the stage with the mechanical stage. The slide is fastened into the mechanical stage by using a small lever located on the mechanical stage. Two knobs located on the side of the mechanical stage are used to move the slide around to locate your specimen; one knob moves the slide from side to side and the other moves the slide forward and backward. The mechanical stage permits precision movements of your slide, especially nice when using the high magnifications. 4. Condenser The condenser, located below the stage, contains a system of lenses that focuses light on your specimen. The condenser may be raised or lowered using the condenser knob. Most microscopes have a built-in light source. Use caution to avoid having the light cord hang where you might trip over it. 5. Iris Diaphragm The iris diaphragm is located on the condenser. The lever of the iris diaphragm is used to adjust the amount of light striking the object being studied. It is critical that you know the proper use of the condenser and iris diaphragm. A common problem with microscope use is having too much light which obliterates the object (more or less like trying to see something while looking directly at the sun). 6. Objective Lenses When proper illumination is provided the resolving power of a microscope depends on the quality of its objective lenses. Our microscopes are provided with 4 objective lenses which are mounted on a rotating turret or nosepiece. As you rotate the turret you will feel the lens "click" into position for proper viewing. If a lens is not in position you will observe only darkness as you look into the microscope. The 4 objective lenses of your microscope and their magnifications are: Scanning lens Low Power Lens High Power Lens Oil Immersion Lens 4x magnification 10x magnification 40x magnification 100x magnification

Note: The magnification of the oil immersion lens requires using the lens with special immersion oil for proper resolution. We will not be using this lens during our class. DO NOT USE THE 100X LENS. Serious damage to the lens or slide might result. THIS IS CONSIDERED EQUIPMENT MISUSE. 7. Ocular The ocular lens, or eyepiece, further magnifies the image formed by the objective lens. It does not improve resolution. Your microscope will probably have a monocular system (one ocular lens). The magnification of the ocular is 10x. Recall that the total magnification of the lens system is the product of the magnification of the ocular times the magnification of the objective lens being used (ocular X objective).

Practice calculating total magnifications: (ocular x objective) Objective Magnification Lens Scanning Low Power High Power Oil Immersion Ocular Magnification Total Magnification

Its good to practice calculating total magnification as you must always include the total magnification (NOT objective or ocular) and the name of the specimen with any/all drawings~ We wont use the oil immersion lens in this class, but you may still calculate its total magnification.

Compound microscope

Exercise II Using the Compound Light Microscope (there will be questions to answer for this section) Slide preparation Two types of slides will be used in this exercise (and in this biology course): permanent slides (prepared slides) and temporary wet mounts (that you make yourself). A. Microscope Orientation using a letter "e" slide 1. Make a wet-mount slide of the letter "e". 2. To place the slide on the stage, retract the stage clip to allow room for the slide to fit. Place the slide on the stage oriented as if you were reading the letter. Gently release, allowing the stage clip to adjust to the size of the glass slide. 3. Observe the letter e under low power (4X). Is the image of the letter "e" upright or reversed as compared to its orientation on the slide? 4. Move the slide a little to the left. Did the image move in the same direction? 5. Center the letter "e" in the field of view and now observe the letter "e" with the low power (10x) lens. Is the image still in focus? 6. Is the "e" still in the center of the field of view? You may have to use the fine adjustment to sharpen the image. 7. Finally rotate the high power (40x) lens into position. Sharpen the focus with the fine adjustment knob. Remember -- never use the coarse adjustment knob with the high magnification objectives -- you might cause damage to the slide or to the lens. How much of the letter "e" do you see? 8. Answer e-slide questions and make your e-slide drawings. 9. Return the letter e slide to the appropriate tray. B. Microscope Depth of Focus using Multicolored Thread Slide 1. Obtain a colored threads slide from the tray at the front counter 2. Under the scanning (4x) lens, adjust the course focus and check which way brings the slide closer to or farther from the lens. 3. Under low (10x) power position the threads so that their region of overlap is in the center of the field of view. 4. Using only the fine adjustment, focus up and down. As you do so you will observe different parts of the threads and different threads become distinct; when one thread is in focus, the others are blurred. By focusing continuously up and down through the slide you can perceive the dimension of depth that is not revealed when you focus at just one point. 5. Determine the order of overlap of the threads on your slide, from top to bottom.

C. Observing Pond Water There are many different types of organisms that live in water and some of the most unusual looking are the microscopic individuals. You will make a wet-mount slide of the available pond water samples: 1. Obtain a slide and cover slip from the front table. 2. Use a dropper to draw pond water from the bottom of the container and place a drop on the slide. *You may see more if you manage to get a small amount of debris in the water. 3. Place a coverslip on top of the drop. 4. Observe the slide under scanning power (4X), low power (10X) and high power (40X). Draw several examples of pond organisms. 5. After completing your drawings, follow the lab-technician instructions for slide-disposal. REMNDER: all drawings must include total magnification~ (not ocular and objectiveTOTAL MAGNIFICATION)

D. Hair Comparison/ Analysis

Hair analysis is one of the most common forensic methods to connect suspects to victims or crime scenes. It requires no high-end molecular biology procedures....just the hair sample, and a microscope. First, you should know some facts about hair: Its basic components are keratin (a protein), melanin (a pigment), and trace quantities of metallic elements. These elements are deposited in the hair during its growth and/or absorbed by the hair from an external environment. After a period of growth, the hair remains in the follicle (an area below the surface) in a resting stage to eventually be sloughed from the body. Hair consists of a shaft that project above the skin, and a root that is imbedded in the skin. A hair grows from the papilla and with the exception of that point of generation is made up of dead, cells.

The cuticle is a translucent outer layer of the hair shaft consisting of scales that cover the shaft. These scales always point from the root end of the hair to the tip end of the hair. There are three basic scale structures that make up the cuticlecoronal (crown-like), spinous (petal-like), and imbricate (flattened/smooth). The medulla is a central core of cells that may be present in the hair. The medulla, when present in human hairs, is generally less than one-third the overall diameter of the hair shaft. The medulla in animal hairs is normally continuous and generally occupies an area of greater than one-third the overall diameter of the hair shaft.

Human hairs are generally consistent in color and pigmentation throughout the length of the hair shaft, whereas animal hairs may exhibit radical color changes in a short distance, called banding. The pigmentation of human hairs is evenly distributed, or slightly more dense toward the cuticle, whereas the pigmentation of animal hairs is more centrally distributed, although more dense toward the medulla.

Follow the instructions (pg 7) to carry out your own hair analysis.

LAB 1- MICROSCOPE USE

Name__________________________________________________

GET PREPARED FOR LAB: read the lab handout and know what we will be doing, and how we will be doing it. There will be a pre-lab quiz! FOR FULL LAB CREDIT: Turn in these answer pages (6-7) at the start of our next lab. Answer all questions and observe/ draw all specimens as directed. With all drawings include the name of the specimen and TOTAL magnification. Make sure your work is legible and complete. At the end of lab, show your work to me and Ill make suggestions on where/ how you can add to it to make sure you get full credit on this first lab! Exercise I: Getting to know your Microscope 1. Calculate total magnification for the THREE lenses we will use in Biology 160:

2. Describe examples of misuse of laboratory equipment, for this lab:

Exercise II: Using the Compound Light Microscope 3. If properly positioned on the stage, the e on your e-slide should look inverted when viewed through the microscope. Explain this phenomenon:

4. Draw the e at all three magnifications: 40x 100x 400x

5. In what order were the colored threads that you observed? TOP __________________ MIDDLE ________________ BOTTOM ___________________

6. Pond water can be really interesting! Draw two organisms, and try to identify them! (look at available field guides or ask your instructor for help with this)

7. How do you know that these organisms are alive?

8. Carry out a hair analysis by plucking one of your hairs (or you may borrow one from your instructor) and observing it using the magnification that provides the best view. Use two slides to make a hair sandwich.

Sketch the view of the shaft.

Locate the primary structures of your hair and choose (circle) the best description for each:
Cuticle scales: Cortex color: Medulla style: Medulla thickness: Medula transparency: flat and smooth protruding or spiky other: ________________ same throughout different colors: _________________________________ broken continuous thick thin transparent semi-transparent opaque

Compare your hair to a second hair. It can be a classmates or one of the animal or wig samples provided. Maybe take a forensic tape sample from your clothing looking for hairs! Specimen: _____________________________________ How does this hair differ from your own? How is it similar?