I. Introduction to the Microscope Perfecting Microscope Skills There are different types of microscopes used by biologists depending on the job they wish to accomplish, including dissecting (or "stereoscopic") microscopes, electron (including both "scanning" and "transmission") microscopes, and compound (or "light") microscopes. All of these microscopes are used to examine specimens and objects difficult or impossible to see with the naked eye. Dissecting microscopes are used to view the surface of relatively larger specimens at low power (typically no more than 10x). They function like elaborate magnifying glasses. One would use a dissecting scope to observe the gross anatomy of a living, preserved, or dissected specimen at greater detail. Electron microscopes are scientific instruments that use a beam of highly energetic electrons to examine objects on a very fine scale (up to several 100,000x). We will be using compound microscopes in our lab today. Compound microscopes are limited by the physics of light to 500x or 1000x magnification. Figure 1 represents a typical compound microscope. A compound microscope consists of two lenses (thus, the name "compound") and the associated hardware to make viewing of specimens easier. The uppermost lens, called the ocular lens, is the part through which a person looks. The lower lens is the objective lens. Usually, several objective lenses are mounted on a turret, allowing rapid changing of objective lenses. The objective lenses on our scopes are 4x (scanning power), 10x (low power), and 40x (high power). The body tube holds the ocular and objective lenses in place. Most microbiological specimens are mounted on glass slides with cover slips on top and placed on the stage. Usually, clips (or clamps) hold the slide firmly on the stage. A light source (thus, the name "light microscope") and a condenser lens are located beneath the stage. Any specimen that you observe must be sliced thin enough so that light can pass through the specimen to be viewed. The condenser focuses the light through a hole in the stage. The condenser usually includes an iris that varies the amount of light passing through the specimen. After passing through the specimen, the light goes through the objective and ocular lenses, and then into the eye of the observer. The total magnification of the image is calculated by multiplying the magnification of the ocular by the magnification of the objective (we will get practice calculating the total magnification later in the lab). As light passes through any object (glass, air, specimens, etc.), it bends. This bending of light is called refraction. The refractive index of a substance is a measurement of the extent that the substance bends light. Excessive refraction can cause distortion of the image. At magnifications of less than 500x, the distortion is minimal. We will not view specimens at any total magnification greater than 400x at our tables, so we will not need to worry about distorted images. But at higher magnifications, the distortion becomes so great that image details are lost. An oil immersion lens helps to remedy this problem by eliminating the air gap between the specimen and the objective lens. Some of your microscopes will have a 4 th objective lens, which is the oil immersion lens. You should never use the oil immersion lens in any BIO 110 or 111 lab! To use an oil immersion lens, a drop of special immersion oil is placed on the microscope slide, and the oil immersion objective lens is maneuvered so 1
that it is touching the oil. Immersion oil has the same refractive index as glass so that the light passes through the slide, specimen, oil and objective lens as if they were a single piece of glass. One would use an oil immersion lens to observe very small specimens, such as bacteria. By the end of today's lab, you will learn how to use all of the parts of the microscope to view specimens of various sizes at different powers (or magnification). This will require practice (your main goal today), as well as simple memorization. To complete this part of the exercise, start with the Parts of a Microscope section below. Do not continue with the rest of today's lab until you are comfortable with all of the parts of a microscope and their functions. Once you have mastered the parts of the compound microscope and their function, we will apply these skills to observe living and prepared slides of single celled protists, such as: Amoeba, Volvox, Paramecium, and whole diatoms. II. Objectives Upon completion of today's lab activities, you should be able to: 1) Demonstrate and describe how to carry a microscope 2) Locate the parts of a compound microscope and describe their functions (see Parts of the Compound Microscope section below for complete list of microscope parts) 3) Define parfocal, wet mount, depth of focus, and field diameter. 4) Describe the procedure for properly focusing a compound microscope 5) Focus an object under scanning, low, and high power using a compound microscope 6) Calculate the total magnification for each objective lens 7) Calculate the field diameter for scanning, low, and high power 8) Estimate the approximate size of specimens in your field of view, given the field diameter for scanning, low, and high power objectives. 9) Use the metric system to make basic measurements of length 10) Convert values between the following units: meters (m), centimeters (cm), millimeters (mm), and micrometers ( m) 11) Utilize the skills listed above to make observations and measurements of unicellular organisms. 12) Describe and demonstrate the proper removal, return, and storage of your microscope III. Parts of the Compound Microscope After the instructor has explained how to carry a microscope, obtain one from the cabinet and place it securely on the table. Identify the following parts on your microscope, and record the answers to the questions in your lab book in the space provided. 1) Ocular lens (or eyepiece): Topmost series of lenses through which an object is viewed. The magnification of the ocular is 10x. 2) Body tube: holds nosepiece at one end and eyepiece at the other end; conducts light rays. 3) Arm: Supports upper parts and provides carrying handle. 2
4) Objectives (objective lenses): a) Scanning power objective: Holds 4x lens used to view the whole slide. b) Low-power objective: Holds 10x lens used to view the object in greater detail. c) High-power objective: Holds 40x lens used to view the object in even greater detail. d) Oil immersion objective (if present): Holds 100x lens and is used in conjunction with immersion oil to view objects with the greatest magnification. Does your microscope have an oil immersion objective?. If so, remember to never use this lens in lab! 5) Coarse-adjustment knob: Knob used to bring object into approximate focus; used only with low-power objective. 6) Fine-adjustment knob: Knob used to bring object into final focus. 7) Diaphragm or diaphragm control lever: Controls amount of illumination used to view the object. 8) Light source: An attached lamp that directs a beam of light up through the object. 9) Base: The flat surface of the microscope that rests on the table. 10) Stage: Holds and supports microscope slides. 11) Mechanical stage: A moveable stage that aids in the accurate positioning of the slide. Does your microscope have a mechanical stage? Yes or No (Circle one). If so, what structures are present to move the stage?. Should you ever use something other than the mechanical stage control knobs to move your stage? Yes or No (Circle one). Explain why in the space provided: 12) Mechanical stage control knobs: Two knobs that are usually located below the stage. One knob controls forward/reverse movement, and the other controls right/left movement. 3
Figure 1. Parts of the Compound Microscope. Ocular (or Eyepiece) Body Tube Objective lenses Arm Stage Clips Stage Diaphragm w/ control lever Light Source Coarse Focus Knob Fine Focus Knob Mechanical Stage Knob Base 4
IV. Rules for Microscope Use Always observe the following rules for microscope use: 1) The lowest power objective should be in position both at the beginning and end of microscope use (in our case, this is the scanning objective). 2) Use only lens paper for cleaning lenses. 3) Do not tilt the microscope when viewing a wet mount. 4) Keep the stage clean and dry to prevent rust and corrosion. 5) Do not remove parts of the microscope. 6) Keep the microscope dust-free by covering it after use. 7) Report any malfunctioning when you notice it. This will benefit you and those who use the microscopes after you! V. Focusing the Microscope Always start with the lowest power objective lens on your microscope (in our case, the scanning objective lens): 1) Turn the nosepiece so that the lowest power lens is in straight alignment over the stage. 2) Always begin focusing with the lowest power objective lens. 3) With the coarse-adjustment knob, lower the stage until it stops. 4) Place a slide of the letter "e" on the stage (your instructor will let you know where these are), and stabilize it with the clips (instructor will demonstrate if necessary). 5) Again, be sure that the lowest power objective is in place. Then, as you look from the side, decrease the distance between the stage and the tip of the objective lens until the lens comes to an automatic stop or is no closer than 3 mm above the slide. If you try to start focusing any other way you risk having the slide and objective lens come into contact! Why would it be a bad idea for the slide and objective lens come into contact? 6) While looking into the eyepiece, rotate the diaphragm (or diaphragm lever) to give the maximum amount of light. 7) Slowly increase the distance between the stage and the objective lens, using the coarseadjustment knob, until the object (in this case the letter e) comes into view, or focus. 8) Once the object is seen, you may need to adjust the amount of light. To increase or decrease the contrast, rotate the diaphragm slightly. 9) Use the fine-adjustment knob to sharpen the focus if necessary. 10) Practice having both eyes open when looking through the eyepieces, as it greatly reduces eyestrain. 5
11) Draw the letter "e" as it appears on the slide (look from the side, not through the eyepieces). Sketch here: 12) Draw the letter as it appears when you look through the eyepieces using the scanning objective. Notes: 13) Move the slide to your right. Which way does the image appear to move? This phenomenon is known as inversion. Compound microscopes are parfocal; that is, once the object is in focus with lowest power (in our case, the scanning power), it should be nearly in focus with the higher power. To go from scanning power to higher power, you must: 1) Bring the object into focus under the lowest power by following the instructions in the previous section. 6
2) Make sure that the object (letter "e") is centered in the field of the lowest objective. 3) Move to the next higher objective (low power, 10x) by turning the nosepiece until you hear or feel it click into place. IMPORTANT - Do not change the focus; parfocal microscope objectives will not hit normal slides when changing the focus if the lowest objective is intially in focus. 4) If any adjustment is needed, use only the fine-adjustment knob. Always use only the fine-adjustment knob with higher powers. On your drawing of the letter "e" that you made for exercise 1, draw a circle around the portion of the letter that you are now seeing with the higher-power magnification. 5) Repeat steps 2-4 with the 40x power objective lens. 6) When you have finished your observations of this slide (or any slide), rotate the nosepiece until the lowest power objective clicks into place, and then remove the slide. VI. Total Magnification Total magnification is calculated by multiplying the magnification of the ocular lens (eyepiece) by the magnification of the objective lens. Calculate the total magnification for each objective in table 1. To calculate the total magnification, multiply the power of the ocular lens by the power of the objective lens. Your table should include the powers of both lenses and the total magnification. Table 1. Total magnification of your microscope. POWER OF OBJECTIVE (x) POWER OF OCULAR = TOTAL MAGNIFICATION Scanning (x) = Low (x) = High (x) = VII. Field Diameter The field diameter (the circle visible through the lens) is the length of the field across the center. The field diameter of the 4x (scanning) objective is 5 millimeters (mm). The field diameter of the 10x objective is 2 mm. The field diameter of the 40x objective is 0.5 mm. Does low power or high power (Circle one) have a larger field of view and allow you to see more of the object? Which power has a smaller field but magnifies to a greater extent? 7
Helpful hint - To locate small objects that are seen on low power, place these objects in the center of the field before rotating to high power. VIII. Estimating the size of specimens in the field of view Once you know the field diameter of your scope at different powers, you can estimate the approximate size (length) of objects in your field of view. To show you how to estimate the size of objects (like cells) in your field of view, let's assume that the field of view when using the 10X objective (100X total magnification) on your microscope is 2 mm. If 8 plant cells extend across the field of view (2 mm), then each cell is 2/8 or 0.25 mm long (see figure 2). Thus, to estimate the size of a specific object in your field of view, you must estimate the how much of the field diameter the object occupies. You can then use basic math or algebra to calculate the estimated size. Remember - the diameter of the field of view changes depending on the power of the objective Figure 2. Estimating the size of an object in the field of view. 8
IX. Preparing a Wet Mount Fresh (wet) mounts of biological material may involve whole organisms, free-hand sections cut with a razor blade of part or all of an organism, or material which is mechanically or chemically teased apart. The material is placed in a drop of water in the center of a clean slide (see figure 3). Some care must be taken in lowering the coverslip in order to avoid trapping air bubbles. The difficulty is overcome by first allowing only one edge of the coverslip to rest on the slide and, while supporting the coverslip with a needle or your finger, slowly lowering it over the preparation. Your instructor will demonstrate the proper technique for making a wet mount. Remember - Whenever you prepare a specimen for observation, the object should always be prepared as a wet mount. Figure 3. Making a wet mount. We will get practice making a wet mount using threads of three different colors. We will use this slide for the next exercise (on depth of focus). To make this wet mount, do the following: 1) Using the scissors and metric ruler at your table, obtain three 1 cm lengths of thread. Each thread must be a different color (e.g., one red, one blue, one yellow). Your instructor will show you where to find the thread 2) Once you have your threads, place one on top of the other on the center of your slide in a pattern as seen below. 9
3) Once your threads are on the slide, add 3-4 drops of water (water should not extend beyond the coverslip). 4) Place the coverslip over the wet mounted threads as described above and demonstrated by your instructor (do not press down on the coverslip). 5) Complete the exercise on depth of focus. X. Depth of Focus The vertical distance that remains in focus at one time is called the depth of focus. Depth of focus is inversely proportional to magnification and field diameter (see figure 4). Figure 4. Changes in image composition coincide with changes in depth of focus. XI. Using the microscope to observe cells Cells serve as the structural building blocks to form tissues and organs, which make up entire organisms. Each cell of a multicellular organism is functionally independent. In other words, all of the functions that an organism needs to carry out in order to live occur at the cellular level. Cells can define their own boundaries and protect themselves from external changes causing internal changes. Cells can use sugars to derive energy for different processes which keep it alive (we will learn about this process, cellular respiration, in Unit III of the lecture portion of this course). Cells contains all the information required for replicating themselves and interacting with other cells to produce a multicellular organism 10
(processes which we will discuss throughout BIO 110 and 111). It may even be possible to reproduce an entire organism from almost any single cell of that organism (we will discuss this during the later part of this course). Biologists divide all cells into two major categories: prokaryotic and eukaryotic cells. Organisms that possess prokaryotic cells are called "prokaryotes." Organisms that possess eukaryotic cells are called "eukaryotes." The only organisms composed of prokaryotic cells are bacteria. Prokaryotes are very simple, and are considered the first organisms to have evolved on our planet. There are no organelles in prokaryotic cells and almost every cellular function is carried out in the cytoplasm of the cell. All other organisms on our planet, aside from bacteria, are composed of eukaryotic cells. The eukaryotes include unicellular algae and protists that live alone or in colonies and multicellular organisms, such as animals, plants, fungi. Eukaryotic cells contain the organelles that compartmentalize the cell. We will learn about cell structure and function in Unit II of the lecture part of this course. In the last part of lab today, you will use the skills we learned in the first portion of the lab to make careful observations of the cells of different taxa. Your main goal today, is to get practice using these important skills which you will need throughout this course and any other biology course you may take in the future. Later in this course, you will need to use these skills to compare and contrast the characteristics of the cells of different biological kingdoms and observed cell division in plants and animals. Remember, today's lab is titled "Perfecting Microscope Skills," and the only way to perfect anything is by practice. So if you struggle initially (which everyone does!), keep at it. We have a variety of living and prepared specimens available for your viewing pleasure, including: Rotifers, Amoeba, Volvox, Paramecium, and whole diatoms. Since it is so early in the semester, you are not expected to already know much about the biology and life history of these cool little critters. However, you will get to observe many of them alive, so it is likely that you may be able to see them feed, move, respond to stimuli and one another, or even mate and reproduce! It is by making careful observations of such behaviors that scientists have learned about the fascinating lives of these inconspicuous, yet extremely common and ecologically important organisms. You will be given the freedom to observe whichever organism you wish. It is recommended, however, that you observe at least 1 organism, so that you can practice making a wet mount. For each organism that you view, you might want to sketch it, and record notes of any interesting observations. For example, keep a keen eye out for the following things: size?, shape?, color?, other aspects of it's appearance?, does it have a thick cell wall?, does it move?, does it have any hairs (cilia) or long tails (flagella)? are they interacting with one another? Has it ingested anything? etc? To observe your selected protist, do the following: 1) make wet mounts or use prepared slides to observe at least one the following organisms: Rotifers, Amoeba, whole diatoms, Volvox, and Paramecium (or any other protists available). 11
2) Sketch each specimen in the circles below, making notes of the magnification you are observing it under and any visible characteristics you observed on each organism (as described above) 3) For each specimen you observe, calculate the field diameter of the field of view you are observing and the approximate size of each individual 4) Notes: Notes: 12
Notes: Notes: 13
XII. Returning your Microscope for Storage - VERY IMPORTANT! Every time you use your microscope in lab, make sure you do the following. Failure to do so may result in loss of points toward your final grade! 1) Remove any slides on the stage. 2) Make sure the scanning objective lens is locked in place. Always return your microscope to the scanning objective when not in use. 3) Return all prepared slides to the correct slot in the slide tray. 4) If you made any wet mounts, make sure to clean the slide and cover slip and return them to your tray. 5) If you break any slides or cover slips, make sure to throw them into the GLASS ONLY trash container (not in the trash cans at the end of your tables). Your instructor will show you the location of the GLASS ONLY trash bin. 6) Center the mechanical stage. 7) Lower the mechanical stage (or raise the nosepiece, depending on the model you have) to maximize the distance between the stage and scanning objective. 8) Turn off the scope (light source). 9) Wrap and tie the electrical cord using the plastic ties provided for the cord. DO NOT wrap the cord around any part of the microscope. 10) Place the dust cover over the microscope. 11) Carry the microscope using the proper technique (as demonstrated by your instructor) to the correct place in the correct storage cabinet. You should place your scope in the bin in the cabinet that corresponds to your station number. 12) Make sure the electrical cord of the microscope is placed on the cabinet shelf, not hanging down. 13) Make sure the arm of the microscope is facing out. When your microscope is put away properly, the number on the microscope base that corresponds to your station number can be seen. 14
Review Questions Upon completion of this lab exercise, answer the following questions: 1) Define parfocal? 2) What is inversion? 3) List four things you should be sure to do before storing a microscope. 4) If your field of view is increasing then your must be decreasing. 5) Can you use any soft cloth to clean a microscope lens? Yes of No (Circle one). Explain. 6) Which adjustment knob is used when first focusing a slide for the first time? 7) If a 100X objective lens is in place and the ocular is 10X, then what is the total magnification? 15