LABORATORY ONE
INTRODUCTION TO THE WAY BIOLOGISTS WORK
To begin our semester of laboratory, we will focus on the way biologists go about their work. Scientists use technology extensively in their activities, and for biologists one essential piece of technology is the compound light microscope. Many of you have probably already learned how to use a microscope and will find this part of the laboratory a review, but be sure that you read the materials carefully. You will need to check yourself on certain skills in microscopy. Be sure that you work carefully through the discussion, learning the parts of the microscope, how to measure under the microscope, and gaining familiarity with our rules for microscope use. We will also learn to use the stereoscopic or dissection microscope. Biologists (and scientists generally) have an orderly way of investigating nature called the scientific method. You will understand the process of science better by looking at components of this method and by actually doing some of the steps. You will carry out a brief scientific enquiry this afternoon, partnering with other students at your bench, and write a report on your results. The report will be due next week.
OBJECTIVES OF LABORATORY:
PART ONE: THE COMPOUND LIGHT MICROSCOPE
Our discussion of the compound light microscope includes: (1) a review of the parts of the instrument, and (2) some experience with prepared slides and wet-mount slides. Rules for using the microscopes are numbered and presented in bold type and are embedded within the procedures. These rules apply to our specific laboratory setting and to our scopes, but most of the rules are very appropriate for microscope use in any setting. Notice, too, that important words are in bold-face type.
Rule 1. You will be assigned a particular microscope that you will use each time you need to use a microscope in a laboratory exercise. Always use your assigned microscope. Always use two hands when carrying the microscope to and from its storage compartment and your lab desk.
Rule 2. Clean the lenses of the microscope with lens paper before and after using it. Use only approved lens paper and cleaning solutions to clean lenses. Note: Kimwipes and paper towels will scratch the lenses.
A. Parts of the Microscope
The compound light microscopes that we will use the Nikon and Zeiss binocular laboratory microscopes. (See Figure 1 and 2.). Our microscopes have a built-in light source. Locate the light switch on the base of the microscope. Notice that the intensity of the light can be adjusted here. Review the parts of the microscope and their functions that are listed below by finding the part on your assigned microscope.
• Oculars (eyepieces or ocular lenses)—these are the lenses through which you view the magnified image. You can adjust the distance between the oculars to fit the distance between your eyes. The magnification capability of the oculars is 10X (or ten times). In the right ocular there is a pointer that should help you to indicate some specific detail of interest to a fellow student or to the instructor.
Rule 3. Leave the ocular lenses in place on the microscope.
Revolving Nosepiece – This part enables you to change magnification of the image. The various objectives are mounted on this wheel and can be snapped into place for viewing at the different magnifications.
Light Source Ocular Tube Condenser Focus Knob Brightness Control Knob ON/OFF Ocular Stage Condenser Head Revolving Turret Objective Lenses Mechanical Stage Y Stage Control Knob X Stage Control knob Course Focus Knob Fine focus Knob
Light Source
Ocular Tube
Condenser Focus Knob
Brightness Control Knob ON/OFF
Ocular
Stage
Condenser
Head
Revolving Turret
Objective Lenses
Mechanical Stage
Y Stage Control Knob
X Stage Control knob
Course Focus Knob
Fine focus Knob
Nikon Microscope
Parts of the Nikon Alphaphot-2 Microscope with Köhler Illumination. 26 June 2003 http://www.eiu.edu/~biology/Facilities/Microscopy/Nikon_YS2-HK.html
• Objectives (objective lenses)—Attached to the revolving nosepiece are four objectives.
The magnification of the objectives is printed on the casing of each. The shortest objective is the scanning or 4X objective. The next longest is the 10X objective or low power. The next longest is 40X or high-power objective, and the longest objective is the 100X objective or the oil-immersion objective. We will not use the 100X objective today or routinely, and we will learn about its proper use later. When this objective is used, immersion oil is placed on the slide with the tip of the objective resting in the oil to reduce the refraction of the light. Note: the Zeiss microscope’s objectives are all the same length. Refer to the side of the objective for correct magnification.
Remember that we have called these scopes, compound microscopes. The word "compound" refers to the fact that there are several lenses involved in making the image that you see. In fact, there are enough lenses involved that the image viewed is upside down and backwards relative to the actual specimen.
• Stage—this is the flat surface on which the slide is placed. Notice the aperture (opening) on the stage through which light passes. Our microscopes are equipped with clips for holding the slide and control knobs for moving the slide into different positions. This device is referred to as a mechanical stage.
• Condenser—underneath the stage is the condenser, a set of lenses that focuses the light. The condenser has a control knob that allows for its adjustment. Normally the condenser should be up as high as it will go towards the stage. The iris diaphragm at the top of the condenser controls the amount of light that is delivered through the aperture of the stage. The diaphragm is adjusted by a lever or a wheel on our microscopes. You can adjust the light coming through the microscope in the following ways: by turning the light switch or by adjusting the iris diaphragm. Higher magnifications usually require more light.
• Focal Adjustment Knobs—focusing the image is accomplished by using the coarse adjustment knob (the larger wheel) with the scanning and low power objectives. The fine adjustment knob (the smaller wheel) is used with the high-power objective and with the oil immersion objective if it is used.
Rule 4. Never use the coarse adjustment knob when focusing with the high-power or oil immersion objectives in place as this can damage the objective or the slide.
determined by multiplying the magnifying power of the oculars (in this case, 10) times the magnifying power of the objective (in this case, 4). Therefore, the overall magnification is 40X. The following magnifications are obtained with our microscopes:
scanning objective (4X) times 10X ocular = 40X magnification
low-power objective (10X) times 10X ocular = 100X magnification
high-power objective (40X) times 10X ocular = 400X magnification
oil-immersion objective (100X) times 10X ocular = 1000X magnification
2. With the crossed threads in sharp focus with the scanning objective in place, turn the revolving nosepiece so that the low-power objective is in place. You should now have to focus very little, if at all, in order to have the image in sharp focus. This property of being able to change objectives with little focusing is called parfocal capability. A microscope that can do this is said to be parfocal. Only good quality microscopes have this capability.
At this power, with the image at 100X magnification, determine which thread is on top of the other two. Using the coarse adjustment knob, make the image go in and out of focus and determine which colored thread's edge comes into focus first with the other threads as background. Have your instructor check your choice. What you have demonstrated for yourself by doing this is that you can detect depth with the microscope. The portion of the specimen on the slide that is in focus at any moment is in the focal plane of the lens system being used. The focal plane has some thickness, and that thickness is known as the depth of field. High-power lenses have shallower depths of field than do low-power lenses.
3. Look at the distance between the tip of the low-power objective and the slide. With the image in clear focus on low-power, turn the high-power objective in place. Now look at the distance between the tip of this objective and the slide. The distance between the tip of the objective and the slide is called the working distance. Note that the greater the magnification of the objective, the smaller the working distance. It is important to remember the small working distances of high power objectives so that you will not damage microscopes or slides. Use the fine-focus adjustment knob to explore the shallow depth of field at high power.
4. Turn the revolving nosepiece so that the scanning or low-power objective is in place, and
remove the thread slide from your microscope.
Rule 5. Remove slides from the microscope only with the scanning objectives in place. Never remove a slide with the high-power or oil immersion objective in place as you might scrape the lens of the objective.
5. Place a slide of diatoms, microscopic organisms from both marine and fresh water environments, on your microscope. With the scanning objective in place, look at the circle of light with the very small algal cells scattered about. Find a group to look at and center this group in the field of view by moving the mechanical stage.
6. Turn the low-power objective into place, and examine the chosen group at the higher magnification. The field of view here has fewer diatoms but larger images.
7. Sketch one or two diatoms in detail. The diameter of the low-power field of view is about 1600 micrometers. A micrometer is 1/1000th of a millimeter.
8. Find a particular diatom to look at, center it in the field of view, and switch to high-power. The diameter of the high-power field of view is about 400 micrometers. By comparing a dimension of your chosen diatom, its length or width, to the diameter of the field of view, you can estimate the size of your diatom in micrometers. Suppose that your diatom covers about half of the field of view; then the estimate of its length will be 200 micrometers (400 micrometers of the field of view diameter divided by 2, because two of these diatoms would fit across the whole field).
9. Estimate the size of several diatoms that you sketched and record their dimensions.
10. Turn the scanning objective into place, lower the stage, and remove the diatom slide from your scope.
11. Not all of the slides that you will use in this laboratory will be prepared slides. Some fresh or live material is best viewed with the microscope using a wet-mount slide. Watch as your instructor demonstrates how to make a wet-mount slide.
12. With a dropper, place a small amount of water on a clean slide. Place a leaf of the water plant Elodea on the slide in the drop of water. Place the edge of a cover slip at the edge of the drop and lower the cover slip slowly to the slide surface. With care you can prepare a wet-mount with few if any air bubbles.
13. Observe your wet-mount preparation at scanning, low-, and high- power. Sketch what you observe and note any movement or interesting features.
14. On the back lab bench, locate the stereoscopic or dissection microscopes. With these microscopes, look at the objects that are available. Objects best viewed with a dissection microscope are much larger than those viewed with a compound microscope and do not require the same preparation. Notice that the image here is clearly three-dimensional and is not inverted or reversed as with the compound scope. Sketch some object seen here.
OVERVIEW
Science textbooks often discuss the methods used by scientists for studying the natural world. You should realize that scientists, biologists among them, do not always depend on a "cookbook" approach to doing science even though they often refer to the scientific method. Sometimes the process of discovery is less formal and more creative. That said, examining aspects of formal scientific methodology is still helpful as a way of understanding how science is done.
One possible scientific method, or outline for discovery, is as follows:
a. Observations are made about the natural world. These observations can be accumulated over a brief period or a much longer period of time and can be made by one or many scientists working together or in separate locations and at different times.
b. Questions are asked or problems are posed concerning these observations.
c. Hypotheses are developed. A hypothesis is a possible answer to a particular question. A hypothesis should be testable or it is not of much use to a scientist. A hypothesis must also be falsifiable—that is, capable of being disproved. For example, after asking yourself whether growing plants gain mass from the soil, you might form the hypothesis, “A potted plant converts soil into stems and leaves.” If you tested this, you in fact would find that your hypothesis was false. Good hypotheses also are stated in precise and narrow terms. The hypothesis “Plants grow fast” is too broad and imprecise to be tested easily.
d. Experiments are set up with controls. The experiments are designed to test predictions that come from the hypotheses that have been proposed. Experiments should be designed so that they can be repeated easily by the researcher and by others. For example, if the hypothesis above is true, you would predict that the mass of the soil would decrease as the plant grows, which is something you and others could easily test.
Controls are comparison trials or ways to eliminate chance from playing a part in the experiments. A control for the hypothesis above should include several pots with equal amounts of soil and no plants. The mass of the soil in each could then be compared with the mass of soil in pots containing plants. Generally, more valid results are obtained when the sample size is as large as possible and replicates are included in the design. In some circumstances in science, especially with historical sciences, such as geology, anthropology, and some fields of biology, experiments are not possible. In such cases, experimental steps may be replaced with careful observations of natural events.
e. Results of experiments (or observations) are recorded carefully and completely. When possible, experiments are designed to be quantitative, i.e., results can be counted or measured in numerical form. Graphs or tables of information are appropriate ways to display results.
f. Conclusions based on the results are formulated. The results are compared to the hypotheses. If the results match the hypothesis, the hypothesis is supported. If the results are negative, the hypothesis is proved false. There is nothing wrong with hypotheses that have been proven false. In fact, progress in science depends on proving hypotheses false and modifying them until they become more and more accurate. We can never prove hypotheses true; we gather more support for them. For the example above, the conclusion would be that plants do not convert soil into stems and leaves; we would reject our hypothesis.
g. Experiments may be repeated or revised, and hypotheses may be altered or rejected. For example, we might modify our earlier hypothesis thus: “A potted plant converts a gas present in air into stems and leaves.”
Procedure:
1. Before beginning your own experiment, make a wet mount slide of one or more individuals of the “water flea,” Daphnia. This little animal is a shrimp-like creature common in many kinds of freshwater habitats. It swims by flicking its large antennae, and feeds by using feathery legs to filter food particles, including bacteria and other small, aquatic animals, from the water. Despite its small size, Daphnia is a complex animal with organs that are in some ways similar to our own. For example, you should be able to easily trace the intestine along the upper, or dorsal, side of the animal. Your instructor will demonstrate, on the large screen at the front of the room, some of the other features of Daphnia, including the heart. Be sure you can locate the heart on your own specimen.
Daphnia cannot regulate its body temperature as we do; most small animals have body temperatures which fluctuate with the environmental temperature. As a result, their activities speed up or slow down according to the changes in the environmental temperature. One of these activities, of course, is the beating of the heart. In addition to temperature, other variables, such as the salt content of the water, might also have an effect on these activities.
2. After making your own observations on the heart of Daphnia, compose a hypothesis about how the heart might be affected by changes in environmental variables, such as temperature, salinity (osmality), or the relative acidity or alkalinity (pH) of the water. It should have all the qualities that have been discussed in the introduction as characterizing a good hypothesis. From the hypothesis, write out a prediction from the hypothesis that could be tested by an experiment.
Then design an experiment to test the validity of your prediction. Ask your instructor or the lab assistant to check your hypothesis and prediction, and to make suggestions about improving your experiment.
3. Using the materials available, carry out your experiment. Gather as much data as time permits.
Assignment:
1. Write a report for this experiment to be handed in at the start of next week's lab. Use the standard four-section format that all scientists use and follow all instructions carefully. Your report will be graded not according to the results of your experiment i.e., whether your hypothesis was supported or disproved, but by how well you adhere to these directions and how effectively you describe your results and their implications.
a. The first section is the Introduction, in which you tell why you did this experiment. Describe the experiences or the observations that led you to pose the question that led to your hypothesis. What did you seek to answer? What did you expect to find? Why did you choose to perform this experiment rather than another one? Be sure to state your hypothesis explicitly in this section.
b. The second section is labeled Materials and Methods. You need not list all the materials you used here. Simply describe, in a narrative paragraph or two, your experimental protocol; this will include mention of all the materials you used. Use the past tense. The purpose of a lab report is to report what you already did so that others can repeat your work. Do not give instructions to "place a goldfish in a bowl," for example; report that "we placed a goldfish in a bowl...." Try to be as precise as possible. Remember that others should be able to repeat your work from just this description, so don't take anything for granted or leave any steps out.
c. The third section is the Results, in which you should include any data you collected, including observations or quantifiable (numerical) data. Organize data in a table or graph. Do not analyze your results in this section.
d. The fourth and final section, the Discussion, is where you offer an analysis of your results. As the title suggests, this is where you discuss your results with reference to your initial hypothesis. Was it supported? Was it disproved? Draw appropriate conclusions about your experiment and discuss them here. Evaluate your work, including, if appropriate, an alternate hypothesis for further experimentation. This is the place to speculate about why the results did not support the hypothesis or about future experiments. What would you do differently if you performed your experiment again? How would you modify or expand your experimental design? What questions arose as a result of your experiment, and what steps would you take to answer them?
Notes on format:
In addition to preparing the report for your experiment, read the next laboratory about analyzing forest communities. Next week’s lab will be outside, so dress for fieldwork in the forest. Long pants, shoes, and socks are required. We will go out even if it is raining.
