Effect of caffeine on daphnia heart rate

Laboratory 3 The Scientific Method

Objectives

· Understand the steps involved in the scientific method

· Define and identify: independent, dependent and control variables

· Calculate the heartbeat of Daphnia under various experimental conditions

· Analyze the data obtained

· Make conclusions regarding the various variables tested and Daphnia’s heart rate

Background Information

All fields of science have one unifying principle that is a common tie among these diverse scientific disciplines. That unifying theme is the scientific method .

The scientific method is simply an organized, methodical, and structured way of observing and/or investigating a situation in an effort to find information about what is being observed. There are six steps to the scientific method.

1. Identification of the situation to be investigated.

This is vital because no progress can be made towards understanding the situation unless one knows exactly what is being investigated. Let’s consider an example. Suppose that you notice (observe) a list of essential nutrients on the label of a box of plant fertilizer. You wonder how plant growth might be affected if plants are deprived of just one of those essential nutrients. You decide to investigate the effect of the lack of potassium on pepper plants.

2. Obtain information about the situation being investigated.

One of the biggest advantages in problem solving is knowing the background information about what is being investigated. This is why researchers do searches of the scientific literature when writing a paper or conducting research. Accordingly, you would go to the library and read as much as you can about plant nutrition and how potassium affects plant growth.

3. Formulation of a hypothesis.

A hypothesis is a possible explanation of the problem or situation based only on what it is known about it so far. The hypothesis must be testable: an experiment must be designed to test its validity. Another important characteristic of a hypothesis is that it must be falsifiable. This means that the hypothesis must make predictions that could be proven false by experimental results. Your first hypothesis might be, “Plants grown in a medium lacking potassium will show some specific signs of malnutrition.

4. Predict the results.

Assuming your hypothesis is correct, you ought to be able to predict the outcome of a situation where your hypothesis was actually applied to the problem. You might now try to imagine how a pepper plant would look when grown in a potassium-free medium. Perhaps there would be obvious changes in the leaves and/or the stem height.

5. Design and conduct an experiment to test the hypothesis.

An experiment is an investigation conducted under very specific conditions in which all variables are controlled except the one being studied. A variable is an event or condition subject to change. In the potassium study, the lack of potassium is the variable being investigated.

If, at the end of the experiment, the hypothesis should be found to be wrong, then it can be modified, further tested or completely discarded. The scientific method commonly results in a long series of repeated testing and hypothesis modification. A hypothesis can never be proven right unequivocally . With more and more experimental evidence to support it, a hypothesis gradually evolves into becoming more and more valid for the situation or problem. The evolution of a hypothesis is based on conducting experiments, making observations, gathering data, etc., all of which are done to investigate the validity and to challenge the hypothesis under consideration.

The design of experiments to test hypotheses requires considerable thought. The variables must be identified, appropriate measures developed, and influences outside of the experimental variables must be controlled. The independent variable is that which will be varied during the experiment; it is the cause. The dependent variable is the effect; it should change as a result of varying the independent variable. Controlled variables are also identified and are kept constant throughout the experiment. Their influence on the dependent variable is not known, but it is postulated that if kept constant they cannot cause changes in the dependent variable and confuse the interpretation of the experiment. For example, suppose you were growing plants with the intention of studying how the amount of water affects their growth. In that case, the independent variable would be the amount of water provided (the variable that you are purposely changing). The dependent variable could be the length of the stem (that is, what changes as the amount of water is purposely changed), and controlled variables would include the amount and quality of light provided, temperature, minerals provided and so on.

Going back to the initial experiment about the role of potassium in pepper plants, you could conduct your experiment based on a technique discovered in the literature search. You could grow your pepper plants hydroponically (in water with plant nutrients and no soil). In your experiment, you would have two groups of plants, each group consisting of six pepper plants of the same variety and all are the same age, size, and general state of health. In addition, both groups of plants would be grown under exactly the same environmental conditions of heat, light, container size, etc. It is important that all of the conditions (except the one being investigated, potassium) be exactly the same for both groups. The only difference between the two groups is that one will be grown with complete nutrients, the other with all nutrients except potassium.

When your experiment is run, the plants should be allowed to grow for a few weeks, after which time the plants would be compared. In this design, the plants growing in the complete nutrient solution serve as the control group, which is the group forming the basis for judging any differences that may appear in the experimental group, the group grown without potassium. A control is essential in any experiment because it reveals any differences in the experimental situation.

6. Form a conclusion based on the results.

The validity of the hypothesis may or may not be determined. Either the results of the experiment support the hypothesis or the results show that the hypothesis needs modification. If you found the control plants to be lush and green with a height increase of three inches since the experiment began, and the experimental plants to have no increase in height, to have weak stems, and to have yellowish leaves with brown spots, you would have supported your hypothesis.

The experiment does not PROVE your hypothesis to be correct beyond all shadow of doubt. What the experiment does show is that under the conditions of the experiment, potassium appears necessary, and the hypothesis is supported.

The scientific method is neither complicated nor intimidating – nor is it unique to science. It is a powerful tool of logic that can be employed any time a problem or question about the fundamental nature of something. In fact, we all use elements of the scientific method to solve little problems every day, but we do it so quickly and automatically that we are not conscious of the methodology. In brief, the scientific method consists of observing, predicting, testing, and interpreting.

You will base today’s experiment on observations of twentieth-century American lifestyles. You have probably observed that when people drink too much coffee, they are often hyperactive. They may be jittery, nervous, and complain about being unable to relax. On the other hand, often when people consume alcoholic beverages, their speech can become slurred, they may lose control of their muscular coordination, and their reactions may slow down. You will be looking at the effect of alcohol and caffeine on Daphnia magna , a small water crustacean. You will evaluate the effects of these drugs by measuring the heart rate of Daphnia when exposed to various concentrations of alcohol and caffeine.

NOTE: All organisms are classified by Latin names that specifically identify them. You must always identify an organism by its proper scientific name so that other scientists know what you are talking about. You must also remember to ALWAYS italicize or underline the Latin names (genus and species) of organisms EVERY TIME YOU USE THEM!

The advantage of studying Daphnia is that they are almost transparent. You can see the heart beating, the squeezing action of the intestine, muscular movements, and occasionally, babies in the brood pouch. Also, because Daphnia is a small, aquatic organism, it makes an excellent subject for studying the effects of drugs on circulation.

Even if you performed all of your experiments carefully, you cannot be certain that the effect you see is due to the drugs. Perhaps the change in heart beat rate that you may observe between the alcohol and caffeine is caused by the heat of the microscope light, or perhaps it is affected by the removal or addition of solutions. Without a control experiment, your data are meaningless. You will begin the experiment by performing the control procedures and getting a base heart rate for Daphnia before it is exposed to the drug solutions.

Activity 3-1: Control Procedure

The control procedure must be performed exactly as the experimental procedure. The only difference is that the variable is omitted. In this case that means that alcohol and caffeine are not added.

1. Capture a living Daphnia and place it in a small drop of water on a depression slide. In order to easily observe and study the Daphnia you will need to obtain 3 threads and arrange the threads on the slide around the Daphnia in order to restrict it to a small area (Figure 3-1).

Fig. 3

-

1

Set up your depression

slide with threads as indicated in

the figure. The black dot

represents the

Daphnia

. This

arrangement will confine the

Daphnia

to a small space and

make it easier for you to identify

Daphnia

the heart.

2. Observe the Daphnia under the LOWEST POWER OBJECTIVE on a compound microscope.

3. Using Fig. 3-2 as a reference, locate the following structures:

· The most obvious structure is the eye.

· The brain is a light-colored organ lying above the eye. The pairs of antennae protrude from the head. These are used for locomotion and to sense the environment.

· Inside the exoskeleton are five pairs of legs. Comb-like gills are attached to some of the legs. When the legs kick forward, they bring a stream of water across the gills and wash bits of food up to the mouth, which lies just beneath the beak.

· From the mouth, the esophagus runs up into the head and then down into the body, where it widens into the stomach, which connects to the intestine.

· The heart lies in the upper part of the Daphnia (above the digestive tract), it is a clear structure and will be contracting rapidly. Before you continue with the lab you will need to take some time observing the Daphnia and make sure you can find the heart .

· In females, a large brood chamber is located behind the heart. Usually it will contain eggs, but occasionally it may be filled with baby Daphnia. The brood pouch will probably not be present in most Daphnia.

4. Working in pairs, one person should keep track of the time while the other person is counting the number of heartbeats in 15 seconds. The heart rate in a healthy Daphnia will be very rapid (3-5 beats per second).

5. You will take 3 readings of normal Daphnia heart rate. After each 15 seconds of observation you must absorb the water from the slide using a KimWipe. To do this, gently place the KimWipe at one edge of the water and all of the water will be absorbed.

Antennae

eye

anus

heart

intestine

egg

ovary

Drawing by Carlos Pacheco

-

Perez, 2004

Figure 3-2. Use this image as a reference for locating the indicated structures and organs in Daphnia.

6. As soon as the water is gone, use a the plastic transfer pipet on your tray to add another drop of distilled water to the Daphnia and count the heart rate for the next 15 second observation period. It is important to not let the Daphnia dry out. However, you must absorb the distilled water between each 15-second reading as this will be the same procedure that you will follow when you are testing the various alcohol and caffeine solutions. In order to maintain good scientific practices, you must follow the exact same procedure for the control that you do for the experiment.

7. When you are finished taking the control measurements, leave the Daphnia in a drop of water.

8. Record your data in Table 3-1 as the control values and calculate the number of beats per minute . One way to do this is to use ratios. For example, if you count 10 beats in 15 seconds, the calculations are as follows:

X beats = 10 beats X = 600

60 seconds 15 seconds; 15 or 40 beats/minute

Another (simpler way) is to count beats for 15 seconds and multiply that by 4.

Calculate the average Daphnia heart rate per minute. Record in Table 3-1.

Applying the Scientific Method
Note: Make sure that you have read the introduction to the lab before attempting to answer these questions.

You will now begin testing the reaction of Daphnia to different concentrations of caffeine and alcohol solutions. All tests will be done by each pair of students in the lab .

Before you begin the experiment, answer the following questions (A-E) which ask you to apply the scientific method to this experiment. If you need more space for your answers, you can use a separate sheet of paper to continue your answers to these questions:

A. Identify the situation that you are investigating.

B. Where can you obtain information about the situation that you are investigating? What kind of information would you look for?

C. Which variable do you think is the independent variable (the one that is varied to invoke a response)? Which variable do you think is the dependent variable (the one that is the effect)? Which variables will be controlled and how will they be controlled?

D. What do you think will happen in this experiment? Predict what results are possible; what you think may realistically happen in this experiment.

Activity 3-2: Measuring the heart rate of Daphnia under two different conditions
1. Use a KimWipe to absorb the water in the depression slide. Place one drop of 1% alcohol on the Daphnia. Wait 1 minute and then count the heartbeats for 15 seconds. Record the data in Table 3-1.

2. After counting the heartbeats for 15 sec, absorb the 1% alcohol solution using a KimWipe as you did earlier. Immediately add 3-5 drops of distilled water to the Daphnia and monitor its heart rate. It is very important that you give the Daphnia time to recover and allow its heart rate to return to normal between each different solution. Turn off the microscope and wait 3-5 minutes. Why is this important step?

3. When the heart rate reaches the average rate observed in the control experiment, you are ready to proceed with the experiment. Absorb the distilled water with a KimWipe and add a drop of 3% alcohol. Wait 1 minute and count the heartbeats for 15 seconds. Record your results in Table 3-1.

4. Quickly absorb the 3% alcohol solution with a KimWipe and add 3-5 drops of distilled water and repeat step 2. When the heart rate returns to normal, continue this process using the 5% alcohol solution. Record your data from these tests in Table 3-1.

5. At the end of the alcohol series of tests, substitute 1% and 2% caffeine solutions for the alcohol concentration solutions and repeat the tests. Make sure to remove the caffeine solutions, add water to the Daphnia, and let the heart rate stabilize between the caffeine solutions. Record your results in Table 3-1.

6. When you are finished with the caffeine series, let your Daphnia recover in water.

Table 3-1. Results from the alcohol and caffeine test series.

Experiment

Beats/15 sec.

BPM

(beats per min.) (Class result average)

Control

180

Alcohol 1%

166

Alcohol 3%

150

Alcohol 5%

129

Caffeine 1%

112

Caffeine 2%

152

Analyzing Data
Results from experiments must be presented in a clear, scientific way. The first lab of the class is the time to learn this step. If tables and graphs are well constructed, they provide a concise summary and allow the reader to see at a glance the pertinent results of the experiment. Use the graph paper provided to plot your results.

Making Graphs
Follow these steps to plot your data with the graph paper included in the lab manual:

1. Decide which variable is the dependent variable and which is the independent variable. The dependent variable is the variable you know as a result of making experimental measurements. The independent variable is the information you know before you start the experiment. It does not change as a result of the dependent variable but changes independently of the other variable.

2. Always place the independent variable on the x-axis (the horizontal axis), and the dependent variable on the y-axis (the vertical axis).

3. ALWAYS LABEL the axes with a few words describing the variable, and ALWAYS INCLUDE THE UNITS of the variable in parentheses after the variable description.

4. Choose an appropriate scale for the dependent and independent variable so that the highest value of each will fit on the graph paper.

5. Plot the data set (the values of y for the particular values of x). In this case you will have 3 graphs: one that includes the results of the alcohol test series, one which includes the results of the caffeine test series, and one which includes the results of the Experimental Beverage series. However, remember that these graphs should all have the same scale so that you can compare the three graphs and that ALL three of them should include the average “control” value.

6. You may choose to draw bar graphs. If you want to draw line graphs instead, draw smooth curves or straight lines to fit the values plotted for any one data set. A smooth line or curve through a set of points is a visual way of averaging out chance variability in data.

7. Every graph should have a legend, a sentence explaining what the figure is about and a TITLE !!!

Interpretation and Conclusions
One of the most important features of scientific inquiry is the exchange of information. Scientists publish experimental results to make them known to others, and they include their interpretations of what those results mean. Write a brief, one paragraph, discussion of this experiment, being careful not to let preconceived notions interfere with an objective analysis. Also be sure to use proper grammar and complete sentences in this discussion.

Further Experiments
The scientific method is a continuing cycle of questions and answers. A good experiment not only answers the question that was originally posed but also gives rise to further questions. As a final assignment, list several questions this experiment raised and the procedures that could be used to test them. Cleaning Up

Return all materials and solutions, and clean up your work area as directed by your laboratory instructor.

References:

1. Dolphin, W.D., Biological Investigations: Form, Function, Diversity and Process,

6th edition, 2002, McGraw Hill

2. Jacklet, A., Laboratory Manual to Accompany Life , 1998, McGraw Hill Companies

3. Skavaril, R., Finnen, M., Lawton, S., General Biology Lab Manual, Investigations into Life’s Phenomena, 1993, Harcourt College Publishers

Figure : Shows effect of Alcohol concentration on Daphnia’s heart rate

Alcohol Bar Graph

0 1 3 5 180 166 150 129
Daphnia heart rate(BPM)

3-2