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1 BIO270 Pre-lab 1 2014

How science ‘works’: An introduction

Design of an experiment to test the effect of bioactive
compounds on Daphnia magna heart rate

Learning Outcomes:

• Compose a testable hypothesis
• Gain experience in designing an experiment incorporating relevant controls and
treatments
• Begin to appreciate the difficulties in designing a reliable and repeatable
experiment
• Develop light microscopy skills (stereo dissection microscope)
• Develop cooperative group skills and scientific writing skills
• Students will experience the ‘way of science’ by participating in a hands-on inquiry
based laboratory

Preparation:

Moyes, CD, Schulte, PM. 2007. Principles of animal physiology. 2
nd
Edition. Toronto:
Pearson Benjamin Cummings. Chapter 3, Cell signaling and endocrine regulation; p.
90 – 141.
Pechenik, J A. 2012. A short guide to writing about biology. 8
th
Edition. Toronto:
Pearson. Chapter 9, Writing laboratory and other research reports; p. 157 – 164, p.
172 -182.
Garside, CS. 2014. Laboratory calculations review. Toronto. p. 1 – 12.

• View the links posted in the Lab 1 folder on Blackboard.
• Carefully read the materials in this file, including the appendices.
• Bring your completed Pre-lab exercise to the Pre-lab (see p. 15).
• Investigate the effects of ethanol, nicotine, and caffeine on heart rate.
• Consider your preliminary experimental design.
• Consult the ‘laboratory calculations review’ file posted in the General Laboratory
Files folder in the Lab Manuals folder on Blackboard.

Note: you will require lab coat, goggles, and closed toed shoes for this
laboratory.

Pre-lab/ Lab Outline

You are a researcher who is tasked with determining whether or not the following
bioactive compounds (ethanol, nicotine, and/or caffeine) have negative or positive
chronotropic effects on the heart rate of the CSB colony of Daphnia magna. Your lab
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group (you and your lab partner) may choose to concentrate on a single bioactive
compound and use multiple treatment levels (perhaps to create a dose-response curve)
or may choose to investigate multiple bioactive compounds at a single treatment level.
This is your choice, it is your experiment! Note that the stock concentrations of the
available bioactive compounds can be found in Appendix 1.

Your goal is to design an experiment using the materials provided to you in the Pre-lab
(see Appendix 1) to answer your chosen question(s). In this introductory file you will
find information regarding experimental design (p. 3-13), some specific experimental
considerations for measuring heart rate in Daphnia (p. 13-14), and a general
introduction to our experimental organism Daphnia magna (Appendix 3; p. 19-23). This
information will be crucial in determining whether or not your experiment will succeed
or fail; review and consider these materials carefully.

Pre-Lab: We expect you to come to your Pre-lab prepared to discuss your preliminary
experimental design including which of the available bioactive compound(s) you would
like to investigate. During your Pre-lab you will have an opportunity to observe and
count the heart rate of Daphnia magna. You will also be provided with the materials
that you will have at your disposal to perform your experiment. This is your pilot study,
take advantage of this time to address some of the experimental design considerations
(see p. 13-14). By the end of the Pre-lab, you and your lab partner should have
discussed your experimental design, tested your experimental design*, and ultimately
come up with a consensus experimental design that you will use in the laboratory. You
should also ensure that you are comfortable working under a stereomicroscope (see
Appendix 2).

* Note: we understand that time is limited in the Pre-laboratory. Your goal is to test
what you can in the time available. Be prepared.

The pre-lab will consist of three parts:

1. Review of the Pre-lab exercise (see p. 15). Note: this must be completed before
you come to your Pre-lab.
2. TA will provide instruction on how to complete your Pre-lab assignment.
3. Time to discuss, consider and test your experimental design with your lab
partner, so come prepared.

After the pre-lab, you must:

1. Individually complete the Pre-lab assignment posted on Blackboard and submit
online by the submission deadline. The assignment includes:

a. Written Materials and Methods section.
b. Answers to questions related to your experimental design.

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2. Individually complete the online quiz which will be based on the material listed in
the Preparation section of this Prelab 1 Manual.

Note: You are encouraged to discuss your experimental design with your TA and your
classmates to get feedback and suggestions during your Pre-lab.

Introduction to Writing in Science:
Science is both a body of knowledge and a process of discovery for building that
knowledge. The body of knowledge has been assembled and continues to be assembled
by the concerted human effort to understand, or to understand better, and explain the
history of the natural world and how the natural world works. The process of discovery
can be carried out through observation of natural phenomena and/or through
experimentation that tries to simulate natural processes under controlled conditions.
Science is exciting, useful, complex, unpredictable, continuous, and is a collaborative
global endeavour.

To be involved in the process of science, it is imperative that a scientist considers what
has already been done (e.g. peer-reviewed literature), assimilates the evidence, openly
communicates ideas, exposes findings to others for critique, and importantly must act
with scientific integrity (e.g. hiding or selectively reporting evidence, plagiarism etc.).

It is important to grasp that although science is powerful, it does have limits. It cannot
make moral or aesthetic judgements, it cannot tell you how to use scientific knowledge,
and it does not devise conclusions about supernatural explanations. Furthermore,
science is not absolute; although scientific data is reliable and supported by evidence,
they can be refined if new techniques become available and new results are reported. It
does not matter how much data you have or how many experiments have been
performed that support your hypothesis. All concepts in science are fundamentally
tentative. As we accumulate evidence our level of confidence in our hypothesis
increases. As more and more evidence accumulates supporting our hypothesis, and no
evidence appears that contradicts the hypothesis, we become more confident in the
data. Newton’s Laws and the theory of evolution are examples of this. We assume that
they are at least very close to the truth; but we never state definitively that they are
the truth. There is no such thing as proof in science. Therefore, science is always
changing and will never be ‘finished’.

Interestingly, the process of scientific discovery is not necessarily complicated or unique
and is certainly not limited to professional scientists working in labs. During our
everyday experiences of deducing that a bicycle will not ride smoothly because of a flat
tire, or that a child is irritable because they have a fever, have fundamental similarities
with classical scientific discoveries. J ust like ‘science’, these experiences all involve
making observations and analyzing all of the available evidence.

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The process of discovery in science has historically and commonly been presented as a
sequence of steps called the scientific method (figure 1). However, the scientific
method severely oversimplifies the ‘real’ process of science. In fact, few, if any
scientists adhere rigidly to this series of steps. Science is a less structured process than
most people realize. Like other intellectual activities, the best science is a process that is
creative, intuitive, and collaborative. Rather than being a simple linear sequence of
steps, science is iterative and not predetermined and manifests itself in many different
forms. Moreover, it necessarily involves attaining input from the scientific community,
and interacting with the society as a whole
(see http://undsci.berkeley.edu/article/scienceflowchart for further details regarding the
real process of ‘science’). Nonetheless, the basic tenets of the scientific method will
provide us with a convenient entry point into the basic process of experimental design.

























Figure 1. Simple example of the Scientific Method.

Listed below you will find a series of steps and experimental design considerations that
you will have to work through before you will be able to successfully design, perform,
and analyze the results of your experiment:


Observation
(Become Aware of)
Ask Question(s)?
Develop a Hypothesis
(Explanation of Trial)
Design and Conduct Experiment
Compare predicted results to experimental results
(Reflect on results and Draw conclusions)
Possible Outcomes
1. Hypothesis is supported
2. Hypothesis is falsified
3. Hypothesis is partially supported
Contemplate and discuss
results (Generate new
questions and hypotheses)
5 BIO270 Pre-lab 1 2014

1. Formulate a Question from an Observation.

A ‘good’ scientist observes, perceives (i.e. sees, hears, smells) and explores the world
around them and ultimately becomes curious about what is happening or why that is
happening. It may stem from recognition of a pattern, departure from a pattern,
analogy with other systems, intuition or imagination. The scientist then formulates a
question about what (s)he observes. It is important that this question be falsifiable by
performing an experiment. Remember that if something is falsifiable it does not mean it
is false; rather, if it is false, then this can be shown by observation or experiment.

For example, you observe that when you drink a cup of coffee, your heart
rate seems to increase. You might then ask the following question:

“Does consumption of caffeine found in coffee increase heart rate?”

2. Develop a Hypothesis.

The proposed explanation for an observable phenomenon is called a hypothesis. It is
based on prior knowledge, general principles, and a review of the scientific literature
and is not simply an educated guess or an opinion. It is essential that any proposed
hypotheses are testable. If a hypothesis is confirmed, it is retained with greater
confidence, but not accepted as true. If falsified, it may be rejected outright as false, or
modified and retested. It is important to remember that hypotheses can be proven
incorrect, but can never be proven or confirmed with absolute certainty. This is because
it is next to impossible to test all given conditions, and in the future someone else may
find a condition under which the hypothesis does not hold true. Our understanding of
biology is always in flux; as novel experiments and new technology force well supported
hypotheses to be modified and retested.

Biological hypotheses are most often stated in terms of the independent and dependent
variables (see section 3a below) that are going to be used in the study. In many cases,
evidence for causality is the aim. If a causes b, we expect, repeatedly (see 3 below), to
find that a change in a, leads to/results in a change in b. The ideal experiment would
involve the measurement of b (dependent (measured) variable), at one or more values
(concentrations, doses, etc.) of the independent variable (a), with results demonstrating
a relationship between them.

In our coffee example, one hypothesis may be:

“Consumption of caffeine found in coffee increases heart rate”. OR more
formally: “If caffeine in coffee causes an increase in heart rate, then
increasing the consumption of coffee will result in a greater increase in
heart rate”. More specifically, the scientist may test how the consumption
of 1-5 cups of coffee affects heart rate.
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As outlined above, when an observation is investigated in science we design a
hypothesis stating that something interesting is happening and then set out to test it.
The essential components of a hypothesis are that it should be testable, falsifiable,
establish the independent and dependent variables, and predict an effect (the direction
of the relationship). Formally, this is referred to as the alternate hypothesis. Traditional
hypothesis testing also requires that the researcher formulate a null hypothesis, which
predicts that the independent variable will not have an effect on the dependent
variable. It is essentially a hypothesis against the prediction stated in our alternate
hypothesis. Predictions are tested in the form of a null hypothesis because science
generally proceeds conservatively, assuming that something interesting is not
happening unless convincing evidence suggests that something interesting might be
happening.

For example:

“The consumption of caffeine found in coffee will not significantly affect
heart rate” OR “There will be no significant effect on heart rate as the
consumption of caffeine increases”.

The purpose of an experiment is to gather evidence in order to decide whether to
accept or reject the null hypothesis. In other words, it determines whether we can
safely say that the independent variable has had an effect on the dependent variable,
or not. See ‘Laboratory Calculations Review’ for a brief discussion of potential statistical
analyses in this laboratory.

3. Designing and Conducting Experiments to test the Hypothesis.

Once you have determined your research question and developed your hypothesis, the
next crucial decision is the problem of how you are going to test your specific
hypothesis. This is really the heart of experimental design.

What is Experimental Design and Why is it important?

Experimental design is the planning of a conceptual framework of the procedure that
enables a researcher to test their hypothesis by reaching valid conclusions about
relationships between independent and dependent variables. It is wise to take time and
effort to design and organize the experiment properly to ensure that the right type of
data, and enough of it, is available to answer the question(s) of interest as clearly and
efficiently as possible. It is about maximizing the amount of information we can gather,
given the resources that we have available. If we have limits imposed upon us with
respect to the system and with the materials provided, then we will have to live with
those limitations. However, if our conclusions are limited by poor experimental design
with what we have at hand then we have wasted time, resources and probably money
also. Because the validity of an experiment is directly affected by its construction and
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execution, attention to experimental design is extremely important. Essentially,
experimental design encompasses careful planning, common sense and biological
insight.

Note: A small quantity of carefully collected data is much better than a large quantity of
poor quality data.

Two key concepts in experimental design for life scientists are to account for any
confounding variables and to minimize random variation.
A confounding variable is an extraneous variable whose presence affects the
variables being studied so that the results you collect do not reflect the actual
relationship between the variables under investigation. Confounding variables can alter
the outcome of a study by influencing the apparent relationship between the
independent and dependent variables. The confounder can either mask a real
relationship between independent and dependent variables or the confounder can make
it seem that there is a relationship when in fact there is not. These variables must be
controlled for so that reliable conclusions can be drawn from the data collected.
Consider our caffeine experiment again. Suppose that we allowed our
study participants to continue to class after their coffee and return after
class to have their heart rate re-measured. One confounding variable here
might be that some of the students had an exam in class whereas the
others did not. Clearly, this could affect the heart rate of those students as
we know that stress tends to raise heart rate.
Extraneous or situational variables which are not controlled by the experimenter, and
are not part of the experiment (e.g. room temperature, solution temperature, lighting,
noise etc.) can become confounding variables if they differ systematically across
experimental conditions and if at all possible should be controlled for.
Random variation (between-individual variation or within-treatment variation)
quantifies the extent to which individuals in a sample differ from each other. Random
variation is ubiquitous in biology due to genetic variation and influence from
environmental factors. This variation makes it difficult to draw valid conclusions from
single observations. For example, consider that we want to determine the characteristic
size of the heart in a particular population of Daphnia. We could not simply measure the
size of the heart of one Daphnia and argue that that size is also valid for all Daphnia in
the population. A researcher needs to measure the size of a representative sample of
the population, allowing them to describe the mean size of the heart and the extent of
variation around that mean.

Life scientists must always be aware of random variation and confounding variables and
must take that into account when designing and carrying out experiments. Two
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important approaches to minimize these are replication and randomization. A third will
be discussed later which is design of carefully controlled experiments.

Replication involves making the same manipulations and taking the same
measurements on a number of different experimental subjects (replicates). Statistics
are based on replication, and are really just a way of formalising the idea that the more
times we observe a phenomenon the less likely it is to be occurring simply by chance.
Replication provides an estimate of experimental error and improves the precision of
the experiment by reducing standard error of the mean. The more replicates we have,
the greater the confidence we have that any difference we see between our
experimental groups is due to the factors that we are testing and not due to chance.
However, we must also take into account the cost of large numbers of replicates (e.g.
financial, time, and animal welfare) and the time available for collection of data.
o How does one select the appropriate number of replicates? There are basically
two approaches: educated presumptions based on similar studies or by carrying
out a formal power analysis. Power analysis is beyond the scope of this course
and as such we will leave the discussion of power analysis for future courses
that you may take.

Randomisation is the process of assigning subjects or objects from the wider
population of all the possible individuals that could be in your sample to a control or
experimental group on a random basis. This is practised to avoid bias in the estimate of
experimental error and to ensure the validity of the statistical tests. Randomisation does
not only apply to the setting up of an experiment, but can equally apply to the order in
which you treat replicates (time of measurement introduced as a potential confounding
factor). For example, will you be as accurate at measuring the heart rate after
observing Daphnia down the microscope for 2 hours as you will be at the beginning of
your experiment? Alternatively, perhaps you become better at taking heart rate
measurements as the experiment nears its end because you discover that you need to
focus on only one specific part of the heart and not the heart as a whole. Whatever the
reason, this means that if you do all the measurements on one treatment group first
and then all those of another treatment group, you risk introducing systematic
differences between the groups because of the changes in accuracy of the methods.
o This is referred to as intra-observer variability: imprecision or inaccuracy
introduced by human error: systematic change in a measuring instrument or
human observer over time, such that the measurement taken from an individual
experimental unit depends on when it was measured in a sequence of
measurements as well as on its own intrinsic properties.
o Inter-observer variability – arises because several observers were used to
gather a body of data. Two observers (or instruments) are not exactly alike,
differences between them can add imprecision and bias, if you are not careful
in your design.
o Observer effects – there may be times when the simple act of observing a
biological system will change the way it behaves. Consider the possibility that
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Daphnia may respond to the fact that the microscope light is shining directly on
them or that you are hovering over the animals. One potential method to deal
with these effects is to allow the animals to acclimatise. Whenever animals are
taken into the lab or moved from one location to another, it is essential to give
them time to settle, so that their behavior and physiology are ‘normal’ when
studies are performed. Don’t forget that an important part of the acclimatisation
might be getting used to the observer, or the handling procedures being used.
o A blind design is one in which the person measuring experimental subjects
has no knowledge of which experimental manipulation was applied or which
treatment group they belong to. These can eliminate any preconceived feelings
about whether the treatment will have an effect or not. If a researcher knows
what they expect will happen in a given experiment, then that prejudice may
bias their assessment. Even if the researcher is not being deliberately
dishonest, which hopefully is the case, there is still the possibility that they will
unconsciously bias the assessment in line with their predictions. Note that with
human studies, a double-blind procedure is often undertaken in which both the
experimental subjects and the researchers are kept unaware of which treatment
group the subjects belong to.

To reduce experimental error, experiments are designed so that test individuals or
experimental units are assigned to treatments in either control groups or experimental
groups randomly and then each group is replicated. In this way, error can be accounted
for and removed or negated by statistical analysis of the results.

The decision about how to test your hypothesis is often the most challenging part of
‘doing’ science. Designing an experiment involves defining variables (explicitly stated in
your hypothesis), outlining a procedure, and determining the controls to be used as the
experiment is performed. Implicit in these decisions regards the type of data you will
collect. Will it be measurements (quantitative), observations (qualitative), or estimates?

A well designed experiment often links a response (dependent variable) to different
levels of the independent variable. These levels are called treatments, and in our coffee
example, they may include different numbers of cups of coffee, and of course
appropriate controls are essential.

a) Defining the variables

In most cases, a scientific experiment must be a controlled experiment. The
experiment will compare/contrast two groups that are exactly the same except
for one condition or variable being tested (this is not trivial and takes careful
planning!). A variable is a factor or characteristic that exists in different degrees
or levels. The two groups are commonly referred to as the experimental
group and the control group. Variables in an experiment should be clearly
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defined and measurable. They are classified as independent, dependent, and
control variables.

• The independent (or manipulated) variable (a) is the variable of
interest, which is intentionally changed by the experimenter. It is often called
the treatment. In our example, caffeine is the independent variable.

• The dependent (or responding) variable (b) is the variable that will be
measured, counted or observed in response to variation of the independent
variable (it depends on the treatment, or independent variable). In our
example, heart rate is the dependent variable.

- The underlying assumption in experimental design is that the independent
variable is affecting the dependent variable. This is only true if all other
variables are controlled. For example, before performing the caffeine
experiment, the researcher must concisely define parameters such as:
volume of coffee consumed, strength of the coffee consumed,
temperature of coffee consumed, time after consumption of caffeine heart
rate will be measured, etc.

• The control variable(s) is/are therefore held constant or whose impact is
removed so that we can ignore it, in order to analyze the specific relationship
between the independent and dependent variables without outside
interference.

An ideal experiment will involve a manipulated independent variable, a measured
dependent variable, and all other variables should remain the same. In practice, this is
actually quite difficult to achieve!

b) Conducting the experiment

The primary assumption that is made in an experiment is that the independent
variable does indeed have an effect on the dependent variable. In order to test
this assumption, you must perform at least two tests:

• One treatment or set of treatments will examine the effect of the
independent variable on the dependent variable (experimental group;
i.e. individuals who drink coffee with caffeine). In a bioassay, the
experimental treatment(s) are often the concentration of the solution to
which the organisms are exposed.
• The other treatment will examine the dependent variable in the absence
of the independent variable (control group; i.e. individuals who drink
coffee without caffeine). Controls are replicates that include all of the
conditions for the experimental treatment except the independent
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variable. Controls attempt to validate that the results are due to only the
independent variable (by canceling out any potential confounding
variables).
o Negative control – group to which no manipulation is applied;
otherwise are treated in an identical manner as the treatment
group. They are exposed to the same conditions as the treatment
groups, except for the variable being tested.
o Positive control – in some cases, the research question will call for
the control group to also be manipulated in some way. This
ensures that there is an effect when there should be an effect, by
using an experimental treatment that is known to produce that
effect (and then comparing this to the treatment that is being
investigated in the experiment).
o Another treatment may examine the solvent used in the experiment
(solvent control group/ placebo; i.e. individuals who drink
water only to see if the act of drinking affects heart rate).

The control treatments serve as benchmarks that allow a scientist to determine
whether the experimental effect is really due to the independent variable.

c) Organization and Analysis of Data

i. Calculate the mean and standard error of the mean (never present raw data).
ii. Decide whether you will use tables, graphs, and/or drawings to organize and
present this summarized data?
iii. Determine if you see any trends or patterns in your data.

Note: If you choose to do inferential statistics, please consult the file ‘Laboratory
calculations review’ in the Lab Manuals folder on Blackboard.
All experimental design decisions involve elements of compromise, and although there
are good and bad experiments, there are no perfect experiments. The best way to carry
out one study will be very different from the best way to carry out another, and
choosing the best experimental system to test your chosen hypothesis will require an
appreciation of both the biology of the system and the pros and cons of different types
of studies. Obviously, the better an experiment is designed, the easier it is to perform
the experiment, obtain data, and draw conclusions. Most successful experiments are
the result of careful planning, attention to detail, multiple attempts, and learning from
past mistakes. The more careful you are about designing and performing your
experiments, the more sense your results will make, and the easier it will be to describe
the results of your experiment.


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4. Reflecting on Results and Drawing a Conclusion based on data obtained.

Once you have executed a well-designed and well-controlled experiment, you are in a
position to use your analyzed data to make a conclusion about your hypothesis. Do the
data you collected refute or support your null hypothesis?
a) If your data refute/falsify the null hypothesis, what else would you like to know
about this question?
b) If your data fail to refute the null hypothesis, you might propose a modified
alternate hypothesis based on what has been learned. Consider whether it was
due to flaws in the experimental design, data collection, analysis etc.?
c) Note that the usual outcome of an experiment is more questions! Remember that
science is iterative and never ‘finished’.

You should be prepared to interpret whatever you discover, regardless of what you
predicted you should find. The purpose of experimental science is to discover the truth,
not to make the data conform to one's expectations.

Other Experimental Design Considerations:

Measurement

Well-designed experiments often involve measuring something (e.g. concentration of
hormone in the blood). When measurements are made, it is important to know both the
accuracy and the precision of your measuring system. Although often used
interchangeably, these two terms are not synonymous: 'accuracy' means the ability of
the method to give an unbiased answer on average (or how close the measured value
is to the actual (true) value), whereas 'precision' is an index of the method's
reproducibility (or how close the measured values are to one another). Ideally your
method should be both accurate (i.e., give the true mean) and precise (i.e., have low
standard deviation). Accuracy and precision contribute to the reliability of your data.

Pilot Study

A pilot study may mean anything from going to the study site and watching for some
period of time or testing your experiment on a limited scale. The aim of a pilot study is
to allow you, the researcher, to become well acquainted with the organism and system
that you will be investigating. During this phase of the project, you will gain information
that will help you to better design your experiment. This phase will provide you a
chance to practice and perhaps validate the techniques you will use in the full study.
Diving straight into a full scale study without any pilot study or preliminary data will
most often provide you with less useful data than you might have collected if the pilot
study or preliminary observations had suggested some early fine tuning.

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In your case, the pilot study will be very short indeed. You will have less than 1 hour in
the Pre-lab to observe the materials available and determine the best experimental
design to answer your question(s). Due to the deficiency of pilot study time, we have
provided some preliminary experimental results and a list of experimental design
considerations below.

Daphnia magna preliminary data:

The results of preliminary experiments below should be taken into account when you
design your experiment. In addition, we have provided you with a list of experimental
design considerations. You should contemplate how you might answer these questions
or account for these issues before you come to your Pre-lab and then test them in the
Pre-lab. Therefore, it is important to come to Pre-lab prepared and to make the most of
your Pre-lab time.

Results from preliminary experiments:

1. 10% ethanol, 50 mM caffeine, and 100 µM nicotine show substantial effects while
higher doses are toxic.

2. Daphnia magna heart rate is temperature sensitive and beats rapidly at room
temperature. It is therefore important to control as best as possible for temperature
changes between animals and solutions (see Table 1). Furthermore, it is very difficult
to measure heart rates at room temperature as the heart beats very quickly. Each
group will be provided with a Daphnia Cooling Chamber to help account for these
difficulties. Our studies show that the Daphnia cooling chamber will cool the petri
dish and small volumes of solutions to 15-18°C (assuming you monitor the chamber
carefully and adjust as necessary).

Table 1. Effect of temperature on Daphnia magna (CSB colony) heart rate (bpm).

Daphnia Temperature (°C)
10 20 30
A 54 120 180
B 48 90 172
C 60 144 252
D 48 108 294
E 66 162 306
F 60 150 240
bpm = beats per minute. Measurements are the mean of three measurements for 10 seconds
multiplied by 6.

3. Two methods of Daphnia immobilization are possible:
a. Use a small volume of bathing solution (5-10 µl)
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b. Add a few strands of cotton wool under or above Daphnia (25 – 100 µl bathing
solution). Adding too many strands will lead to the Daphnia having to be
sacrificed…this means a few strands!

4. The heart rate of Daphnia magna may be erratic due to stress. You must do your
best to minimize stress.

Below are some of the issues to consider in your design, answers to which
should be clearly addressed in your Materials and Methods (expect that more
will come up!):

• How will you transfer your Daphnia to your testing dish?
• How will you restrict movement of Daphnia to ensure that you can clearly and
reliably observe and count the heart rate?
• How will you maintain a temperature conducive to heart rate measurement?
• What will be treatment and/or treatment levels?
• How will you remove and add treatment solutions?
• How can you be sure that the concentration of independent variable under the
microscope is the same as the intended concentration?
• How will you ensure that you are testing the effect of the test compound and not the
effect of induced temperature changes? Consider acclimation.
• How long will you count the heart rate for? All data should be presented per minute.
• What will be your control(s)?
• How will you ensure a reliable, repeatable baseline heart rate measurement prior to
addition of test solutions?
• How many replicates do you need to feel confident in your heart rate estimates?
• Consider if the amount of time spent looking down the microscope will affect the
accuracy of your measurements (negatively or positively)? Will you randomize the
sequence of measurements?
• Will you apply several treatments to the same individuals, or apply different
treatments to different individuals. In the first case we probably will use fewer
individuals, but they would need to be kept under experimental conditions for a
longer period of time and handled more often. The second case would necessitate
the use of more animals, but keeping them for less time and handling them less
often. Which case is better? There is no simple answer, but the pros and cons should
be considered before carrying out an experiment.
• How will you analyze and present your data?

You should also think about the following questions (although these should
not be directly addressed in the Materials and Methods section):

• What is the question you want to answer? Will your experimental design allow you to
answer the question?
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• Based on the literature, can you make a prediction about the effects you might
observe?
Pre-lab 1 Exercise:
Consider the following Observation:
Individuals who live in houses with smokers seem to have a higher incidence
of cancer.
Come up with a question regarding this observation?
_____________________________________________________________________
Determine the Variables.
Dependent: _____________________ Independent: _____________________
Determine the Experimental Group: _________________________________________
Identify a treatment that makes the question testable.
_____________________________________________________________________
Identify an outcome that makes the question falsifiable:
_____________________________________________________________________
Determine a control Group:
_____________________________________________________________________
Put the above elements together to form a complete falsifiable hypothesis
_______________________________________________________________________
State your null hypothesis:
______________________________________________________________________
List any confounding variables that you would like to eliminate so that results could be
assumed to be as a result of the independent variable.
______________________________________________________________________
16 BIO270 Pre-lab 1 2014


Appendix 1.

Provided Materials


Cell and Systems Biology (CSB) Daphnia colony (max 15 per pair of students)
Plastic wide-mouthed pipettes (this is essential to minimize damage to Daphnia when
transferring)
P
20
, P
200
, P
1000
pipettors
1.5 mL eppendorf tubes (consider your dilutions before you come to lab)
Petri dish
Depression slides
Daphnia Cooling Chamber
Ice
Stereomicroscope and light
Cotton Wool
Kim wipes
Filter paper
Double distilled water
100 μM Nicotine Solution
50 mM Caffeine Solution
10 % Ethanol Solution
Daphnia rehabilitation chamber
Thermometer
Stopwatch
Sharpies
Gloves




17 BIO270 Pre-lab 1 2014

Appendix 2.

How to Use a Stereomicroscope

A stereomicroscope is a low-power compound microscope that produces a stereoscopic
image. It is widely used in many fields of science, industry and medicine. To perform to
its maximum potential, the stereomicroscope must be properly set up.

Instructions:

1. Set the microscope in a comfortable position and turn on the light. Place an object
onto the stage plate. A coin or any other flat, detailed object will do nicely.
2. Adjust the eyepieces for the correct interpupillary distance. Do this by bringing the
eyepieces closer together or farther apart until you observe a single field of view.
3. Set the diopter adjustment rings on both eyepieces to the zero position.
4. Use the zoom control to set the highest magnification. Bring the image into focus
with the focus control.
5. Set the zoom to the lowest magnification. The image might be slightly out of focus.
Do not adjust the focus with the focus knob.
6. Use the diopter adjustment on each eyepiece to bring the object into sharp focus.
Once you have a clear image of the object, the microscope is now "parfocal". This
means that as the microscope is zoomed from high to low magnification the image
will stay in focus throughout the entire range. Note that each individual will have a
different setting.



Figure X. Leica MS5 Stereomicroscope.
Focus Knob
Zoom Control Knob
Eyepiece Diopter
18 BIO270 Pre-lab 1 2014

Appendix 3.
Physiological Effects of Environmental Bioactive Molecules:

Note: for general background on our experimental organism, Daphnia magna, please
refer to Appendix 4 of this file.

Multicellular organisms utilize a variety of different molecules to transmit signals
between their cells. There is considerable variation in both the structure and function of
these molecules (e.g. proteins, neurotransmitters, gases etc.). These molecules may
carry signals over long distances or may act locally to convey information between cells.
Signaling molecules also differ in their mode of action on their target cells. Some are
able to cross the plasma membrane and bind to intracellular receptors in the cytoplasm
or nucleus, whereas the majority bind to receptors expressed on the target cell surface.
One universal feature of signaling molecules is that they all act as ligands that bind to
specific receptors.

Heart rate in crustaceans can be altered by many factors. Neurotransmitters,
temperature, and bioactive compounds can all have an effect. Neurotransmitters
presumably act on the organism’s heart rate through the nervous system in a
parasympathetic-like or sympathetic-like manner. This can lead to either a positive or
negative chronotropic effect on heart rate based on the function of the neurotransmitter
in question. Due to the fact that crustaceans are poikilotherms, temperature has a
major effect on heart rate. Lower temperatures tend to decrease the heart rate
whereas high temperatures tend to increase heart rate due to the increase in metabolic
activity and higher rate of chemical reactions within the body. Numerous bioactive
molecules have also been shown to cause chronotropic effects on crustacean heart rate
(e.g. Crustacean cardioactive peptide and Crustacean myosuppressin).

Until recently, little was known about endogenous signaling molecules in Daphnia. The
recent sequencing and public release of the Daphnia pulex genome (Colbourne et al.
2011) and transcriptome (Dircksen et al. 2011) have led to the identification of
numerous peptidergic, aminergic, gas and small molecule transmitter pathway proteins
in Daphnia. To date, little data exists regarding the effects of these bioactive
compounds, or their agonists or antagonists, in Daphnia. In fact, some of the data
which has been published is contradictory. For example, epinephrine has been shown to
accelerate the heart rate (Baylor 1942) whereas Bekker and Krijgsman (1951) indicated
that epinephrine slowed the heart rate.

Your task in this Pre-lab is to design an experiment to test the effect of one or more of
the provided bioactive compounds (ethanol, caffeine, nicotine) on the heart rate of the
CSB colony of Daphnia magna.


19 BIO270 Pre-lab 1 2014

Appendix 4.

Our Model Organism: Daphnia magna
(Kingdom Animalia; Phylum Arthropoda; SubPhylum Crustacea)

Daphnia sp are a commonly employed model organism because they are widely
distributed, common and easily collected animals that survive and reproduce well in
culture. Furthermore, it’s model organism status is due to its ability to rapidly adapt
morphologically, physiologically, and behaviorally to environmental challenges. As a
result, they have been intensively studied from many points of view and a substantial
literature is based upon them. For example, Daphnia has served as a standard organism
for aquatic toxicity testing for decades. Recently, the Daphnia peptidome has been
identified and the genome of Daphnia has been sequenced (Dircksen et al. 2011;
Colbourne et al. 2011) which adds to the validity and utility of Daphnia serving as a
model organism.

Daphnia sp. are one of the most common crustaceans found in lakes, ponds and slow-
moving streams. Daphnia belong to the Branchiopoda, a primitive group of crustaceans
characterized by flattened leaf-like legs. The branchiopods are classified within the
Cladocera, whose laterally compressed bodies are enclosed by an uncalcified shell,
known as the carapace. This distinctive transparent carapace has a double wall,
between which hemolymph flows and body cavity is located. Cladocerans are commonly
referred to as "water fleas". The name “water flea” comes from the fact that they move
by making jerky (jumpy) movements through the water. These movements are guided
by powerful 2
nd
antennae that are often longer than the rest of the body and is easily
visible by the naked eye. The stroking action of the antenna move the animal toward
the surface. Then it pauses for a short while, falling toward the bottom of the pond,
before the next stroke.

Daphnia sp. range from 0.5 mm – 5 mm in size and the majority are planktonic filter
feeders, ingesting mainly unicellular algae and various sorts of organic detritus including
protists and bacteria. Beating of the flattened leaf-like legs produces a constant water
current through the carapace which brings such material into the digestive apparatus.
The thoracic appendages are equipped with microscopic setae that are instrumental in
filtering alimentary particles and in capturing planktonic prey. The trapped food
particles are formed into a food bolus which then moves down the digestive tract until
voided through the anus located on the ventral surface of the terminal appendage.
Much of the motion you will observe in Daphnia is feeding.

Daphnia are poikilotherms (cold-blooded animals) and therefore do not thermoregulate.
As a result, ambient temperature will have a great effect on physiological processes.
Growth, reproduction, and response to stimuli may all be temperature-dependent.
Anatomical description (see figure 2 for details):

20 BIO270 Pre-lab 1 2014

- Eye: compound eye is controlled by ciliary muscles with nerve connections to the
brain
- Intestine: where ground up food particles are digested
- Legs: used for collecting food, delivering oxygenated medium to the respiratory
surface, and stabilizing the animal
- Antenna: used for swimming and sensing the environment
- Heart: pumps hemolymph around the body
- Carapace (protective outer shell): transparent
- Brood chamber: incubating young that hatch from large yolk filled eggs




Figure 2. Daphnia pulex Anatomy. Retrieved from http://www.biyoportal.com/portal/haberler/zooloji/60-
genom-dizisi-tamamlanan-kuecuek-bir-model-organizma-daphnia-pulex.html,
and http://www.evolution.unibas.ch/ebert/publications/parasitismdaphnia/ch2f1.htm, J uly 18,
2012.

Life cycle:

In a good-quality environment, most Daphnia are female and reproduce
parthenogenetically, without breeding, through a natural form of cloning. When
reproducing this way, the eggs do not get fertilized; as a result, young are exact copies
of their mothers. The unfertilized eggs develop into live embryos inside the brood
chamber and the young are released into the environment within two to three days.
Daphnia bear on average, ten live young per individual. Generation after generation of
females can be born in this way, with new females reproducing as early as four days
old at intervals as often as every three days, up to twenty five times in their lifetime.
Clearly, a healthy Daphnia population can quickly increase.

When the environment becomes stressful (e.g. winter), Daphnia sp. adapt by producing
male as well as female embryos. Once they become mature, these individuals breed
and produce fertilized eggs, termed winter eggs, that are encased in a tough protective
A B
21 BIO270 Pre-lab 1 2014

extra shell layer called the ephippium. These are released from the female's body and
must go through several cycles of freezing and thawing before they will hatch. When
conditions improve again, the egg producing generations begin producing live young
parthenogenetically and the male sex dies out completely until required when
conditions again degenerate.

It is difficult to distinguish male and female Daphnia. The two sexes are practically
identical except that males are generally smaller in size, have larger antennules, and
the first legs have a stout hook used in clasping the female during mating. Rather than
trying to identify males to detect stressful environments, it is much simpler to inspect
the brood chambers of females. The presence of eggs encased in protective shells is a
sign of unfavorable environmental conditions. If the brood chambers are full of embryos
or eggs with no protective coating, you can safely conclude that the Daphnia are not
stressed. Why do Daphnia respond to stress by reproducing sexually rather than
through cloning? In this way, they produce young that are not exact copies of their
mothers, and some of these young may be better adapted than others to the stressful
environment in which they will live. In addition, the fertilized eggs are enclosed in tough
shells which help to protect them until the environment becomes favorable. This is a
useful adaptation for organisms that live in ponds or other bodies of water that may dry
up or freeze for part of the year. Although the adults will die, their eggs are well
adapted to surviving until the environment becomes favorable. For Daphnia, cyclical
parthenogenesis (parthenogenesis is interrupted at intervals by sexual reproduction)
seems to combine the best aspects of the two modes of reproduction.

General Circulation and Gas Exchange:

Most animals need to obtain oxygen (O
2
) and nutrients from the environment and
eliminate carbon dioxide (CO
2
) and other metabolic waste products. Diffusion and bulk
flow are the two mechanisms by which aerobic animals accomplish these tasks. Bulk
flow refers to the transfer of oxygen (and CO
2
and other metabolic wastes) in a mass of
air or liquid (e.g. water or blood) by the movement of that mass. Diffusion is the
process whereby oxygen and other molecules of liquids or gases move from regions of
high concentration to regions of low concentration as a result of their random thermal
motion.

Diffusion is only effective over short distances (sufficient for gas exchange in single-
celled organisms), so its relevance in multicellular organisms is usually restricted to
transport processes across thin physical barriers such as the respiratory surfaces and
within tissues. Bulk flow, in contrast, often dominates the transport of nutrients,
hormones, and gases in moving respiratory media and circulating body fluids.
Ventilatory and circulatory bulk flow tend to link long transport distances that may exist
between the environment and the respiratory surfaces and between the respiratory
surfaces and the tissues.

22 BIO270 Pre-lab 1 2014

Circulatory System:

The open circulatory system of Daphnia consists of a heart, hemocoel, and hemolymph;
no blood vessels are present. The hemocoel contains a series of lacunae or sinuses
between and surrounding organs; one of which, the pericardial sinus, surrounds the
heart. The hemolymph (no distinction between blood and interstitial fluid) comes into
direct contact with the tissues in these spaces and it is here that chemical exchange
occurs between the hemolymph and cells of the body. The distinctive two chambered
heart is located in the dorso-anterior part of the thorax, dorsal to the intestine, and
anterior to the brood sac. It is small, slightly flattened, elongated and thin walled. In
fact, it is only a single cell layer thick over much of its surface (Stein et al. 1966). The
heart beats rapidly (180-450 bpm at room temperature) and is relatively easily counted
through the transparent carapace. Contractions of the heart force blood anteriorly
through the arterial ostiole into the hemocoel of the head from which it flows
posteriorly into the thorax via three hemocoelic channels. The two lateral channels each
serve one side of the carapace whereas the median channel runs ventral to the gut and
gives off branches to the thoracic appendages. Hemolymph enriched with oxygen
returns to the heart, via the pericardial sinus, through the lateral ostial valves that open
during dilation of the heart and close during systole. Contraction of the heart also
generates a pressure gradient that draws the oxygenated hemolymph into the
pericardial sinus and ultimately into the heart via the lateral ostioles. The circulation of
hemolymph around the hemocoel is aided by body movements that squeeze the
sinuses. Consider how this setup differs from the vertebrate closed circulatory system
(see figure 2).

Figure 3. Circulatory Systems. From Moyes and Schulte, 2007.
The heartbeat of most crustaceans is neurogenic. Neurogenic heart muscle has no
endogenous rhythmical properties and is driven by periodic pacemaker bursting activity
of the cardiac ganglion situated in the heart. Interestingly, the ganglionic pacemaker
23 BIO270 Pre-lab 1 2014

common in most crustaceans is lacking in Daphnia and therefore the heart of Daphnia
is most likely myogenic, like vertebrate hearts (Prosser 1942; Stein et al. 1966;
Steinsland 1982). Moreover, it has been demonstrated that there exists similarity in
heart ultrastructure between Daphnia and vertebrates (e.g. striated myofibrils have Z-,
A-, I- and H banding as well as a regular sarcoplasmatic reticulum and an extensive t-
tubular system (Stein et al., 1966). As a result of these similarities, it has been
suggested that Daphnia sp. could serve as a convenient testing ground for potential
vertebrate bioactive molecules.

Gas Exchange

Although gill respiration and intestinal respiration have been suggested for gas
exchange surfaces in Daphnia, the consensus now seems to be that they use
integumentary respiration. Interestingly, the name “Branchiopoda”, the gill feet, implies
that the epipodites (branchial sacs) are the respiratory organs for gas exchange. Pirow
et al. (1999) have shown that gas exchange occurs primarily across the inner wall of
the carapace. The limbs, in association with the carapace form a suction-pressure pump
which constantly delivers oxygenated water for respiration (and for feeding).
Presumably, this form of respiration is possible because of the large surface-to-volume
ratio and thin-walled integument of Daphnia. To support bulk oxygen transport,
Daphnia have the extracellular respiratory protein hemoglobin (Hb), a multi-subunit,
multi-domain macromolecule. In response to environmental changes (oxygen
concentration, temperature), the Hb concentration varies up to about 20-fold. In highly
aerated water, the blood is transparent. When grown in stagnant hypoxic water the
animal produces large amounts of hemoglobin in its blood giving the animal a reddish
pink hue.

24 BIO270 Pre-lab 1 2014

References

Baylor ER. 1942. Cardiac pharmacology of the cladoceran, Daphnia. Biol. Bull. Woods
Hole 83: 165-172.
Bekker J M, Krijgsman BJ . 1951. Physiological investigations into the heart function of
Daphnia. J . Physiol. 115:249-257.
Colbourne J K, Pfrender ME, Gilbert D, Thomas WK, Tucker A, Oakley TH, Tokishita S,
Aerts A, Arnold GJ , Basu MK et al. 2011. The ecoresponsive genome of Daphnia
pulex. Science 331, 555-561.
Dircksen H, Neupert S, Predel R, Verleyen P, Huybrechts J , Strauss J , Hauser F,
Stafflinger E, Schneider M, Pauwels K, Schoofs L, Grimmelikhuijzen CJ P. 2011.
Genomics, transcriptomics, and peptidomics of Daphnia pulex neuropeptides and
protein hormones J . Proteome Res. 10, 4478–4504.
Friberg-J ensen U, Nachman G, Christoffersen KS. 2010. Early signs of lethal effects in
Daphnia magna (Branchiopoda, Cladocera) exposed to the insecticide cypermethrin
and the fungicide azoxystrobin. Env. Toxicol. Chem. 29(10): 2371-2378.
Moyes CD, Schulte PM. 2007. Principles of animal physiology, 2
nd
Ed. Toronto: Pearson
Benjamin Cummins.754 p.
Pirow R, Wollinger F, Paul, RJ . 1999. The sites of respiratory gas exchange in the
planktonic crustacean Daphnia magna: an in vivo study employing blood
haemoglobin as an internal oxygen probe. J . Exp. Biol. 202: 3089-3099.
Stein RJ , Richter WR, Zussman RA, Brynjolfsson G. 1966. Ultrastructural
characterization of Daphnia heart muscle. J . Cell Biol. 29(1): 168-170.
Understanding Science. 2012. University of California Museum of Paleontology. 7
August 2012 <http://www.understandingscience.org>.




Daphnia Background Links

Daphnia Genome:
http://wfleabase.org
http://genome.jgi-psf.org/Dappu1/Dappu1.home.html
http://animaldiversity.ummz.umich.edu/site/accounts/classification/Daphnia_pulex.html
http://www.sciencemag.org/content/331/6017/555.abstract
http://ngm.nationalgeographic.com/2011/08/visions-now-next#/next/2

Heart Beat Video:
http://www.ebiomedia.com/the-biology-classics-daphnia-heart.html
http://www.vidacollection.org/browse/browseRecords/detail?recordId=949&