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The Scientific Method, Research Tools, and Techniques.

Objectives
In this lab you will learn:
How to utilize the scientific method
How to make observations and collect data
How to test an hypothesis using simple statistics

Scientific Method
Modern science began during the Age of Enlightenment, which swept through Europe in
the late 16
th
and early 17
th
centuries. Although science itself was performed in all parts
of the world and in various manners long before then, the philosophical advancements
that were brought about in the Age of Enlightenment codified a logically sound
methodology that is the basis for all modern scientific knowledge. The methodology,
naturally, is referred to as the scientific
method.

The scientific method is a process that starts with careful observation of the natural
world. From these observations, a hypothesis is formulated to explain the observation.
The hypothesis is used to make predictions, and these are tested through
experimentation. The experimental results are then interpreted to either support or
reject the hypothesis. This process repeats, until the hypothesis is either highly
supported or rejected.

The scientific method is based on deductive reasoning championed by Francis Bacon
(1561!1626), a philosopher and scientist from the Age of Enlightenment. Deduction is a
philosophical process in which you can conclude that something is true because it is a
logical extension of other things you know to be true. For instance, because you know
that there are two sides to a coin, you can deduce that the odds of attaining a head is
50%. Induction, on the other hand, is a form of reasoning in which multiple, consistent
observations yield a conclusion. In our coin example, if you were to flip a coin 10 times
and attained 10 heads, you might inductively conclude that a flipped coin always lands
on heads. A conclusion based on inductive reasoning, however, is easily negated: a
single tail in our example would disprove our conclusion. Often induction is used to
develop hypotheses, because hypothesis development is based on observations. These
hypotheses are in turn tested through experimentation and rejected or supported
through deductive reasoning.

More specifically, the scientific method employs what is known as a hypothetico!
deductive (or hypothesis!prediction) approach. In this logical method, a specific
hypothesis is formulated to explain a particular phenomenon. The hypothesis is
designed to make predictions about the phenomenon you observed. This hypothesis is
tested with an experiment designed to permit deduction about that phenomenon. The
experimental results will either support or refute your hypothesis, allowing you to draw
conclusions about the truth or accuracy of your hypothesis. This process can be
ongoing ad infinitum, allowing you to learn a great deal about a single phenomenon.
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The formulation of a good hypothesis is an important part of the scientific method. A
hypothesis must give a testable explanation of the observations you have made of a
particular phenomenon. For example, perhaps you have noticed that your dog is fond of
chasing some of your neighborhood cats. You may make a hypothesis to test, for
example, whether your dog chases neighborhood cats more than out!of!town cats,
black cats more than tabby cats, more cats in the morning than in the afternoon, etc. All
of these can be tested experimentally and will provide information about your dog and
its relationship to cats. However, it is important to realize that there are hypotheses that
cannot be tested, that are outside the scope of scientific inquiry. It would be impossible
to test, for example, whether your dog chases cats because it hates them or if it chases
cats because it likes them.

It is much easier to reject a hypothesis than it is to accept one. Recall our discussion on
deductive vs. inductive reasoning. A single observation that disproves a hypothesis
nullifies the validity of that hypothesis, whereas multiple observations that support it may
not be sufficient to demonstrate that it is true. You might hypothesize that all crows are
black, because every crow you have ever seen has been all black. But take a journey to
Europe and you will observe a crow with a white stripe around its neck and shoulders.
Your idea that all crows are black can be nullified with a single observation of black!
and!white crow.

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This is why it is common in the scientific method to try to deductively refute competing
hypotheses rather than gathering inductive support for your hypothesis. The competing
hypothesis states that the factor you are testing experimentally has no effect. For
example, if you hypothesize that birds prefer red berries, your competing hypothesis
should be that berry color has no effect. This competing hypothesis is called the null
hypothesis, and very often experiments are designed to reject the null. By rejecting the
null hypothesis, you are in fact supporting your own hypothesis. Say you were to
hypothesize that drug abuse during pregnancy causes low fetal birth weight. It would be
easier to disprove the null hypothesis: that drug abuse during pregnancy has no effect
on fetal birth weight. You could then experimentally sample birth weights of drug
abusers and non!drug abusers. If, based on your experimental data, you were able to
reject the null hypothesis that drug abuse has no effect on birth weight, then your
alternative hypothesis is supported. You were able to deductively reject the null
hypothesis (no effect).

An important thing to remember about science is that it is an ongoing enterprise, where
hypotheses are constantly tested and retested. The field of scientific knowledge
therefore is always changing as new ideas are supported and old ideas are rejected. A
hypothesis that withstands a high degree of scrutiny through many experiments can be
called a theory a hypothesis that has withstood a great deal of scrutiny and has great
predictive power but even theories can be overthrown with a single observation. In
1919, for example, Einsteins theory of relativity disproved the physics of Isaac Newton
that had robustly explained so many natural phenomena regarding the movement of
heavenly objects for over 200 years. Einsteins hypothesis was that space and time
were curved, whereas in Newtonian physics (the kind you may have learned in high
school), space was flat and time linear. In 1919, an expedition was conducted by the
British Royal Society to Brazil and the island of Principe off the western coast of Africa
to observe a total solar eclipse. Einsteins hypothesis predicted that in those two places,
astronomers would be able to observe stars that were located directly behind the sun
because the light from those stars would curve around the mass of the sun. The Royal
Society astronomers undertook the expedition and were able to observe light from the
stars behind the sun. That single observation was sufficient to disprove Newtons theory
of physics. Such is the nature of science: nothing can ever be proven. Even the most
robust of theories must be revised in light of new evidence. However, this is also the
strength of science. It is not dogmatic in its beliefs; it constantly evolves so that our level
of knowledge only improves with each experiment that is performed.


Next time you are walking to school, sitting outside studying, or walking to class take a
look around and try to watch some of the animals you see on campus (trust me there
are more than just hungry squirrels). Formulate a hypothesis regarding something you
have seen. State your null hypothesis, and try to design a simple experiment to address
this hypothesis. While you dont have to turn this in, you may be asked to present your
hypothesis in lab this week, so make sure to take some time to look around and think
about what you see.


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Observing and Quantifying Animal Behavior

Making an interesting Observation
Most investigations of scientific questions begin with an interesting observation. People
have noticed that some species of ground squirrels exhibit a very peculiar behavior
when they encounter anything that is saturated in snake scent. For example, California
ground squirrels and rock squirrels will chew on shed snake skins and apply it to their
fur by licking their bodies. This behavior has been called snake scent application
(SSA), and is also found in other rodents (e.g., chipmunks and mice). Have a quick look
at this behavior now.

In this lab exercise you will make observations, record data, quantify observed
behavioral patterns, and learn a simple statistical test to evaluate your hypothesis. You
will watch videos of a behavior exhibited by two species of ground squirrels to
determine if the SSA behavior to various regions of the body is random.

Asking a Question and Developing a Testable Hypothesis
Intriguing observations about behaviors often lead to questions about how or why they
occur. For instance, we may wonder how rodents apply snake scent do they apply the
scent to the different parts of their bodies in a particular order, or is the sequence
random? Do they apply scent to one region of their body more than others, or do they
cover all regions of the body equally?

We also may wonder about the function of applying the scent. Rattlesnakes have been
a major predator of ground squirrels for millions of years and this coexistence has led to
the evolution of several unique anti-predator strategies in ground squirrels. For instance,
in some species of ground squirrels adults are resistant to rattlesnake venom and will
actively harass and attack these predators.

Group Exercise
A) Rodents are a major prey source for snakes why do you think rodents apply their
predators scent? Tentative explanations to your question are called hypotheses.
Within your group, brainstorm as many hypotheses as possible to answer the
question about function above. List your hypotheses. Are all of them testable? Give
at least one example of a non!testable hypothesis.



B) Choose one testable hypothesis and suggest specific ways to test the hypothesis.
What predictions would you make to support or refute the hypothesis?



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In this lab we will focus on the SSA behavior and investigate whether SSA applied
randomly over the squirrels bodies. We will be making observations of two different
Spermophilus species (S. beecheyi and S. variegatus).

Write your hypothesis and null hypothesis below.




Why might you expect SSA to be non-random? While you are thinking about this
consider some things about the ecology of squirrels: where do they live, where are they
likely to be found in their environment (i.e. on the ground, rocks, in trees), could there be
a benefit of applying snake scent to some regions versus others on the body?

You will be collecting data on more than one species, do you expect different patterns
between species? What other things did you consider when trying to address this
question?



Testing Your Hypothesis
In this section, you will test the hypothesis that SSA behavior is random with respect to
areas of the body. That is, whether the squirrels simply apply the snake scent or
whether they apply the scent to specific areas of their bodies.

You will watch videos of SSA behavior from two species of ground squirrel, California
ground squirrels (S. beecheyi) and rock squirrels (S. variegatus). To obtain the videos, a
researcher staked out a shed skin of a sympatric rattlesnake species (Crotalus atrox for
rock squirrels, Crotalus oreganos for California ground squirrels) and surrounded it with
a minimal bait trail (sunflower seeds) to attract the squirrels to the skin. The squirrels
were marked with numbers (fur dye) to identify individuals. Each video represents a
different individual. The study sites
were in Winters, CA (California ground squirrels) and Caballo, NM (rock squirrels) in
county or state parks where squirrels were used to cars. The squirrels were videotaped
from a car, ~20!30 m away.

Observations and Data Collection

The behavior you will be scoring is what body regions ground squirrels apply snake
scent to after chewing shed skins. You will be tallying snake scent application on the
following body regions:





'


Head Tail





Front Legs Flank





Hind legs



Figure 1: Ground squirrel body sections
http://pad1.whstatic.com/images/thumb/d/d3/Draw-a-Squirrel-Step-19.jpg/550px-Draw-a-Squirrel-Step-19.jpg

You will record the frequency of application behavior for both species. Make
observations for four individuals of each species. You will find the video clips on the
desktop of the computer (labeled SSA video clips). Choose four from each species
(labeled Clip b# for S. beecheyi, and Clip v# for S. variegatus), watch each of these and
record the total frequency for each body area for all focal individuals in Table 1 below.
At the end, you should have data collected for four different individuals of each species,
eight individuals total.


Table 1. Observed SSA behavior for two species of squirrel.
Species Indiv. Flank Head
Front
Leg
Hind
Leg Tail
Row
Total
S. beecheyi 1
2
3
4
Total
S. variegatus 1
2
3
4
Total




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Interpreting Your Results
The data collected from the videos allows us to statistically test whether SSA behavior
is random or not. The statistical method employed is the chi-square test. The chi-square
(X
2
) test compares the observed behaviors to what would be expected if the behaviors
were randomly distributed. The basic Chi-square formula used to compute the statistic
is:


where: o = observed frequency, and e = an expected frequency, asserted by the null
hypothesis

For example, let us say we were to observe 100 SSA behaviors, divided into 4 groups:
the flank, the tail base, the middle tail, and the tail tip. If the behavior were truly random,
we would expect those 100 behaviors to be distributed evenly among our 4 body parts,
25 each. This gives us our expected values for our chi!square calculations. These
expected values represent our null hypothesis; in this case, our null hypothesis is that
squirrel snake scent application behavior is randomly distributed over the body (hence
25 expected touches to each of the 4 body parts). If our statistical analysis allows us to
reject this hypothesis, we may conclude that SSA behavior is non!random. (NOTE:
Your data is divided into 5 body regions, not 4. The expected number of touches to a
particular body region therefore would be your total number of observations divided by
5.)

In our 100 SSA observations, let us say we see squirrels lick their flanks 21 times, their
front legs 22 times, their hind legs 29 times, and their tail 28 times. We would see that
there is some variation between our observed and expected values. Although a chi!
square calculation is simple enough to do as a single!line equation, some might find it
helpful to organize your data into a table like the ones below. (You will need to do these
calculations for both species).

Flank Front Leg Hind Leg Tail Total
Expected 25 25 25 25 100
Observed 21 22 29 28 100

To begin our chi-square calculations, we subtract the observed values from our
expected values and then square them:
Flank Front Leg Hind Leg Tail Total
Expected 25 25 25 25 100
Observed 21 22 29 28 100
o-e -4 -3 4 3
(o-e)
2
16 9 16 9

)
Next, we divide the squares of our expected-observed observations by the expected
value:
Flank Front Leg Hind Leg Tail Total
Expected 25 25 25 25 100
Observed 21 22 29 28 100
o-e -4 -3 4 3
(o-e)
2
16 9 16 9
(o-e)
2
/e 0.64 0.36 0.64 0.36

Finally these values are summed giving a chi-square value of 2.

By itself, a !
2
value of 2 is quite meaningless. We need to determine whether or not this
indicates a significant deviation from our expected distribution. Therefore, once our !
2
value is obtained, we need to compare it to a table of critical values (like that shown
below), which will allow us to determine whether our observed data deviates
significantly from our expectations due to random chance. The first step in this process
is determining the degrees of freedom we have in our sample data. Degrees of freedom
refer to how many independent pieces of data are assembled in our final data set. In our
case, there are 4 body parts in our data set. Only three of these are independent,
because if we were given three pieces of the data table, we would be able to determine
what the fourth was. As such, the fourth value is not independent and cannot be
included in our degrees of freedom, leaving us with a df of 3. Generally speaking for
simple data sets, the degree of freedom is one less than the number of observed
classes of data.

Table of Chi-squared Critical Values:
Degrees of
Freedom
0.10 0.05 0.025 0.01 0.001
1 2.706 3.841 5.024 6.635 10.828
2 4.605 5.991 7.378 9.210 13.816
3 6.251 7.815 9.348 11.345 16.266
4 7.779 9.488 11.143 13.277 18.467
6 9.236 11.070 12.833 15.086 20.515

In our table of critical values, we see that the critical !
2
value to reject our null
hypothesis is 7.815 at a confidence of 0.05 (an arbitrary standard cutoff, although others
are sometimes used) with 3 degrees of freedom. This means that our !
2
score would
have to exceed 7.815 in order for us to reject our null hypothesis (that SSA body
application is random). In our case, a chi!square value of 2 does not allow us to reject
this hypothesis. Our data therefore cannot be said to be significantly different from
random.