The Scientific Method: Hypothesis Formulation,
Experimental Design and Data Analyses
Explore the validity of your previously formulated RQ’s by posing one or more experimentally testable hypotheses. A hypothesis differs from the RQ in that it is a tentative answer to the question under investigation. A good (although not the only) way to state a hypothesis (in preparation for an experimental test) is to phrase it as a predictive hypothesis. Using the format common in deductive reasoning, a predictive hypothesis would imply that if “X” is true, then “Y” will be an expected consequence. For example,
If beetroots are
incubated in ethanol, then betacyanin will leak from the cells
The cause is the incubation of
beetroot in ethanol. The effect is the leakage of
betacyanin. When a hypothesis is phrased to
predict a cause and effect relationship, two sets of variables are created:
those associated with cause (dependent variables) and those associated with
effect (independent variables). Thus, incubation in ethanol is the independent variable
and leakage is the dependent
variable. This means you are hypothesizing that leakage depends
on if the beetroot is incubated in ethanol or not.
Note that the independent variable can be manipulated,
for example, by varying the concentration of ethanol and the length of
incubation time. This allows you to investigate the nature of the cause and
effect relationship between the dependant and independent variables. For
example, you can determine if it is linear, exponential or shows a threshold
(non-linear type) response.
In order to
test a predictive hypothesis, an experiment must have experimental units
assigned to at least two groups: the experimental group, and the control
group. Because organisms are very complex, designing the proper controls in
an experiment is the most difficult part of experimental design. The
experimental group and the control groups should be treated as identically as
possible. The difference between the two groups is that the independent
variable to be tested (e.g., ethanol) should only be applied to the
experimental group and controlled in the control group (e.g., water).
What To Do Today
1.
Discuss in your lab group the RQ’s
from last week and the hypotheses formulated from them. Identify one hypothesis
you would like to test. What are the dependent and independent variables
associated with your hypothesis. Get input from your instructor before you
proceed to step 2.
2.
Design an experiment to test this hypothesis. Write
an outline of the procedure in your lab notebook. Make sure you can identify
both the control and experimental groups. You will also need to
construct a data collection table in your lab notebook. Make sure the
instructor OK’s your experimental design before you proceed.
3.
Carry out the experiment making sure to record the
data in the data collection table.
4.
Plot a graph showing the relationship between the dependent
and independent variables. For betacyanin concentration
use the following unit: μM.
5.
Determine if your hypothesis was accepted or
rejected based upon the outcome of the experiment.
6.
Critically analyze your experiment using the “Experimental
Design Critique” sheet.
Tools You Will Need
How to make up dilutions of stock
solutions using the mass balance equation
1.
Decide
what volume and what concentration of dilutions you will need. As
an example, assume you need to make 5 ml of a 20% solution.
2.
Determine
the concentration of the available stock solution (assume it is 80%). Also find
out what solvent the stock is dissolved in (water).
3.
The mass balance equation is:
C1V1
= C2V2
C2
is the concentration you desire (20%)
V2
is the volume you desire (5 ml)
C1
is the concentration of the stock (80%)
V1
is what you solve for and represents the volume of stock you will
need to make your dilution. You will need to add enough water to this
amount to make up a final volume of 5 ml, your desired volume. So:
How do you calculate the V1 volume?
V1
= C2V2/C1
How
much water will you need to make up C2?
5-1.25
= 3.75 ml
How
do you make the dilution?
Add 1.25 ml stock to 3.75 ml of water.
How to convert betacyanin absorbance
into μM concentration
1. Using the
spectrophotometer, measure the absorbance of betacyanin (at λmax) in each of your samples from both
control and experimental groups.
2. Calculate betacyanin concentration using
the following formula:
A = εcL
A
= absorbance at λmax
ε = 38,000 L/mole-cm (absorptivity coefficient)
L = 1.2 cm (path length)
c = concentration in M
3. Solve the equation for “c”. The units will be in M. Convert to μM.