western
Physics and Astronomy

                                   
Scientist's Apprentice
Objective To model a realistic scientific investigation in a biological context.
Participants Teams of up to six.
Materials At the Olympics you can expect to find:

Standard laboratory glassware and supplies as mentioned in the protocols given above: blenders, beakers, pipettes, graduated cylinders, test tubes and racks, forceps, stop watches etc.

Water baths at about 1^oC, 37^oC, 60^oC, 100^oC, Hydrogen peroxide (1%), Cold distilled water, Crushed ice, Lab Coats, gloves and safety glasses

Catalase Extracts from:

Fruit from the Tree of the Knowledge of Good and Evil
The Liver of Smaug the Dragon
Leaves from Jack's Bean Stock

All extracts will be provided as 1/10, 1/100 and 1/1000 fold dilutions.

We strongly recommend that teams wishing to enter this event attend the workshop. Details are given in the General Information booklet.

You may bring supplies of your choice for use in presentation of your data and conclusions. No additional paper or equipment will be allowed.
Rules

In preparation for the event, teams should become proficient in working as a team with the basic catalase extraction and assay protocol. You should peruse the list of tissues and equipment that will be available and develop a hypothesis/question for which an answer can be found on the day of the Olympics. Teams should arrive with two copies of an experimental design, one to work from and one for judges, intended to test a prediction made by their hypothesis/question. The design should be in the form of a Protocol that can be used to direct your work "at the bench".

The Protocol should:
(i) clearly outline the major steps of the investigation.
(ii) indicate the important volumes, times, temperatures etc.
(iii) note potential safety hazards,
(iv) include an appropriate table to receive data as they become available.

One hour will be available for teams to conduct their investigation and display their data and conclusions.

Judging

Judges will award points using the following criteria:

Criterion
Points
Does the protocol show the important information in logical order?
3
Are safety concerns highlighted?
2
Does the design include appropriate controls and replicates?
5
Is the hypothesis testable by the suggested experimental design?
10
Does the group work smoothly and carefully, without careless accidents or oversights?
10
Are the data displayed effectively?
10
Are the conclusions convincingly supported by the data?
10


Background Scientists are obsessed with the unknown. Regardless of discipline, they are all out to discover and characterize the contents of the next "Black Box" that Nature provides. Students of Science, however, are often obsessed with the known: the teacher's notes, the textbook, the expected outcome of a demonstration. Some students may come to believe that to be a scientist is to be an engorged organic encyclopedia.

Although it is often essential to be aware of knowledge that has been discovered previously, this background information is only one of many tools used by scientists to make discoveries. Technical tools including various optical instruments and electronic devices expand our ability to observe Nature. Mathematical tools help to organize and analyze observations and create simulated model systems. One of the most tried and true tools of the science trade is a methodological one usually called The Scientific Method. The Scientific Method can be thought of as a circular process driven at different times by insightful questioning, sound experimentation and/or reliable observation.

The most useful scientific questions are those for which answers can be obtained given the tools available. Questions are often expressed as hypotheses that make testable predictions. The most useful experiments are those that generate observations that are clearly consistent, or clearly inconsistent, with the predictions. The inherent variability in Nature ensures that any single observation is unreliable. The most useful data are actually accumulations (e.g. averages) of multiple, repeatable, observations.

For example, imagine that you have noticed that, of 50 sunflower seedlings on a classroom window sill, all are bent toward the window. A number of hypotheses could be developed as an explanation of this observation: "Sunflower seedlings bend toward glass."; "Sunflower seedlings bend away from people."; "Sunflower seedlings bend toward the brightest light." etc. All of these hypotheses make predictions that are testable given the tools available. It is important to consider all of the variables that may reasonably influence the outcome of an experiment. If you were testing the tendency of seedlings to bend away from people, the experiment must be designed such that only the important variable (presence or absence of people) is different from one group of plants to the other. The amount of light, the presence of glass etc. must be kept constant for all groups of plants. Observations should be made carefully and consistently and then displayed in an appropriate table, graph, drawing or photograph. In order for your data to be most convincing to skeptical colleagues, they should include determinations on a number of plants and should show that similar effects occur repeatedly under the same conditions.

Conceptual Tools Required The model system for this event will be the activity of an enzyme called catalase. Enzymes are biological catalysts that mediate thousands of chemical reactions that occur in living cells. Enzymes are protein or RNA molecules (or a mix of the two) with a unique 3-dimensional structure that makes them specific for a certain reaction. The substance to be acted upon, the substrate, binds to a specific region on the enzyme called the "active site". The enzyme converts the substrate to the reaction products in a process that often involves multiple chemical steps.

Catalase is found in most aerobic organisms. It is one of a cluster of antioxidant enzymes which act in concert to protect cells from damage caused by highly reactive oxygen species that are generated during oxidative metabolism. Catalase functions mainly to prevent the accumulation of hydrogen peroxide. Specifically, the catalase-mediated enzymatic reaction enhances by 100,000,000-fold, the conversion of hydrogen peroxide (H202) to water and oxygen according to the following reaction:

2H202 + catalase -> 2H20 + 02 + catalase

Technical Tools A simple assay for catalase activity can be conducted by exploiting the evolution of oxygen from the enzymatic breakdown of hydrogen peroxide. The following protocol outlines a crude yet rapid and effective procedure for extracting catalase from any soft tissue.


a. Place 50g of chopped tissue, 50 ml of cold distilled water and a small amount of crushed ice into a pre-chilled blender. Homogenize for 30 seconds at high speed.

b. Filter the extract through 4 layers of cheese cloth into a beaker. Transfer the extract to a 100 ml graduated cylinder and add cold distilled water to bring the volume to 100 ml. This extract is arbitrarily designated to contain 100 "units" of enzyme per ml. Transfer the catalase extract to a beaker and keep on ice.

Once the catalase extract has been prepared, a variety of predictions can be tested regarding activity under different conditions of enzyme concentration, enzyme source, substrate concentration, pH, temperature, light etc.
For example, to test the hypothesis that "the activity of catalase is directly proportional to the amount of enzyme present":

i. Label 4 beakers as follows: 100, 75, 50, and 0 units per ml. Prepare 40 ml of the enzyme at each of these concentrations by mixing the stock solution with distilled water as follows:

ml of stock enzyme (S) + ml cold distilled H2O (W) = units per ml (U)

S
W
U
40
0
100
30
10
75
20
20
50
0
40
0

Save the undiluted enzyme for testing other parameters.

ii. Keep these catalase preparations on ice. Label 4 large test tubes to correspond to each of the enzyme concentrations.

iii. Into each dispense 10 ml of 1% hydrogen peroxide solution. Keep these at room temperature.

iv. Using forceps, immerse a filter disk half way into the catalase solution.

v. Allow 5 seconds for the enzyme to be absorbed by the filter disk then remove and drain the disk on paper towel for 10 seconds.

vi. Drop the disk into the tube containing the substrate. Oxygen released from the catalytic reaction will become trapped in the disk causing it to float to the surface of the solution. Measure the time (t), in seconds, it takes for the disk to float to the surface from the moment the disk touches the solution.

vii. Remove the disk from the substrate and repeat the procedure using a new filter disk. You should do at least 2 trials for each enzyme concentration and average the results.

viii. Record your data in a table and calculate the rate of enzyme activity. Rate of enzyme activity is equal to the inverse of the time (average) it takes for the disk to float: R = 1/t.

(What would be the "control" for this experiment? How do you know that the disk is rising because of enzyme activity, rather than just non-biological chemical reactions in the extract?)

Source London District Science Olympics. This event was designed by Tom Haffie.

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