GENERATION AND TESTING OF HYPOTHESES USING SUGARS
The Scientific Method is often presented in textbooks as a series of steps (a particular algorithm) that one follows in a "cookbook" fashion from initial observation to generation of a strongly supported theory. One of the parts of this method is proposing a hypothesis that might explain the observed phenomenon. What is often not emphasized, is that after proposing a hypothesis and conducting an experiment(s) to test it, it may be necessary to revise the hypothesis and do additional experiments to test the revision. This process of testing and revision continues until the hypothesis fits all available data. After further verification by experimentation a theory can be formulated. In today's laboratory you will have an opportunity to propose a hypothesis, perform an experiment to test it, and then revise your hypothesis if necessary and perform additional experiments to test your revision(s).
Yeast cells (Saccharomyces cerevisiae) use sugars as carbon and energy sources during aerobic and anaerobic respiration. One of the products of anaerobic respiration is carbon dioxide gas. Not all sugars can be metabolized by yeast. To ascertain if yeast cells can metabolize a particular sugar anaerobically, simply allow the yeast access to the sugar and look for production of carbon dioxide. The shape (structure) of the sugar molecule determines if it can or cannot be used by yeast. Since the respiratory enzymes recognize only certain parts of the sugar molecule, if these portions of the sugar molecule are not present then the yeast cells will be unable to metabolize the molecule.
In this laboratory you will propose a hypothesis regarding which part(s) of the sugar molecule is/are recognized by the respiratory enzymes. You will then perform simple experiments to test your hypothesis. Based upon your experimental results you will revise your hypothesis if necessary, and perform additional experiments to test your revision. When you complete the exercise you should have arrived at a workable hypothesis that fits all of the available data.
Take careful note of these points when constructing hypotheses:
· the C and OH groups affect the digestibility of the sugars, not the H atoms
· carbon 1 is always at top, thus carbon 5 or 6 is at bottom.
· Whether the -OH and =O groups are on the left or right can influence digestibility of the sugar. The exception is the OH group around Carbon 6 – this OH group can spin around the molecule’s axis- so don't worry about position.
PROCEDURES
TRIAL 1:
Observation: You will work as groups of two. Your instructor will provide you with a yeast suspension that has been warmed to approximately 35o C and 5% solutions of fructose and galactose that have also been warmed to 35o C. In addition, you will be provided with two fermentation tubes that will be used to determine if a given sugar is metabolized. Your instructor will explain how to properly fill the fermentation tube with your yeast suspension and sugar solution. After receiving instruction on its use, mix 10 ml of your yeast suspension with 12 ml of one of the sugar solutions provided. Repeat this process to fill the second fermentation tube with yeast and the remaining sugar solution. Watch what happens during the next 10 minutes. Evolution of carbon dioxide indicates that the yeast is using the sugar for energy. The structural formulas for fructose and galactose, the first sugars you are to test, are shown below:
Fructose Galactose
Is the yeast able to utilize either fructose or galactose? How did you determine this?
Hypothesis Formulation: Propose a hypothesis that explains which part(s) of the sugar molecule was/were recognized by the enzymes of yeast. Write your hypothesis below:
Six additional sugar solutions are available for you to test. The structural formulas for these sugars are shown below:
Glucose Sorbose Mannose Xylose Ribose Arabinose
TRIAL 2:
Hypothesis Testing: Choose one of the sugars shown above that, based upon your hypothesis, you think will be metabolized and one that, based upon your hypothesis, will not be broken down. Record your predictions in the space below being sure to indicate why you think that one will metabolize the sugar and one will not.
Prediction:
Data Collection: Record your observations in the space below and then compare your hypothesis with the data you collected. NOTE!! A HYPOTHESIS SHOULD ALWAYS BE COMPARED TO DATA, NOT VICE VERSA!! THE DATA ARE REAL AND CANNOT BE CHANGED WHILE HYPOTHESES ARE ONLY IDEAS AND ARE SUBJECT TO REVISION IF THEY DO NOT AGREE WITH THE REAL DATA!!
Results:
Hypothesis Revision: If your first hypothesis was rejected (incorrect) then go back and look at the structural formulas once again and revise your original hypothesis. Your revised hypothesis must explain your observations of what the yeast enzymes were recognizing when they metabolized, or did not metabolize, the four sugars that you have tested thus far. Record your revised hypothesis below. If your first hypothesis was not rejected, then explain that in the space below and test the hypothesis again in trial 3.
Revised Hypothesis:
TRIAL 3
Testing of Revised Hypothesis: Repeat the experiment using two different sugars, OR,
If your first hypothesis was correct, then retest it using different sugars to confirm that your original idea was correct and that other factors were not involved. In either case, record the sugars you selected to test in the space below and the results of your experiment using them. State your predictions, and your results below.
Prediction:
Results:
TRIAL 4:
Testing of Revised Hypothesis: If your results support your revised hypothesis then test the two remaining sugars to confirm that you have a workable hypothesis supported by all of the available data. If necessary, modify your hypothesis once more before testing the last two sugars. If you have to revise your revision then record your new hypothesis in the space provided below.
Revised Hypothesis:
Prediction:
Results:
Final Hypothesis:
At the end of the experiments each group should have arrived at a reasonable hypothesis to explain metabolism of monosaccharides by yeast cells. You should have arrived at one of three possible hypotheses that your instructor will share with the entire class. It is impossible to distinguish between these hypotheses based upon the experiments you were able to conduct.
Proposing New Experimental Tests: Design two sugar molecules that could be used to distinguish between the hypotheses arrived at by various groups in the lab. Draw the structural formulas for these sugars below.
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