• design and implement and experiment

• dependent variable, control, repeated trails
• differentiate between qualitative and quantitative data -set up data tables
• graph data and establish relationships
• perform statistical analysis on the data to determine trends
• perfect experimental techniques involving microreactions

Introduction: We have already had a discussion about the importance of enzymes in biology. We will now focus on a particular enzyme that most living things (including yourself) produce. The enzyme is catalase. This enzyme is produced to decompose hydrogen peroxide (H2O2) into water (H2O) and oxygen (O2) gas. Hydrogen peroxide is toxic to living organisms, this is why you put it on wounds to kill germs. "Catalases are used for applications where hydrogen peroxide has to be removed, such as in contact lens cleaning systems, for bleaching of textiles and hair, and in industrial processeses that generate hydrogen peroxide." (Horn and Pierce, Experiment with Enzymes) Baker's yeast, Saccharomyces cereviae, also produces the catalase enzyme. You will use the bread yeast as a source of catalase enzyme to perform a number of experiments teaching you the basics of experimental design and statistical analysis

Materials: (per class)

• 300 ml warm water (37C)
• 500ml beaker or flask
• 1 teaspoon (1 package bread yeast)
• 1 teaspoon of sugar (sucrose) (per lab station)
• 10 ml of water in a dixie cup -10ml of hydrogen peroxide in a cup
• 10 ml of yeast suspension in a cup -filter paper dics (holes punch holes of filter paper)
• transfer pipette (1ml plastic)
• forceps
• paper towel
• a 24-well plate
• clock with second hand
• graduated cylinder
• sharpie

Procedure: Part I :Exploration A. Calibration of volume (10ml dixie cups)

1) Get a dixie cup, black permanent maker (Sharpie) and the graduated cylinder.

2) Fill the graduated cylinder with tap water to the 10ml mark (remember about the meniscus!)

3) Pour the water from the cylinder into the dixie cup.

4) Mark on the outside of the cup a mark indicating 10 ml.

5) Repeat procedure with the two other dixie cups

A. Calibration of volume (24-well reaction plates)

1) Take one of the dixie cups you just calibrated and fill with 10ml of tap water.

2) Use the transfer pipette and count how many drops it takes to fill one well. Record the results in a Data Table in your results section.

3) Have your partner fill the cell next to yours, counting the drops to fill it. Record the results in your data table.

4) Use the transfer pipette and count how many drops it takes you to half fill one of the cells. Record results in your data table.

5) Have your partner count how many drops it takes them to half fill one of the cells.

6) Repeat steps 2-5 two more times. Record your observations in your data table.

7) Did you come out with the same number of drops each time? __________________________

8) Did your partner come out with the same number of drops as you? __________________

9) Why would there be a difference between each person's results? _________________________________________________________________________________________________

10) After you and your partner have decided on just how many drops it really takes to fill one cell then put that exact amount of drops into a graduated cylinder and determine the volume of one cell in ml. Record it in your results.

Results: Part I: Exploration A. Calibration of volume (24-well reaction plates) Set up your data table here. Leave as much space as you need.

Volume of one cell in ml:___________________________

Procedure: Part I-Exploration B. Yeast Assay

(You need 10ml of water, 10ml of yeast suspension, 10ml of hydrogen peroxide, filter paper holes, forceps, 24-well reaction plate, clock with second-hand)

"You have been provided with a suspension of living yeast cells. Soak a filter paper disc in the yeast suspension, then place the disc at the bottom of a well containing hydrogen peroxide.

Observe.

You have also been given some water to use for making dilutions of hydrogen peroxide. Explore this system, using different concentrations of peroxide in the wells. Record as many observations as you can. Record what you do, and then record what you see. It is improtant to record observations of everything that you do, and everything that you see. Record both qualitative and quantitative observations. An example of a qualitative observation would be: The yeast suspension looks cloudy and cream-colored. An example of a quantitative observation would be: it took the disc 10 seconds to rise from the bottom of a well that had a mixture of 10 drops of water plus 10 drops of peroxide." (Horn and Pierce, Experiment with Enzymes)

B. Yeast Assay Results. Use as much space as you need. THE NEXT CLASS SESSION: DISCUSS CLASS RESULTS!

Part II. Experimental Design

We have learned several things from our Exploration activity. Can you name some of them?

Scientists must carefully design their experiment to measure exactly the reaction or behavior in question. To do so, they must identify the independent and dependent variables. The independent variable is the treatment that is changed and the dependent variable is the resulting reation or behavior from that treatment. For example, to study the effect of fertilizer on plant growth. The independent variable would be the amount of fertilizer applied to the plant and the growth rate of the plant would be the dependent variable. All other variables must be kept the same. The independent variable is plotted on the x-axis and the dependent variable on the y-axis. Scientists must always have a control. The control has no treatment at all. In the case of the plants, no fertilizer added at all. At least three repetitions (3 trails) as a minimum is required to get the accurate trend. Scientists use statistical analysis (Mean, median, mode, standard deviation) to determine the result of the treatment.

Use the five question technique (see Experimental Design Book) to design an experiment to determine the "Effect of Temperature on Yeast Catalase Enzyme).

Effect of Cold Temperature on Yeast Catalase Enzyme

Effect of Hot Temperature on Yeast Catalase Enzyme

RECORD YOUR HYPOTHESIS HERE:

RECORD THE PROCEDURE HERE. (Use as much space as you need)

RECORD YOUR RESULTS (both qualitative and quantitative) HERE. BE SURE TO HAVE BOTH TABLES AND GRAPHS! (Use as much space as you need!)

5 Part III. Experimental Design: "Effect of Temperature on Catalase Enzyme Activity" Discussion/Conclusion

1. Was your hypothesis correct? Why or why not?

2. What could you do differently next time to improve your results?

3. What were some qualitative results you recorded?

4. What were some quantitative results you recorded?

5. What type of relationship exhists between temperature and catalase activity? (Hint: Get your math teacher to help you find the right equation for you graph!)