Cellular respiration experiment with yeast and balloons

Lab Manual

*Adapted from Online BIO100A Survey of Bioscience Laboratory Manual Version 4.0 by Michael Maxwell and Omar Clay.

Midterm Lab Report Conduct the experiment by following the detailed instructions below. Fill out the tables and answers the questions below to guide your experiment and organize your results and conclusions. Take pictures of your experiment prior to beginning once you have all of your equipment set up, in the middle of the experiment and at the end of the experiment. Record your detailed observations and results throughout the experiment. Once you have conducted the experiment and completed the questions/tables, write a 3 page lab report in essay format (MLA) summarizing the experiment, results and conclusions. See grading rubric.

Cellular Respiration Materials needed Active dry yeast (alternatively Rapid rise or instant yeast) Six small bottles- .5L plastic water bottles (alternatively test tubes) Candy Thermometer (alternatively oven/meat thermometer) Note: must be capable of reading temperatures between 100 -130 F. Six same sized balloons (5” diameter or bigger) Safety goggles (recommended) Indelible marker Sucrose (sugar) String Ruler Watch/ Clock Measuring Cup Measuring spoons Kitchen pot Optional Materials Vinegar Ammonia pH paper Beef bouillon Fructose or honey Sugar substitute (e.g. NutraSweet)

Introduction In this experiment, you will investigate cellular respiration. By monitoring the volume of CO2 gas produced and the growth of a yeast medium (dependent variables), you will investigate the role of sugar, temperature, and another independent variable of your choosing in cellular respiration. You will also learn about yeast, cellular respiration, experimental design and estimating measurement uncertainties.

Cellular Metabolism Cellular respiration is a set of biochemical reactions essential to cellular metabolism. In this process, sugars inside of cells are broken down into adenosine triphosphate (or ATP) which serves as a source of

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cellular energy. Cellular respiration also produces carbon dioxide (CO2) and other products. The production of CO2 is the chief way that CO2 enters the atmosphere and is thus a primary aspect of the carbon cycle; the other primary leg of the carbon cycle is photosynthesis which captures CO2 from the atmosphere and uses solar energy to produce sugars.

The first step in cellular respiration occurs in the cell cytoplasm and is called glycolysis (Figure 3.2). In glycolysis, a single glucose (sugar) molecule is broken down into two pyruvate molecules and two ATP molecules.

There are two principle types of cellular respiration (Figure 3.2). First, in the presence of oxygen, aerobic respiration can take place in the cell's mitochondria, where pyruvate and oxygen are used to produce CO2 and 36 ATP. The mechanisms by which this occurs are the Krebs cycle and the Electron transport chain.

Second, in the absence of oxygen, cells can engage in anaerobic respiration which takes place in the cytoplasm. One form of anaerobic respiration occurs in the muscle cells of animals when they are using more oxygen than the blood can supply. This is known as lactic acid fermentation because it produces lactic acid. Ultimately, lactate can be used to produce some more ATP. Some fungi and bacteria also use this biochemical pathway. Yeast and some bacteria engage in another form of anaerobic respiration known as ethanol fermentation. This process does not provide more ATP but does produce CO2, ethanol (drinking alcohol) and maintains the biochemical conditions necessary for continuing glycolysis in the cytoplasm.

Figure 3.2. Cellular respiration. [Figure from Johnson and Raven, 2004, Biology, Holt Rinehart and Winston, p. 110]

Yeast Yeast is a single-celled fungus. These cells are typically 3-4 microns in size, but don’t let that

fool you into thinking that they are not important to humankind. A particularly import kind of yeast is Saccharomyces cerevisiae. These organisms are used to ferment the sugars of grains (wheat, barley, rice, corn, etc.) and other foods (e.g., grapes) to produce alcoholic drinks (whiskey, beer, wine, etc.) and to make bread rise. Yeast is also used in the production of some cheeses, and is being used in the field of bioremediation and ethanol fuel production. Yeast-like fungi are also normal inhabitants in the mouth, vagina, skin, and intestines.

Measurement Accurate measurement of the natural and physical world is fundamental to science. Modern scientists use the metric system in their measurements. The metric system was developed in France in the late 1700s, and is now commonly used in most countries. A main advantage of the metric system is that it is based on multiples of 10. This is much easier than the English system of 12 inches to the foot, 16 ounces to the pound, and 16 cups to the gallon. Americans are often unfamiliar with some of the basic units of the metric system; thus, it can present a difficulty. In this laboratory, you will develop an understanding of the metric system and use it to measure common objects. You will also learn about how to report uncertainty in your measurements. Estimating measurement uncertainty is a critical component of scientific reporting that you will use in later labs. Metric units The basic measurements of the metric system are:

1. Length, expressed in meters (m). 2. Mass (weight), expressed in grams (g). 3. Volume (capacity), expressed in liters (l).

These units have the following English equivalents. One meter is roughly 3 feet (1 yard), or slightly longer than one pace. One gram is very light; one paperclip is about 1 gram, while a nickel is about 5 grams. Standard weights are often expressed in kilograms (1,000 grams). One kilogram is roughly 2 pounds. One liter is roughly 1 quart, so there are about 4 liters in 1 gallon. For each of these units, large or small amounts are expressed by prefixes that reflect multiples of 10, as the following table shows.

Using these prefixes, the following exact conversions can be made:

Temperature

The metric unit of temperature is degrees centigrade (º C). The Celsius temperature scale was developed by the Swedish astronomer Anders Celsius in 1742. Nearly every country in the world uses the Celsius system. It can be confusing for traveling Americans when the morning news announces a "nice day" of 24 degrees. This sounds cold, but 24º C is actually 75º F. Temperature relates to the average energy of movement (kinetic energy) of molecules in the environment.

Uncertainty Estimation A key aspect of accurate measurement and reporting of measurement is estimating the precision of your measurements. For instance, when someone reports that it took twenty minutes to cook a particular item, do they mean it took twenty minutes plus or minus a minute? Or do they mean twenty minutes plus or minus five minutes? This kind of information is very important if you are trying to exactly replicate previous experiments. Thus, in addition to reporting a measurement, scientists try to estimate the range of values that they are certain captures the object being measured. In this measurement lab, you will estimate the uncertainty in your measurements. A good way to do this is to make and record the measurement and then come back and make the measurement again. If this is done several times, you will get a good idea of how precisely you can measure the given object. No scientific measurement is exact, as every measurement technique has limits. In scientific parlance, the uncertainty in a measurement X is often referred to as ΔX (called "delta X"). To calculate uncertainty:

1. Take 3 measurements of the same item. Write down each of these. 2. Calculate the mean (average) of the three measurements (measurement 1 + measurement 2 +

measurement 3)/3 3. Calculate the standard deviation of the

measurements https://www.mathsisfun.com/data/standard-deviation-calculator.html 4. Calculate the square root of n (sqrt(3)) 5. Calculate the uncertainty by dividing the standard deviation by the square root of n.

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Experiment: Investigation of CO2 production from cellular respiration in yeast 0. Read through the following instructions thoroughly before beginning your experiment. Exercise caution and be mindful of safety as you work with hot water and plastic bottles. 1. Collect all of your materials in a clean safe place. Decide what alternate experimental conditions you will examine. Ideas for alternate experimental conditions (e.g., sugar substitute, pH change, yeast conditioning, temperature) are described more fully below. 2. Make sure that your thermometer and five plastic bottles will all fit inside of your kitchen pot (Figure 3.1). Make sure that your balloons fit tightly on your bottle necks (Figures 3.4 and 3.5). You may also want to tie your bottles together with string so that they will not float in the bath water.

3. Make sure that each bottle is clean and dry. Add 2 teaspoons of yeast to each bottle with the possible exception of #6, which will depend upon your alternative choice. Figure 3.4. Labeled, loaded, and ready to grow. 4. Fill each bottle with 1/4 cup of water. Be sure that each receives the same kind and quantity of water. For instance, if you use tap water, use it with all of the bottles. Write down what kind of water you use. If you are going to run an experiment on pH and have pH paper, measure the pH of the water. 5. Record the independent variables (i.e., the conditions under experimental control) for each yeast population (bottle). The independent variables are Sugar, Yeast, and Water in Table 3.1. Do not measure the height (in cm) of the yeast solution until Step 8.

6. Notice that Bottles #2 and #3 have identical conditions. This will allow you to gauge the precision of your experimental method. Bottle #6 is to be used in an experiment of your choosing. You should change only one of the independent variables and keep the others constant. For instance, you might choose to investigate whether yeast can grow as well with something other than sugar, such as beef bouillon, NutraSweet, saccharine or a higher quantity of sugar (e.g., 2 teaspoons). If you do this, be sure to keep the yeast quantity constant at 2 teaspoons and heat the bottle like the others. Other ideas include: altering the pH of the water by adding a measured amount of ammonia or vinegar; changing the amount of yeast in the bottle; microwaving the dry yeast before trying to grow them (microwaving might kill the yeast), or seeing if the yeast can grow in the cold by placing Bottle #6 in the refrigerator instead of the warm water bath. 7. Replace the caps on each bottle and swirl the solutions thoroughly to ensure that the yeast cells are well distributed in the solution. 8. Remove the caps and replace them with balloons. Stretch the mouth of each balloon over the mouth of each bottle (Figure 3.5). Make sure that the balloon/bottle connection is secure; if necessary, use tape or string. Measure the height of the yeast solutions in the bottles and record them in Table 3.1 (they should all be pretty much the same).

9. Fill the pot with water to a height a just a bit deeper than the height of the water in the bottles (this way the bottles won’t float too much when you place them in the bath). Place the thermometer such that you can safely monitor the temperature. Heat the water to 110F (slightly warmer than a hot spa). During the experiment, continually monitor the temperature and adjust the burner such that the water temperature is kept relatively constant at 110F. Be careful of steam. 10. Place bottles # 2-5 in the warm water and maintain the temperature between 100 and 120 F for ~20 minutes. Bottle # 6 may or may not be placed in the bath, depending upon your choice of experimental treatment. Bottle #1 should be left sitting at room temperature (it will serve as a temperature treatment). Monitor the experiment carefully. Do not let the bath temperature exceed 120F. 11. After 20 minutes, turn off the burner and remove all of the bottles. Make a quick examination of the results and note the volume of the balloons (large, medium, small, none), the growth of the yeast medium (much, moderate, little, none). Record these quick observations in Table 3.2. 12. Use a string to take various measurements of each balloon. Be careful not to dislodge the balloon while you measure it. Record your measurements in Table 3.3.

a) Estimate the diameter of each balloon (when looking at the balloon, diameter is the linear distance from edge to edge). Since the balloons are closer to ellipsoids (Figure 3.5) than spheres, you will need to measure both the long vertical axis parallel to the body of the bottle and the shorter dimension perpendicular to the bottle body.

b) Wrap a string around the horizontal circumference of the balloon at its fattest point. The string should be horizontal (i.e. parallel with the ground). Mark the string where it meets itself and measure the circumference (C) of the balloon in cm by laying the string along the side of a ruler. Record the length of the horizontal circumference (C) in Table 3.3.

c) Estimate the accuracy of your measurement of the circumference by measuring twice. You should be able to be confidently say that the circumference C of the balloon is your measured value plus or minus ΔC. Include +- uncertainty in your results.

d) For the long axis radius (R), you cannot wrap the string all the way around because of the bottle. In this case, use the string to estimate the distance from the top of the balloon to its base at the bottle neck (i.e. half of the circumference). The radius of the balloon on this axis is R = measured value/3.14. Estimate your uncertainty in making this measurement (ΔR). Record these measurements in Table 3.3.

e) Measure the new heights of the yeast solutions. Record in Table 3.3. Include +- uncertainty for all values.

Record Your Observations & Answer the Following Questions to Guide your Lab Report 1. List the following experimental materials. A) Kind of yeast used: B) Kind of water used:

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C) Average temperature of the water bath during the experiment: D) Average room temperature during the experiment (estimate if necessary): E) Duration of yeast solutions exposure to bath:

2. Record your results in Tables 3.1 - 3.4.

3. In Table 3.4, record yeast growth and estimated volume of each balloon on Bottles 1-6. a) Yeast growth = New height (in Table 3.3) - Original height (in Table 3.1) b) If the balloon did not inflate, it has a volume of zero. c) To estimate the volume of each balloon, use the following formula for the approximate volume of an ellipsoid with a horizontal circumference C and long axis radius R

(from Table 3.3): d) To estimate the fractional uncertainty in the volume, use this formula:

4. Describe the experimental questions in this yeast activity.

5. Describe what is measured by the balloon volume. How does it correlate with yeast growth? Be specific.

6. Compare Bottles # 2 & 3. Are they very different? Discuss the utility of having a duplicate measurement when considering the precision of your experimental technique.

7. Compare Bottles # 1 to 2 & 3 and discuss the effect of temperature on cellular respiration in yeast.

8. Compare Bottles # 2, 3, 4, 5 and discuss the effect of sugar on cellular respiration in yeast.

9. Discuss results obtained with your experimental Bottle #6 in comparison with the other experimental conditions.

10. Describe your conclusions, thoughts about what you learned about cellular respiration, and/or things that went wrong.