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    Labrespiration1 opt Labrespiration1 opt Presentation Transcript

    • CELL RESPIRATIONIntroduction to cell respiration laboratory This laboratory consists of three sets of experiments thatillustrate the oxidation of glucose for the production of ATP. 1.) The photosynthesis vs. respiration experiment illustrates the importance of respiration to plants. 2.) Fermentation products will be tested for to demonstrate the importance of yeast in the fermentation process. 3.) An enzymatic reaction of the Krebs cycle will be studied to show how enzyme activity may be manipulated by changing substrates. • 100’s of free ppt’s from www.pptpoint.com library
    • CELL RESPIRATIONIntroduction to comparison of photosynthesis and respiration It is well known that in the presence of light plants performphotosynthesis. It is less well understood that at the same time plants arealso performing cell respiration. To demonstrate this we will determine whether CO2 is consumedor produced as Elodea is placed in either a light or dark environment.The change in CO2 will be detected by the pH indicator phenol red. Think about the chemical equations for respiration andphotosynthesis. Which one releases CO2 causing an increase and which one usesup CO2 which will cause a decrease?
    • CELL RESPIRATIONIntroduction to comparison of photosynthesis and respiration (cont.) Phenol red is yellow under acidic conditions (high H ionconcentration), pink under basic or alkaline conditions (low H ionconcentration) and orange under neutral conditions. A change in CO2 will cause a directly proportional change in Hion. If the CO2 concentration decreases, the H ion concentration willalso decrease and the solution will change to pink. If the CO2 concentration increases, the H ion concentration willalso increase and the solution will change to yellow.
    • CELL RESPIRATIONExperimental procedure for comparison of photosynthesis andrespiration Step 1: Fill four test tubes approximately 2/3 full with tap water and add 15 drops of phenol red. What tap water should turn pink. Is Glendora tap water acidic or basic? Step 2: Adjust the pH of the tap water to neutral, orange by gently (remember this word gently) blowing into the solution with a straw. Repeat this procedure with all four tubes. It is important that all four tubes are the same orange color at the beginning of the experiment.
    • CELL RESPIRATIONExperimental procedure for comparison of photosynthesis andrespiration (cont.) Step 3: Place a piece of Elodea (cut end up) in two of the tubes. Each set of tubes will contain one with Elodea and a control without.
    • CELL RESPIRATIONExperimental procedure for comparison of photosynthesis andrespiration (cont.) Step 4: Place two tubes, one with Elodea and one without between the lights on the counter.
    • CELL RESPIRATIONIntroduction to fermentation Fermentation is a pathway for the oxidation of glucose thatproduces ATP in the absence of oxygen. Fermentation is similar torespiration because it begins with glycolysis. Fermentation differs fromrespiration because it does not involve the Krebs cycle or an electrontransport chain. Fermentation yields waste products other than CO2 and H2O. Inhumans, fermentation yields lactic acid and in yeast fermentation yieldsethyl alcohol in addition to CO2. Since fermentation lacks a Krebs cycle and an electron transportchain, the only energy products come from glycolysis. The 2 NADH areused to produce the alcohol or lactic acid. The total amount of ATPproduced from one molecule of glucose is 2.
    • CELL RESPIRATIONExperimental procedure for fermentationTests performed by the Instructor: Test for carbon dioxide (CO2)production: The instructor willdemonstrate the reaction between CO2 and barium carbonate by using astraw to breath into a tube of barium carbonate. Note the reaction whenthe instructor performs this activity. Students will not perform thisactivity because barium carbonate is toxic. The instructor will then place a tube from a beaker containingyeast in a sucrose solution into a second tube of barium carbonate to testfor CO2 production by the yeast.
    • CELL RESPIRATIONExperimental procedure for fermentation (cont.)Tests performed by the Instructor (cont.): Test for ethyl alcohol production: The instructor willassemble a distillation apparatus to collect alcohol from both flasks. Theflasks will be heated to separate the alcohol from the water. Thedistillate will be tested for the presence of alcohol as described in the labmanual. This flask contains This flask sucrose alone. contains =====> yeast and sucrose. <=====
    • CELL RESPIRATIONExperimental procedure for fermentation (cont.)Tests performed by the Student: Controls for alcohol production: You will prepare two tubes(Tube 1 and Tube 2) that will be used as controls for the presence ofalcohol. Tube 1 will be a negative control because it will not containalcohol. This tube will give a negative result for the presence of alcohol. Tube 2 will contain alcohol and will give a positive result for thepresence of alcohol. You will use these tubes to interpret the results of the alcoholtests performed by the instructor.
    • CELL RESPIRATIONIntroduction to the Krebs Cycle and the Action of Succinic (Acid)Dehydrogenase. Succinic acid dehydrogenase is and enzyme that oxidizessuccinic acid into fumaric acid as part of the Krebs cycle. During thisreaction the coenzyme FAD is reduced to FADH2. This reaction isshown as Figure 7.4 in your lab manual. This reaction represents a typical enzymatic reaction thatinvolves a substrate (succinic acid), an enzyme (succinic aciddehydrogenase), and a product (fumaric acid). We will use methylene blue as an indicator that this reaction istaking place. Methylene blue will remove the electrons from FADH2and turn from blue to colorless. Thus we will be able to determine therate of the reaction by noting how long it takes the methylene blue to
    • CELL RESPIRATIONIntroduction to the Krebs Cycle and the Action of Succinic (Acid)Dehydrogenase. We will use methylene blue as an indicator that this reaction istaking place. Methylene blue will remove the electrons from FADH2and turn from blue to colorless. Thus we will be able to determine therate of the reaction by noting how long it takes the methylene blue toturn from blue to clear. The methylene blue The methylene blue will clear as FADH2 will start out blue. ===> is produced. <====
    • CELL RESPIRATIONExperimental procedure for The Krebs Cycle and the Action ofSuccinic (Acid) Dehydrogenase. Tube 1: Place approximately 2 cm of liquid hamburger into atest tube. Then add 10 drops of succinic acid and 3 drops of methyleneblue to the hamburger and mix. Note the time as you mix. This is atimed experiment and it is important to know the time the reactionstarted. Once the reagents are mixed do not mix them again (Yourinstructor will explain why). Not the time when the only blue remainingis a thin band at the surface of the reagents. Tube 2: Place approximately 2 cm of liquid hamburger into atest tube. Then add 10 drops of distilled water instead of succinic acidand 3 drops of methylene blue to the hamburger and mix. Note the timeas before and watch for the color to change.
    • CELL RESPIRATIONExperimental procedure for The Krebs Cycle and the Action ofSuccinic (Acid) Dehydrogenase (cont.). Tube 3: Place approximately 2 cm of liquid hamburger into atest tube. Then add 10 drops of malonic acid instead of succinic acidand 3 drops of methylene blue to the hamburger and mix. Note the timeas before and watch for the color to change. Tube 4: Add 5 ml of water to the test tube instead of liquidhamburger. Then add 10 drops of succinic acid and 3 drops ofmethylene blue to the hamburger and mix. Note the time as before andwatch for the color to change.Suggestions: Rinse the mixing rod between tubes. Mix all the tubes at the same time. Do wait for tube 1 tofinish reacting before starting tube 2, tube 3, or tube 4.
    • CELL RESPIRATIONExperimental procedure for The Krebs Cycle and the Action ofSuccinic (Acid) Dehydrogenase (cont.). Results: Three of the four tubes should clear. The time requiredfor each of the three tubes should be different. To determine why thetimes are different you should consider the only variable used in this partof the experiment. One of the tubes should not change. Can you guesswhich one and why? Your results should look like this picture.