BIOL 2051 Lab Final Exam Study Guide Experiment 21 Catalase Production VOCABULARY Enzymes proteins that serve as biological catalysts Substrate the substance that an enzyme reacts with Extracellular enzymes enzymes that act outside of the cell in which they are produced They degrade large molecules in to units that are small enough to be transported across the cell membrane and into the cell where they can be useful Endoenzymes act inside the cell to degrade molecules to simpler molecules releasing energy or synthesizing molecules that are needed by the cell Inducible or adaptive enzymes enzymes produced only if the appropriate substrate is available Constitutive enzymes enzymes that are essential to the cell and produced at all times Morphology the cell size shape and arrangement of a bacterium Physiology an organism s metabolic capabilities including the enzymes it produces and how it reacts biochemically Catalase enzyme that breaks hydrogen peroxide into water and molecular oxygen produced by essentially all organisms that use oxygen for respiration BACKGROUND INFORMATION Hydrogen peroxide is a byproduct of aerobic metabolism but is toxic so must be broken down Catalase breaks down the toxic hydrogen peroxide into water and molecular oxygen 2 H 2O2 catalase Oxygen production is observed due to the production of bubbles Catalase production is used to differentiate between organisms especially Staphylococcus and Streptococcus 2 H 2O O2 PROCEDURE POSSIBLE PRACTICAL 1 Add a drop of hydrogen peroxide H2O2 to a slide 2 Aseptically transfer cells of the organism to be tested to the hydrogen peroxide drop and mix together 3 Observe for production of bubbles record results RESULTS Organism Staphylococcus epidermidis Streptococcus mutans Appearance of Test Bubbles produced No bubbles produced Does this organism produce catalase Yes No Streptococci are strictly fermentative and DO NOT produce catalase while staphylococci are aerobic and DO produce catalase QUESTIONS 1 What is the reagent that serves as the substrate in the catalase test 2 What type of metabolism is associated with catalase production 3 For what product is one testing when using the catalase test A hydrogen peroxide A aerobic metabolism A Oxygen 4 Would you expect anaerobes to produce catalase Explain your answer A No because anaerobes cannot live in an oxic environment and catalase is used to break down hydrogen peroxide into water and oxygen 5 Describe the appearance of a positive test and a negative test A A positive catalase test will immediately form bubbles when the bacterial cells are mixed and a negative catalase test will not form bubbles at all 6 What does a positive or negative test indicate about the metabolism of the cells tested A A positive test indicates that the cells use aerobic metabolism while a negative test indicates that the cells do not use aerobic metabolism and may be fermentative or anaerobic If your catalase test was positive to what 2 possible genera could the organism belong A Staphylococcus and Micrococcus 7 8 What is the equation catalyzed by the enzyme catalase A 2 H 2O2 catalase 2 H 2O O2 9 Would you expect your cells to produce catalase Explain your answer A Yes because humans are obligate aerobes and use aerobic respiration Hydrogen peroxide therefore would be toxic to our cells Experiment 22 Oxidase Production BACKGROUND INFORMATION Oxidase enzymes are made by organisms that produce energy during aerobic respiration The production of oxidase enzyme is used to identify organisms that can use oxygen such as o Aerobes o Facultative anaerobes o Microaerophiles negative Oxidase tests are used to differentiate medically relevant genera o Vibrio and Pseudomonas oxidase positive from Escherichia and Salmonella oxidase o Neisseria and Moraxella oxidase positive from Acinetobacter oxidase negative Test results must be observed immediately due to false positives from oxidation from the air Cytochrome oxidase transfers electrons to O2 to form H2O or H2O2 as the final electron transfer in electron transport of aerobic respiration There are different types of cyt oxidases of which cyt c oxidase is one Shown in the figure is cyt bo3 oxidase in E coli PROCEDURE Inoculate a plate of TSA with a straight line s of bacteria to be tested Invert and incubate 1 2 After bacteria has grown use a micropipettor and a new tip to transfer 200 l of oxidase reagent to the line of bacteria 3 A positive result is seen within seconds as the organism that produces cytochrome oxidase will turn blue purple where the oxidase reagent was added The other microorganism will have little or no color change indicating the absence of cytochrome oxidase TSA Tryptic Soy Agar MEDIA RESULTS Organism Escherichia coli Pseudomonas putida Color Observed Right after the Addition of Oxidase Reagent White Purple blue Oxidase Positive or Negative Negative Positive QUESTIONS 1 What is the function of cytochrome oxidase A It catalyzes the oxidation of a cytochrome a member of the membrane electron transport chain and the subsequent reduction of molecular oxygen to either H2O or H2O2 2 Why are strict aerobes oxidase positive A Because oxidase is an enzyme that is critical to aerobic cellular respiration specifically the final reduction of oxygen in the electron transport chain 3 The oxidase test is used to differentiate among which groups of bacteria A Vibrio and Pseudomonas which are oxidase positive from Escherichia and Salmonella which are oxidase negative Also Neisseria and Moraxella which are oxidase positive from Acinetobacter which is oxidase negative 4 What is the function of the test reagent in this procedure A It serves as an artificial substrate to be oxidized by cytochrome oxidase in the presence of oxygen Experiment 23 The Hydrolysis of Polysaccharides VOCABULARY Monosaccharides single sugar units i e glucose Oligosaccharides two or more sugar units i e sucrose Polysaccharides larger polymers of sugar units and may serve as structural components or important reserve carbon and energy sources Cellulose polymer of glucose used as a structural component and an energy source 1 4 glycosidic bond joins glucose residues in cellulose global carbon cycle entails biosynthesis and biodegradation of cellulose cellulase produced by some microorganisms to decompose cellulose aerobic with oxygen anaerobic without oxygen thermophile can degrade cellulose at high temperatures glycogen polymer of glucose that is the reserve energy polysaccharide