BIOL 3150 Lab5 - ,youshould 1 2 ,P1 3 Knowhowtoru

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55 LABORATORY 5 – Food Microbiology and Microbial Genetics NB: This week (and in the remaining weeks), you will be working on more than one experiment/project at the same time. Therefore, it is essential that you: 1. Read over the laboratory procedures carefully before coming to the lab. 2. Come prepared to start work immediately. 3. Organize your time efficiently. 4. Keep detailed notes on your results and observations for each experiment/project. EXERCISE 1: Food Microbiology (Part 2). Characterization of the Unknown Coliform (Work in Pairs) Food products contain a wide variety of microorganisms, many of which are capable of growing on EMB agar. To determine whether a particular EMB positive microorganism is a coliform and to classify it with respect to its genus and species , isolated colonies of the organism are usually Gram stained and subjected to a battery of biochemical tests. Two simple biochemical tests commonly used to classify microorganisms are the catalase and oxidase tests . These tests determine if a particular organism can tolerate molecular oxygen (O2) and use cytochrome c to transfer electrons to O 2 during aerobic respiration. Certain forms of molecular oxygen such as the superoxide radical (O 2 ) are toxic to all living cells. Aerobic organisms can detoxify these dangerous forms of oxygen (most possess a superoxide dismutase enzyme that adds protons to the superoxide radical to form hydrogen peroxide): 2O 2 + 2e 2O 2 + 4H + 2H 2 O 2 Since hydrogen peroxide itself is also toxic, most aerobic microorganisms produce the enzyme catalase , converting this chemical into water and oxygen. 2H 2 O 2 2H 2 O + O 2 Objectives for Week 5 After completing these exercises, you should: 1. Know how to analyze isolated organisms for catalase and oxidase activity. 2. Know how to prepare a lysate of the Escherichia coli transducing phage, P1. 3. Know how to run PRC gel. Reading: Madigan et al (2015): Ch 4: pp 114; Ch 10: pp 301 304; Ch 27: pp 801 802 and 810 811; Ch 31: pp 909 920.
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56 Most enteric bacteria are catalase positive. Some (but not all) aerobic microorganisms use the electron transport chain (ETC) to transfer e to O 2 during respiration. To determine if this pathway is being utilized, microorganisms can be tested for the presence of the enzyme oxidase . In this test, if oxidase is present, electrons are transferred from the reduced form of the substrate tetramethyl p phenylenediamine (TMPD) to the oxidized form of cytochrome c, which will be available to accept electrons if the ETC is functioning. The oxidized form of TMPD forms a dark violet or purple color (oxidase positive) while the reduced substrate remains colorless (oxidase negative). The oxidase test is used in clinical laboratories to distinguish between the oxidase positive organisms such as Pseudomonas, Neisseria and Vibrio and the oxidase negative “enterics”. This week, you will carry out Gram stains, catalase tests and oxidase tests on an unknown to determine if it is a possible coliform.
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  • Winter '13
  • MichaelGadson
  • Microbiology, Genetics, oxidase, R‐top agar, catalase positive control, P1vir phage

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