BIO15 Lab Manual_MF_Sp2016.pdf

BIO15 Lab Manual_MF_Sp2016.pdf

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Laboratory Manual for Bio 15 – General Microbiology Saddleback College Department of Biological Sciences Version Specific for Professor Monica Friedrich Revised January 2016 J. Clark, M. Friedrich, E. Ininns, A. Sirulnik, A. Bandekar, and R. Wrightsman
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2 Table of Contents Table of Contents ........................................................................................................................................ 2 Laboratory Safety ....................................................................................................................................... 4 Map of the Laboratory ............................................................................................................................... 8 Setting Up Your Laboratory Notebook .................................................................................................. 11 Compound Microscope ............................................................................................................................. 14 Care of the Microscope ............................................................................................................................. 18 Pouring Agar Plates .................................................................................................................................. 21 Ubiquity of Microorganisms .................................................................................................................... 23 Media Preparation .................................................................................................................................... 26 Aseptic Transfer ........................................................................................................................................ 28 Preparing a Bacterial Smear .................................................................................................................... 32 Simple Stain ............................................................................................................................................... 34 Gram Stain ................................................................................................................................................. 36 Negative Stain ............................................................................................................................................ 39 Capsule Stain ............................................................................................................................................. 41 Schaeffer-Fulton Endospore Stain .......................................................................................................... 43 Inclusion Body Stain ................................................................................................................................. 45 Streak Plate Technique ............................................................................................................................. 46 Acid Fast Stain .......................................................................................................................................... 50 Measurement with the Microscope ......................................................................................................... 52 Unknown Project ....................................................................................................................................... 55 Selective and Differential Media .............................................................................................................. 71 Bacterial Pigmentation Demonstration ................................................................................................... 73 Aseptic Use of the Serological Pipette ..................................................................................................... 74 The Viable Plate Count Method of Counting Bacteria ......................................................................... 76 Effects of Disinfectants and Antiseptics .................................................................................................. 81 Effects of Antibiotics ................................................................................................................................. 83 Bacterial Motility ...................................................................................................................................... 86 Oxygen Requirements and the Cultivation of Anaerobes and Microaerophiles ................................ 87 Digestive Exoenzymes ............................................................................................................................... 89 Effects of Temperature ............................................................................................................................. 91 Effects of Ultraviolet Light ....................................................................................................................... 94
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3 Carbohydrate Fermentation .................................................................................................................... 96 Oxidase Test ............................................................................................................................................... 99 Hydrogen Sulfide Production using Peptone Iron agar ...................................................................... 100 Catalase Test ............................................................................................................................................ 102 Urease Test ............................................................................................................................................... 103 Lysine and Ornithine Decarboxylase Tests .......................................................................................... 105 Deoxyribonuclease (DNase) Activity ..................................................................................................... 107 IMViC Tests ............................................................................................................................................. 108 Nitrate Reduction .................................................................................................................................... 110 Phenylalanine Deaminase Test .............................................................................................................. 113 Bacterial Agglutination ........................................................................................................................... 114 Rapid Plasma Reagin (RPR) Test .......................................................................................................... 116 Multitest ................................................................................................................................................... 117 Coagulase Test ......................................................................................................................................... 122 ELISA ....................................................................................................................................................... 123 Yeast Fungi .............................................................................................................................................. 125 The Germ Tube Test ............................................................................................................................... 128 Filamentous Fungi ................................................................................................................................... 129 Bacteriological Examination of Water .................................................................................................. 130 Live Protozoans (The Wet Mount Technique) ..................................................................................... 134 Journey into the “Final Unknown” ....................................................................................................... 135 Final Unknown Report Sheet ................................................................................................................. 137 Parasitic Protozoa I ................................................................................................................................. 139 Parasitic Protozoa II ............................................................................................................................... 140 Parasitic Helminths ................................................................................................................................. 141 Study Guide - Laboratory Comprehensive Exam ............................................................................... 142 Appendix A: Results Charts ................................................................................................................... 144 Appendix B: Discussion Questions ........................................................................................................ 156 Appendix C: Supplemental Info on the Unknown Project Paper ...................................................... 164 Appendix D: Supplemental Info on the Final Unknown Project ........................................................ 168 Appendix E: Lab Notebook and Quiz Checklist ................................................................................. 169
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4 Laboratory Safety Our laboratory work in microbiology involves the use of pathogens and/or potential pathogens of either animals or plants. Our primary concerns are to learn and practice safe laboratory techniques, and to follow procedures that permit the safe handling of all microorganisms. These procedures include aseptic procedures (techniques used to prevent contamination of microbiological cultures). These procedures are performed each laboratory period and can only be performed in a laboratory that is properly cleaned both before and after use. As well, some laboratory procedures will involve using potentially hazardous chemicals and/or reagents. Proper chemical handling and use is to be strictly observed. Please see below for a list of procedures and guidelines that will minimize microbiological contamination during the
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