Microbiology lab manual May2019.pdf - Sierra College Biological Sciences Department MICROBIOLOGY Bio Sci 4 and 8A\/8B LABORATORY MANUAL Lead instructor

Microbiology lab manual May2019.pdf - Sierra College...

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Unformatted text preview: Sierra College Biological Sciences Department MICROBIOLOGY Bio. Sci. 4 and 8A/8B LABORATORY MANUAL Lead instructor: Sasha Warren May, 2019 Copyright 2019 1 ________________________________ (Your Name) I have read the information provided in the Introduction and Student Obligations section and in the Safety, Sanitation, Storage and Discard Procedures sections of this laboratory syllabus. I fully understand my obligations with respect to this course and agree to the best of my ability to meet these expectations. Signed ______________________________________ Date ___________________________ 2 TABLE OF CONTENTS Sections/Topics: Page #: Table of Contents...................................................................................................................... 3 Introduction to Microbiology....………………..................................................................... 9 Suggested Laboratory Procedures and Safety Regulations ............................................ 11 Suggested Study Techniques................................................................................................ 15 Semester Project……………………………..……………………………………..…..……. 17 Semester Project Write-Up Guidelines…………………………………..…………. 18 Ex. 1: Effectiveness of Handwashing……………………………………………….…..….. 21 WORKSHEET – Exercise 1 …………………………………………………............ 25 Ex. 2A: Microscopic Technique & Examination of Microorganisms................................. 27 Ex. 2B: Microscopic Measurement........................................................................................ 33 Ex. 2C: Observation and Scientific Illustration.................................................................... 36 WORKSHEET – Exercise 2…………………………………………………………. 39 Ex. 3: Culture of Microorganisms & Media Preparation..................................................... 41 WORKSHEET – Exercise 3…………………………………………………………. 45 Ex. 4: Aseptic Technique & Streak Plate Preparation.......................................................... 47 Ex. 5: Pure Culture Technique ………………………………………….............................. 51 WORKSHEET – Exercises 4 & 5………………………………….………………… 55 Ex. 6A: Staining of Microorganisms -- Direct vs Indirect Staining.................................... 57 Ex. 6A Supplement: Bacterial Cellular Morphology & An Introduction to the Bergey’s Manual ………………………………………………………………………….......... 61 WORKSHEET – Exercise 6A…………………………………..……………………. 65 Ex. 6B: Staining of Microorganisms -- Gram Stain ……………......................................... 67 Ex. 6B Supplement: The KOH Test: Differentiation of Gram Positive & Negative Bacteria Without Staining …………………………………………......................... 70 WORKSHEET – Exercise 6B & 6B Supplement……...……………………….….... 71 Ex. 6C: Staining of Microorganisms – Acid-Fast Stain…………………………………… 75 WORKSHEET – Exercise 6C.………………………………………………………. 77 Ex. 6D: Staining of Microorganisms – Endospore Stains, Capsule Stains & Flagella ..... 79 WORKSHEET – Exercise 6D……………………………….……………………….. 85 3 Table of Contents (continued) Morphological Unknown Assignment ..............................................................................89 REPORT FORM – Morphological Unknown……………………………………… 91 Ex. 7: Introduction to Cyanobacteria................................................................................... 93 Ex. 8: Introduction to Fungi ……......................................................................................... 95 Ex. 9: Introduction to Microscopic Algae ........................................................................... 101 Ex. 10: Introduction to Protozoa......................................................................................... 105 Ex. 11: Introduction to Multicellular Parasites .................................................................. 111 Ex. 12: Food Microbiology …………………………………………...................................117 WORKSHEET – Exercise 12..…………………………………………………..…. 125 Ex. 13: Determination of Microbial Numbers .................................................................... 129 WORKSHEET – Exercise 13..………………………………………..…………… 133 Ex. 14: Use of Selective & Differential Media & Replica Plating Technique …............. 135 WORKSHEET – Exercise 14..…………………………………..………………… 139 Ex. 15A: Physiological Unknown #1: Introduction & the O/F Test.. ........................... 141 REPORT FORM – Physiological Unknown #1 ………………………………….. 147 Ex. 15B: Physiological Unknown #1: Oxidase, Pigment Production, Carbohydrate Slant, Catalase, Coagulase, Esculin Hydrolysis and Hemolysis tests ……….... 155 Ex. 15C: Physiological Unknown #1: SIM, Citrate, Urea Hydrolysis, MR-VP and Lysine Decarboxylation tests…………………………………………………......... 161 Ex. 15D: Physiological Unknown #1: Carbohydrate Deep Test ...................................... 169 Ex. 15E: Additional Physiological Tests: Gelatin hydrolysis, Starch hydrolysis, Salt tolerance, Nitrate Reduction/Denitrification and CAMP tests ………………... 171 Ex. 16A: Physiological Unknown #2: Introduction & Polymerase Chain Reaction …. 177 REPORT FORM – Physiological Unknown #2 ………………………………….. 183 Ex. 16B: Physiological Unknown #2: Automated Nucleotide Sequencing & Electropherogram Evaluation ……………………………….…………...……… 187 Ex. 16C: Genomics & Bioinformatics ………………......……….……………………...…191 Ex. 17: The Miniscreen - Rapid Isolation of Plasmid DNA .............................................. 193 Ex. 18: Gel Electrophoresis of DNA Samples ..................................................................... 197 WORKSHEET – Exercises 17 & 18……………………………………………….. 4 201 Table of Contents (continued) Ex. 19: Restriction Endonuclease Digestion of DNA & RFLP…................................... 203 WORKSHEET – RFLP Assignment ........................................................................207 Ex. 20: Transformation of E. coli With Plasmid DNA...................................................... 209 WORKSHEET – Exercise 20 ……………………….………………………....…... 213 Ex. 21A: Introduction to Viruses and Phage Typing ……………………………………215 WORKSHEET – Exercise 21A ……………………………………………………..219 Ex. 21B: Isolation and Purification of Coliphages from the Environment......................221 WORKSHEET – Exercises 21B….…………………………………………...…......225 Ex. 21C: Bacteriophage Reproduction and Plaque Formation………..………………... 227 WORKSHEET – Exercise 21C…………………………………………………….. 229 Ex. 22A: Microbial Control Methods ……………………………………………............... 231 WORKSHEET – Exercise 22A…..………………………………….………..…… 237 Ex. 22B: Antimicrobial Sensitivity Testing - Kirby-Bauer Test ……………………........241 WORKSHEET – Exercise 22B..….…………………………………………...….. 245 Ex. 23: Diagnostic Immunology – The Rapid Strep Test................................................247 WORKSHEET – Exercise 23…….………………………………...….…………… 251 APPENDIX SECTION........................................................................................................ 253 Appendix A - Correct Use of Micropipettes.................................................................... 253 Appendix B – Enrichments for Selected Bacteria from the Environment .................... 255 Appendix C – Preparation of Fungus Slide Culture Chambers....................................... 261 Appendix D – Additional Procedures for Cultured Foods ........................................... 265 Appendix E – Bacteriological Examination of Water …………....................................... 269 Reference Literature on Microbiology ................................................................................ 273 Answers to Laboratory Syllabus Questions ....................................................................... 275 5 6 This copy of the Microbiology Laboratory Syllabus belongs to: ____________________________________________ Class Day and Time: _____________________________________ Instructor: _____________________________________ 7 8 INTRODUCTION TO MICROBIOLOGY (Bio. Sci. 4 & 8A-8B) Introduction and Student Obligations Microbiology is a one-semester five-unit course (Bio. Sci. 4) or a two-semester five-unit course (Bio. Sci. 8A-8B) designed as an introduction to the structure, physiology, classification and significance of microorganisms, with an emphasis on bacteria and viruses. It is recommended for students majoring in the allied health field, sanitation, human environmental sciences, physical education, or biological sciences, and is not recommended for non-life science majors. The course includes two 1.5-hour lectures, and two 3-hour laboratories each week (or one 1.5-hour lecture and one 3-hour laboratory each week, extended over two semesters) to be attended by all students. Grades for this course will be determined by the student’s overall performance in the class, with the distribution of letter grades being based on total point accumulation. Lecture exams, laboratory exams, a lecture final and a semester project will comprise the basis of points for grade determination; however, points will also be given for homework assignments, the identification of unknown organisms assigned in the laboratory, and for quizzes given frequently throughout the semester. Quizzes are given to insure instructors that students are understanding and keeping up with the material being covered in class; and as a device to encourage attendance. Individual instructors will specify the point values for exams, quizzes and assignments, as well as the percentage requirements for obtaining letter grades. If you are unable to attend class, it is your responsibility to obtain the information and/or assignments missed. Since questions included on quizzes and exams will pertain to information provided in class (lectures and or laboratory presentations), obtaining in-class information will be essential to your success. If you know that you will miss school for an extended period of time, please contact the school or your instructor (phone or email), and apprise us of your absence. Habitual tardiness is not recommended since students coming to class late will miss lecture information and tend to disrupt the class. Tardy students may also forfeit their opportunity to take regularly scheduled quizzes. Microbiology instructors do not like to give makeup quizzes/exams. They are a hassle, and can be unfair to the people who take quizzes/exams on time. If you know in advance that you must miss a quiz/exam, please arrange to take it early. If you miss an exam, and do not notify your instructor of your absence, a makeup exam will be given at the instructor’s discretion, and you can expect to be given an essay-type makeup. Individual instructors will specifiy policies regarding late assignment, quizzes, and exams. Laboratory exams are even more difficult to makeup...PLEASE, DO NOT MISS LABORATORY EXAMS! Note: If you wish to gain the most from the lecture material given, you should complete all reading assignments (lecture and laboratory) before coming to class on the date they are assigned. 9 Notebooks are almost mandatory in every college class, and Microbiology is no exception. A complete set of lecture notes, lab exercises, drawings, quizzes, exams, and handout materials will provide you with the best possible source of information to study for exams. Keeping all test and quiz papers will also provide you with an accurate record of your performance in the class. We suggest that you obtain a ring binder, and keep all of your class papers together and in order. This will alleviate considerable confusion as the class progresses and paper materials accumulate. Students enrolled in microbiology classes are considered to be serious about their educational goals and highly motivated. Our job as instructors is to meet student expectations by helping to provide a safe and comfortable learning environment and by facilitating education through a variety of means. To this end, different learning styles will be addressed, successful study techniques will be recommended and students will be encouraged to work cooperatively for the benefit of all participants. Students are expected to be familiar with information provided in the Sierra College, Student Rights and Responsibilities Handbook and to avoid behaviors requiring disciplinary action. Behavior contrary to student success including disruptive conversation/activity, willful damage to laboratory equipment or materials, theft, cheating, fraud and plagiarism will not be tolerated. Academic honesty is taken seriously and conscientious students are encouraged to report dishonest activities to their instructors. Internet resources including the Biological Sciences Department web page, Canvas, NCBI and other sites can be of great educational assistance and students are strongly encouraged to gain the computer skills necessary for their utilization. Since some students have limited computer availability at home, the college provides access to computers in the Learning Resources Center (Library) and in the microbiology laboratory during open lab times. This department strives to maintain an open lab policy allowing students access to laboratory facilities, equipment and instructional personnel during non-scheduled hours. Open lab times will vary from semester to semester, but typically include interlaboratory periods Monday-Friday, when the laboratory is not scheduled for class use. Please check the schedule on the laboratory door for exact times. Students entering the laboratory during such periods are expected to follow established rules, to use equipment/materials appropriately and to treat other individuals and their lab preparations with courtesy and respect. 10 SUGGESTED LABORATORY PROCEDURES AND SAFETY REGULATIONS Suggested Procedures 1. PREPARE AHEAD OF TIME: Read over each new laboratory exercise before coming to class so you are familiar with the materials and procedures to be used. 2. WAIT FOR INSTRUCTIONS: The instructor will begin each laboratory period with a short introductory session providing specific instructions. Wait for these instructions before beginning protocols and ask questions if there are parts of the exercise you do not understand. Make sure you know what you are expected to do before you do it. 3. KEEP ACCURATE RECORDS: Record observations of all laboratory data when first collected and determine results as indicated. Make sketches or drawings where applicable. Record any variations in procedures or materials used, times required, etc. 4. COMPLETE ALL EXERCISES: It is your responsibility to complete all laboratory assignments, some of which will require data collection over several lab sessions. Do not expect the instructor or other students to collect data for you. 5. TEST YOUR UNDERSTANDING: Answer the questions at the end of each exercise to test your understanding of the laboratory material covered. Recognize that the same or similar questions often appear on laboratory exams. 6. TAKE TIME TO REVIEW: After completing each exercise, reread that portion of the laboratory syllabus to make sure you thoroughly understood the procedures being carried out, and/or the basic principles being demonstrated. 7. BE ALERT: Pay attention to instructional notices posted in the laboratory, they are there for your benefit. Some Safety Regulations 1. WORK ON A CLEAN SURFACE: Clean the surface of your laboratory table with the laboratory disinfectant provided at both the beginning and the end of each lab session. Note - It is your responsibility to keep your disinfectant container filled and ready for use. A supply of additional disinfectant is available in the large plastic container at the back of the laboratory. 2. MINIMIZE CLUTTER: To avoid problems with stains, toxic chemicals, or burners, keep your tabletop free of non-essential materials at all times during the laboratory. Place your books and folders on the floor or in the table shelf provided. 11 3. BE CAREFUL WITH FIRE: Take note of the locations of Bunsen burners in this laboratory and recognize that they are usually lit when laboratories are in session. Be aware of the potential hazard presented by lose hair, fuzzy sweaters, papers and other flammable materials. Never leave your lighted burner unattended, and avoid placing it in a position likely to bring it into contact with other students. 4. WEAR PROTECTIVE CLOTHING: Since students will often work with potential pathogens and a variety of materials that may cause permanent stains, it is strongly recommended that a lab coat or other protective covering be worn. In the event of contaminating spills, this clothing can be removed and disinfected. Provisions will be made to keep protective clothing at school until the end of the semester. Since broken glass is an occasional hazard, bare feet are prohibited. 5. PROTECT YOUR VISION: Wear safety glasses whenever you are heating liquids in tubes or working with materials that might splatter and cause eye injury. Know the location of the emergency eyewash station and how to use the eyewash correctly. 6. CLEAN UP SPILLS: Clean up accidental microbial spills using an abundant quantity of laboratory disinfectant. Avoid handling broken glass or other materials that might cause injury (sharps). Place broken glass in the glass boxes and slides, tubes or pipettes in the containers provided within the discard area. Do not place sharps in the waste paper receptacles. 7. DISPOSE OF SOLID WASTES PROPERLY: Place all used culture tubes in the plastic baskets or racks provided (keep them vertical please), discard Petri plates in the metal bins indicated and any contaminated flasks/beakers on the shelves available in the discard area. Remove all tape labels from glass containers (tubes, flasks, etc.). Do not place culture tubes, flasks or beakers in the metal bins, never dispose of culture containers in trash receptacles, and do not pour contaminated liquids down sink drains. 8. LEARN AND USE ASEPTIC TECHNIQUE: Remember that you will be working with a variety of potentially pathogenic microorganisms in this laboratory, and that you should practice aseptic techniques whenever dealing with living cultures. 9. AVOID CONTACT WITH PATHOGENS: Avoid unnecessary exposure to potential pathogens by following a few simple rules; do not consume food or drinks while working in the microbiology laboratory, avoid nail-chewing or other hand-to-mouth activities, and never pipette by mouth. 10. AVOID INFECTION: Report any accidents or injuries (cuts, burns, etc.) to your instructor as quickly as possible. First aid materials are available, and all precautions should be taken to avoid possible infection. 12 11. BE INFORMED: Know the locations and appropriate use of eyewash stations, glass containers, fire extinguishers, fire blanket, first aid kit, MSDS folders and clean up materials for spills/broken glass. Know the proper exits to use and procedures to follow in the event of an emergency evaculation. 12. USE COMMON SENSE: This facility, the laboratory equipment and supplies are available for your educational use. Generally be careful and think about what you are doing. If you need help, please contact the instructor. Sanitation, Storage and Discard Procedures The use of these procedures aids in the maintenance of uncontaminated cultures (especially important once you begin working with unknowns), and prevents the spread of viable, possibly pathogenic microorganisms. They are designed to protect everyone working within this laboratory from unnecessary exposure, and should be taken seriously. 1. Before beginning your work with any live microbial culture, clean the countertop with the micro-disinfectant provided. Spread the disinfectant in a thin film over the counter surface with a paper towel, allow it to stand for a moment and then wipe it off. At the end of the laboratory period, after all materials have been put away or properly discarded, repeat the counter cleaning procedure, and wash your hands thoroughly. 2. The incubation and storage of live microorganisms ...
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