Microbiology for the Health Sciences Second Edition Spring 2018 “A person who won’t read has no advantage over one who can’t.” -Mark Twain “Computers are useless. They can only give you answers.” -Pablo Picasso 1 Table of Contents Lab Title Page # Introduction to Microscopy and Microbes 12 Cultivation of Microbes; Microbes in the Environment and on the Skin 25 Isolation of Bacteria by Dilution Techniques 31 Gram Stain 44 Acid Fast and Endospore Staining 54 Morphological Unknown 66 Microbial Growth; Differential, Selective and Enriched Media 76 How does the atmosphere affect microbial growth? 85 Carbohydrate Metabolism 96 Bacterial Transformation 105 Ames Test and UV radiation induced mutation 112 Control of Microbial Growth; Physical Methods 120 Chemical Methods of Control; Disinfectants and Antiseptics 126 Antimicrobial Drugs 132 How Does Antibiotic Susceptibility Differ Between Fungi and Bacteria? 142 Epidemiology: how a cold spreads through the dorm or lecture hall 149 Contaminated Spray Bottle and Water Contamination 155 Genus Unknown 163 Immunology; Innate Immunity 173 Blood Typing: ABO and Rh Blood Group Systems 180 White Blood Cell Identification 189 Rapid Antigen Detection Test for Group A Streptococcus 195 Public Service Announcement: Communicating Science to the Public 201 Universal Precautions in Action, Specimen Transport 207 Dipstick Urinalysis for the Detection of Urinary Tract Infections 213 Bacteria of the Gastrointestinal (GI) Tract 219 2 Note from the authors We want to impart onto you that a solid understanding of microbiology is valuable in all aspects of life and is absolutely critical in many fields including nursing, dietetics, respiratory therapy, health science, and health education & promotion. You are ultimately responsible for your learning. Those of you who enter the field of biology in some capacity whether it be the health sciences, basic sciences, or industry will be entering professions that will rapidly change during the course of your professional career. As such, you will need to be a life-long learner to remain current in your understanding of science and up to date on best practices. Take a genuine interest in your field and try to learn all you can while you’re still in school but you will continue to learn for the rest of your careers. We attempted to make this lab manual as clear, concise, engaging, and clinically relevant as possible. Please email David Huston ([email protected]) and Doug Bernstein ([email protected]) if you have suggestions or complaints regarding the lab manual that can be corrected for future semesters. Our goal is make the labs relevant to the health sciences and also interesting. Some of the labs that we will be performing are experiments in the truest sense of the word. As such, we as instructors and you as students have no idea exactly what the results will be. The great physicist Enrico Fermi stated, “There are two possible outcomes: if the result confirms the hypothesis, then you've made a measurement. If the result is contrary to the hypothesis, then you've made a discovery.” We will not always know what the result will be when we perform an experiment but your goal as students will be to interpret the results that you get to the best of your ability and draw conclusions from these results. Furthermore many of the labs we will perform will take multiple days. We will do the experiment one day and look at the results during the next lab period. This is intentional and unavoidable as microbes require many hours to grow. We want to continue to improve this lab manual in the future. If you spot anything that is problematic or could be improved, please email David Huston ([email protected]) and Doug Bernstein ([email protected]). Use the subject heading “Regarding BIO 113 Lab Manual” and indicate specific page numbers if possible. For questions about your grade, missed labs, or if you are struggling in the lab, please contact your professor or lab GA. Good luck and have a great semester! Doug and David 3 Laboratory Safety Guidelines • Read the appropriate section in your lab manual before you come to lab and watch the appropriate videos posted on Blackboard. Make sure that you understand the science behind the lab that you will be performing. If something is unclear, I strongly encourage you to reread parts that you do not understand. If things are still unclear, read about the subject in your textbook and online. If things are still unclear, talk to your professor during office hours or your GA before lab begins. Contact your professor or GA if you will be unable to meet with them during these times. • Again, read the lab manual and watch the Blackboard videos and make sure you understand their contents before you come to lab. There are pre-lab questions you will need to answer prior to coming to lab. These may be collected at the beginning of lab for credit. • Show up to lab on time. The lab periods are short, and there is a lot to do. In lab, be efficient, but be safe. Never compromise on safety. • Wash your hands with soap before and after lab. Sanitize your lab station before and after lab. Wear gloves when handling bodily fluids. If you come into contact with bodily fluids of another person, notify your lab GA immediately. If bodily fluids are spilled on the laboratory bench, cover them with paper towels and then soak with bleach or other similar disinfectant for 15 minutes. Discard waste in the appropriate containers. Ask your lab GA if you have questions. • Be a good lab citizen, make sure you place everything back where you found it when you are done. Reagents are expensive. Use what you need for the lab, but try not to be wasteful. • Label all of your plates with your name, lab section, and date. Place plates upside down in the incubator to prevent condensation from accumulating on the surface of your agar. If you use oil immersion, please clean the oil off of your microscope using a KimWipe or microscope paper when you are done. (Don’t use paper towels!!!!) • It is important that you follow all of the instructions that your lab GA provides. Although it may be tempting, do not play with fire. Turn off the gas when you are not using it. Make sure alcohol containers are not near open flames. Do not drink from the alcohol containers, you will not get drunk but you will get diarrhea and expelled from lab. • Whenever heating a tube point away from yourself and others. • Keep long hair pulled back during lab even when rock music is playing. Do not eat, drink or smoke in the lab. Avoid touching your face. Avoid touching the faces of others. • Do not wear open toed shoes or sandals in lab. If you do your lab instructor will ask you to leave the lab. • Make sure you know where the safety shower, eye wash station, fire blanket, fire extinguisher, fire alarms are and how to use them. 4 • In this lab, you will be working with several antibiotics. If you have known allergies to these antibiotics, please let your professor and/or lab GA know as soon as possible. • The microbial strains that you will be working with in this lab should not cause disease under normal circumstances. If you are immunocompromised, pregnant, or have open wounds, please let your professor and/or lab GA know and special precautions can be taken. • Despite best efforts to prevent accidents, they do happen. Notify your lab GA immediately, and take the appropriate steps to minimize damage. We understand that accidents happen. Just don’t attempt to hide them. If you agree to abide by these rules of lab and the rule of common sense, please sign below ____________________________________ Date_________________ 5 Microscope Care and Use Contract 1. When you are finished using the oil immersion lens, move stage to lowest position. 2. Remove slide from stage clips. 3. Wipe oil off oil objective (100X) with lens paper. Clean/dry lens and stage with lens paper. 4. Put scanning power (4X) objective in center position. 5. Turn off light. 6. Unplug and wrap up power cord neatly. 7. Replace dust cover, if available. 8. Store microscope in correctly numbered cabinet with stage down and 4X objective in center position. Carry microscope with both hands at all times. I have read and I agree to abide by the above rules for microscope maintenance at all times. If I am unsure of a rule I will ask the lab instructor for assistance. I understand that these microscopes are used by many students in many classes and as such must be maintained with the upmost care. Failure to abide by these rules will have a negative impact on my grade. Students may be held responsible for the cost of repairs or replacement if they are found to be handling the equipment improperly. X_________________________________ 6 Laboratory Safety Guidelines (student copy, to keep for your reference) • Read the appropriate section in your lab manual before you come to lab and watch the appropriate videos posted on Blackboard. Make sure that you understand the science behind the lab that you will be performing. If something is unclear, I strongly encourage you to reread parts that you do not understand. If things are still unclear, read about the subject in your textbook and online. If things are still unclear, talk to your professor during office hours or your GA before lab begins. Contact your professor or GA if you will be unable to meet with them during these times.