Microbiology Lab Manual: Discovering Your Tiny Neighbors Lab Manual for Microbiology 22 Mt. San Antonio College Revised July 2018 Assembled by Chris Briggs, from numerous sources Photo of Luke Jerram's blown glass sculpture by Matteo De Stefano/MUSE Science Museum, Trento, Italy. – 1 – Table of Contents Orientation and Safety Part 1: Microscopy Exercise 1: The Microscope and Bacteria Part 2: Handling, Growing, and Simple Staining Bacteria Exercise 2: Culturing your Environment Exercise 3: Media and Aseptic Techniques Exercise 4: Simple Stains Part 3: Differential and Structural Staining Exercise 5: Gram Stain, Acid-Fast Stain, and Spore Stain Identification of Unknown Bacteria Part 4: Eukaryotic Microorganisms Exercise 6: Fungi Exercise 7: Protists Exercise 8: Parasitic Worms and Arthropod Vectors Part 5: Cultivation and Identification of Bacteria Exercise 9: Pure Culture Techniques Exercise 10: Isolation of Pathogens Exercise 11: More Tests of an Unknown Bacterium Exercise 12: Rapid Bacterial Identification Exercise 13: Summary of UTI Results Part 6: Control of Microbial Growth Exercise 14: Susceptibility to Antibiotics, Dyes, and Metals Exercise 15: Heat, Cold, Drying, and Radiant Energy Exercise 16: Antibacterial Products Part 7: Epidemiology and Immunology Exercise 17: HIV Epidemic and Detection with ELISA Part 8: Microbial Genetics Exercise 18: Bacterial Transformation Part 9: Water and Milk Microorganisms Exercise 19: Testing Water and Milk – 2 – Acknowledgements This lab manual is largely the result of work by Mt. San Antonio College microbiology instructors. Many of the lab exercises were developed by Dr. Lou Shainberg, who taught at Mt. SAC for 45 years, from 1962 until he died in 2006. Lou was a role model, mentor, and beloved colleague of Mt. SAC microbiology professor Cindy Anderson. Cindy has further developed this lab manual in her many years of teaching. Some of the text and several images have been adapted from an open-source lab manual by Joan Petersen and Susan McLaughlin at CUNY Queensborough Community College. In addition, this manual has benefited from valuable input and suggestions from Mt. SAC microbiology instructors Monica Lee, Dr. Raminder Kaur, Dr. Jessica Spitzer, Dr. Kaushiki Menon, and Nan Shea. The labs also rely on the careful attention of our laboratory technicians Donna Lee, Ana Jara de Araya, Naomi Velarde-Jang, and Brandon Jacoby. And to our students, welcome to the exciting world of microbiology! Please enjoy your exploration and investigation. I am always interested to hear your feedback on this manual. – Chris Briggs, Professor and Course Coordinator – 3 – Orientation and Safety Welcome to Microbiology Lab! -------------------------------------------------------------------------------------------------------------------------- Student preparation Doing well in lab requires regular attendance. Students who miss more than two weeks of lab (or three days in winter or summer sessions) will be dropped from class. Read the exercise before coming to lab. Bring your lab manual to each lab meeting. Reach out to your classmates to develop friendships. The class will be even more enjoyable that way. Labs will fill the entire lab period, so plan to stay until the end. Useful references Lab manual Course textbook A Photographic Atlas for the Microbiology Laboratory, by Leboffe and Pierce. This contains full- color photographs. www.microbelibrary.org and www.microbeworld.org. These contain images, videos, and more. -------------------------------------------------------------------------------------------------------------------------- Safety instructions 5. Avoid all hand-to-mouth, finger-to-mouth, and finger-to-eye contact. (Break the habit Our lab can be an interesting and exciting place, of licking your fingers to turn pages, or of but it includes potential hazards. Improper putting pencils in your mouth.) Never handling of chemicals, equipment, or microbial apply cosmetics or insert contact lenses in cultures is dangerous and can result in injury or the laboratory. infection. The instructions below are designed to keep you safe in the laboratory. Please read them 6. Wash hands, and wipe down bench area carefully. If you have any questions about safe with disinfectant prior to working. Before laboratory practices, ask your instructor. you leave the lab for the day wipe down your bench area with disinfectant and then General preparedness wash your hands. Wash your hands at any time during the lab if you think you may 1. Come to lab on time and prepared for that have contaminated them. Wipe any day's experiments. If you miss oral surfaces or equipment with disinfectant instructions, this can be dangerous to you immediately if you suspect contamination and to your classmates. with living cultures. 2. Make sure to carefully read through the 7. Keep your desk and floor free of non- entire procedure before beginning an essential materials at all times (including experiment in the lab. This will help cell phones, since they do not respond well prevent you from making mistakes that to being soaked in disinfectant). could compromise your safety. 8. Know where the fire extinguisher, 3. Follow all directions given by the emergency eye/face wash and shower are instructor. Bring any safety concerns to the located in our laboratory. Make sure chairs attention of the instructor. are not blocking the room’s exits. 4. No eating, drinking, or smoking at any 9. To remain in the lab, you must use proper time. Do not bring food or drink items into personal protective equipment. This the lab. includes a lab coat that goes to your knees – 4 – (long sleeved and buttoned/closed in dedicated broken glass container. front), closed-toe shoes that cover the top of your foot, and clothing that covers your 18. Immediately report any incidents such as legs (no shorts or short dresses). Any open cuts or burns to the instructor. wounds should be covered and protected before entering the lab. Open-toed shoes 19. Do not put contaminated instruments, (sandals, flip-flops etc.) cannot be worn in such as inoculating loops, needles, or lab. pipettes, on bench tops. Loops and needles must be sterilized by incineration. 10. Wax pencils and permanent markers are useful for writing on glassware and 20. Never pipette by mouth. Proper pipetting microscope slides. Please do not write on is carried out with the aid of a mechanical any white frosted portions of glassware, pipetting device, and the cotton plug in the because the marks are very difficult to top of the disposable pipettes is to be left remove. alone. 11. Never remove media, equipment, or When using Bacti-Cinerators bacterial cultures from the lab. 21. Bacti-Cinerators will reach optimum 12. For liability and safety reasons, no visitors sterilizing temperature (1500°F/815°C) are allowed in the laboratory. after 10 minutes. Please turn off the Bacti- Cinerator at the end of lab, but leave it When working with bacteria or plugged in. (The plugs last longest when hazardous chemicals the wires are not tugged.) 13. Loose clothing and long hair must be tied 22. Do not let your inoculating loops or back while working to avoid inadvertent needles stay inside the Bacti-Cinerator contamination. unattended! They can fall out, and will weaken rapidly if heated too long. 14. Wear gloves and eye protection whenever hazardous chemicals or living cultures are Disposal and clean-up handled. 23. The most critical (and most expensive) 15. Always handle cultures with care. Keep piece of equipment in the microbiology tubes in a test tube rack to prevent laboratory is the microscope. If you expect accidental spillage. Hot test tubes should to see specimens through the microscope, be handled with test tube holders. it must be kept clean and in good condition. Instructions for the use and care 16. Treat all living cultures of microorganisms of the microscope can be found in Lab 1 of (bacteria, yeast, etc.) as potential the lab manual. Report any problems with pathogens. Avoid spilling or spreading the your microscope to your instructor. microorganisms. Place all used materials in the appropriate waste containers 24. Do not use tape on plates or reusable designated for cultures (to be autoclaved). materials because the tape is too difficult Use the techniques specified by the to remove. instructor for handling microorganisms. 25. Remove rubber bands before discarding 17. If there is a spill, immediately cover spilled test tubes or plates. cultures or broken culture tubes with paper towels and then saturate the paper 26. Dispose of contaminated plates, pipettes, towels with germicide. Notify your and cotton swabs in the autoclave instructor. After 15 minutes of reaction container (“burn box”). time, remove the towels and dispose of them in a manner indicated by your 27. Dispose of contaminated test tubes in the instructor. Broken glass is swept up with wire baskets provided. brush and dustpan, and discarded into a – 5 – 28. Dispose of all glass coverslips and broken- non repairable slides in the broken glass container. 29. Live mounts of hazardous organisms must be soaked in germicide for 15 minutes, before washing with soap and water. (Stained slides can be washed with soap without soaking in germicide first, since organisms on stained slides are killed by the staining process.) Clean and dry all slides before returning them to their proper boxes. 30. Remove oil from prepared slides with lens paper. 31. Any papers on the floor at the end of the laboratory period are to be picked up and discarded in the wastebasket. The same is true for your laboratory bench area. 32. DO NOT put plates, tubes, swabs, slides, pipettes, pipette tips, or broken glass into the regular garbage. These items need to be disposed of properly. Throwing potentially contaminated items into the regular garbage is a safety issue for students, instructors, lab technicians, and the cleaning staff. If these items are found in the regular garbage the ENTIRE BAG OF GARBAGE must be autoclaved before disposal. If you are unsure about where an item should go, feel free to ask your instructor.