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Kleyn−Bicknell: Front Matter Preface © The McGraw−Hill Microbiology Experiments: Companies, 2003 A Health Science Perspective, 4/e P r e f a c e To the Student ten to use as little expensive media and equipment as possible. The manual contains more exercises A microbiology laboratory is valuable because it ac- than can be done in one course so that instructors tually gives you a chance to see and study microor- will have an opportunity to select the appropriate ganisms firsthand. In addition, it provides you with exercises for their particular students and class. We the opportunity to learn the special techniques hope that the instructors find these laboratories an used to study and identify these organisms. The enjoyable component of teaching microbiology. ability to make observations, record data, and ana- lyze results is useful throughout life. Acknowledgments It is very important to read the scheduled exercises before coming to class, so that class time can Wewould like to acknowledge the contributions of be used efficiently. It is helpful to ask yourself the the lecturers in the Department of Microbiology at purpose of each step as you are reading and carrying the University of Washington who have thought- out the steps of the experiment. Sometimes it will fully honed laboratory exercises over the years until be necessary to read an exercise several times be- they really work. These include Dorothy Cramer, fore it makes sense. Carol Laxson, Mona Memmer, Janis Fulton, and Conducting experiments in microbiology labora- Mark Chandler. Special thanks to Dale Parkhurst tories is particularly gratifying because the results for his expert knowledge of media. We also thank can be seen in a day or two (as opposed, for instance, the staff of the University of Washington media to plant genetics laboratories). Opening the incuba- room for their expertise and unstinting support. tor door to see how your cultures have grown and We also want to thank Eugene and Martha how the experiment has turned out is a pleasurable Nester, Nancy Pearsall, Denise Anderson and moment. We hope you will enjoy your experience Evans Roberts for their text Microbiology: A Human with microorganisms as well as acquire skills and un- Perspective.This text was the source of much of the derstanding that will be valuable in the future. basic conceptual material and figures for our laboratory manual. And with great appreciation, many To the Instructor thanks to our editor, Deborah Allen, for her sugges- tions, assistance, and ever cheerful support. The manual includes a wide range of exercises— Additional thanks to Meridian Diagnostics in some more difficult and time-consuming than oth- Cincinnati for their generous offer to make diag- ers. Usually more than one exercise can be done in nostic kits available for some exercises. We also a two-hour laboratory period. In these classes, stu- thank the following instructors for their valuable dents can actually see the applications of the prin- input on the revision of this manual. ciples they have learned in the lectures and text. We have tried to integrate the manual with the Reviewers text Microbiology: A Human Perspective, Fourth Edition by Eugene Nester et al. Barbara Beck Kathleen C. Smith The exercises were chosen to give students an Rochester Community Emory University opportunity to learn new techniques and to expose and Technical College Evert Ting them to a variety of experiences and observations. Mark Chatfield Purdue University Calumet It was not assumed that the school or department West Virginia State College Robert Walters had a large budget, thus exercises have been writ- James Madison University vii Kleyn−Bicknell: Front Matter Laboratory Safety © The McGraw−Hill Microbiology Experiments: Companies, 2003 A Health Science Perspective, 4/e L a b o r a t o r y S a f e t y Tobe read by the student before beginning any lab- 9. Avoid creating aerosols by gently mixing oratory work. cultures. Clean off the loop in a sand jar 1. Do not eat, drink, smoke, or store food in the before flaming in the Bunsen burner. laboratory. Avoid all finger-to-mouth contact. 10. If a culture is dropped and broken, notify 2. Never pipette by mouth because of the danger the instructor. Cover the contaminated of ingesting microorganisms or toxic chemicals. area with a paper towel and pour disinfec- 3. Wear a laboratory coat while in the laboratory. tant over the material. After ten minutes, Remove it before leaving the room and store it put the material in a broken glass container in the laboratory until the end of the course.* to be autoclaved. 4. Wipe down the bench surface with disinfectant 11. Carefully follow the techniques of handling before and after each laboratory period. cultures as demonstrated by the instructor. 5. Tie long hair back to prevent it from catching 12. When the laboratory is in session, the doors fire in the Bunsen burner or contaminating and windows should be shut. A sign should be cultures. posted on the door indicating that it is a 6. Keep the workbench clear of any unnecessary microbiology laboratory. books or other items. Do not work on top of 13. Be sure you know the location of fire the manual because if spills occur, it cannot extinguishers, eyewash apparatus, and other be disinfected easily. safety equipment. 7. Be careful with the Bunsen burner. Make sure 14. Wash your hands with soap and water after that paper, alcohol, the gas hose, and your any possible contamination and at the end of microscope are not close to the flame. the laboratory period. 8. All contaminated material and cultures must 15. If you are immunocompromised for any reason be placed in the proper containers for (including pregnancy), it may be wise to autoclaving before disposal or washing. consult a physician before taking this class. *Other protective clothing includes closed shoes, gloves (optional), and eye protection. viii Kleyn−Bicknell: Front Matter Laboratory Safety © The McGraw−Hill Microbiology Experiments: Agreement Companies, 2003 A Health Science Perspective, 4/e L a b o r a t o r y S a f e t y A g r e e m e n t Tobe read by the student before beginning any lab- 9. Avoid creating aerosols by gently mixing oratory work. cultures. Clean off the loop in a sand jar 1. Do not eat, drink, smoke, or store food in the before flaming in the Bunsen burner. laboratory. Avoid all finger-to-mouth contact. 10. If a culture is dropped and broken, notify 2. Never pipette by mouth because of the danger the instructor. Cover the contaminated of ingesting microorganisms or toxic chemicals. area with a paper towel and pour disinfec- 3. Wear a laboratory coat while in the laboratory. tant over the material. After ten minutes, Remove it before leaving the room and store it put the material in a broken glass container in the laboratory until the end of the course.* to be autoclaved. 4. Wipe down the bench surface with disinfectant 11. Carefully follow the techniques of handling before and after each laboratory period. cultures as demonstrated by the instructor. 5. Tie long hair back to prevent it from catching 12. When the laboratory is in session, the doors fire in the Bunsen burner or contaminating and windows should be shut. A sign should be cultures. posted on the door indicating that it is a 6. Keep the workbench clear of any unnecessary microbiology laboratory. books or other items. Do not work on top of 13. Be sure you know the location of fire the manual because if spills occur, it cannot extinguishers, eyewash apparatus, and other be disinfected easily. safety equipment. 7. Be careful with the Bunsen burner. Make sure 14. Wash your hands with soap and water after that paper, alcohol, the gas hose, and your any possible contamination and at the end of microscope are not close to the flame. the laboratory period. 8. All contaminated material and cultures must 15. If you are immunocompromised for any reason be placed in the proper containers for (including pregnancy), it may be wise to autoclaving before disposal or washing. consult a physician before taking this class. *Other protective clothing includes closed shoes, gloves (optional), and eye protection. I have read and understood the laboratory safety rules: __________________________________________________________ ______________________ Signature Date ix Kleyn−Bicknell: I. Basic Microbiology Introduction to © The McGraw−Hill Microbiology Experiments: Microbiology Companies, 2003 A Health Science Perspective, 4/e PART ONE I BASIC MICROBIOLOGY N T R O D U C T I O N to Microbiology When you take a microbiology class, you have an breaking down dead plant and animal material into opportunity to explore an extremely small biologi- basic substances that can be used by other growing cal world that exists unseen in our own ordinary plants and animals. Photosynthetic bacteria are an world. Fortunately, we were born after the micro- important source of the earth’s supply of oxygen. scope was perfected so we can see these extremely Microorganisms also make major contributions in small organisms. the fields of antibiotic production, food and bever- A few of these many and varied organisms are age production as well as food preservation, and pathogens (capable of causing disease). Special more recently, recombinant DNA technology. The techniques have been developed to isolate and principles and techniques demonstrated here can identify them as well as to control or prevent their be applied to these fields as well as to medical tech- growth. The exercises in this manual will empha- nology, nursing, or patient care. This course is an size medical applications. The goal is to teach you introduction to the microbial world, and we hope basic techniques and concepts that will be useful to you will find it useful and interesting. you now or can be used as a foundation for addi- Note: The use of pathogenic organisms has been tional courses. In addition, these exercises are also avoided whenever possible, and nonpathogens designed to help you understand basic biological have been used to illustrate the kinds of tests and concepts that are the foundation for applications in procedures that are actually carried out in clinical all fields. laboratories. In some cases, however, it is difficult As you study microbiology, it is also important to find a substitute and organisms of low patho- to appreciate the essential contributions of mi- genicity are used. These exercises will have an ad- croorganisms as well as their ability to cause dis- ditional safety precaution. ease. Most organisms play indispensable roles in Introduction to Microbiology I–1 1 Kleyn−Bicknell: I. Basic Microbiology Introduction to © The McGraw−Hill Microbiology Experiments: Microbiology Companies, 2003 A Health Science Perspective, 4/e NOTES: Kleyn−Bicknell: I. Basic Microbiology 1. Ubiquity of © The McGraw−Hill Microbiology Experiments: Microorganisms Companies, 2003 A Health Science Perspective, 4/e 1 EXERCISE Ubiquity of Microorganisms Getting Started Definitions Microorganisms are everywhere—in the air, soil, Agar. A carbohydrate derived from seaweed used and water; on plant and rock surfaces; and even in to solidify a liquid medium. such unlikely places as Yellowstone hot springs and Colony. A visible population of microorganisms Antarctic ice. Millions of microorganisms are also growing on a solid medium. found living with animals—for example, the Inoculate. To transfer organisms to a medium to mouth, the skin, the intestine all support huge pop- initiate growth. ulations of bacteria. In fact, the interior of healthy plant and animal tissues is one of the few places Media(medium, singular). The substances used free of microorganisms. In this exercise, you will to support the growth of microorganisms. sample material from the surroundings and your Pathogen. An organism capable of causing disease. body to determine what organisms are present that will grow on laboratory media. Sterile. The absence of either viable An important point to remember as you try to microorganisms or viruses capable of grow organisms, is that there is no one condition or reproduction. mediumthat will permit the growth of all microor- Ubiquity. The existence of something ganisms. The trypticase soy agar used in this exer- everywhere at the same time. cise is a rich medium (a digest of meat and soy products, similar to a beef and vegetable broth) and Objectives will support the growth of many diverse organisms, but bacteria growing in a freshwater lake that is 1. To demonstrate that organisms are very low in organic compounds would find it too ubiquitous. rich (similar to a goldfish in vegetable soup). How- 2. To demonstrate how organisms are grown on ever, organisms that are accustomed to living in our laboratory culture media. nutrient-rich throat might find the same medium lacking necessary substances they require. Reference Temperature is also important. Organisms asso- ciated with warm-blooded animals usually prefer Nester et al. Microbiology: A human perspective, temperatures close to 37°C, which is approximately 4th ed., 2004. Chapter 4. the body temperature of most animals. Soil organisms generally prefer a cooler temperature of 30°C. Organisms growing on glaciers would find room Materials temperature (about 25°C) much too warm and Per team of two (or each individual, would probably grow better in the refrigerator. depending on amount of plates available) Microorganisms also need the correct atmos- phere. Many bacteria require oxygen, while other Trypticase soy agar (TSA) plates, 2 organisms find it extremely toxic and will only Sterile swabs as needed grow in the absence of air. Therefore, the organ- Sterile water (about 1 ml/tube) as needed isms you see growing on the plates may be only a Waterproof marking pen or wax pencil small sample of the organisms originally present. Exercise 1 Ubiquity of Microorganisms 1–1 3 Kleyn−Bicknell: I. Basic Microbiology 1. Ubiquity of © The McGraw−Hill Microbiology Experiments: Microorganisms Companies, 2003 A Health Science Perspective, 4/e Procedure Figure 1.1 Plates labeled on the bottom for ubiquity exercise. First Session 1. Each pair of two students should obtain two petri plates of trypticase soy agar. Notice that Name Name Date Date the lid of a petri plate fits loosely over the 37°C 25°C bottom half. Source 1 Source 2 Source 1 Source 2 2. Label the plates with your name and date using Source 3 Source 4 Source 3 Source 4 a wax pencil or waterproof marker. Always label the bottom of the plate because sometimes you may be examining many plates at the same time and it is easy to switch the lids. 3. Divide each plate in quarters with two lines on the back of the petri plate. Label one plate 37°C and the other 25°C (figure 1.1). Note: For best results, the plates incubated at 37°C 4. Inoculatethe 37°C plate with samples from should be observed after 2 days, but the plates at your body. For example, moisten a sterile swab room temperature will be more interesting at about with sterile water and rub it on your skin and 5–7 days. If possible, place the 37°C plates either in then on one of the quadrants. Try touching the refrigerator or at room temperature after 2 days so your fingers to the agar before and after that all the plates can be observed at the same time. washing or place a hair on the plate. Try 1. Examine the plates you prepared in the first whatever interests you. (Be sure to place all session and record your observations on the used swabs into an autoclave container or report sheet for this exercise. There will be bucket of disinfectant after use.) basically two kinds of colonies:fungi (molds) 5. Inoculate the plate labeled 25°C (room and bacteria. Mold colonies are usually large temperature) with samples from the room. It is and fluffy, the type found on spoiled bread. easier to pick up a sample if the swab is Bacterial colonies are usually soft and moistened in sterile water first. Sterilewater is glistening, and tend to be cream colored or used so that there will be no living organisms yellow. Compare your colonies with color in the water to contaminate your results. Try plates 1 and 2. sampling the bottom of your shoe or some 2. When describing the colonies include: dust, or press a coin or other objects lightly on a. relative size as compared to other colonies the agar. Be sure to label each quadrant so that b. shape (round or irregular) you will know what you used as inoculum. c. color 6. Incubate the plates at the temperature written d. surface (shiny or dull) on the plate. Place the plates in the incubator e. consistency (dry, moist, or mucoid) or basket upside down. This is important f. elevation (flat, craterlike, or conical) because it prevents condensation from 3. There may be surprising results. If you pressed forming on the lid and dripping on the agar your fingers to the agar before and after below. The added moisture would permit washing, you may find more organisms on the colonies of bacteria to run together. plate after you washed your hands. The explanation is that your skin has a normal Second Session flora (organisms that are always found growing on your skin). When you wash your hands, Handle all plates with colonies as if they were po- you wash off the organisms you have picked tential pathogens. Follow your instructor’s direc- up from your surroundings as well as a few tions carefully. layers of skin. This exposes more of your normal flora; therefore, you may see different 4 1–2 Exercise 1 Ubiquity of Microorganisms Kleyn−Bicknell: I. Basic Microbiology 1. Ubiquity of © The McGraw−Hill Microbiology Experiments: Microorganisms Companies, 2003 A Health Science Perspective, 4/e colonies of bacteria before you wash your Note:In some labs, plates with molds are hands than afterward. Your flora is important opened as little as possible and immediately in preventing undesirable organisms from discarded in an autoclave container to prevent growing on your skin. Hand washing is an contaminating the lab with mold spores. excellent method for removing pathogens 5. Follow the instructor’s directions for that are not part of your normal flora. discarding plates. All agar plates are 4. (Optional) If desired, use these plates to autoclaved before washing or discarding in the practice making simple stains or Gram stains municipal garbage system. in exercises 4 and 5. Exercise 1 Ubiquity of Microorganisms 1–3 5 Kleyn−Bicknell: I. Basic Microbiology 1. Ubiquity of © The McGraw−Hill Microbiology Experiments: Microorganisms Companies, 2003 A Health Science Perspective, 4/e NOTES: Kleyn−Bicknell: I. Basic Microbiology 1. Ubiquity of © The McGraw−Hill Microbiology Experiments: Microorganisms Companies, 2003 A Health Science Perspective, 4/e Name Date Section 1 EXERCISE Laboratory Report: Ubiquity of Microorganisms Results Room Temperature (about 25˚C) Plate Plate Quadrant 1 2 3 4 Source Colony appearance 37˚C Plate Plate Quadrant 1 2 3 4 Source Colony appearance Questions 1. Give three reasons why all the organisms you placed on the TS agar plates might not grow. Exercise 1 Ubiquity of Microorganisms 1–5 7