Front Matter Hints and Precautions the different t sof dilution.This includes a variety of Additional information on what to watch out for.what Instructor's Guide Laboratory Report An instructor's guide has been prepared for the labora e Various pedagogical techniques are used for recording to the questionsin this manual Finally,it is our hope that this manual will serve Review Questions le to (1)introd at the end of each labo the so tha another:(2)provide a solid foundation for furthe study for those electing a career in science;and niques presented in each exercise. (convey somethi ng of the mean Dilution Ratios Used in This Manual We appreciate the many comments off ered to ther colons (:)or shills().but note there is a difference of to continue to improve this laboratory manual we invite constructive comments from those using it.Please con- parts,with a total of 3 parts.Thus,equals 1:1,but 1:equals Dilution Problems John P.Harley Lansing M.Prescott Preface
Harley−Prescott: Laboratory Exercises in Microbiology, Fifth Edition Front Matter Preface © The McGraw−Hill Companies, 2002 Preface ix Hints and Precautions Additional information on what to watch out for, what can go wrong, and helpful tidbits to make the experiment work properly are presented in accompanying boxes. Laboratory Report Various pedagogical techniques are used for recording the obtained results. This part of the exercise can be turned in to the instructor for checking or grading. Review Questions Review questions are located at the end of each laboratory report. These were written so that students can test their understanding of the concepts and techniques presented in each exercise. Dilution Ratios Used in This Manual According to the American Society for Microbiology Style Manual, dilution ratios may be reported with either colons (:) or shills (/), but note there is a difference between them. Ashill indicates the ratio of a part to a whole; e.g., d means 1 of 2 parts, with a total of 2 parts. Acolon indicates the ratio of 1 part to 2 parts, with a total of 3 parts. Thus, d equals 1:1, but 1:2 equals h. Dilution Problems Since dilution problems are such an integral part of any microbiology course, Appendix A gives an overview of the different types of dilution. This includes a variety of practice problems. Answers are provided. Instructor’s Guide An instructor’s guide has been prepared for the laboratory manual and is available on our web site at www.mhhe.com/prescott5. This guide provides answers to the questions in this manual. Finally, it is our hope that this manual will serve as a vehicle to (1) introduce the complexity and diversity of microorganisms and their relationships to one another; (2) provide a solid foundation for further study for those electing a career in science; and (3) convey something of the meaning, scope, and excitement of microbiology as a significant perspective from which to view the world. We appreciate the many comments offered to us over the years by both faculty and students. In our desire to continue to improve this laboratory manual, we invite constructive comments from those using it. Please contact us through the Cell and Molecular Biology Editor, McGraw-Hill Publishers (www.mhhe.com/prescott5). John P. Harley Lansing M. Prescott
Front Matter Ackmowledgments ACKNOWLEDGMENTS Our special thanks go to the following reviewers, Raymond B.Otero whose comments proved very helpful to us: Ghayasuddin Ahmad Seton Hall University Duquesne University School of Pharmacy Marcia Pierce Mary A.Anders Gustavus Adolphus College Jackie Reynolds Richland College Nancy Ricker University of Nebraska-Lincoln o College Geoffrey W.Gearner Ivan Roth University of Georgia Dana Kolibachuk Tho University of Connecticut Kentucky University Glendon Miller Wichita State University products
Harley−Prescott: Laboratory Exercises in Microbiology, Fifth Edition Front Matter Acknowledgments © The McGraw−Hill Companies, 2002 x Our special thanks go to the following reviewers, whose comments proved very helpful to us: Ghayasuddin Ahmad Seton Hall University Alberta M. Albrecht Manhattanville College Mary A. Anderson Gustavus Adolphus College Susan T. Bagley Michigan Tech University Paul Blum University of Nebraska–Lincoln Geoffrey W. Gearner Morehead State University Robert J. Kearns University of Dayton Dana Kolibachuk Rhode Island College David Mardon Eastern Kentucky University Glendon Miller Wichita State University Rita Moyes Texas A&M University Raymond B. Otero Eastern Kentucky University Norbert A. Pilewski Duquesne University School of Pharmacy Marcia Pierce Eastern Kentucky University Ralph J. Rascati Kennesaw State College Jackie Reynolds Richland College Nancy Ricker Capilano College Ivan Roth University of Georgia Julie J. Shaffer University of Nebraska at Kearney Thomas Terry University of Connecticut Robert Twarog University of North Carolina A special thanks also goes to Kay Baitz, KEY Scientific Products, 1402 Chisholm Trail, Suite D, Round Rock, Texas 78681, for all of her help with the KEY products. ACKNOWLEDGMENTS
Front Matte 8a ORIENTATION TO THE LABORATORY: RULES OF CONDUCT AND GENERAL SAFETY Many of the microorganisms used in this course may h.identification and proper disposal of be pathogenic for humans and animals.As a result different types of waste intecung yourselr ornvone nyone n the ness that endangers others may be subject to immedi- ate dismissal from the laboratory.If doubt arises as to agreement indicating that the student has d and understands the safety rules of the nom k.good lab practice,including returning materials to proper locations.proper care A student successfully completing basic micro- 2.Protective procedures,including biology will demonstrate the ability to explain and practice safe 1.Microbiological procedures,including contact lenses).and using such equipment in a.reporting all spills and broken glassware to appropriate situations the instructor and receiving instructions for b. b.methods for aseptic transfer c.minimizing or containing the production of 3.Emergency procedures,including d.washing hands prior to and followin laboratories and at any time contamination is fire extinguishers,chemical safety showers. suspected b. h。 and end covers of this laboratory manual c.following proper steps in the event of an g.disinfecting lab benches prior to and at the emergency conclusion of each lab session
Harley−Prescott: Laboratory Exercises in Microbiology, Fifth Edition Front Matter Orientation to the Laboratory: Rules of Conduct and General Safety © The McGraw−Hill Companies, 2002 xi ORIENTATION TO THE LABORATORY: RULES OF CONDUCT AND GENERAL SAFETY Many of the microorganisms used in this course may be pathogenic for humans and animals. As a result, certain rules are necessary to avoid the possibility of infecting yourself or other people. Anyone who chooses to disregard these rules or exhibits carelessness that endangers others may be subject to immediate dismissal from the laboratory. If doubt arises as to the procedure involved in handling infectious material, consult your instructor. In 1997, the American Society for Microbiology, through its Office of Education and Training, adopted the following on laboratory safety. Each point is considered essential for every introductory microbiology laboratory, regardless of its emphasis. A student successfully completing basic microbiology will demonstrate the ability to explain and practice safe 1. Microbiological procedures, including a. reporting all spills and broken glassware to the instructor and receiving instructions for cleanup b. methods for aseptic transfer c. minimizing or containing the production of aerosols and describing the hazards associated with aerosols d. washing hands prior to and following laboratories and at any time contamination is suspected e. never eating or drinking in the laboratory f. using universal precautions (see inside front and end covers of this laboratory manual) g. disinfecting lab benches prior to and at the conclusion of each lab session h. identification and proper disposal of different types of waste i. never applying cosmetics, including contact lenses, or placing objects (fingers, pencils) in the mouth or touching the face j. reading and signing a laboratory safety agreement indicating that the student has read and understands the safety rules of the laboratory k. good lab practice, including returning materials to proper locations, proper care and handling of equipment, and keeping the bench top clear of extraneous materials 2. Protective procedures, including a. tying long hair back, wearing personal protective equipment (eye protection, coats, closed shoes; glasses may be preferred to contact lenses), and using such equipment in appropriate situations b. always using appropriate pipetting devices and understanding that mouth pipetting is forbidden 3. Emergency procedures, including a. locating and properly using emergency equipment (eye-wash stations, first-aid kits, fire extinguishers, chemical safety showers, telephones, and emergency numbers) b. reporting all injuries immediately to the instructor c. following proper steps in the event of an emergency
Front Matter tand Genen 1.train faculty and staff in proper waste stream riment being performed,this will save you much ratory period management effort during the actual labo 2.provide and maintain necessary safety equipment ratory experiments begin with a brief and information resources he location rials and in 3.train faculty,staff,and students in the use of ormi el eyou edure derstand the the principle involved. The co the work in th e System(WHMIS)requires that all hazardous in coverage of subiect matter.to save time and ex- substances,including microorganisms,be labeled pense,and to enco in a specific man addi ere mu Many of the ende precautions a MSDS given in- with every chemical sod have read the above rules and understand All laboratory work can be done more effectively and efficiently if the subject matter is understo ore coming to the la Signature gins.Know how each exercise is to he done and what Date Orientation to the Laboratory:Rules of Conduct and General Safety
Harley−Prescott: Laboratory Exercises in Microbiology, Fifth Edition Front Matter Orientation to the Laboratory: Rules of Conduct and General Safety © The McGraw−Hill Companies, 2002 xii Orientation to the Laboratory: Rules of Conduct and General Safety In addition, institutions where microbiology laboratories are taught will 1. train faculty and staff in proper waste stream management 2. provide and maintain necessary safety equipment and information resources 3. train faculty, staff, and students in the use of safety equipment and procedures 4. train faculty and staff in the use of MSDS. The Workplace Hazardous Materials Information System (WHMIS) requires that all hazardous substances, including microorganisms, be labeled in a specific manner. In addition, there must be a Material Safety Data Sheet (MSDS) available to accompany each hazardous substance. MSDS sheets are now supplied with every chemical sold by supply houses. The person in charge of the microbiology laboratory should ensure that adherence to this law is enforced. All laboratory work can be done more effectively and efficiently if the subject matter is understood before coming to the laboratory. To accomplish this, read the experiment several times before the laboratory begins. Know how each exercise is to be done and what principle it is intended to convey. Also, read the appropriate sections in your textbook that pertain to the experiment being performed, this will save you much time and effort during the actual laboratory period. All laboratory experiments will begin with a brief discussion by your instructor of what is to be done, the location of the materials, and other important information. Feel free to ask questions if you do not understand the instructor or the principle involved. Much of the work in the laboratory is designed to be carried out in groups or with a partner. This is to aid in coverage of subject matter, to save time and expense, and to encourage discussion of data and results. Many of the ASM’s recommended precautions are represented by the specific safety guidelines given inside the cover of this laboratory manual. I have read the above rules and understand their meaning _ Signature _ Date
Front Matte SUMMARY OF UNIVERSAL PRECAUTIONS AND LABORATORY SAFETY PROCEDURES Universal precautions instruments after procedures.To prevent needlestick injuries.needles should not be recapped.purposely or broken by hand,rem gens,blood and body-fuid precautions inges and needles should be consistently used for all patients. theyare used.disposab scalpel blades,and other sharp items should be health-care workers shouldouus e-res ers to 4 eed o with bloodorotherbody fuids of any patient is gency mouth-to-mouth resuscitation,mouthpieces. s,mu membranes. d fo incgoedibodobodyndids.and 5.Health-care workers who have exudative lesions for performing venipuncture and other vascular or weeping dermatitis should refrain from all Gloves sh direct patient care and from handling patient-care 6.The follow cedure should be used to clean droplets of blood or other body fuids to prevent gloves and any other ary barners.(2) exposure of m usmembranes o els i edures that are likely to (3)Disinfect the area with either a commercial EPA-approved germicide or household bleach 2.Hands and other ces should be washed nte).I Shouldbediluted washed immediately after gloves are removed. 24 hours old.When dealing with large spills or 3.All health-care worke those containing sharp objects such as broken glass. prevent injuri s caused by needk st cov the spil with cedures:when cleanin used instruments:durin a1:10 bleach solution and allow it to stand forat disposal of used needles:and when handling sharp least 10 minutes.Finally clean as described above. xiii
Harley−Prescott: Laboratory Exercises in Microbiology, Fifth Edition Front Matter Summary of Universal Precautions and Laboratory Safety Procedures © The McGraw−Hill Companies, 2002 xiii SUMMARY OF UNIVERSAL PRECAUTIONS AND LABORATORY SAFETY PROCEDURES Universal Precautions Since medical history and examination cannot reliably identify all patients infected with HIV or other bloodborne pathogens, blood and body-fluid precautions should be consistently used for all patients. 1. All health-care workers should routinely use appropriate barrier precautions to prevent skin and mucous-membrane exposure when contact with blood or other body fluids of any patient is anticipated. Gloves should be worn for touching blood and body fluids, mucous membranes, or non-intact skin of all patients, for handling items or surfaces soiled with blood or body fluids, and for performing venipuncture and other vascular access procedures. Gloves should be changed after contact with each patient. Masks and protective eyewear or face shields should be worn during procedures that are likely to generate droplets of blood or other body fluids to prevent exposure of mucous membranes of the mouth, nose, and eyes. Gowns or aprons should be worn during procedures that are likely to generate splashes of blood or other body fluids. 2. Hands and other skin surfaces should be washed immediately and thoroughly if contaminated with blood or other body fluids. Hands should be washed immediately after gloves are removed. 3. All health-care workers should take precautions to prevent injuries caused by needles, scalpels, and other sharp instruments or devices during procedures; when cleaning used instruments; during disposal of used needles; and when handling sharp instruments after procedures. To prevent needlestick injuries, needles should not be recapped, purposely bent or broken by hand, removed from disposable syringes, or otherwise manipulated by hand. After they are used, disposable syringes and needles, scalpel blades, and other sharp items should be placed in puncture-resistant containers for disposal. 4. Although saliva has not been implicated in HIV transmission, to minimize the need for emergency mouth-to-mouth resuscitation, mouthpieces, resuscitation bags, or other ventilation devices should be available for use in areas in which the need for resuscitation is predictable. 5. Health-care workers who have exudative lesions or weeping dermatitis should refrain from all direct patient care and from handling patient-care equipment. 6. The following procedure should be used to clean up spills of blood or blood-containing fluids: (1) Put on gloves and any other necessary barriers. (2) Wipe up excess material with disposable towels and place the towels in a container for sterilization. (3) Disinfect the area with either a commercial EPA-approved germicide or household bleach (sodium hypochlorite). The latter should be diluted from 1:100 (smooth surfaces) to 1:10 (porous or dirty surfaces); the dilution should be no more than 24 hours old. When dealing with large spills or those containing sharp objects such as broken glass, first cover the spill with disposable toweling. Then saturate the toweling with commercial germicide or a 1:10 bleach solution and allow it to stand for at least 10 minutes. Finally clean as described above