LABORATORY WORKBOOK IN GENERAL MICROBIOLOGY Student name ________________________________ Faculty _____________________________________ Group ___________ Minsk BSMU 2017 2 МИНИСТЕРСТВО ЗДРАВООХРАНЕНИЯ РЕСПУБЛИКИ БЕЛАРУСЬ БЕЛОРУССКИЙ ГОСУДАРСТВЕННЫЙ МЕДИЦИНСКИЙ УНИВЕРСИТЕТ КАФЕДРА МИКРОБИОЛОГИИ, ВИРУСОЛОГИИ, ИММУНОЛОГИИ ЛАБОРАТОРНЫЙ ПРАКТИКУМ ПО ОБЩЕЙ МИКРОБИОЛОГИИ LABORATORY WORKBOOK IN GENERAL MICROBIOLOGY 6-е издание Минск БГМУ 2017 C l a s s 1. Methods in diagnostic microbiology. Microscopic method of examination. Basic morphological forms of bacteria. Simple methods of staining. 3 УДК 576.8(811.111)-054.6(076.5)(075.8) ББК 52.64 (81.2 Англ-923) Л12 Рекомендовано Научно-методическим советом университета в качестве лабораторного практикума 01.12.2016 г., протокол № 3 А в т о р ы: доц. В. В. Слизень; доц. Е. Ю. Кирильчик; доц. Ж. Г. Шабан; доц. Д. А. Черношей; доц. Т. А. Канашкова Р е ц е н з е н т ы: канд. мед. наук, доц. В. Э. Бутвиловский; канд. мед. наук, доц. А. М. Близнюк Лабораторный практикум по общей микробиологии = Laboratory workbook in gen- Л12 eral microbiology : лаб. практикум / В. В. Слизень [и др.]. – 6-е изд. – Минск : БГМУ, 2017. – 82 с. ISBN 978-985-567-636-3. Содержит информацию для подготовки к практическим занятиям по разделу «Общая микробиоло- гия». Приведены схемы, алгоритмы, справочные сведения, методики выполнения лабораторных работ. Первое издание вышло в 2010 году. Предназначен для студентов 2-го курса медицинского факультета иностранных учащихся, обучаю- щихся на английском языке. УДК 576.8(811.111)-054.6(076.5)(075.8) ББК 52.64 (81.2 Англ-923) ISBN 978-985-567-636-3 © УО «Белорусский государственный медицинский университет, 2017 C l a s s 1. Methods in diagnostic microbiology. Microscopic method of examination. Basic morphological forms of bacteria. Simple methods of staining. 4 CONTENT P r a c t i c a l c l a s s № 1. Methods in diagnostic microbiology. Microscopic method of examination. Basic morphological forms of bacteria. Simple methods of staining ........................ 4 P r a c t i c a l c l a s s № 2. Microscopic method of examination. The morphology and fine structure of bacteria. Differential methods of staining ............................................................ 14 P r a c t i c a l c l a s s № 3. The topic: Microscopic method of examination. The morphology and fine structure of bacteria. Differential methods of staining ..................................................... 25 P r a c t i c a l c l a s s № 4. Microscopic method of examination. The morphology of the spirochetes, actinomyces, rickettsiae, chlamydiae, mycoplasmas. ............................................ 31 P r a c t i c a l c l a s s b № 5. Antimicrobial measures. Methods of sterilization and disinfection. Asepsis and antisepsis. Bacteriological method of laboratory diagnosis of infection diseases. Techniques of microbial pure cultures isolation and maintenance. ........................................... 36 P r a c t i c a l c l a s s № 6. Bacteriological method of laboratory diagnosis of infection diseases. Techniques for pure culture identification. ........................................................................... 53 P r a c t i c a l c l a s s № 7. Molecular Basis of Bacterial Genetics. Molecular methods of infection diseases diagnosis and bacterial genetic investigations ............................................. 59 P r a t i c a l c l a s s № 8. Ecology of microorganisms. Methods of human normal flora investigation. Infections. ................................................................................................... 61 P r a c t i c a l c l a s s № 9. Antibiotic susceptibility testing of microorganisms. application of laboratory animals in microbiology. ................................................................................... 69 P r a c t i c a l c l a s s №10. Credit 1 ―Morphology and physiology of microorganisms. Infection.‖ ...................................................................................................................... 80 C l a s s 1. Methods in diagnostic microbiology. Microscopic method of examination. Basic morphological forms of bacteria. Simple methods of staining. P r a c t i c a l c l a s s № 1. Methods in diagnostic microbiology. Microscopic method of ex- amination. Basic morphological forms of bacteria. Simple methods of staining Suggested reading for self-study: History of the department microbiology, virology, immunology; main spheres of activity and trends in research. Design and equipment of microbiological laboratory, biosafety levels. Basic rules of work in microbiological laboratory (biosafety in work with class II biohazards). Uni- versal precautions in work with burners and electric supplies. Taxonomy of microorganisms: classification and nomenclature. Modern approaches to taxonomy of micro- organisms. Taxonomic ranks. Vars (types), strains, clones, pure cultures. Basic morphological forms of bacteria. Morphological characteristics of cocci, rods and spiral-shaped bacte- ria. Microscopic method of examination: tasks, procedure, evaluation of the method. Bright-field light micro- scope: components and proper use of the microscope. Smear preparation and fixation. Simple methods of staining. The technique of oil immersion microscopy. _______________________________________________________________________________________ UNIVERSAL PRECAUTIONS AND LABORATORY SAFETY PROCEDURES General regulations 1. Restrict or limit access to the laboratory when working. 2. Eating, drinking and smoking are forbidden at all times in the laboratory. 3. Use protective clothing (lab coat) obligatory during all work activities to protect clothing from con- tamination or accidental discoloration by staining solutions. 4. If necessary use personal protective equipment (gloves, face protection, eye protection). 5. Do not place anything in your mouth while in the laboratory. This includes pencils, food, and fin- gers. Learn to keep your hands away from your mouth and eyes. 6. Place all extra clothing, unnecessary books, purses, backpacks in an appropriate place. The laborato- ry work area must be kept free from unnecessary things. 7. Decontaminate work surfaces daily. 8. Maintain insect and rodent control program. Precautions during work 1. Work according to plan and fill in all the required reports in laboratory manual and have them signed by the tutor. 2. Mechanical pipetting devices should be used for manipulating all liquids in the laboratory. Mouth pipetting must not be done. 3. Flame wire loops and needles before and immediately after transfer of cultures. Do not move through the laboratory with a loop or pipette containing infectious material. 4. Be careful with the burner flame. 5. Use of needles and syringes should be limited to situations in which there is no alternative, and the recommendations for preventing injuries with needles should be followed. 6. Minimize splashes and aerosols during laboratory procedures. 7. Avoid contamination of benches, floor, and wastebaskets during experiment. 8. Blood and other body fluids from all patients should be considered infective. All persons processing blood and body-fluid specimens should wear gloves. Gloves should be changed and hands washed after completion of specimen processing. 9. Slides and coverslips are glass. Do not cut yourself when using them. Dispose of any broken glass in the appropriately labeled container. Precautions after work 1. Label all experimental material with: a) your name; b) date; c) specimen; d) microorganism name. 2. Return all reagents, cultures, and glassware to their appropriate places after experiment. 3. Contaminated materials used in laboratory tests should be decontaminated before reprocessing. Spe- cial receptacles will be provided for infectious materials and used glass slides. Place all discarded cultures and contaminated glassware into these receptacles. Tall jars filled with a germicide will be provided for pi- pettes. 4. After completing laboratory activities wash your hands thoroughly, using disinfecting soap and re- move your protective clothes. 6 Precautions after accidents 1. Laboratory work surfaces should be decontaminated with an appropriate chemical germicide after a spill of blood, other body fluids, microbial cultures 2. The following procedure should be used to clean up spills of blood or blood-containing fluids: i. put on gloves and any other necessary barriers. ii. wipe up excess material with disposable towels and place the towels in a container for sterilization. iii. disinfect the area with either a commercial germicide or household bleach (sodium hypochlorite). 3. When infectious material is accidentally spilled, cover it immediately with a disinfectant and notify your instructor at once. 4. When infectious material is accidentally spilled on skin, wash it with antisepticat first and then with soap thoroughly and notify your instructor at once. _______________________________________________________________________________________ Biosafety - combinations of laboratory procedures, laboratory facilities and safety equip- ment that protect workers, ―products‖, co-workers, lab support personnel, environment when work- ing with potentially infectious microorganisms (tab. 1.1). A biosafety level (BSL) - is the level of the biocontainment precautions required to isolate dangerous biological agents in an enclosed facility. As of 2006, there are four safety levels. Higher numbers indicate a greater risk to the external environment. A biological