許勝傑 INTRODUCTION TO MICROBIOLOGY MICROBIAL NUTRITION, GROWTH, AND CONTROL MICROBIAL METABOLISM 王永樑

1. 微生物學 Prescott, Harley and Klein’s "Microbiology" seventh edition (2008)Carter J and Saunders V.  Virology: Principles and Applications (2007)  許勝傑 INTRODUCTION TO MICROBIOLOGY MICROBIAL NUTRITION, GROWTH, AND CONTROL MICROBIAL METABOLISM 王永樑 MICROBIAL MOLECULAR BIOLOGY AND GENETICS DNA TECHNOLOGY AND GENOMICS 期中考 劉世東、黎欣白、趙玫 THE VIRUSES 期中考 劉世東、陳怡原、謝絹珠 THE DIVERSITY OF THE MICROBIAL WORLD 期中考 劉世東、謝絹珠 MICROBIAL DISEASES AND THEIR CONTROL FOOD AND INDUSTRIAL MICROBIOLOGY 期末考

2. What is microbiology? • study of organisms too small to be clearly seen by the unaided eye (i.e., microorganisms) • these organisms are relatively simple in their construction and lack highly differentiated cells and distinct tissues Chapter 1 The History and Scope ofMicrobiology The Importance of Microorganisms • most populous group of organisms and are found everywhere on the planet • play a major role in recycling essential elements • source of nutrients and some carry out photosynthesis • benefit society by their production of food, beverages, antibiotics and vitamins

3. Classification schemes • five kingdom scheme includes Monera, Protista, Fungi, Animalia and Plantae with microbes placed in the first three kingdoms • three domain alternative, based on a comparison of ribosomal RNA, divides microorganisms into Bacteria(true bacteria),Archaeaand Eucarya (eucaryotes) Members of the microbial world • procaryotic cells lack a true membrane-delimited nucleus • eucaryotic cells have a membrane-enclosed nucleus, are more complex morphologically and are usually larger than procaryotic cells

4. Domain Archaea – all procaryotic Domain Bacteria – all procaryotic • most are single-celled • most have peptidoglycan in cell wall • can survive broad range of environments • most are non-pathogenic and play major role in nutrient recycling • cyanobacteria produce oxygen as a result of photosynthesis • procaryotic • distinguished from Bacteria by unique ribosomal RNA sequences • lack peptidoglycan in cell wall • many found in extreme environments • no pathogenic species known

5. Viruses Domain Eucarya – all eucaryotic • animals, plants and eucaryotic microorganisms • Microorganisms include protists (unicellular algae, protozoa, slime molds and water molds) and fungi • Most are larger than procaryotic cells • acellular • smallest of all microbes • cause a range of diseases including some cancers

6. Discovery of Microorganisms • Antony van Leeuwenhoek (1632-1723) • first person to observe and describe microorganisms accurately

7. But could spontaneous generation be true for microorganisms? • John Needham (1713-1781) • his experiment: mutton broth in flasks  boiled sealed • results: broth became cloudy and contained microorganisms • Lazzaro Spallanzani (1729-1799) • his experiment: broth in flasks sealed  boiled • results: no growth of microorganisms The Conflict over Spontaneous Generation • spontaneous generation • living organisms can develop from nonliving or decomposing matter • Francesco Redi (1626-1697) • disproved spontaneous generation for large animals • showed that maggots on decaying meat came from fly eggs

8. Louis Pasteur (1822-1895) • his experiments • placed nutrient solution in flasks • created flasks with long, curved necks • boiled the solutions • left flasks exposed to air • results: no growth of microorganisms

9. Final blow to theory of spontaneous generation • John Tyndall (1820-1893) • demonstrated that dust carries microorganisms • showed that if dust was absent, nutrient broths remained sterile, even if directly exposed to air • also provided evidence for the existence of exceptionally heat-resistant forms of bacteria

10. The golden age of microbiology (1857-1914) • Many disease producing organisms discovered • Microbial metabolism studies undertaken • Microbiological techniques refined • A better understanding of the role of immunity and ways to control and prevent infection by microbes The Role of Microorganisms in Disease • was not immediately obvious • establishing connection depended on development of techniques for studying microbes • once established, led to study of host defenses - immunology

11. More evidence… • M. J. Berkeley (ca. 1845) • demonstrated that the great Potato Blight of Ireland was caused by a water mold • Heinrich de Bary (1853) • showed that smut and rust fungi caused cereal crop diseases • Louis Pasteur • showed that the pébrine disease of silkworms was caused by a protozoan Recognition of the Relationship between Microorganisms and Disease • Agostini Bassi (1773-1856) • showed that a disease of silkworms was caused by a fungus

12. Final proof… • Robert Koch (1843-1910) • established the relationship between Bacillus anthracis and anthrax • used criteria developed by his teacher Jacob Henle (1809-1895) • these criteria now known as Koch’s postulates • still used today to establish the link between a particular microorganism and a particular disease Other evidence… • Joseph Lister • provided indirect evidence that microorganisms were the causal agents of disease • developed a system of surgery designed to prevent microorganisms from entering wounds as well as methods for treating instruments and surgical dressings • his patients had fewer postoperative infections

13. Koch’s postulates • The microorganism must be present in every case of the disease but absent from healthy individuals. • The suspected microorganism must be isolated and grown in a pure culture. • The same disease must result when the isolated microorganism is inoculated into a healthy host. • The same microorganism must be isolated again from the diseased host.

14. Other developments… • Charles Chamberland (1851-1908) • developed porcelain bacterial filters used by Ivanoski and Beijerinck to study tobacco mosaic disease • determined that extracts from diseased plants had infectious agents present which were smaller than bacteria and passed through the filters • Infectious agents were eventually shown to be viruses The Development of Techniques for Studying Microbial Pathogens • Koch’s work led to discovery or development of: • agar • petri dish • nutrient broth and nutrient agar • methods for isolating microorganisms

15. Other developments… • Pasteur and Roux • discovered that incubation of cultures for long intervals between transfers caused pathogens to lose their ability to cause disease • Pasteur and his coworkers • developed vaccines for chicken cholera, anthrax, and rabies Immunological Studies • Edward Jenner (ca. 1798) • used a vaccination procedure to protect individuals from smallpox NOTE: this preceded the work establishing the role of microorganisms in disease

16. More developments… • Emil von Behring (1854-1917) and Shibasaburo Kitasato (1852-1931) • developed antitoxins for diphtheria and tetanus • evidence for humoral immunity • Elie Metchnikoff (1845-1916) • discovered bacteria-engulfing, phagocytic cells in the blood • evidence for cellular immunity

17. The Development of Industrial Microbiology and Microbial Ecology Additional Developments… • Louis Pasteur • demonstrated that alcohol fermentations and other fermentations were the result of microbial activity • developed the process of pasteurization to preserve wine during storage • Sergei Winogradsky (1856-1953) and Martinus Beijerinck (1851-1931) • studied soil microorganisms and discovered numerous interesting metabolic processes (e.g., nitrogen fixation) • pioneered the use of enrichment cultures and selective media

18. Some Important Events in the Development of Microbiology

23. Microbiology has basic and applied aspects • Basic aspects are concerned with individual groups of microbes, microbial physiology, genetics, molecular biology and taxonomy • Applied aspects are concerned with practical problems – disease, water, food and industrial microbiology The Scope and Relevance of Microbiology • importance of microorganisms • first living organisms on planet • live everywhere life is possible • more numerous than any other kind of organisms • global ecosystem depends on their activities • influence human society in many ways

24. Microbiology actually represents many fields of study • Examples • medical microbiology is concerned with diseases of humans and animals • immunology is concerned with how the immune system protects a host from pathogens • microbial ecology is concerned with the relationship of organisms with their environment • microbial genetics and molecular biology are concerned with the understanding of how genetic information functions and regulates the development of cells and organisms

25. More challenges and opportunities… • biofilms • genome analysis • microbes as model systems • assessment of implications of new discoveries and technologies The Future of Microbiology:Challenges and opportunities for future microbiologists • infectious disease • new and improved industrial processes • microbial diversity and microbial ecology • less than 1% of earth’s microbial population has been cultured

26. Home work: Give a scientist working on microbiological problems had Nobel Prize.

27. Chapter 2 The Study of Microbial Structure: Microscopy and Specimen Preparation Lenses and the Bending of Light • light is refracted (bent) when passing from one medium to another • refractive index • a measure of how greatly a substance slows the velocity of light • direction and magnitude of bending is determined by the refractive indices of the two media forming the interface

28. Lenses • focus light rays at a specific place called the focal point • distance between center of lens and focal point is the focal length • strength of lens related to focal length • short focal length more magnification

30. The Light Microscope • bright-field microscope • dark-field microscope • phase-contrast microscope • fluorescence microscope

31. The Bright-Field Microscope • produces a dark image against a brighter background • has several objective lenses • parfocal microscopes remain in focus when objectives are changed • total magnification • product of the magnifications of the ocular lenses and the objective lenses

32. Microscope Resolution • ability of a lens to separate or distinguish small objects that are close together • wavelength of light used is major factor in resolution shorter wavelength  greater resolution

33. The Dark-Field Microscope • Image is formed by light reflected or refracted by specimen • produces a bright image of the object against a dark background • used to observe living, unstained preparations • For eucaryotes has been used to observe internal structures • For procaryotes has been used to identify bacteria such as Treponema pallidum, the causative agent of syphilis

34. The Phase-Contrast Microscope • enhances the contrast between intracellular structures having slight differences in refractive index • excellent way to observe living cells • Especially useful for detecting bacterial components such as endospores and inclusion bodies that have refractive indices different from that of water

36. The Differential Interference Contrast Microscope (DIC) • creates image by detecting differences in refractive indices and thickness of different parts of specimen • excellent way to observe living cells • Live, unstained cells appear brightly colored and three-dimensional

37. The Fluorescence Microscope • exposes specimen to ultraviolet, violet, or blue light • specimens usually stained with fluorochromes • shows a bright image of the object resulting from the fluorescent light emitted by the specimen • Has applications in medical microbiology and microbial ecology studies

39. Fixation • preserves internal and external structures and fixes them in position • organisms usually killed and firmly attached to microscope slide • heat fixation – routine use with procaryotes • preserves overall morphology but not internal structures • chemical fixation – used with larger, more delicate organisms • protects fine cellular substructure and morphology Preparation and Staining of Specimens • increases visibility of specimen • accentuates specific morphological features • preserves specimens

40. Dyes and Simple Staining • dyes • Ionizable dyes have charged groups • basic dyes have positive charges • acid dyes have negative charges • chromophore groups • chemical groups with conjugated double bonds • simple stains • a single stain is used • use can determine size, shape and arrangement of bacteria

41. Gram staining Differential Staining • divides microorganisms into groups based on their staining properties • divides bacteria into two groups based on differences in cell wall structure

42. Acid-fast staining • particularly useful for staining members of the genus Mycobacterium e.g., Mycobacterium tuberculosis – causes tuberculosis e.g., Mycobacterium leprae – causes leprosy • high lipid content in cell walls is responsible for their staining characteristics

43. Staining Specific Structures • negative staining • e.g., capsule stain used to visualize capsules surrounding bacteria • capsules are colorless against a stained background • endospore staining • double staining technique • bacterial endospore is one color and vegetative cell is a different color • flagella staining • mordant applied to increase thickness of flagella

44. Electron Microscopy • beams of electrons are used to produce images • wavelength of electron beam is much shorter than light, resulting in much higher resolution

45. The Transmission Electron Microscope (TEM) • electrons scatter when they pass through thin sections of a specimen • transmitted electrons (those that do not scatter) are used to produce image • denser regions in specimen, scatter more electrons and appear darker

46. Specimen Preparation • analogous to procedures used for light microscopy • for transmission electron microscopy, specimens must be cut very thin • specimens are chemically fixed and stained with electron dense material

47. Other preparation methods • shadowing • coating specimen with a thin film of a heavy metal

48. freeze-etching • freeze specimen then fracture along lines of greatest weakness (e.g., membranes)

49. The Scanning Electron Microscope • uses electrons reflected from the surface of a specimen to create image • produces a 3-dimensional image of specimen’s surface features

50. Newer Techniques in Microscopy • confocal scanning laser (CLSM) microscopy and scanning probe microscopy • have extremely high resolution