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Chapter 1 The History and Scope of Microbiology 1 What is microbiology? Biology is the study of living organisms Micro = very small Microbiology is the study of microbes, which are extremely small (microscopic) living organisms and certain non-living entities these organisms are relatively simple in their construction and lack highly differentiated cells and distinct tissues 2 The Importance of Microorganisms - most populous group of organisms are found everywhere on the planet and are essential for life play a major role in recycling essential elements Microorganisms are involved in the decomposition of dead organisms and waste products (fertilizers as returning inorganic nutrients to the soil, Nitrates, Phosphates etc) Saprophytes or decomposers are organisms that live on dead and/or decaying organic matter, source of nutrients and some carry out photosynthesis, Photosynthetic algae and bacteria (such as cyanobacteria) produce much of the oxygen in our atmosphere (more than the plants) The use of microbes to clean up toxic wastes and other industrial waste (oils) products is known as bioremediation 3 benefit society by their production of food, beverages, antibiotics and vitamins Algae and bacteria serve as food for tiny animals; they are important links in food chains Microbes that live in the intestinal tracts of animals aid in the digestion of food and produce beneficial substances E. coli, vit K, B1 For many years, microorganisms have been used as “cell models”; the more that scientists learned about microbial cells, the more they learned about cells in general In genetic engineering, a gene or genes from one organism is/are inserted into a bacterial or yeast cell; the cell that receives the new gene(s) is then capable of producing the gene product(s) coded for by the new gene(s) The use of living organisms or their derivatives to make or modify useful products or processes is call biotechnology 4 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 5 Living microbes are known as cellular microbes or microorganisms; examples include bacteria, archaea العتيقة, some algae, protozoa األوليات, and some fungi Non-living microbes are known as acellular microbes or infectious particles; examples include viroids, prions, and viruses Microorganisms are ubiquitous (they are found virtually everywhere) 6 Classification schemes five kingdom scheme includes Monera األصليات, Protista الطالئعيات, Fungi, Animalia and Plantae with microbes placed in the first three kingdoms Six kingdom classification (bacteria) ? viruses three domain alternative, based on a comparison of ribosomal RNA, divides microorganisms into Bacteria (true bacteria), Archaea and Eucarya (eucaryotes) The small ribosomal subunit is composed of only one rRNA molecule, which is coded for by a gene called the 16S rRNA gene in procaryotes and the 18S rRNA gene in eucaryotes 7 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 Domain Archaea – all procaryotic procaryotic distinguished from Bacteria by unique ribosomal RNA sequences lack peptidoglycan in cell wall many found in extreme environments no pathogenic species known Unusual metabolic characteristics, methanogenes 8 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 Viruses (animals and bacteria), viroids (plants), virusoids (plants, hepaitis), prions(proteins) acellular smallest of all microbes 10000 smaller than bact cause a range of diseases including some cancers 9 Discovery of Microorganisms Antony van Leeuwenhoek (16321723) first person to observe and describe microorganisms accurately 10 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 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 11 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 12 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 (endospores) 13 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 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 14 Recognition of the Relationship between Microorganisms and Disease Agostini Bassi (1773-1856) showed that a disease of silkworms was caused by a fungus 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 15 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 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 16 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. 17 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 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 18 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 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 19 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 20 The Development of Industrial Microbiology and Microbial Ecology 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 Additional Developments… 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 21 First Microorganisms on Earth • • • • Fossils of primitive microorganisms date back about 3.5 billion years ago. Candidates for the first microorganisms on Earth are archaea and cyanobacteria LUCA Infectious diseases of humans and animals have existed for as long as humans and animals have inhabited the planet. Earliest known account of pestilence occurred in Egypt in about 3180 BC. 22 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 ecology influence human society in many ways diseases, benefits, life 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 23 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 24 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 More challenges and opportunities… biofilms genome analysis microbes as model systems assessment of implications of new discoveries and technologies 25 26 Chapter 2 The Study of Microbial Structure: Microscopy and Specimen Preparation 27 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 29 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 S. aureus Copyright © 2011 Wolters Kluwer Health | Lippincott Williams & Wilkins S. aureus and red blood cells as seen by light microscopy (photomicrograph) 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 35 Darkfield Microscopy of Treponema pallidum (the bacterium that causes syphilis) 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 37 38 39 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 40 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 41 42 43 Preparation and Staining of Specimens increases visibility of specimen accentuates specific morphological features preserves specimens 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 44 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 45 Differential Staining divides microorganisms into groups based on their staining properties Gram staining divides bacteria into two groups based on differences in cell wall structure 46 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 47 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 48 Electron Microscopy beams of electrons are used to produce images wavelength of electron beam is much shorter than light, resulting in much higher resolution 49 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 50 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 51 Other preparation methods shadowing coating specimen with a thin film of a heavy metal 52 •freeze-etching freeze specimen then fracture along lines of greatest weakness (e.g., membranes) 53 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 54 Newer Techniques in Microscopy confocal scanning laser (CLSM) microscopy and scanning probe microscopy have extremely high resolution 55 Confocal Microscopy laser beam used to illuminate a variety of planes in the specimen computer compiles images created from each point to generate a 3dimensional image used extensively to observe biofilms 56 57 58 59 Scanning Probe Microscopy scanning tunneling microscope steady current (tunneling current) maintained between microscope probe and specimen up and down movement of probe as it maintains current is detected and used to create image of surface of specimen 60 Scanning Probe Microscopy atomic force microscope sharp probe moves over surface of specimen at constant distance up and down movement of probe as it maintains constant distance is detected and used to create image 61