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INTRODUCTION TO MICROBIOLOGY MICROBIOLOGY • The study of organisms too small to be seen without magnification – Bacteria – Viruses – Fungi – Protozoa – Helminths (worms) – Algae – Some multicellular parasites Pioneers of Microbiology • Robert Hooke, UK (1665) – Proposed the Cell Theory – Observed cork with crude microscope – All living things are composed of cells • Spontaneous generation – Some forms of life could arise spontaneously from non-living matter • Francesco Redi, IT (1668) – Redi’s experiments first to dispprove S.G. Pioneers of Microbiology • Antoni van Leeuwenhoek, DE (1673) – First observed live microorganisms (animalcules) • Schleiden and Schwann, DE – Formulated Cell Theory: cells are the fundamental units of life and carry out all the basic functions of living things • Pasteur, FR and Tyndall, UK (1861) – Finally disproved S.G. Pioneers of Microbiology • Louis Pasteur (1822-1895), Chemist – Fermentation (1857) – Pasteurization: heat liquid enough to kill spoilage bacteria (1864) – Vaccine development – rabies – Proposed the germ theory of disease – Proposed aseptic techniques (prevent contamination by unwanted microbes) – Director of Pasteur Institute, Paris (1894) Pioneers of Microbiology • Joseph Lister, UK (1867) – Used phenol (carbolic acid) to disinfect wounds – First aseptic technique in surgery • Robert Koch, DE (1876) – Postulates – Germ theory (1876) – Identified microbes that caused anthrax (1876), tuberculosis (1882) and cholera (1883) – Developed microbiological media & streak plates for pure culture (1881) Koch’s Postulates • The specific causative agent must be found in every case of the disease. • The disease organism must be isolated from the lesions of the infected case and maintained in pure culture. • The pure culture, inoculated into a susceptible or experimental animal, should produce the symptoms of the disease. • The same bacterium should be re-isolated in pure culture from the intentionally infected animal. Branches of Microbiology • Bacteriology: study of bacteria • Mycology: study of fungi • Parasitology: study of protozoa and parasitic worms • Virology: study of viruses – Beijerinck, NE: discovered intracellular reproduction of TMV; coined the term “virus” (1899) Branches of Microbiology • Chemotherapy – Treatment of disease by using chemical means – Antibiotics produced naturally – Synthetic drugs – Paul Ehrlich (1878) – used arsenic compounds to fight disease – ‘magic bullet’ • Immunology: study of immunity – Edward Jenner, UK: developed vaccination (1798) – Metchnikoff, RU: discovered phagocytes (1884) – Paul Ehrlich, DE: theory of immunity (1890) Branches of Microbiology • Chemotherapy – Alexander Fleming, Scotland (1928) discovered penicillin – Selman Waksman, Ukraine (1944) discovered streptomycin • Problems – Toxicity of drugs => Selective toxicity – Resistance of bacteria to drugs Branches of Microbiology • Recombinant DNA Technology –Recombinant DNA –Genetic engineering/biotechnology –Microbial genetics – mechanism by which microbes inherit genes –Molecular biology – structure and function (expression) of genes –Molecular epidemiology/diagnostics MICROBES ARE INVOLVED IN • • • • • • Nutrient production & energy flow Decomposition (bioremediation) Production of foods Production of drugs & vaccines Genetic engineering Causing disease 12 MICROORGANISM CLASSIFICATION • Microorganisms and all other living organisms are classified as prokaryotes or eukaryotes. • Prokaryotes are probably the smallest living organisms, ranging in size from 0.15 um (mycoplasmas) to about 2.0 um (many of the bacteria). • Viruses and subparticles such as prions are considered neither prokaryotes nor eukaryotes because they lack the characteristics of living things, except the ability to replicate. Prokaryotes • • • • • • Cell Wall Teichoic Acids LPS Endospores Circular DNA Plasmids Classification Schemes • Traditionally these have been inferred on the basis of morphologic or biochemical characteristics. • Schemes have recently been revised based on the degree of genetic (DNA, RNA) similarity between different species. • Genus and species are of primary importance in designating a microorganism. • The correct format for naming an organism is genus (capitalized, italicized, or underlined), species (lowercase, italicized, or underlined): Escherichia coli (abbreviation, E. coli). Size of Bacteria • • • • • • Average bacterial cell diameter is 0.5 - 2.0 um. Surface Area ~12 square um Volume is ~4 cubic um Surface Area to Volume is 3:1 Typical Eukaryote Cell SA/ Vol. is 0.3:1 Food enters through SA, quickly reaches all parts of bacteria • Eukaryotes need structures & organelles Shapes of Bacteria • Spherical (Cocci) – Chain = Streptococcus – Cluster = Staphylococcus • Rod Shape (Bacilli) – Chain = Streptobacillus • • • • • • Coccobacilli Comma shape (Vibrios) Spirillum Spirochete Square Star Bacterial Structures • • • • • • • • Flagella Pili Capsule Cell Wall - Lipopolysaccharides - Teichoic Acids Plasma Membrane Cytoplasm - Genetic materials - Ribosomes Inclusions Spores Extracellular Polymeric Substance (EPS) • Polysaccharide on external surface: Capsule, Glycocalyx, or Slime (Antigen) • EPS does not take ordinary stains, is not necessary for survival of the cell, and may be lost upon continuous cultivation • Adherence of bacteria to surfaces (S. mutans and enamel of teeth) • Prevention of Phagocytosis (Complement cannot penetrate sugars) The Cell Wall • The cell wall of bacteria is a complex, semi-rigid structure that is made up of peptidoglycan (mucopeptide or murein), responsible for the shape of the cell. • It differs between gram positive and gram negative bacteria • In most gram-positive bacteria the cell wall consists of many layers of peptidoglycan forming a thick rigid structure. • By contrast, gram-negative cell walls contain only one (or very few) layers of peptidoglycan. Cell Wall • Peptidoglycan Polymer (amino acids + sugars) • Unique to bacteria • Sugars - N- acetylglucosamine (NAG) - N- acetylmuramic acid (NAM) • D form of Amino acids used not L form – Hard to break down D form • Amino acids cross link NAG & NAM Cell Wall • Gram positive Bacteria - Peptidoglycan - Teichoic ( ribitol or glycerol residues) and Teichuronic (Sugar acids) acids; wall and membrane Teichoic acids supply cell with magnesium. - Polysaccharide • Gram negative Bacteria - Peptidoglycan - Lipoprotein - Outer membrane - Periplasmic space The Outer Membrane • Gram-negative cells possess an outer membrane that is composed of lipoproteins, lipopolysaccharides, and phospholipids. • The outer membrane helps some organisms evade phagocytosis, provides a barrier to certain antibiotics, and confers properties of virulence (endotoxin). Lipopolysaccharide (LPS) • Endotoxin or Pyrogen – Fever causing – Toxin nomenclature • Endo - part of bacteria • Exo - excreted into environment • Structure – Lipid A – Polysaccharide • O Antigen • Gram negative bacteria only – Removed by Alcohol/Acetone LPS (cont’d.) • Appearance of Colonies – Mucoid = Smooth (lots of LPS or capsule) – Dry = Rough (little LPS or capsule) • O Antigen of Salmonella and E. coli – 2,000 different O Ags of Salmonella – 100’s different O Ags of E. coli • E. coli O157 • O Ags differ in Sugars, not Lipid A LPS (cont’d) • Functions – Toxic; kills mice, pigs, humans • G - ve septicemia; death due to LPS – Pyrogen; causes fever • DPT vaccination always causes fevers – Adjuvant; stimulates immunity • Heat Resistant; hard to remove • Detection (all topical & IV products) – Rabbits (measure fever) – Horse shoe crab (Amoebocytes Lyse in presence of LPS) Cell Wall Summary • Unique to bacteria • 20-40% of bacterial cell weight • Determines shape of bacteria • Prevents osmotic rupture • Target for some antibiotics (Penicillin) Cell Membrane • The plasma membrane encloses the cytoplasm of the cell and provides selective permeability for nutrients to enter. • Phospholipid Bilayer • Water can penetrate • Flexible • Not strong, ruptures easily – Osmotic Pressure created by cytoplasm Cytoplasmic Structures • 80% Water {20% Salts-Proteins) • DNA is a single long circular molecule of doublestranded DNA “bacterial chromosome”. – More efficient; grows quicker – Mutations allow adaptation to environment quicker • Plasmids; small circular transferable, doublestranded DNA molecules – Antibiotic Resistance • Bacteria also contain transposons • Ribosomes function as the site of protein synthesis. • No organelles (Mitochondria, Golgi, etc.) Appendages of Bacteria • Some bacteria have flagella which are long filamentous appendages that can propel the cell. • Many gram-negative bacteria possess hair-like appendages that are used for attachment rather than for motility. These are divided into two types, fimbriae and pili. • Fimbriae enable a bacterial cell to adhere to surfaces (including other cells) while pili join bacterial cells in preparation for the transfer of DNA from one cell to another. Flagella • Motility - movement • Swarming occurs with some bacteria – Spread across Petri Dish – Proteus species most evident • Arrangement basis for classification – Monotrichous; 1 flagella – Lophotrichous; tuft at one end – Amphitrichous; both ends – Peritrichous; all around bacteria Mono- or Lophotrichorus Pilli • Short protein appendages – smaller than flagella • Adherence of bacteria to surfaces – E. coli has numerous types • K88, K99, F41, etc. – Antibodies to will block adherence • F- Pilus; used in conjugation – Exchange of genetic information F- Pilus for Conjugation Endospores • When essential nutrients are depleted, certain gram positive bacteria (e.g., Clostridium and Bacillus), form “resting” cells called endospores. • These endospores contain condensed nuclear material and protein and can survive extreme heat, lack of water, and exposure to toxic chemicals. • When growth conditions permit, the cell will germinate into a dividing bacterium. Endospores • Resistant structure – Heat, irradiation, cold – Boiling >1 hr • Takes time and energy to make spores • Location important in classification – Central, Subterminal, Terminal • Bacillus stearothermophilus -spores – Used for quality control of heat sterilization equipment • Bacillus anthracis - spores – Used in biological warfare