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Microbiology and Immun (Sci)
MIMM 211
Benoit Cousineau

Introduction to microbiology (MIMM 211) Dr. Benoit Cousineau  Lyman Duff Medical Building, room 617. (514) 398 – 8929 [email protected] Lee-Hwa Tai (TA)  [email protected] th Text : Prescott, Harley and Klein’s – Microbiology 7 Edition by Willey, Sherwood and Woolverton. McGraw-Hill, New York. Computer support (1) : • Course outline o Important dates st o Midterm  Wednesday October 21 . In class. Lectures 1-15 only. • All lectures in PDF format. • Audio and visual recording of lectures by Instructional Communication Centre. Course evaluation : 1. Midterm Exam (50Q)  25% of course grade (L1-15) 2. Final Exam (150Q)  75% of course grade (L16-39) Multiple-choice questions. What is microbiology? (1) Microbial biology, (2) biology of microorganisms, (3) study of microscopic organisms. Chapter 1, the history and scope of microbiology Microbio : size of organisms studied (cannot be seen by the eye). set of techniques used to study the organisms (microscope, grow pure cultures) Microorganisms can be found everywhere in various conditions (-40 to 300 C). They are associated to humans (gut, skin). They can be useful to humans (food, biotechnology) or harmful (import historical impacts such as plague, malaria, AIDS). Study perspective : • Gross morphology; • Fine structure; • Nutrition; • Reproduction; • Physiology; • Genetics; • Classification; • Evolution; • Distribution; • Intersections with other living things and environment. Can be found in the 3 kingdoms of life : (1) bacteria, (2) archaea, (3) Eucarya. Members of the microbial world • Procaryotic or prokaryotic cell  Pro = Before. Karyon = Nucleus. No enclosed DNA. o Bacteria o Archaea • Eucaryotic or eukaryotic cell  Eu = true. o Algae o Fungi o Protozoa • Non-living microorganisms o Prokaryotic and eukaryotic viruses Microorganisms share common threads of life • Can grow (increase in size) • Metabolism o Need energy to grow (consume – transform – store) o Consume nutrients o Excrete wastes • Motion (moving itself or having internal motion) • Reproduction (create identical entities that are separate) • Respond to stimuli (measure properties of their environment and act upon certain conditions) The CELL is the basic structural unit of life. Microorganisms are usually unicellular. Historical perspectives • Lucretius and Girolamo Fracastoro suggested that invisible organisms cause disease. • 1590-1608 : Johannes Jansen develops the first microscopes. • 1665 : first description and depiction of a microorganism by Robert Hooke using a microscope : the reproductive structures of the microfungus Mucor. • 1674 : discovery of bacteria and protozoa by Anthony van Leeuwenoek. o Small hand-held microscopes (50x to 300x). o Discovered invisible creatures he called animalcules. o Found them everywhere (water, soil, teeth scrapings, excrements). o Found that animalcules are alive.  Increase in numbers and move  “Appeared” in certain materials  Spontaneous generation (abiogenesis) : spontaneous formation of living things from inanimate matters. • Aristotle, Descartes, Newton and other scientists believed in the spontaneous generation. • Origin of many organisms was thought to be spontaneous: invertebrates, rats, flies, etc. • In 1665, Francesco Redi showed that fly larvae can only develop in meat that fly can reach. • Is it different for microorganisms? Does decomposition form microorganisms or do microorganisms cause decomposition? Introduction to microbiology (MIMM 211) Course 2 Historical Perspectives • John Needham boiled mutton broth, put it in a flask and sealed it. Microorganisms appeared and he suggested that his experiments were supporting spontaneous generation. • Lazzaro Sapllanzani put broth in a flask, sealed it and boiled it. No microorganisms appeared and he claimed that he disproved spontaneous generation. • Supporters of the spontaneous generation suggested that spontaneous generation probably required air. • In 1859, the French Academy of science sponsored a competition to prove/disprove this theory. • Louis Pasteur, in 1861, boiled meat broth in a flask and curved its neck. Air could freely enter but not dust. No microorganisms grew. This experiment disproved spontaneous generation. Microbiology became an experimental science as opposed to an observational science. (Pasteur’s swan-necked flasks) • In 1877, John Tyndall supported Pasteur’s finding by demonstrating that dust carries microorganisms. o Let dust settled in a closed box containing tubes filled with broth. o Interior of the bax coated with glycerin : traps dust particles. o Air could enter but dust was trapped. No microorganisms. • Role of microorganisms in disease o Lucretius and Fracastoro suggested that invisible organisms cause disease. o People believed that diseases were caused by (1) supernatural forces, (2) poisonous vapors called miasmas, (3) imbalances between the four humors of the body (blood, phlegm, yellow bile, black bile). o Agostino Bassi first showed that a microorganism (fungus) can cause disease in silkworms. Beginning of the “germ theory of disease”. o In 1845, Berkeley showed that the great potato blight of Ireland was caused by a fungus. o Pasteur showed that silkworms were parasitized by a protozoan in the French silk industry. They then started to raise caterpillars from eggs produced by healthy moths. o In 1867, Joseph Lister heat sterilized his instruments and used phenol on surgical dressings and on patient wounds to prevent infections.  Influenced by Pasteur’s work : showed that heat and phenol could kill.  Highly successful in preventing infections. o In 1876, Robert Koch demonstrated that a bacterium caused anthrax.  Injected healthy mice with material from diseased animals. Mice became ill.  Transferred anthrax through a series of 20 mice. They all got sick.  Incubated a piece of spleen from the 20 mouse in beef broth (bacteria grew producing endospores).  Isolated endospores from the bacterial culture were injected into healthy mice (developed anthrax). o This lead to Koch’s Postulates:  The microorganism must be present in every case of the disease but absent from healthy organisms.  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. o Development of techniques to study pathogens  Robert Koch wanted to isolate suspected pathogens. • Difficult to work with liquid (mixed) cultures. • Grow bacteria on a solid surface (clonal cultures leading to colonies). • He tried on slices of boiled potatoes (bad growth). • He tried on solidified culture media (media + gelatin). o Gelatin is degraded by microorganisms. o Gelatin melts at 37, optimal growth temperature. • Fannie Hesse (Koch’s assistant’s wife) suggested agar (seaweed powder). • Richard Petri (Koch’s assistant) developed the Petri dish. • To culture and isolate human pathogens, Koch developed media similar to body fluids (meat extracts and protein digests). • Using these techniques and media Koch isolated tuberculosis, numerous other human pathogens were rapidly isolated in different laboratories. • Beginnings of immunological studies o Edward Jenner immunized people against smallpox using the cowpox virus (fluid from cowpox blisters).  He heard that dairymaids were protected from smallpox. o Louis Pasteur created the first attenuated vaccine.  Grew pure culture of pathogens (anthrax, Bacillus anthracis).  Attenuate them; 42-43 degrees with potassium bichromate.  They did not cause disease but conferred immunity. o DE Salmon and Theobald Smith found that killed microbial cells were effective as vaccines. o Emil von Behring discovered humoral immunity. Antibodies could be produced in blood against the diphtheria toxin and could neutralize the toxin and be protective against infection. o Elie Metchnikoff discovered cellular immunity: phagocytes could engulf disease-causing bacteria. o Rabies vaccine and the Pasteur Institute in Paris  Attenuated by growth in an abnormal host • H Brains and spinal cords from dead rabbits were dried. • Joseph Meister had been bitten by a rabid dog. He received 13 injections from Pasteur over a 10-day period. The boy survived. • Industrial microbiology and microbial ecology o Pasteur discovered that microorganisms are responsible for fermentation (chemical instability of sugar)  Beet sugar to alcohol produced less alcohol and sour taste.  Yeast was being replaced by lactic acid bacteria. o Sergey Winogradsky oxidated iron, sulfur and ammonia by soil bacteria to obtain energy; transformation of carbon dioxide to organic matter. o Beijerinck isolated nitrogen-fixing bacteria and sulfate-reducing bacteria. o Both Wino and Beij introduced the enrichment-culture technique and use of selective media. Classifying organisms : Phylogeny vs Taxonomy • Taxonomy o Artificial classification of organisms. o Solely based on visible similarities. o Still used to name organisms. • Phylogeny o Natural classification of organisms. o Reflects evolutionary relatedness between organisms. Phylogeny (evolutionary relationship between organisms) • Before the concept of evolution, organisms were grouped based on morphological similarities, no relationship between the groups. • Many fossils of animals and plants were found and used to suggest the appearance of different groops, built evolutionary family trees. • Microorganisms were left out until late 60’s. o No microbial fossils. o Very similar shaped. • Use ubiquitous gene sequence to compare m-o and construct a universal phylogenetic tree of life. Evolution of early schemes for classifying microbes • Tradition early schemes (before 1866) o Plants and animals • Ernest Haeckel’s proposal (1866) o Plants, animals, microorganisms • Edouard Chatton (1937) o Eucaryotes and procaryotes • Roger Stainer and CB … o • Five-kingdom scheme of Robert Whittaker (1959) o Monera, Protista, Fungi, Plantae and Animalia • Three-kingdom of Carle Woese and colleagues (1977). They used a molecular approach (16S ribosomal RNA gene was found in every organism). o Bacteria, Archaea and eucaryotes o These three groups are distant. o Length of branches depends on the distance between two species. Endosymbiotic theory of evolution • Procaryotes do not contain organelles (mitochondria, chloroplasts); • Almost all eucaryotes have organelles. o Plants have both mito and chloro; o All other eucaryotes only have mito. • Mitochondria and chloroplasts resemble prokaryotes o Approximately the same size (1*10^-6 m) o The only organelles containing DNA (active genome) o Mitochondrial and chloroplast genomes are circular. o They contain 70S ribosomes (80S in eucaryotes) o Double membranes resemble cytoplasmic and outer membranes of gram-negative bacteria. However they do not have the cell envelope. o They multiply and divide by binary fission. • Lynn Margulis proposed in 1981 the endosymbiotic theory: A primitive eukaryotic cell engulfed an ancient prokaryote to create the first eukaryotic cell. o First prokaryote appeared 3.5 billion years ago o First eukaryote appeared only 1 billion years ago o The two cells continued to evolve in a mutually beneficial symbiotic relationship for 1 billion years. o The prokaryotic cell benefited from having a sheltered environment rich in nutrients. o The eukaryotic cell benefited from containing an organism that produced energy (ATP) by respiration (mitochondria) or by photosynthesis (chloroplast). o Mitochondria are the descendants of an alpha proteobacteria (gram-negative, Rickettsia prowazekii)  RP is an obligate intracellular parasite. o Chloroplasts are the descendants of a cyanobacteria (prochloron) • More recent endosymbiosis (an evolutionary snap shot) o Eucaryotes phagocyte prokaryotic cells. o Need time to co-evolve and create a stable new organism. • Aphids and related insects: a recent endosybiotic relationship (200 million years) o The endosymbiont still has its gram-negative double membrane and cell envelope, phylogenetically closely related to E.coli o Stable endosymbiotic event  The endosymbiont can no longer grow outside its host  Aphids are also dependant of their endosymbiont, they die if the endosymbiont is killed (antibiotic) Building a phylogenetic tree • Get the sequence of an ubiquitous gene (like the 16S ribosomal RNA gene) for all the organisms to be included in the phylogenetic tree. • Align the gene sequences using a sequence alignment program. • Feed the aligned sequences to a phylogenetic algorithm. o Find which sequences share the highest level of homology and link closest neighbors. o Create phylogenetic tree, branching the organisms related to the similarity of their gene sequence. o The length depends on the evolutionary distance. Bacterial taxonomy • Science of taxonomy or systematic includes o Classification (biological class.) o Nomenclature (how to n ame organisms) o Identification (visible characteristics) • Goals, stability, predictability, build larger groups, study one member and learn about group The hierarchical scheme of classification • 18 century, Carolus Linnaeus, Linnaeus scheme of classification. • Based on the comparison of visible characteristics. • Darwin… o Kingdom or domain o Phylum or division o Class o Order o Family o Genus o Species (basic taxon) How bacteria are named. Basic taxon in bacterial taxonomy is the species. The names are binomial and are a binary combination of Latin words. They consist of a genus name and a single epithet. The genus name has an initial capital letter. Species epithet has initial lower case. Species name is always italicized. They tell us about the organism (appearance, habitat, characteristic property, scientist name). Concept of a species in bacteriology. • A collection of strains that share many stable properti
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