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MICROBIOLOGY MIMM211 (Biology of Microorganisms) Lecture 2 Dr. Benoit Cousineau Department of Microbiology & Immunology McGill University Learn about cutting-edge research over lunch with cool profs September 14 - 18, 2009 11:30 AM Redpath Museum More info + schedule: www.mcgill.ca/science/ Historical perspectives (3) Spontaneous generation or abiogenesis: formation of living things from inanimate matters • Aristotle (384-322 B.C.), Descartes, Newton and numerous other scientists believed in spontaneous generation • Origin of many organisms: invertebrates, rats, flies, etc. • In 1665, Francesco Redi (1626-1697) showed that fly larvae can only develop in meat that fly can reach • Different for microorganisms? decomposition form microorganisms or microorganisms cause decomposition? 1 Historical perspectives (4) • John Needham (1713-1781) boiled mutton broth, put it in a flask and sealed it. Microorganisms appeared and he suggested that his experiments were supporting spontaneous generation • Lazzaro Spallanzani (1729-1799) put broth in a flask, sealed it and than boiled it. No microorganisms appeared and he claimed that he disproved spontaneous generation • Supporters of “spontaneous generation” suggested that maybe spontaneous generation require air Historical perspectives (5) • In 1859, the French Academy of Science sponsored a high profile competition to prove or disprove the theory of spontaneous generation • In 1861, Louis Pasteur (1822-1895) boiled meat broth in a flask and curved the neck of the flask, air could freely enter the flask but not dust, no microorganisms developed. When he tilted the broth into the neck of the flask, microorganisms appeared in broth ⇒ Disproved spontaneous generation and microbiology became an experimental science as opposed to an observational science Pasteur’s swan-necked flasks 2 Historical perspectives (6) • In 1877, John Tyndall (1820-1893) supported Pasteur’s finding by demonstrating that dust carry microorganisms - He let dust settled in a closed box (dust-free box) containing tubes to be filled with broth - The interior of the box was coated with glycerin to trap dust particles - He filled the tubes with broth and boiled it - Air could freely enter the box through curved tubes preventing dust entry and resuspension of the settled dust in the box - Like in Pasteur’s experiments, no microorganisms appeared even after long periods of time Tyndall’s dust-free box Historical perspectives (7) Role of microorganisms in disease • Lucretius (98-55 B.C.) and Girolamo Fracastoro (14781553) suggested that invisible organisms cause disease • In general, people believed that diseases were caused by - Supernatural forces - Poisonous vapors called miasmas - Imbalances between the four humors of the body: Blood, phlegm (mucus), yellow bile (choler), black bile (melancholy) 3 Historical perspectives (8) Role of microorganisms in disease (2) • Agostino Bassi (1773-1856) first showed that a microorganism (fungus) can cause disease in silkworms, beginning of the “germ theory of disease” • In 1845, M.J. Berkeley showed that the great potato blight of Ireland (1800s) was also caused by a fungus • Pasteur showed that silkworms were parasitized by a protozoan in the French silk industry, they started to raise caterpillars from eggs produced by healthy moths Historical perspectives (9) Role of microorganisms in disease (3) In 1867, Joseph Lister (1827-1912) heat sterilized his instruments and used phenol on surgical dressings and directly on patient wounds to prevent infections - He was influenced by Pasteur’s work, showed that heat and phenol can kill microorganisms - Highly successful in preventing infections during surgical interventions - Suggested the role of microorganisms in disease • Historical perspectives (10) Role of microorganisms in disease (4) • In 1876, Robert Koch (1843-1910) demonstrated that a bacterium (Bacillus anthracis) was causing anthrax - Injected healthy mice with material from diseased animals (mice became ill) - Transferred anthrax through a series of 20 mice (all got sick) - Incubated a piece of spleen from mice #20 in beef broth (bacteria grew, produced endospores) - Isolated endospores from the bacterial culture were injected into healthy mice (developed anthrax) 4 Historical perspectives (11) Koch’s postulates: relationship between a microorganism and a specific disease • 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 Historical perspectives (12) Development of techniques to study pathogens • Robert Koch wanted to isolate suspected pathogens - Difficult to work with liquid cultures (mixed cultures) - Grow bacteria on a solid surface, each colony (accumulation of millions of bacteria) would originate from a single cell, clonal cultures (pure culture) - He tried on slices of boiled potatoes (bad growth) - He tried on solidified culture media (media + gelatin) - Gelatin is degraded by microorganisms - Gelatin melts at 37°C, optimal growth temperature for human pathogens Historical perspectives (13) Development of techniques to study pathogens - Fannie Eilshemius Hesse (wife of one of Kock’s assistant) suggested agar (seaweed powder) - Richard Petri (Kock’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 Mycobacterium tuberculosis, numerous other human pathogens were rapidly isolated in different laboratories 5 Petri dish E. coli colonies on a Petri dish Historical perspectives (14) Beginnings of immunological studies • Edward Jenner (1749-1823) immunized people against smallpox using the cowpox virus (fluid from cowpox blisters) -He heard that darymaids were protected from smallpox • Louis Pasteur created the first attenuated vaccine (1880s) - Grew pure culture of pathogen (anthrax, Bacillus anthracis) - Attenuate them; 42-43°C, potassium bichromate - They did not cause disease but confer immunity (immunological protection) - Avian cholera, anthrax, rabies Historical perspectives (15) Beginnings of immunological studies (2) • D.E. Salmon and Theobald Smith found that killed microbial cells were effective as vaccines • Emil von Behring (1854-1917) discovered humoral immunity, antibodies could be produced in blood against the diphteria toxin (neutralize the toxin) and be protective against infection • Elie Metchnikoff (1845-1916) discovered cellular immunity, phagocytes (blood leukocytes) could engulf disease-causing bacteria and provide immunological protection 6 Historical perspectives (16) Beginnings of immunological studies (3) Rabies vaccine and the Pasteur Institute in Paris • Rabies vaccine: attenuated by growth in an abnormal host - Brains and spinal cords from dead rabbits were dried - Joseph Meister (9 year-old boy) that had been bitten by a rabid dog received from Pasteur 13 injections of increasingly virulent preparations over a 10 day period - The boy surprisingly survived - In gratitude for Pasteur’s development of vaccines, people from around the world contributed to the construction of the Pasteur Institute in Paris Historical perspectives (17) Industrial microbiology and microbial ecology Pasteur discovered that microorganisms are responsible for fermentation (chemical instability of sugar) - Beet sugar to alcohol, less alcohol and sour taste - Yeast was being replaced by lactic acid bacteria • Sergei N. Winogradsky (1856-1953) oxidation of iron, sulfur and ammonia by soil bacteria to obtain energy; transformation of carbon dioxyde to organic matter • Beijerinck (1851-1931) isolated nitrogen-fixing bacteria and sulfate-reducing bacteria • Beijerinck and Winogradsky introduced the enrichmentculture technique and use of selective media • Classifying organisms: Phylogeny vs Taxonomy • Taxonomy - Artificial classification of organisms - Solely based on visible similarities - Still used to name organisms • Phylogeny - Natural classification of organisms - Reflect evolutionary relatedness between organisms 7 Phylogeny evolutionary relationship between organisms • Before the concept of evolution, organisms were grouped based on morphological similarities (taxonomy), no relationships between the groups • Many fossils of animals and plants were found and used to suggest the appearance of the different groups, built evolutionary family trees • Microorganisms were left out until late 1960s - No microbial fossils - Very similar shapes • Use ubiquitous gene sequence to compare microorganisms and construct a universal phylogenetic tree of life (include microorganisms) 8