Introduction to Microbiology Revision Notes PDF

Introduction to Microbiology MICROBES Microbiology: study of microbes (an organism so small, it cannot be seen with the naked eye) - Bacteria (bacteriology) - Viruses (virology) - Fungi (mycology) - Protozoa (parasitology) Most infections dise...

Introduction to Microbiology MICROBES Microbiology: study of microbes (an organism so small, it cannot be seen with the naked eye) - Bacteria (bacteriology) - Viruses (virology) - Fungi (mycology) - Protozoa (parasitology) Most infections diseases are caused by microbes - 15 million deaths per year (26% total global deaths) pathology antibiotic rests cute Which enhance opportunistic adaptable dynamic , & , , , Importance of Microbes x[ - Disease - pneumonia, influenza, diarrhoeal diseases, AIDS, TB, malaria, measles, syphilis, hepatitis, tropical diseases - Environmental – treatment of wastewater, oil spills, landfill I - Industry ~ Food: brewing, baking, dairy Medicine: antibiotics, amino acids, insulin, HGH - Agriculture – enhance soil fertility and plant growth, combat pests and disease Why Study Microbiology? Biomedical Science - Infectious disease – treatment, management & prevention - Bacteria as laboratory / industrial tools – brewing, baking, antibiotics, proteins Forensic Science - Bioterrorism & biocrimes - Microbes as a tool in forensic investigation  6 Subgroup of Microbes 1. Bacteria 2. Archaea vabpaf 3. Algae 4. Fungi 5. Protozoa > - parasites 6. Viruses Universal Tree of Life need to know * only there are 3 domains * pro pro prokaryotic Koch’s Postulates Crules A particular micro-organism causes a particular disease * not all pathogens 1. Causative micro-organism must be present in every case of disease, but absent from follow these healthy organisms Miles 2. Suspected micro-organism must be isolated & grown in pure culture ↓ some can't 3. Cultured micro-organism should cause disease when introduced into a healthy host be cultured 4. Same micro-organism must be isolated again from diseased host *General guidelines only - Some infectious agents are clearly responsible for disease even though they do not fulfil all of the postulates e.g. cholera, polio, HIV, hepatitis C, herpes simplex  Microbiology Today to disease a not human side effects least amount of - Chemotherapy: drugs/antibiotics must be selectively toxic > - induced immunity - Immunology: vaccine development to prevent infectious diseases -artificially - Basic Biology: starting point for understanding the fundamental properties of organisms (e.g. metabolism & genetics) - Genetic Engineering & Genomics: synthesis of human proteins, delivery systems for gene therapy BASIC METHODS IN MICROBIOLOGY Types of Microscopy Size of Micro-organisms Schemantics esurface (scanning) (transmission) micrometre xm = ↓ transmits through & see inside  Electron Microscope Greater resolving power than light microscopes - Beam of electrons, instead of light - Electromagnetic lens, instead of glass - Heavy metals (absorb electrons), instead of dyes - Samples held in a vacuum (dead cells only) Transmission (TEM) – viewing internal ultra-structures Scanning (SEM) – viewing microbial surfaces Staining Bacteria are almost transparent under a microscope - Simple stains: make bacteria visible (generally a simple basic dye) waxy cell wall (capsule ↑ to differentiate - - DiHerential stains: gram strain, Acid Fast stain (mycobacteria (w/ colours) between stained red) -Carbolfuschin ↓Derulous - waxy layer resists decolarisation h cannot Gram stain 2 different 'bugs' In - Special stains: stain specific structures (flagella, endospores) fail to stronger identify requires · capsule Gram Staining geta Developed by Hand Christian Gram in 1884 - Di]erentiates bacterial species into 2 groups - Gram positive = purple à thick cell wall outer membrane - Gram negative = pink à thin cell wall - > ontop of this cell wall *Based on chemical & physical properties of a bacteria’s cell wall * not all bacteria mycoplasma A capsule & flagella = can be gram ↳ not all have cell wall stained virulence factor or have waxy surface ↓ vective virulence enhances ↓ gene pathology tuberculosis all purple ↓ ↓ allows causes cell wall gram-i to shrink & trap alc breaks outer. ↳ crystal violet Stain membrane- > washes & stains inner cell wall away crystal violet to gram +: does nothing cell wall -> stays violet Culturing Micro-Organisms Necessary to isolate single pure cultures (colony of bacteria arising from a single cell) from mixed cultures) Culture Media Di]erent microbes have vastly di]erent environmental & nutritional needs - Fastidious (‘fussy’) bacteria need specific organic ingredients to grow - Source of ingredients may be known (eg. blood) but exact nutritional requirements are undefined ↳ Media can be liquid (broth) or solid (contain sugar) - Solid media – ideal for isolating pure cultures - Liquid media – ideal for culturing large quantities *Suitable environment also necessary (temperature, pH, [oxygen]) Types of media: 1. Defined synthetic media: exact pure chemical with exact composition known 2. Complex media Extracts from natural materials (eg. beef, blood, casein, soybeans) Precise chemical composition unknown BASAL MEDIA - Nutrient broth: 3g beef extract, 5g peptone (hydrolysed casein), 8g NaCl in 1L water - Nutrient agar: as above but solidified with 1.6% agar Media Types – Special Culture Media Enriched media > - addition of additional solutes on top of basal media - Contains all the necessary ingredients to grow better a desired bacteria but also a multitude of bacterial species (e.g. Tryptic Soy agar & Nutrient agar; blood agar; chocolate agar) DiHerential media - Includes ingredients, such as chemical indicators, that produce observable di]erences between species of bacteria (e.g. Blood agar) ↓ differentiate & red color ↑ gone > - - bita naemolysis : bacteria destroy : RBCs > - apha" completely : partially breakdown > gamma - : no destruction Selective media - Contains ingredients that inhibit growth of some bacteria, but enhance growth of target organism - Medium is often formulated so that it is both selective & di]erential (e.g. MacConkey agar & Mannitol Salt agar) ↑ I -explode listen to rosmotic gradient recording Destroy Staf most bacteria can survive in salt Selected ina eat sugar (mannitol ↑ acidity when eat sugar ↓ activate pH indicator ↓ yellow Bacteria STRUCTURE & FUNCTION Prokaryotes huce no ↑ - An organism whose DNA is not enclosed within a nuclear membrane - Also lack other membrane-bound organelles > - bacteria cell size = smaller than excarya eg. mitochondria, golgi apparatus, endoplasmic reticulum - Bacteria & archaea are prokaryotes - Algae, fungi, protozoa, animals & plants are eukaryotes Common Features of All Cells - Enclosed by a lipid bilayer - Require ATP for energy - Cell “blueprints” stored as genes/DNA read to prod amino acids Constant genetic code - , - ↓ proteins - Cell structure & function controlled by proteins - DNA ® RNA ® proteins - Proteins synthesized by ribosomes General Prokaryotic Cell Structure burst systemic eprevent cause ebacteria > can atoxin - inflammation (pressure from cytoplasm) > - antibiotic which disrupts sellpiccan ↳ metabolic reactions like eukaryotes & resp for cell · shape in eucarya unlike 2 prokaryotes ↳ DNA attached to plasma memb. > - not bound - floating in cytoplasm > - conjugation : horizontal genetic > - elongated = fimbria transfer  Comparison of Bacteria & Eucarya unicellular don't use antibiotic awa whichtargetcel component of micoplasma peptidoglycan ↳ no cell wall introns (sequenceofGenomena smaller which cannot be & proteins call coding) translated into ↳sinall I mRNA can have I gene = ImRNA are only exons into from multiple = I protein proteins genel into (coding ~ smaller & lighter bound to EM Roug floating. some ↳ antibiotic Amore know about diff. - more target & exploit diff wout harming us stair Gram can't ↑ Bacterial Envelopes thickwayser a pin component pidiy sacid-fast cera stain > - tuberculosis Outer Membrane Lipid bilayer om - Inner = phospholipids - Outer = mostly lipopolysaccharide (LPS)  * penicillin targets cell wall d facing penicillin , when Gram- LPS found only in Gram-negative bacteria has natural - LPS allows only water & gases to pass through membrane resistance All other molecules must pass through (porin) pores because - LPS is an endotoxin (toxin within cell wall of bacteria) in humans Outer membrane provided protection - Gram-negative bacteria tend to be more resistant to toxins tenhances disease factor evirulent Capsule ↓ - Most bacteria secrete slimy substances that become the outermost layer of the attachment & cell organised protection from organised a cles immune system Referred to as a capsule, slime layer or glycocalyx (sugar Cup) ↓ be capsule can than bigger > - huge capsule macrophage ↓ I can't eat it - Highly variable structures dependent on the species:.. In thickness, rigidity & composition Function: - Protection from drying out I - Protection from phagocytosis, cell lysis - Protection from oxygen (anaerobe bacteria) - O Adherence virulence not on External Structures bind to host evivulance factor -enhance pathology Vf - Pili: hairlike appendages used for attachment Mainly common in Gram-negative bacteria Vf - Flagella: corkscrew-shaped projection(s) Rotate & propel the cell Important for chemotaxis · run > - Votate anticlockwise · tumble - "clockwise  - Chemotaxis: movement of bacteria according to a chemical stimulus From a low-to-high nutrient/favourable environment Away from a toxin or repellent also: aero-, photo-, magnetotaxis ↓ suspended animation more to a for centuriesa a environment a response to bad a canStayaSpore Endospores surround we multiple layers a & reduction of size a germination some bacteria - A resting structure formed inside some bacteria - - Resistant to unfavourable environmental conditions: Extreme heat, dehydration, toxic chemicals, radiation L Toughest biological structures to destroy - Commonly found in soil & water - Tend to be Gram-positive - Human pathogens - Bacillus anthracis (anthrax) - Clostridium (botulism, tetanus, gas gangrene) Archaea - Single-celled organisms lacking nuclear membrane, thus prokaryotes - Re-classified in 1977 as a separate domain Based on separation from other prokaryotes on 16S rRNA phylogenetic trees - Similar to bacteria in most aspects of cell structure & metabolism - However, archaeal genetic transcription & translation are more similar to those of eukaryotes than bacteria - Display unique features in their lipid composition ↓ Unseen in bacteria & eukaryotes Ether  env. love extreme a Many are Extremophiles - Thermophiles – live in high temperature environments - Methanogens – live in anaerobic environments, produce methane 204 - Halophiles – live in extreme salt environments Each group has unique biochemical features which can be exploited for use in biotech industries. - Extreme molecular stability of thermophile enzymes - Novel C1 pathways (methanogens) - Synthesis of organic polymers (halophiles) Comparison of Bacteria, Archaea & Eucarya CLASSIFICATION 3 Domain System Taxonomy - Science of naming & classifying organisms Classification of organisms in an ordered system that indicates natural relationships - Modern biological classification derived from the scheme devised by Carolus Linnaeus (1735) + Scientific Nomenclature Each organism designated 2 names (binomial nomenclature) - 1st = genus; 2nd = species - Printed in italics, handwritten underlined e.g. Escherichia coli; Staphylococcus aureus; Salmonella typhi; After a term is used once the genus can be replaced by its initial - E.coli, S.typhi, etc. - Variants of species may be names such as: E.coli O157 *Variant: Genetic variant or subtype of the same species - Same species of bacteria isolated from a dieerent location or at a dieerent time - Altered by mutation or genetic exchange - antibodies not used to Concept of Species Related organisms that can freely interbreed - Share a common gene pool - New species appear when some members of an existing species change or become geographically isolated So they can no longer breed with the rest of the group This definition does not apply to prokaryotes à prokaryotes are asexual - Sexual exchange of genes is not an essential part of prokaryotic life cycle - Genetic exchange of genes amongst bacteria is sporadic & can occur between distantly related organisms - *A *Prokaryotic ‘species’ are therefore defined only by the similarities of its members & not by capacity to interbreed  Microbial Classification Traditional Characteristics - Morphology (individual shape, group arrangement, Gram-stain) - Biochemical Creations 'bugs' can do - Physiological - Serological (use of antibodies to identify 'bugs') Modern Characteristics technology - Comparing genetic material (DNA and/or RNA) Shape & Arrangement Physiological & Biochemical Classification Physiological - Aerobic, anaerobic or both? - Temperature range, pH, osmotic strength Biochemical I - Carbon source Able to utilise specific carbon sources to support growth fermentation of glucose, lactose, mannitol, etc. - Enzyme Activity menemon Enzyme Activity Indole Test - Tests the presence of Tryptophanase Separate E.coli (positive) from Klebsiella- Enterobacter-Hafnia-Serratia (mostly negative) Coagulase Test coagulase - Tests the ability to clot plasma Separate S.aureus, from other Staphylococcus spp. Catalase Test - Ability to decompose hydrogen peroxide to water & O2 Found in most aerobes & facultative anaerobes (main exception is Streptococcus spp.), absent in anaerobes Serology Study of serum (non-cellular fraction of blood) - Serum contains antibodies, which are highly specific, targeting specific microbes (even strains of same species) - Sera that inactive particular bacterium is called antisera - Antiserum against capsular polysaccharide Antisera is used in slide agglutination test & fluorescent antibodies Comparing Genomes All the genetic material (DNA & RNA) of an organism - Key to modern taxonomy is the organism’s genome - Percent G + C - DNA hybridization - DNA sequencing Ribosomal RNA genes 2 · · Protein encoding genes o small difference in Sequence * protozoa , fungi ↳ diff. between bacteria  Viruses Origin of Viruses Viruses are too simple to have evolved first - Rely on host cells, which must have pre-existed Current theory: “Genes on the loose” - Evolving from genes in the cells they now infect Degenerated or escaped 75% of human viruses believed to have been “passed” from animals à new viruses (eg. HIV & SARS) spread quickly in modern human populations - Due to rapid travel & hospital environments (amplification of infection), proximity to mass producing animals (+/- with poor hygienic conditions) Are Viruses Alive? extremely * dependent Possess genes, they replicate, they evolve & are adapted to particular hosts & habitats on host - However, viruses cannot capture & store free energy entities - They are not functionally active outside their host cells intracellular obligate Viruses must make use of the host cellular machinery to reproduce and survive (ie. obligate intracellular parasites) - A virus becomes part of a living system only after it has infected a host cell & its genome becomes integrated with that of the cell - Replicate only through the metabolic activities of infected cells Therefore viruses are classified as non-living infectious entities  viruses Viruses animal ↑ Can infect prokaryotes & eukaryotes - Prokaryote virus = bacteriophage especifically range host -little Most viruses infect only specific types of cells in one host (called specific viruses); if not specific, called generalist viruses > - diverse - Host range is determined by specific host attachment sites and cellular factors - Recognise cell membrane proteins (receptors), which have an alternate function in “normal” cell physiology -protein Genome protect M Viruses contain either RNA or DNA enclosed in a protein coat (capsid) - Double or single stranded nucleic acid viruses > - can have double & stranded DNA single - Some contain additional lipoprotein envelopes & a single compliment immuneMeit , double RNA stranded syst ↑ weakness Viral Structures - veloped Agentkohee envirul as i immune syst. A naked virus : genome surrounded by only capsid ⑳ d out cylindrical scherical classify based on how shaped Multiplication of Bacteriophages a regulated/sequential assembly step A know how assemble : can intervene w/ medication > - babrus a conjugation & spread ↓ can provide antibiotic resistant genes Inansduction 1. Attachment - Phage attaches by tail fibers to host cell 2. Penetration - Phage lysozyme opens cell wall (via Lysozyme), tail sheath contracts to force tail core & DNA into cell 3. Biosynthesis > - DNA rep. - Production of phage DNA & proteins 4. Maturation - Assembly of phage particles 5. Release - Phage lysozyme breaks cell wall  Alternate Bacteriophage Cycles (Lytic vs Lysogenic) ↓ Clytic bacteriophages: lysogenic : DNA conditions are poor (no either destroy bacteria or leave it spare food) - not multiply ↓ bacteria" still multiply tho if kill bacteria - kill comebacktooa of self ↓ bac. infect a combine w/ bacterial DNA (latent ↓. & bacteria when gets food will viral DNA integrated w/ chromosomal bac. multiply with virus DNA DNA virus multiplies t canput u ↓ bacteria daughter cells have incorporated ↓ DNA circularizes > - bacteria a has exonuclease > - immune response viral DNA inside chromosomes productive / ↳ exonuclease can't disrupt viral DNA bursts open Animal Virus Cycle 1. Attachment - Viruses attach to cell membrane released 2. Penetration enzymes - By fusion, endocytosis or adsorption evival 3. Uncoating > takes of - capsid inside cell > - release genome inside cell - By viral or host enzymes 4. Biosynthesis DNA - Production of nucleic acid & proteins or IRNA - Mechanism depends of type of nucleic acid present 5. Maturation - Nucleic acid & capsid proteins assemble 6. Release - By budding (enveloped viruses) or rupture  of genomola apnylogeneticusesequence Viral Taxonomy - Order: name end in -virales - Family: name end in -viridae - Genus: name end in -virus - Species: group of viruses sharing same genetic information & host Common names are used for species - Subspecies: are designated by a number Taxonomy based on 1. Nucleic acid type, 2. Replication strategy, 3. Morphology Molecular & Genetic Composition of Viral Families a ↓ > - equivalent to mRNA has to to nucleus go some uncoat in cytoplasm or nucleus ↓ one tiny ↓ can go through wh pore /translatiaim nuclear capsid transcription o bacterioCytol in phage- uncoat in l nicer  it of cell rules in follow s replicating Viral Gene Expression coali aquich ·ower Gets more complicated if the virus has minus-strand RNA & when virus needs to replicate RNA to package into new virions RNA Virus Reproductive Strategies 1. why strand is template to form new pos strand. We neg. to form pos template 2. to form neg etemp. of new enzyme in hybrid form integraceinto chromosomal RNA when edestroy a integrate nucleus einside - > - like lyogenic hybrid Rules: 1. +RNA is required to make proteins 2. a viral genome need to be replicated to end up with the same initial genome (e.g. –ssRNA after replication ends up as also –ssRNA) 3. if you generate a ssRNA, this must be generated from a dsRNA 4. forming a ssRNA requires an enzyme called transcriptase merase 5. forming a dsRNA requires an enzyme called replicase N ENOHymerase ↓ to form double VIRAL DIESEASE Common Viral Diseases HIV Structure - Retrovirus; 2 single-strands of RNA; contains 9 genes a assembly of virus Including reverse transcriptase, integrase, protease - Recognises CD4, CCR5 & CXCR4 receptors on immune cells ↓ ↓ CD4+ T-cells, macrophages, dendritic cells core of to immunity adaptive immunity *Simple genome à proteins: less sites for potential drug targets Transmission 3 major routes od transmission 1. Sexual transmission: unprotected 2. Exposure to contaminated blood: IV drug use, blood transfusion 3. Transmission from infected mother to foetus or suckling infant Infection - RNA into cell - Reverse transcription to proviral DNA - Integration (integrase) into host genome - Host production of virion components - Immature proteins cleaved into mature by protease - Assembly of new virus - Budding from host cell - Repeats the cycles uncoating S vival protease AIDS-Related Diseases Examples - Pneumocystis carinii pneumonia - Candidiasis - Kaposi’s sarcoma - AIDS dementia complex - CMV retinitis - HIV causes Acquired Immunodeficiency Syndrome (AIDS) intersul - Progressive failure of the immune system Know Leading to life-threatening opportunistic infections & malignant neoplasms - AIDS first recognised in 1981 - HIV identified in 1983 - 35 million deaths attributed to AIDS - At present approx. 40 million peope infected with HIV 1/3rd global AIDS deaths occur in 3rd world countries HIV Test & Treatment *Simple genome à proteins: less sites for potential drug targets HIV Test - Based on Enzyme-Linked Immunosorbent Assay (ELISA) - Detects antibodies against HIV in patient serum - No vaccine or cure at present - Current treatments however help control symptoms, impair disease progression: Highly active antiretroviral therapy (HAART): Nucleoside analogues, protease ↳looks like a nucleoside inhibitors, anti-infectives & anti-neoplastic drugs ↳ When reverse transcriptase incorp false -DNA. = not stable & isn't rep. ↳ due to reduced immunity Prolong life expectancy survival à 1996: 39yrs old (contracted around 20yrs old) à 2011: up to 70 yrs old *Combination treatments & multiple drug regiments are necessary as HIV is highly mutable & can become resistant  Influenza Virus (A, B, C) - Orthomyxoviridae, enveloped minus-strand RNA - Shape & genome sequence is highly variable Segmented genome (8 separate pieces) - Common symptoms: fever, sore throat, muscle pains, severe headache, coughing & weakness & fatigue, serious cases → pneumonia Two types of protein spikes 1. Hemagglutinin (HA): mediates viral attachment to host cells 2. Neuroaminidase (NA): degrade sialic acid in host membrane to release progeny virus Influenza is always in the community, emerging as pandemics a few times each hundred years à 1918 (50 million deaths), 1957 & 1968. - Once infected the host is immune from that particular strain M selrnigen gene Synthesis antibodies that inactivate HA & NA fan M - However, HA & NA RNA sequences are constantly changing à influenza virus is highly mutable, dimerent strains become dominant -modif for rock - Antigenic drift creates influenza viruses with slightly-modified antigens earian wr sometimeval.don't Genetic mutations sumicient enough to necessitate annual flu vaccine will be live - Antigenic shift generates viruses with entirely novel antigens Host infected with 2 dimerent strains, progeny is a mix of RNA from both Strains jumping from animals to humans ↓ animal & human flu mix ↓ humans about animal's flu's impact in know little  Flu vaccine contains 3 most dominant strains - Contain dead or inactivated virus or purified viral proteins - Influenza virus is highly mutable, dimerent strains become dominant Latent Viral Infection Following 1st infection, symptoms disappear - But virus remains in asymptomatic host cells for long periods - Once reactivated à recurrent or new symptoms (priviledge site Herpes simplex virus 1 A immune sys. can't reach nerves suspended amination - Latent in nerve cells of lips & mouth - Reactivated during fever, cold, sun, stress (immunocompromised Older - Cold-sores o become sys a immunea down Varicella zoster virus – Chicken pox - Laten in nerve cells of body, reactivates to cause shingles inte corgive you prase integrate : Oncogenic Viruses  Eukaryotic Pathogens FUNGI fungi can be pathogens ↳resp for infection. fungi = opportunistic d immunocompromised pretty resistant antifung. fungi - to Fungi – Biotechnology > - minority are problematic - Source of antibiotics Penicillium spp. (beta-lactam antibiotics) - Fermentation industries Baking Brewing Wine production - Blue cheeses Fungal Forms - Yeasts: single-celled, reproduce asexually through budding - Moulds & fleshy fungi: long filamentous structures (hyphae) - Dimorphic Fungi: grow as either yeast or mould depending on environmental single cell or multi conditions bod = Blastomyces dermatitidis, Candida spp. simmunocomp can inhale them e *Spores: reproductive cells that are dispersed by wind, capable of germinating & producing a new mycelium à can sexual or asexual  - glucosewout A cell wall : d-glucan d targetted by medication Fungi – Mycology - Study of fungi = mycology - Found in wide range of environments (many are soil borne) - Diverse groups of eukaryotes Multicellular: moulds & fleshy fungi (eg. mushroom) Single-cellular: yeasts can't be dry I e don'tuse - Heterotrophic (absorb nutrients in solution), non-phototrophic, most are aerobic; Saprophytic (obtain nutrients from decaying organic matter) L Ox. used as final acceptor of elec. d -eitogetherember next week - - cell walls contain cellulose & /or chitin (N-acetyl - glucosamine polymers covering -class. based on how reproduce d fungi & humans eukary. - target differenced Fungi Reproduction Fungi reproduce in many diLerent ways - Asexual, sexual or both strategies at diLerent times Asexual Reproduction Sexual Reproduction - DiLerent to that of animals or plants Involves production of sexual spores (gamates Pathogenic Fungi - Most fungi are plant pathogens Few are human/animal pathogens - Fungal infections typically spread by spores - Enter the body through inhalation (typically soil borne) or damaged skin) - Person-to-person contact - Soil (spores) or insects bound with spores Food-based Mycotoxins - Secondary toxic metabolites formed by moulds >200 known mycotoxins Infects cereals, nuts, figs, spices, coLee, dried fruits 1. Aflatoxin B1 (AFB1): produced by Aspergillus Flavus and Parasiticus - Potent carcinogens, associated wih liver cancer 2. Ochratoxin A (OTA): produced by by (Penicillium spp) (P. verrucosum) & Aspergillus spp (A. niger; A. carbonarius; A. ochraceus) - kidney damage in humans & is a potential carcinogen 3. Patulin: produced by Penicillium spp. & Aspergillus spp. - potentially carcinogenic, damage immune, enterocytes & nervous system. In apples, compotes. Most countries have adopted detailed screening of raw commodities for mycotoxins to limit exposure Hallucinogenics Ergot (Claviceps purpurea) - Fungus that infects grains or rye & related grasses - Contains lysergic acid alkaloids (LSD precursors) ergotoxines; ergotamine; ergometrine - Ergotism aLlicted 100,000’s people in Europe during Middle Ages (ingestion of contaminated grain) Vasoconstriction, gangrene, uterine contractines, nausea, convulsions, seizures, madness, hallucinations, death Magic Mushrooms (eg. Psilocybe cubensis) - Principle active compounds are psilocybin & psilocin Similar to serotonin ALGAE - Phylogenetically diverse (prokaryotes & eukaryotes) - Metabolically unified – photosynthesis - Most are aquatic or live in moist conditions Single – (eg. phytoplankton) or multi-cellular (eg. seaweeds) - Distinct from higher plants as they lack tissue diLerentiation and are simple plants (no leaves, roots, connective tissues) - Motile due to flagella Pathogenic Algae Very few are pathogenic - Prototheca spp. Colourless algae Infection rare even after high exposure Bursitis (inflammation of the joints) - Alexandrium tamarense & Karenia brevis produce a potent neurotoxin which : accumulates in shellfish – paralysis if eater Red tide ‘blooms’ PARASITES An organism that lives in or on the living tissue of a host organism at the expense of that host à usually refers to protozoa & helminths only 1. High prevalence in tropical regions 2. Organisms activate immune responses but immune system unable to get rid of inflection à can last for years in insectsa then to humans leg. -start move 3. Elaborated lifecycles Protozoa - Non-photosynthetic, unicellular eukaryotes - Despite uni-cellularity, complex organisation - Pinnacle of unicellular complexity eg. Paramecium spp. > - cilliate protozoa - Cilia – cell motility & sweep food into oral groove - Cell Mouth - where food enters adivision - Anal Pore - disposes of waste ↓ everything - Contractile Vacuole - contracts & forces extra water else out of cell - Trichocysts - used for defence - Gullet - forms vacuoles for food storage - Macronucleus - larger nucleus, performs normal cell functions - Micronucleus - smaller nucleus, responsible for cell Pathogenic Protozoa > - fecal oval route multiple flagella ↳ motile ↳ GIT -chew up wall/desiminate in other organs (actin filama ↳nose brain colfac bub elongation - eat. - motile not motile cats Protozoa causing intestinal diseases Protozoa causing blood and tissue invasions Toxoplasma gondii – toxoplamosis (from cat faeces) - Non-motile parasite - Elaborate lifestyles: cat is definitive host; human intermediate host - In humans in first time infected, mother may pass on T.gondii to foetus (transplacental) - Blindness, hydrocephalus, jaundice, eye lesions & neurological problems justKnow t cycle Plamodium spp – malaria - P. falciparum; P. vivax; P. ovale; P. malariae - Tropical/sub-tropical infectious disease Symptoms - Destruction of RBCs by merozoites Synchronised release, every 48hrs, expect for P. malariae every 72hrs metab. - heat -no no - During attack: cyclic chills, high fever, drenching sweat - Anaemic, tachycardia, comeI & death *Np current vaccine; anti-malarial drugs = quinine or artemisinin derivatives Lifecycle of Plasmodium into esplit ferta & T gam. released into blood Ciliate Protozoa - Often have macronucleus & micronucleus Macronucleus: polypoid, vegetative growth & cell division Micronucleus: sexual reproduction - Cilia for locomotion & feeding - Often commensal organisms of animals (eg. ruminants) - Only 1 known pathogenic ciliated protozoon: Balantidium coli Causes inflammation of colon humans Causes diarrheal - -animals Transmission: F-O-R & zoonotic (esp pigs) No prevention Diagnosis trophozoites/cysts in stool (feces Arthropods & Helminths Not micro-organisms. But they can be vectors and reservoirs of micro-organisms that: 1. Can cause parasitic / infectious disease 2. Can transmit infectious disease Note: microbiology & infectious diseases go hand-in-hand chance Microbial infections transmitted by arthropod vectors climate in a e ↓ move mostcounti D · new ↓ ThatPocket  Arthropods infected fly lands on meat- Helminths (Metazoa - parasitic macroscopic worms) Flat untain s D Metabolism: Bacterial METABOLISM - Metabolism is a process by which cells utilise nutrients from local environment & via biochemical reactions - Derives su9icient energy & building blocks for synthesis of new cells, whilst maintaining life - Eukaryotes: occurs mainly in mitochondria - Prokaryotes: occurs in cytoplasm Extremely diverse, especially in comparison with eukaryotes Catabolism - Metabolic breakdown of complex molecules, accompanying release of energy - Complex molecules are broken down, to obtain ‘building block’ molecules Anabolism (opposite of catabolism) - Constructive park of metabolism, where complex molecules are built up Therefore, metabolism is the sum of biological reactions (both catabolic & anabolic) O O Human pathogenic bacteria are chemoheterotrophs - Derive energy from the breakdown of organic nutrients - Use this energy both for resynthesis & secondary activities cannot let free e - erelease damage cells Le can Bacteria oxidize nutrient substrates by means of either 1. Cellular respiration (aerobic & naerobic) ↳ ox· final acceptor ↳ something else 2. Fermentation of e- Note: human pathogenic bacteria can be classified in terms of their O2 requirements & tolerance nearorse ↓ Strict anarobe nowhere Bacteria are good experimental models (eg. E. coli) - Easy to culture - Short generation time (approx. 18min) - Genetics are well characterised (mutants) - Models for eukaryotic metabolism of how bugs strong level of complexity work don't need R just to get nutrients in -250 reactions of least. ato The Metabolic “Factory” know indepth Production of identical new cells - Requires: 5 metabolic tasks to double cell mass (in E. coli) (shown below) - With: “plan” (DNA) Raw materials (available nutrients) “fuel/driving force” (ATP & reducing power) 1. Transport of nutrients into cell (250 reactions) - Across cytoplasmic membrane, concentrate in cytoplasm 2. Catabolism (406 reactions) em -factorswhichtae - Nutrients used to produce precursor metabolites, ATP & reducing power nutrients 3. Biosynthesis (438 reactions) - Small molecule synthesis (including building blocks) from precursors 4. Polymerisation (482 reactions) - Link together building blocks, form macromolecules 5. Assembly (7 reactions) - Assemble macromolecules Bringing Nutrients into Cell Uptake of nutrients: easMy · - First barrier is outer membrane call = gal Semi-permeable watera Role of porins (di9usion of nutrients) Transport down a concentration gradient - Cell wall (peptidoglycan; no barrier) - Periplasm (role of binding proteins) -trap - Second barrier is cytoplasmic membrane Role of transporters (facilitated di9usion, active transport or group translocation) Win act Nutritional Classes of Microbes feed on T mos comp. J most d · bacter Central Metabolism A need to have up NAD present freed e-d form NADH to pick 3 pathways central to metabolism a creating reducing factor in derob Or and not needO, ↓. a -create At NaDH 1. Glycolysis > - break down of Sugar > occurs in cytoplasm 2. Krebs cycle (aka tricarboxylic acid, TCA or citric acid cycle) 15 carbon 3. Pentose phosphate > - give ATP > - feed glycolysis ribose 5 phosphate multiplye create for cells that start w/ glucose a G Phosphat dimp. ↳ make DNA & makes NaDPH All pathways yield energy - Directly (ATP synthesis ) and/or - Indirectly (NADH synthesis) ause ↳breaking don i T Catabolism - All cells require carbon, nitrogen, phosphate, sulfate etc… - Diverse range of carbon compounds utilised Supplies cell with: 1. Precursor metabolites for building blocks à (12 precursors for E. coli) - Starting materials for making all the cell’s biochemical parts - 6 precursor metabolites made via glycolysis; 4 via Krebs cycle, 2 via pentose phosphate pathway 2. Stored energy in the form of ATP 3. Reducing power (NADH or NADHP) FADH ATP can be generated by 1. Substrate level phosphorylation = & 2. Chemiosmosis Prokaryotes: occurs at cytoplasmic membrane Eukaryotes: occurs as mitochondrial membrane Importance of electron transfer chain & terminal electron acceptor Respiration works with a final acceptor 1. Glycolysis oxidises glucose and form: - ATP molecules - Reducing factors NADH (from NAD+) - Ends wth Pyruvic acid (3 C) 2. Krebs cycle starting with Acetyl CoA and form: - ATP molecules (more precisely GTP) - Form additional NADH but also PADH2 3. Electron Transport chain (ETC) - 3.1 captures all electrons from: All NADH (of glycolysis and Krebs) All FADH2 - 3.2 hand the electrons over to a final acceptor (in example below O2) Chemiosmosis (Electron Transport Chain) ET energya creates & through protonmore - A: NADH donates hydrogen atoms - B: Electron transport chain transfers protons (H+) to outside of cell, creating a gradient - Protons (H+) flow back through the membrane, energy of their movement is used by ATPase enzyme to phosphorylate ADP, forming ATP *O2 as final electron acceptor = Aerobic Metabolism ⑧ od more At man Anaerobic Metabolism S Generation of ATP in the absence of O2 1. Anaerobic Respiration - Uses an alternative final electron acceptor than O2 - Eg. nitrate, sulfate, fumarate - Less e9ective than aerobic respiration, so less ATP produced 2. Fermentation - Generates less ATP than aerobic respiration 2 ATP vs. 38 ATP in E.coli using glucose Yeasts ® ethanol + CO2 Lactic acid bacteria ® lactic acid Prevent glycolysis from stopping by producing NAD+ (shown by red dot) Fre feechay - - respiration no - ↓ nobr no & et Every time fermentation regenerates NAD+, every time glycolysis can ‘sweep’ down its biochemical reactions and produce another set of ATP à imagine fermentation regenerates 1000NAD+, you will actually produce 2x 1000 ATP; the organism can have energy this way and survive. In case of no respiration: no final acceptor is available Respiration vs Fermentation unches a lot stri a put him. MICROBIAL GROWTH Phases of Growth 1. Lag Phase - Prepare for growth 2. Exponential Phase - Cell numbers double at regular (maximal intervals) 3. Stationary Phase - Growth ceases, cells become smaller, synthesize ‘starvation’ proteins, enabling cells to become more resistant to damage 4. Death Phase - Live cell numbers decline Describing Growth Times - Doubling time: period required for cells in a microbial population to enlarge, divide & produce 2 new cells from each E. coli: under optimal lab conditions = 18min , in the intestine = 12hrs Growth in laboratory does not mimic nature - As nutrients are depleted & toxic metabolic products accumulate, growth conditions become less favourable, doubling time increase, eventually growth stops - Growth rate: number of doubling times per hour d = 20min double time : rate growth Requirements for Microbial Growth Growth Factors reac. Catalyse - Essential metabolites that microbe is unable to synthesize Purines & pyrimidines: required for synthesis of nucleic acids (DNA & RNA) Amino acids: required for the synthesis of proteins Vitamins: needed as coenzymes & functional groups of certain enzymes Note E. coli = no growth factor required à L. citrovorum = all 20 amino acids, purine, pyrimidines & 10 vitamins - Growth factors are not metabolized directly as sources of carbon or energy assimilated y cells to fulfil their specific role in metabolism. IMPORTANT: Auxotroph is a mutant strain of bacteria that requires some growth factor not needed by the wild-type (parent) strain - eg. E.coli strain that now required tryptophan in order to grow would be called a tryptophan auxotroph, designated E.coli trp- Trace elements: K+, Mg2+, Ca2+, Cu2+, Zn2+, Fe2+/Fe3+, Co2+,Mn2+ Major Elements: C, O, N, P, S Bacteria & Oxygen ↓ don'tweaccept is aout car Non-Nutrients - Temperature Psychrophiles (cold lovers) Mesophiles (moderate temperature lovers) Thermophiles (heat lovers) - pH Acidophiles Alkaliphiles - Hydrostatic pressure Barophiles - Osmotic strength Halophiles Measuring Microbial Growth measure ten 1. Measuring cell mass - Determine dry weight -centrifuge - clear today know if - Turbidity (spectrophotometer) 2. Measuring cell numbers don't diae b or - - Total cell count (microscopy, Coulter counter) - Viable count (plate count, filtration count, MPN) 3. Measuring metabolic activity Turbidity: content in a tube absorb light from source and detector measure such absorbance. I refracted Coulter Counter - Electronic Total Cell Count Cells pumped through a small pore (15-μm) Pore is part of an electrical circuit As each cell passes through pore, conductivity of circuit drops A drop in conductivity means a cell is counted - Fast, but requires pure cultures Foreign particles (blood cells, soil) can be erroneous counted Counts both alive & dead cells - Need methods for viable cell counting Total Cell Count (PetroR-Hausser Chamber) Explanations: - We have a solution of cells of unknown amount - We pour a sampling on a grid chamber volume of 0.02 mm3 - Microscopy counting with the knowledge of the chamber dimensions will enable a good estimation of initial amount of cell of the solution How is this initial cell number estimation performed? - The 1 mm2 Grid contains 25 large squares You count visually how many cells in ONLY one large grid (15 cells) - Grid chamber volume: Cover slip is 0.02 mm above the grid Grid is 1 mm2 Volume of the grid chamber is 1 mm x 1 mm x 0.02 mm = 0.02 mm3 - You must now determine the No. bacteria per ml (cm3) You know that 15 x 25 cells are found in 0.02 mm3 (the chamber volume) So in 1ml (or 1 cm3 ), you must multiply (15x25) by (50(to get to 1mm3) x 1000 (to get to 1cm3) ) = 18,750,000 = 1.9 x 107 cells/ml Filtration Count (Viable Cells) Similar to plate count method, but we can assess small cell numbers in large liquid volumes or in air Plate Count criable cells - How many bacteria are in 1mL of original culture? - Which plate provides the most accurate to count? - What is its dilution factor? Microbial Genetics DNA Structure of DNA 3 + g ↑ 5 - 3 - 2 strands of DNA (anti-parallel) - Double helix - Eukaryotes: DNA stored in nucleus - Prokaryotes: DNA stored in cytoplasm Nucleotides base- > Sugar- > phosphate - Hydrogen bonds between strands - Neighbouring deoxyribose connected On each strand a phosphate bonds the 3’ of one deoxyribose to the 5’ of the next deoxyribose Base pairing in G-C grich - G and C (3 H bonds) otA packed = - A and T ( 2 H bonds’’ RNA-has Ox Ox DNA-no Roles of DNA 1. DNA Replication - Process by which DNA double helix unwinds & makes a copy of itself - Needs to be copied accurately - Necessary for cell division 2. Gene Expression - Information stored in DNA is used to tell the cell what to do Bacterial Genetics - No nuclear membrane - Single circular chromosome - No intronic DNA - Can also possess extra-chromosomal DNA Known as plasmids Cells can have ≥1 copy of plasmid Replicate independently of main chromosome Genetic Information Genome - Total DNA of a cell - In bacteria: most have single circular chromosome, some are linear  Plasmids - Small, circular, extra-chromosomal DNA\ - Encode beneficial factors a gene tracticin -transmipt hamid have - Resistance factors (antibiotic) genetic novizontal - Conjugative plasmids (or F factor) Transfer to other cells Genotype: genetic makeup/blue-print vonment -envi epigenetics Phenotype: appearance & function DNA Replication Semi-conservative - After replication each chromosome consists of: 1 old template strand 1 newly synthesised strand à complementary to template Replication fork - Multiple enzymes processing the following DNA unwinds > - helicas Exposes nucleotides Synthesize (DNA polymerase [green dot]) the new strand in pone direction only – 5’ to 3’ pocane on Bacterial Chromosomes Replication of circular chromosome 1. Origin of replication - Bubble forms, DNA unwinds 2. Replication occurs in both directions - 2 replication forks 3. Continues until replication forks meet 4. Strands separate - Producing 2 chromosomes  TRANSCRIPTION & TRANSLATION Gene Expression Eukaryotes - DNA in membrane-bound n