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BIOL 241 Notes - After Midterm.docx

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University of Waterloo
BIOL 241
Barbara Butler

BIOL 241 – Applied Microbiology Lecture 9 – Purification of Drinking Water Monitoring of Water Quality - Water courses = sources of raw water for drinking - Waterborne pathogens cause gastrointestinal upset - Transmitted by fecal-oral route (cycle) Potential Pathogens - Bacteria - Viruses - Protozoa Monitor for Pathogens - Too many - Often low in test waters - Technical issues - Use indicator organisms for monitoring purposes Ideal indicators - Member of intestinal microbiota of warm-blooded animals - Equally resistant as pathogens to environmental insults, disinfection in water and wastewater treatment plants - Present in greater numbers than pathogen - Non-pathogenic - Should multiply in the environment Indicator Organisms - Coliform group – facultatively aerobic, gram-negative, nonspore-forming, rod-shaped, ferment lactose with gas formation - Growth-based, operational definition - Classic coliform test conducted by most portable number (MPN) procedure - Widely-used alternate is membrane filtration procedure E. coli  not a perfect indicator but not bad for enteric bacterial pathogens Defined Substrate Technology - Based on pocession of/expression of certain genes (enzyme activity) - Coliforms – possess gene for B-galactosidase (grow on lactose) - Convert MUGal  fluorescent product - E. coli possess gene for B-glucuronidase - Convert flurogenic/chromogenic substrate - MUG  fluorescent product - IBDG  insoluble blue compound Filter From Drinking Water Sample - On MI agar for 24h at 35°C examined under UV light - M = MUGal and I = IBDG - Total coliform and cell can be counted Waterborne Microbial Diseases - Epidermics of waterborn disease often show pattern of cases consistent with common source of infection (e.g. water supply) - Untreated water = problem Cholera (Vibrio cholerae) - Transmitted through contaminated water - Gram-negative, curved rod - Free-living in coastal waters, adhering to normal microbiota Disease - Initiated when ingested bacteria attach to epithelial cells of small intestine - Begin to grow and release enterotoxin - Causes high dehydration rates Typhoid (Salmonella typhi) - Most common route of transmission  via water - Carrier is individual that harbours organism but shows no disease symptoms - S. typhi: gram-negative rod Cryptosporidiosis and Giardiasis - Cysts or oocysts of these parasitic protozoa found in most surface waters - Giardia lamblia: flagellated protozoa, infects animals, humans - Environmentally resistant cyst is infective agent - Cryptosporidium parvum infects variety of warm-blooded animals - Ingested oocytes germinate, trophozoites growth within epithelial cells of stomach, intestine - Chronic diarrhea in individuals with impaired immunity Legionellosis - May be asymptomatic - Pontiac fever: mild cough, mild sore throat, mild headache, self-limiting - Legionellosis: type of pneumonia, more likely to infect the elderly - Lives happily in cooling towers, air conditioning systems, hot water tanks, whirpool spas, etc - No person-person transmission Waterborn Viral Diseases - Cause gastroenteritis - Cause eye throat infections - Hepatitis viruses Amebiasis Entamoeba histolytica - transmitted primarily by water - Excysted trophozoites grow on/in intestinal mucosal cells - Common in tropics, subtropics Naegleria fowleri - Free-living amoeba - May enter through nose, burrow into brain  multiples Lecture 10 – Microorganisms and Foods - Spoilage of food - Methods of food preservation - Use of microorganisms in food production - Foodborne disease-causing microorganisms Food Spoilage - Any change in appearance, smell, or taste of food product that makes it unacceptable to the consumer - Food not necessarily unsafe, but generally regarded as unpalatable Foods = organic material  suitable as microbial media - Perishable foods (meat, eggs, milk, most fruits, vegetables) - Semiperishable foods (potatoes, nuts, and some apples) - Stable or nonperishable foods (sugar, rice, flour, dry breans) Water activity: measure of available water Water activity = volume of air in equilibrium with substance or solution / volume of pure water Bacterial Growth in Batch Culture - Rate of exponential growth affected by temperature, oxygen, pH, nutritive calue of food, natural antimicrobial substances - Extent of lag phase depends on contaminating organism - Aim of food preservation: extend lag phase indefinitely Food Preservation Control of Temperature Storage at Cold Temperatures - Refrigeration at 5C, freeze to -20C (-80C) - Microbial growth slows but does not stop under low temp. conditions - Psychrotolerant microorganisms may spoil refrigerated foods - Freeze-thaw sequence alters physical structure of many foods Heat Processing - Cooking markedly reduces microbial load; must avoid reinoculation - Canning produces food that is stable indefinitely without refrigeration - Pasteurization designed to reduce microbial load, kill pathogens Low temperature hold (LTH): 62C for 30 min High temperature, short time (HTST): 71C for 15 sec -Aseptic Food Processing: juice boxes, milk products Increase Acidity of Food - Adding acidulant (pickling) - By encouraging fermentation by microorganisms Lactic acid bacteria: milk  yogurt, cheese, sour cream Acetic acid bacteria (Acetobacter) Propionic acid bacteria: flavour and holes characteristic of Swiss Cheese Decrease Water Activity of Food - Drying: natural sunlight, oven; maybe conjunction with smoking - Lyophilization (freeze-drying): camping rations - Addition of Sugar/Salt: sugar – mainly fruits; salt – mainly meats and fish Irradiation - Cobalt 60 or Cs 137 sources - Food does not become radioactive - Reduction in microbial load; to limit contamination by pathogens - Sterilization Modified Atmostphere Packaging - Use of impermeable films, evacuation, elevation of carbon dioxide content - Strategies of effective food preservation often employ multiple hurdles Use of Microorganisms in Food Production, Other Industrial Processes - Various food fermentationdedes - Beverage alcohol: ethanolic fermentation: glucose  ethanol + 2Wwater - Vinegar production Biotransformation - Cortisone production using microorganism - Rhizopus nigricans will catalyze biotransformation of progesterone - Primary metabolite: one that is formed during growth phase of microorganism - Secondary metabolite: one that is formed near end of growth phase, nearing stationary phase Making Vinegar - Requires some source of alcohol and certain strains of acetic acid bacteria - Strict aeroes, the strains of use do not oxidize their organic electron donors completely to carbon dioxide and water Vinegar Generator - Adequate aeration is important - Fluid recirculated until desired acetic acid content is reached - Other materials in starting material, produced in microbial reaction, contribute to vinegar flavour Vitamin Sysntheses Vitamin B12 Natural Synthesis is exlusively by microorganisms B12 deficiency can lead to pernicious anemia in humans Riboflavin Needed for flavin synthesis FAD, FMN, coenzymes that participate in oxidation-reduction reactions Production of Enzymes - Often exploit extracellular enzymes (excenzymes) - Specifically of enzyme’s catalytic reaction useful in bioconversions - Extremophile enzymes (extremozymes) remain functional under harsh conditions Detergents: amylases, proteases, lipases, reductases from Bacillus licheniformis Taq polymerase: thermostable DNA polymerase from Thermus aquaticus used in polymerase chain reaction (PCR) Invertase: sucrose-digesting enzyme Glucose isomerase: high fructose sweetener from corn, wheat, potato starch - Immobilization of soluble enzymes allows large-scale reactions under continuous conditions Scale-Up - Oxygen transfer is much more difficult to obtain in large-scale - Mixing and aeration more easily accomplished in small volumes - Methods of temperature control must change with scale-up - Sterilization protocols must change with scale-up - Bioengineers: skilled in gas transfer, fluid dynamics, mixing, thermodynamics Downstream Processing - Removal of biomass from culture medium, concentration and purification of product from spent medium - Harvesting and fractionation of biomass to recover desired product, separation from cellular debris, concentration and purification of product Lecture 11 – Biotechnology Biotechnology: the use of living organisms to carry out defined chemical processes for industrial or commercial application - Bioremediation of hydrocarbon, solvent, pesticide spills, bioleaching of metals - Production of foods, food additives, alcohol, antibiotics - Bioconversion, biocatalysts Current usage: implies organism used to carry out process genetically manipulated in vitro: molecular biotechnology Taq polymerase: produced in E. coli strain engineered to carry and express T. aquaticus gene Before the Molecular Age - Introduction of random mutations + screening for mutants with desired traits - Industrial strain development (penicillin production): numerous mutations achieved spontaneously, by irradiation by chemical exposure AEC = lysine analog; will bind to aspartokinase and shut down pathway - Mutants of Brevibacterium flavum that over-produce lysine obtained by looking for AEC-resistant cells - Site-directed mutagenesis to modify aspartokinase gene  catalytic function of aspartokinase unaffected; capacity of covalent modification by lysine (or AEC) eliminated - Site-directed mutagenesis: a technique whereby a gene with a specific mutation can be constructed in vitro 1. Clone into single-stranded vector 2. Add synthetic oligonucleotide with one base mismatch 3. Extend single strand with DNA polymerase 4. Transformation Means to Move DNA Around Plasmid pER322 – an early cloning vector that replicates in E. coli - Origin of replication allows independent replication - Single cleavage sites for various restrictions - Tetracycline resistance gene - Ampicillin resistance gene Origin of DNA replication: to maintain the number of plasmid within the cell Host bacteria + cloned plasmid = selective marker - Insertion of foreign DNA at BamHI site - Tetracycline resistance gene is thus inactivated - Transformants carrying foreign DNA are ampicillin resistant but tetracycline sensitive Transofrmation: transfer of genetic information via free DNA Moving DNA Around: transformation and conjugation transduction - Only works with bacteria cells - Hard techniques to work with - Inject DNA into host cell = integrate into host genome then express Bacteriphage lamba (modified) as a cloning vector Transduction: transfer of host genes from one cell to another by a virus Electroporation: use of an electric pulse to enable cells to take up DNA - Millisecond-length pulses open small pores in cell membranes - DNA can move into/out of the cells via pores Microprojectile “DNA gun”: shoot DNA into cell Expression of human genes in prokaryotes requires a method of dealing with introns - Eukaryotes: extron and intron - Prokaryote: no split gene Reverse transcriptase: copies information from RNA to DNA - the cDNA derived from the processed human mRNA could be used to clone in prokaryotic systems Expression vector pSE420 - Polylinker: insert desired DNA - Amp resistance - Trc promoter - lacO (operator) 1. Growth to high concentration of microbes 2. Inducer added to turn it on 3. Cloned gene expressed and product produced Production of the Human Hormone Insulin by Genetic Engineering Problem: active insulin is comprised of only parts of polypeptide encoded by insulin gene Insulin gene codes for: preproinsulin  proinsulin (inactive peptide)  insulin (active; polypeptide A-polypeptide B connected by disulfide bridges) Human Proinsulin - Interconnecting peptide must be cleaved to produce active insulin Biotechnology Solution: a) Human insulin gene is small  chemically synthesized the DNA code to produce proinsulin, rather than isolating gene from human tissue b) Proinsulin produced then converted to insulin via enzymatic cleavage, disulfide bond formation Lecture 12 – Foodborne Diseases Gastroenteritis: acute inflammation of gastrointestinal tract Especially small intestine, or large intestine (enterocolitis) Diarrhea: a result of Decreased absorption of fluid from intestinal tract Increased secretion of fluids derived from blood into the intestinal tract Enterotoxins: tonxins that affect GI tract Typically exotoxins (protein excreted from bacterial cells) Stimulate secretion of water and salts  intestinal lumen Food Poisoning (intoxification): disease tha results from ingestion of foods containing performed microbial toxins Food Infection: active infections resulting from ingestion of pathogen-contaminated foods Staphilococcal Food Poisoning Staphyloccocus aureus: small gram positive rod - common on skin, in nasopharynx - Certain strains may produce heat-stable enterotoxins Enterotoxins: Types A, B, C1, C2, D, E - Type A most frequently associated with food poisoning - entA gene is chromosomal, types B, C encoded on mobile genetic elements Superantigens: activate general inflammatory response in intestine - Massive fluid loss S. aureus will grow aerobic and anaerobic conditions Do not compete well with other microorganisms in food Symptoms - Onset 1-6 after consumption; duration of illness typically less 12 h - Gastroenteritis characterized by severe nausea, vomiting - Moderate diarrhea - Usually no fever Foods Involved - Milk products, custards, cream puff fillings, sandwich spreads, poultry stuffing, potato salad - Problem is often “post-proessing” contamination Control - Regrigeration below 6-7C - Cooking kills microorganisms - Toxin can tolerate 30 min of boiling Botulism - Rare; fatality rate is high Clostridium botulinum - Gram positive sporeforming rod, strict anaerobe - Normally inhabits soil, water; spores may contaminate foods before honest - Spores may initiate growth, toxin production in improperly processed foods - Botulism food poisoning results from ingestion of toxin Toxin
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