BSC242 week of 2/15 notes
BSC242 week of 2/15 notes BSC 242
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This 22 page Class Notes was uploaded by Alexandra on Saturday February 20, 2016. The Class Notes belongs to BSC 242 at University of Alabama - Tuscaloosa taught by Daryl W. Lam in Winter 2016. Since its upload, it has received 23 views. For similar materials see Microbiology and Man in Biology at University of Alabama - Tuscaloosa.
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Date Created: 02/20/16
Microbial Growth 02/09/2016 ▯ Growth= increase in number of cells NOT cell size Physical requirements for growth o Temp (In C) 0°C=32°F examples psychrophiles: -10° to 20° psychrotrophs: -10° to 30° mesophiles: 10° to 50° (meso think middle) thermophiles: 40° to 70° hyperthermophiles: 65° to 110° Min growth temp Optimum growth temp Maximum growth temp o Improper cooling It is major reason for food-borne illnesses Leaving cooked foods at room temp too long Refrigerating large quantities of food in deep containers o pH most bacteria grow between pH 6.5 and 7.5 molds and yeasts grow between pH 5 and 6 acidophiles grow in acidic environments (below pH 7) o osmotic pressure microbes obtain nutrients from their environment Hypertonic environments (increased salt/sugar) cause plasmolysis Plasmolysis: cell loses water Extreme or obligate halophiles require high osmotic pressure (dead sea bacteria) Facultative halophiles tolerate high osmotic pressure (staphylococci) Chemical requirements for Growth o Carbon Used for structural organic molecules, energy source Heterotrophs use organic carbon sources (hetero=different, troph=feeder) Autotrophs (photosynthetic organisms) use CO to mak2 sugars o Nitrogen In amino acids, proteins, nucleic acids Most bacteria decompose proteins Some bacteria use NH 4+ or NO 3- A few bacteria use N in2nitrogen fixation o Sulfur In amino acids (cys, met), thiamine and biotin Most bacteria decompose proteins 2- Some bacteria use SO 4 or H2S o Phosphorus In DNA, RNA, ATP and membranes (phospholipids) PO 43-is a source of phosphorus o Trace elements Inorganic elements required in small amounts Usually as enzyme cofactors Ex Zinc, iron, magnesium, iodine o Oxygen (O ) 2 Obligate aerobe Requires molecular oxygen to live Facultative anaerobe Can grow w/ or w/o molecular oxygen Obligate anaerobe Does not use molecular oxygen & is killed in its presence Aerotolerant anaerobes Does not use molecular oxygen but is not affected by its presence Microaerophiles Grows best in an environment with less molecular oxygen than is normally found in air Toxic forms of Oxygen (pgs 155-156) - Superoxide free radicals: O 2 O 2 2 H —superoxide dismutase—> H O 2 2 + O 2 2- Peroxide anion: O 2 (toxic) 2 H O —catalase—> 2 H O + O 2 2 + 2 2 H 2 2 2 O —peroxidase—> 2 H O 2 Singlet oxygen: O -1 2 Very reactive, causes damage to cells Hydroxyl radical (OH) Very reactive o Organic growth factors (“essential” in diet) Organic compounds obtained from environment Vitamins, amino acids, purines, and pyrimidines ▯ Culture Media Culture medium o Nutrients prepared for microbial growth o Must contain proper Nutrients (C, N, energy sources) pH salt conc Temp Oxygen requirements o Types Broth Used for large # of cells in small place Agar slant Used for transport & sustaining of cultures Agar deep Used for motility & O 2equirement Agar plate Used for isolated colonies Agar Complex polysaccharide Used as solidifying agent for culture media in Petri plates, slants, and deeps Generally not metabolized by microbes Liquefies at 100°C Solidifies ~40°C o Chemically defined media exact chemical composition is known o Complex media extracts and digests of yeasts, meat, or plats (nutrient broth, nutrient agar) Sterile o No living microbes (including endospores) Inoculum o voluntary introduction of microbes into medium Contamination o Involuntary introduction of microbes into medium Culture o Microbes growing in/on culture medium ▯ Anaerobic Culture Methods Reducing media o Contain chemicals (thioglycolate or oxyrase) that combine O 2 and deplete it o Heated to drive off O 2 Anaerobic jar o Chemicals produce H and CO2which rem2ve O by combining 2 it in presence of a catalyst with H to 2orm water Anaerobic chamber Oxyrase: combines with O with H and removes O as water is 2 2 2 formed ▯ Capnophiles require High CO (Low O 2 2 Candle jar (candle generates CO 2 CO –2acket ▯ Selective (S) Media Clinical specimens are often contaminated by other microbes Suppresses unwanted microbes and encourages growth of desired microbes Examples o Media containing antibiotics (only resistant ones grow) o Sabouraud’s Dextrose Agar (low pH suppresses bacteria and favors yeasts) ▯ Differential (D) Media Makes it easy to distinguish colonies of different microbes from each other Examples o Blood agar (clear zones around Streptococcus pyogenes from throat swabs) o X-gal plates (lactose positive colonies appear blue; lactose negative colonies appear white) [pg 248 Figure 9.11] ▯ Mannitol salts Agar plates (MSA) S: high NaCl inhibits many microbes D: pH indicator (phenol) turns yellow to indicate acid from fermentation ▯ Eosin-Methylene Blue Agar (EMB) S: dyes inhibit Gram-positives D: lactose fermenters turn dark; E. coli gets a green shade also ▯ Enrichment Media Encourages growth of desired microbe ▯ Type Purpose Chemically defined Growth of chemoautotrophs and photoautotrophs; microbiological assays Complex Growth of most chemoheterotropic organisms Reducing Growth of obligate anaerobes Selective Suppression of unwanted microbes; encouraging desired microbes Differential Differentiation of colonies of desired microbes from others Enrichment Similar to selective media but designed to increase numbers of desired microbes to detectable levels Definitions Pure culture (axenic) Contains only 1 species or strain Colony (CFU: colony-forming unit) Population of cells arising from a single cell or spore ▯ ▯ Preserving Bacteria Cultures Deep-freezing: -50° to -95°C Lyophilization (freeze-drying): frozen (-54° to -72°C) and dehydration in a vacuum ▯ ▯ Reproduction in Prokaryotes (asexual) Binary fission o Bacteria o Organism separate to form 2 new organisms o Cytoplasm is split evenly o Steps Cell elongates and DNA is replicated Cell wall and plasma membrane begin to grow inward Cross-wall forms completely around divided DNA Cell separate (a) a diagram of the sequence of cell divison (b) a thin section of a cell of Bacillus licheniformis starting to divide Budding o Yeast (only a few bacteria) o New organism is from old organisms and cytoplasm splits unevenly Conidiospores (actinomycetes) Fragments of filaments ▯ ▯ Generation time: time it takes a cell to divide (or population to double) Ex: began with 5 cells, ended with 160 cells. How many generations? 5*2=10 *2=20 *2=40 *2=80 *2= 160 (5 generations) ▯ Important graph ▯ ▯ ▯ Direct Measurements of Microbial Growth Plate counts: perform serial dilutions of a sample o inoculate Petri plates from serial dilutions o spread and pour plates o after incubation, count colonies on plates that have 25-250 colonies ▯ Filtration Direct microscopic count ▯ Estimating Bacterial Numbers by Indirect Methods Turbidity (spectrophotometry) o Measuring light passing through Metabolic activity Dry weight ▯ ▯ Chapter 7 ▯ The control of Microbial Growth Sepsis Refers to microbial contamination Asepsis The absence of contamination Aseptic technique Used in lab to prevent contamination of solutions and cultures (aseptic surgery techniques prevent microbial contamination of patients) Sterilization Removal of all microbial life (including endospores) Commercial sterilization Enough heat to kill Clostridium botulinum endospores Disinfection Removal of pathogens from surfaces Antisepsis Removal of pathogens from living tissue Degerming Removal of microbes from a limited area (like alc. wipe before shot) Sanitization Lower microbial counts on eating utensils Biocide/Germicide Kills microbes Bacteriostasis Inhibiting, not killing, microbes Bacterial populations die at a constant logarithmic rate ▯ Effectiveness of antimicrobial treatment Depends on o Number of microbes o Environment (organic matter, temp, biofilms) o Time of exposure o Microbial characteristics Biofilms o Matrix of polysaccharides, DNA and protein=slime o Bacteria that are grouped together in communities that provide great benefits from that grouping o Biological systems: functional communities o Usually attached to rocks, teeth or mucous membranes (single species or diverse group) o Share nutrients and are protected from harmful factors o Conjugation of genetic material between organisms DNA is sucked into community (discussed in later ch) o Super resistant to microbicides ▯ Actions of Microbial Control Agents Alteration of membrane permeability Damage to proteins o Stop metabolic reactions and damage offspring Damage to nucleic acids ▯ Physical methods of microbial control Heat o Thermal death point (TDP) Lowest temp. at which all cells in a culture are killed in 10 min. o Thermal death time (TDT) Time to kill all cells in a culture o Moist heat denatures proteins Autoclave: steam under pressure Pressure and temperature in autoclave Standard autoclave run= 121C (15psi) for 15 min Steam must directly contact all surfaces of solid objects Steam Sterilization Steam must contact item’s surface Pasteurization reduces spoilage organisms and pathogens Equivalent treatments o 63°C for 30min (classic pasteurization) o high temp, short time (HTST): 72°C for 15sec o ultra high temp (UHT): 140°C for <1sec (sterilizes) Dry heat sterilization kills by oxidation o Flaming o Incineration o Hot-air sterilization Filtration Removes microbes High pressure Denatures proteins Low temp Inhibits microbial growth Refrigeration Deep freezing Lyophilization Desiccation (absence of water) Prevents metabolism Osmotic pressure Causes plasmolysis Dried microbes are a problem in hospitals Radiation damages DNA o Ionizing radiation Kills microbes on certain meats, vegetables, spices, plasticware, mail, some medical supplies (x-rays, gamma rays, etc.) High degree of penetration bc short wavelengths o Nonionizing radiation (UV) light Germicidal lamps found in hospitals, some vaccines are treated with UV Low degree of penetration bc longer wavelength ▯ Chemical Methods of Microbial Control Principles of effective disinfection o Conc. of disinfectant o Organic matter present o pH o time o temp evaluating a disinfectant o use-dilution test metal rings dipped in test bacteria are dried dried cultures are placed in disinfectant for 10 min at 20°C rings are transferred to culture media to determine whether bacteria survived treatment o disk-diffusion method can see zone of inhibition (halos, rings of diffusion) ▯ Types of Disinfectants Phenol and Phenolics (Lysol and Amphyl) o Disrupt membranes and proteins o Active in organic matter and effective against mycobacteria Bisphenols (hexacholorphene, Triclosan) o Disrupt plasma membranes Biguanides (Chlorhexidine) o Disrupt plasma membranes o Combined with detergent or alcohol in surgical scrubs Halogens o Often effective against mycobacteria and endospores o Affect enzyme activity o Iodine Binds to proteins Available as a Tincture of Iodine (iodine in alcohol) or as the Iodophor (e.g. Betadine) o Chlorine Biocidal activity is based on formation of hypochlorous acid (HOCl, a strong oxidizer) when in water (e.g. Clorox bleach) Used in water treatment Chlorine dioxide gas is used to treat large areas Alcohols (ethanol, isopropanol) o Denature proteins, dissolve lipids o Can enhance effectiveness of other agents (e.g. tinctures) o Used to degerm skin Heavy Metal (Ag and Cu) o Denature proteins (combines with sulfhydryl groups) o 1% silver nitrate used in eyes of newborns (before antibiotics available) o silver impregnated wound dressings and catheters o copper sulfate is used to kill green algae in ponds, pools, and fish tanks Surface-active agents or surfactants o Decrease surface tension of molecules Soap Degerming Acid-anionic detergents Sanitizing Quaternary ammonium compounds Bactericidal, denature proteins, Cationic detergents disrupt plasma membrane o Soaps emulsify the oily film on skin together with any debris and this mix gets washed away with water (scrubbing) o QUATS are derivatives of the ammonium ion; probably affect the membrane. Some pseudomonades can grow in quat solutions. Quats are not useful against mycobacteria or endospores. Bactericidal against gram-positive bacteria and less active against gram-negative bacteria Chemical food preservatives: retard spoilage o Organic acids Inhibit metabolism Sorbic acid, benzoic acid, and calcium propionate Control molds and bacteria in foods and cosmetics o Nitrite Prevents endospore germination: meat products o Antibiotics Nisin and natamycin prevent spoilage of cheese Safe for human consumption Aldehydes: very effective antimicrobial o Inactivate proteins by cross-linking with functional groups (-NH2, -OH, -COOH, -SH) o Glutaraldehyde, formaldehyde, and ortho-phthalaldehyde (OPA) o Effective on endospores and mycobacteria Gaseous Sterilants o Denature proteins o Ethylene oxide (used in a chamber) o Penetrates well o Very effective on all microbes and endospores o Cross-linking of DNA and proteins (stops vital cellular functions) o Toxic to humans and a suspected carcinogen Peroxygens o Oxidizing agents o O (ozone), H O (peroxide), benzoyl peroxide (acne treatment), 3 2 2 peracetic acid (a sporocide) o Not good for open wounds o Catalase in human tissues readily inactivates hydrogen peroxide producing oxygen gas ▯ CH 27 ▯ ▯ Microbial diversity Microbes live in variety of habitats because of their abilities to o Use variety of C and energy sources o Grow under different physical conditions Extremophiles (mostly in archaea) live in extremes o pH (extreme acidophiles) o temp (extreme thermophiles) o salinity (extreme halophiles) ▯ Mycorrhizae Myco=fungus Rhiza=root Fungi living in close association with plant roots Extend surface area of roots Commercial uses o Helped the pine tree grow taller and more roots to absorb more nutrients ▯ Biogeochemical Cycles Saprophytes obtain nutrients from dead organic matter Symbiosis is the interaction among organisms that live together o Both mutually benefit Soil is a vibrant community of billions of organisms (e.g. bacteria, fungi, earthworms) that are responsible for much of the recycling of elements Factors o C, N, S, P Importance o Carbon cycle main component of biological compounds and minerals Ocean: C moves between the atmosphere and the ocean through diffusion Aquatic plants: absorb C through photosynthesis (step 1) Terrestrial plants: remove C from the atmosphere through photosynthesis Fish, other marine life and animals: obtain C through consumption (heterotrophs): energy and CO2 released through respiration C is returned to H2O and air through respiration When the organisms dies, C is decomposed and transferred through the ocean floor by sedimentation C is in rocks and is incorporated in fossil fuels. Burning the fossil fuel releases the CO2 back into the atmosphere Carbon dioxide gets released during metabolism of carbohydrates by heterotrophs The released carbon dioxide is now available to be used by autotrophs to make carbohydrates Carbon dioxide also gets released by complete oxidation of the organisms themselves after death Burning of fossil fuels produces carbon dioxide at levels that are not easily balanced by nature, presumably leading to global warming Methanogens use carbon dioxide as a final electron acceptor to make methane Methanotrophs oxidize methane to get energy and make carbon dioxide Both methane and carbon dioxide are greenhouse gases Methane is a greenhouse gas just like carbon dioxide but it is about 20 times more effective in trapping heat. So if there is too much methane in the atmosphere then it will trap more heat leading to more global warming o Nitrogen cycle amino acids, proteins, bases of nucleic acids and chlorophyll molecules Important for life, proteins made from it, protein=growth Nitrogen is needed for proteins, nucleic acids, etc. Nitrogen must be fixed and combined into organic compounds 4 parts: ammonification, nitrification and denitrification, nitrogen fixation (deamination: amino groups of amino acids are removed and converted into ammonia (NH3)) ammonification: release of ammonia nitrification: oxidation of the N in the ammonium ion to produce nitrate (nitrosomonas & nitrobacter, 2-step process) denitrification: energy from nitrogen is fully oxidized=no usable energy, but can be used as an electron acceptor by microbes metabolizing other organic energy sources in the absence of atmospheric oxygen leads to loss of N in atmosphere (pseudomonas, NO3-(nitrate ion)NO2- (nitrite ion)N2O(nitrous oxide)N2(nitrogen gas) o it’s a circle, can go both ways nitrogen fixation only a few species of bacteria can use N gas directly as a nitrogen source=nitrogen fixation conversion of N gas to ammonia o needs Nitrogenase (very very limited) o types of microorganisms that can do this rhizosphere (free-living) symbiotic (root nodules) relationship between plant and bacteria (FOCUS ON THIS) proteins and waste products ——microbial decomposition— amino acids amino acids (-NH2) ——ammonification— ammonia (NH3) ammonium ion (NH4+) ——nitrosomonas— nitrite ion (NO2-) (nitrification) nitrate ion (NO2-) ——nitrobacter— nitrate ion (NO3-) (nitrification) nitrate ion (NO3-) ——pseudomonas— N2 (denitrification) N2 ——nitrogen-fixation (nitrogenase)— ammonia (NH3) ▯ o Sulfur cycle proteins, cofactors, fossil fuel production Resembles nitrogen cycle: lots of oxidations Sulfides: most reduced form (H2S) H2S: source of energy for some autotrophic bacteria that convert it into sulfur granules and fully oxidized sulfates (SO42-) Burning fossil fuels (in factor) it comes out as SO2 When it mixes with water, it comes down as acid rain (H2SO3) o Phosphorous cycle plant and microbial growth, nucleotides, bones (calcium phosphate), tooth enamel, exoskeleton, phospholipids Important for DNA, RNA, ATP Exists mainly as PO43- No atmospheric step in the phosphorous cycle Most phosphorous is in the ocean ▯ Bioremediation: use of microbes to detoxify or degrade pollutants; enhanced by nitrogen and phosphorus fertilizer ▯ Bioaugmentation: addition of specific microbes to degrade of pollutant ▯ Composting: arranging organic waster to promote microbial degradation ▯
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