chapter 6

chapter 6 - Chapter 6: Chapter 6: Microbial Growth...

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Unformatted text preview: Chapter 6: Chapter 6: Microbial Growth Environmental Requirements Environmental Requirements Physical and Chemical Factors Temperature pH Osmotic pressure Oxygen availability Hydrostatic pressure Radiation Temperature Requirements Temperature Requirements Type Psychrophiles Mesophiles Thermophiles Range 0­20°C Optimum 15°C 15­45°C 20­45°C 45­70°C 60°C Hyperthermophiles 70­120°C 90°C Temperature Requirements Temperature Requirements Temperature Requirements Temperature Requirements Psychrophiles: Cause spoilage of foods while refrigerated Include most human pathogens (body is 37°C) Found in compost heaps Hydrothermal vents, hot springs (above 104°C) Mesophiles: Thermophiles Hyperthermophiles: pH Requirements pH Requirements Type Acidophiles Neutrophiles Alkalophiles Range pH 1.0 – 5.5 pH 5.5 – 8.0 pH 8.5 – 11.5 Acidotolerant and alkalotolerant microbes can persist for short periods under these conditions, but are unable to reproduce pH Values of Some Environments pH Values of Some Environments Acidic 1 2.5 3.5­4.5 6 8 9 10 Basic 11 ammonia gastric juices vinegar peaches, tomatoes peas, corn, shrimp seawater alkaline lakes/soils soap solutions household pH Requirements pH Requirements Large changes in [H+] can: Disrupt the cytoplasmic membrane Inhibit enzymes and transport proteins Most bacteria are neutrophiles Even in acido­ or alkalophiles, cytoplasmic pH remains neutral Keeping the Cytoplasmic pH Neutral Keeping the Cytoplasmic pH Neutral Antiport exchange of K+ for H+ in neutrophiles (Na+ for H+ in alkalophiles) Synthesize proteins under acidic conditions Acid shock proteins use ATP to actively transport H+ out of the cell Fermentation produces acids Putrefaction produces ammonias Export wastes to the environment Osmotic Pressure Osmotic Pressure Moderate halophile Require NaCl; found in marine habitats Require NaCl; found in hypersaline habitats like the Great Salt Lake Can grow in salty situations, but grow better without the NaCl; skin bacteria Requires sugars; yeasts & molds Extreme halophiles Osmotolerant Saccharophiles Osmotic Pressure Osmotic Pressure Effect of Osmotic Pressure Effect of Osmotic Pressure Osmotic pressure and water activity are inverse Solution has low water activity = high osmotic pressure Low water activity draws water out of cells via osmosis to dilute external [solute] Water is essential to macromolecule breakdown High water activity may lyse cells by drawing too much water into cell cytoplasm via osmosis Cell wall offers great protection from this Mechanosensitive channels in plasma membrane open Regulating Osmotic Pressure Regulating Osmotic Pressure To prevent growth, reduce water availability inside the cytoplasm Add solutes (sugars, salts) Lyophilization (freeze­drying) Dessication (drying) Oxygen Availability Oxygen Availability Major groups based on O2 use and tolerance: Obligate aerobes Facultative anaerobes Obligate anaerobes Aerotolerant anaerobes Microaerophiles Obligate Aerobes Obligate Aerobes Undergo aerobic respiration: O2 required as a terminal electron acceptor No other respiratory paths available In a broth medium: Growth only near top of liquid Limited by the penetration of dissolved O2 from the atmosphere Facultative Anaerobes Facultative Anaerobes In the presence of O2: Undergo aerobic respiration using O2 as terminal electron acceptor Producesmore ATP = more growth capable Undergo anaerobic respiration or fermentation paths less ATP produced, and hence less growth than aerobic In the absence of O2: Facultative Anaerobes Facultative Anaerobes In a broth medium: Growth will be densest at top where O2 is available Growth will occur throughout the depth of the medium via anaerobic respiration or fermentation, or both Obligate Anaerobes Obligate Anaerobes Undergo anaerobic respiration: Ions such as NO3­ are required as a terminal electron acceptor No other respiratory paths available In a broth medium: Limited by the penetration of dissolved O from the atmosphere­ very toxic Growth only near bottom of tube 2 Obligate Anaerobes Obligate Anaerobes O2 presence causes the formation of toxic compounds (superoxides, free radicals) Disrupt cytoplasmic membranes & other cell components Do not have enzymes capable of converting toxic compounds to harmless ones: Superoxide dismutase (SOD): converts superoxides to peroxides Catalase: breaks down hydrogen peroxide to H2O and O2 Peroxidase: converts peroxides to water and NAD+ Aerotolerant Anaerobes Aerotolerant Anaerobes Ignore O2 No toxic effects of O2 due to SOD presence Use fermentation or anaerobic respiration for ATP production In a broth medium: Grow throughout the depth of the medium­ no denser region at surface Microaerophiles Microaerophiles Too high of O2 content is damaging Low enzymes = inability to adequately prevent damage from toxic species Require 2­10% O2 concentration Normal atmosphere is 20% Micro­environments with aerobically respiring consortia reduce [O2] to tolerable range Many respiratory pathogens are microaerophiles Oxygen Use/Tolerance Groups Oxygen Use/Tolerance Groups Hydrostatic Pressure Hydrostatic Pressure Barophiles (piezophiles): optimal growth rate where pressure > atmospheric pressure Pressure­adapted microbes growing at higher temperatures are mostly Archaea Pressure­adapted microbes growing at moderate and cold temperatures are mostly Bacteria Adaptation to pressure is not too extreme Slight genomic differences between pressure­ adapted vs. normal atmosphere isolates Radiation­ Ultraviolet Rays Radiation­ Ultraviolet Rays Damages DNA base pair bindings to produce mutations like thymine dimers Mutations will result indirectly in cell death Inability to replicate chromosome Inability to correctly transcribe mRNA Radiation­ Ultraviolet Rays Radiation­ Ultraviolet Rays UV light DNA damage can be repaired: Photoreactivation Blue light energizes a specific enzyme which breaks the thymine dimers Allows normal cross­helix base­pairing by hydrogen bonding Dark reactivation Thymine dimers are excised by endonucleases Missing bases in the DNA sequence are replaced by other endonucleases Ionizing Radiation Ionizing Radiation Cause atoms to lose electrons Include X­ray and gamma radiation Low levels cause mutation and can indirectly cause cell death High level exposure causes direct cell death Breaks H­H bonds, oxidizes double bonds, breaks ring structures, polymerize some molecules Often used as a sterilizing treatment What is Microbial Growth? What is Microbial Growth? Defined as an increase in number Achieved by: Budding Binary Fission Cell duplicates its components, then shares them between 2 daughter cells Daughter cells independent when septum forms betweencell ‘halves’ Bacterial Cellular Growth Cycle Bacterial Cellular Growth Cycle C phase = chromosome replication D phase = delay period Nucleoid partitioning Septation begins Cytokinesis = septation complete Bacterial Culture Growth Cycle Bacterial Culture Growth Cycle Log vs. Arithmetic Scales Log vs. Arithmetic Scales Conversion to log scale compresses the distance between data points evenly. Bacterial Culture Growth Cycle Bacterial Culture Growth Cycle Culture Growth Phases: Lag Log (Exponential) Stationary Death (Decline) Easily measured during growth in liquid media by spectroscopy or densitometry Bacterial Culture Growth Cycle Bacterial Culture Growth Cycle Lag phase: Metabolically active but NO increase in number Adaptation: induce enzymes needed; synthesize new ribosomes, ATP, and cofactors; replicate chromosome Repair cellular components, increase in cell size Unbalanced growth­ rates of synthesis of cell components varies with one another Length of entire phase varies w/ species & environmental conditions Bacterial Culture Growth Cycle Bacterial Culture Growth Cycle Log (Exponential) phase: Population doubles each generation Generation (doubling) time ranges from 7 min to 20 hr – average is 20 min Growth is asynchronous­ not all cells divide at exact same time Growth rates are saturable; limited to [celluar enzyme] Balanced growth­ all cellular constituents made at constant rates to one another Bacterial Culture Growth Cycle Bacterial Culture Growth Cycle Log (Exponential) phase: Rapid expansion with 20­min generation time: also called doubling time Population doubles in number every 20 minutes) 0 m in 2 0 m in 4 0 m in 1 h r 2 h r 3 h r 4 h r 5 h r 6 h r 1 0 ce lls 2 0 ce lls 4 0 ce lls 8 0 ce lls 6 4 0 ce lls 5 , 1 2 0 ce lls 4 0 , 9 6 0 ce lls 3 2 7 , 6 8 0 ce lls 2 , 6 2 1 , 4 4 0 ce lls Bacterial Culture Growth Cycle Bacterial Culture Growth Cycle Stationary phase: Curve horizontal: population growth ceases New cells made at same rate as old cells die (growth rate = death rate) Reasons for stationary phase: Nutrient limitation or O2 limitation Accumulation of toxic wastes Cell density Bacterial Culture Growth Cycle Bacterial Culture Growth Cycle Stationary phase: Very common in nature (oligotrophic) Not simply a time when things run out and cell enters a stasis­ we see changes in: Gene expression: starvation proteins and other proteins, as well as antibiotics, are produced Peptidoglycan cross­linking Nuceloid condensation Endospores formed by certain species Changes make them more resistant to unfavorable conditions Bacterial Culture Growth Cycle Bacterial Culture Growth Cycle Death (Decline) phase: Number of viable cells decreases exponentially Constant number of cells die per hour Usually a logarithmic, but not always so clear Bacterial cell death is defined by the inability to grow (reproduce) Death (Decline) phase: Bacterial Culture Growth Cycle Bacterial Culture Growth Cycle What causes cell death or loss of viability? Build up wastes/toxins and poor environmental conditions for survival Survival of the fittest to reproduce Viable But Not Culturable (VNBC) Temporarily unable to grow under lab conditions but resuscitate upon entry into different environment Programmed Cell Death Certain % of cells that commit suicide to provide nutrients to survivors Bacterial Death/Lossof Viability Bacterial Death/Loss of Viability Continuous Culture Systems Continuous Culture Systems Continuous growth = a constant state of the growth curve; often exponential phase Achieved in a chemostat chamber: Fresh growth medium added at same rate as spent medium and cells are removed Removes pressure of limiting nutrient Also removes toxic waste build­ups Growth rate is adjusted by exchange rate Model Chemostat Model Chemostat Population level and generation time are controlled by dilution rate Increase dilution = increase generation time because less of limiting nutrient is available; density kept low­ most energy used for maintenance, not reproduction Decrease dilution = decrease generation time because little limitation of nutrient; density also increase Measuring Microbial Growth Measuring Microbial Growth By cell number By cell mass Viable vs. Total Direct Microscopic Counts Coulter Cell Counters Viable Counts Calculations and conversions Total cell weight, or by individual chemical (carbon, protein, etc.) Turbidity Direct Microscopic Counts Direct Microscopic Counts Petroff­Hauser Counting Chamber Accepts fixed volume (0.1 ml) Count number of cells per volume Cannot distinguish live from dead cells Calculations done to determine original cells/ml Coulter Counter Coulter Counter Automated counting device Microbial suspension directed through a small hole the size of an individual cell Change in electrical resistance when cell passes through the hole = 1 cell counted Can’t distinguish live vs. dead cells or cells from small particles of debris Viable Counts Viable Counts Measures colony­forming units rather than cells (due to possible clumping) Seek statistically “countable” plate having 30­ 300 colonies Serial dilution, plate count, membrane filters Does NOT necessarily count all living bacteria present in the sample­ just those able to grow under certain conditions given Used with spread and pour plates, also membrane filtration Viable Counts Via Membrane Filtration Viable Counts Via Membrane Filtration Turbidity (Cell Mass) Turbidity (Cell Mass) Done by measuring the amount of light scattered by a cell More mass = more scatter (proportional) Uses a spectrophotometer to measure optical density (OD) of the cells Create standard curves to determine population density based on turbidity Turbidity (Cell Mass) Turbidity (Cell Mass) Low population of cells = low scatter = low OD High population of cells = high scatter = high OD Top scale = % transmittance Bottom scale = optical density Turbidity (Cell Mass) Turbidity (Cell Mass) Create standard curves to determine population density based on turbidity Done in conjunction with viable plate counts initially OD700nm is plotted against the number of viable cell counts taken at the same time points to produce the standard curve Future growth can be estimated from this established relationship Species and environmental conditions must be identical as when the original curve was produced Standard Curve Standard Curve ...
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