Chapter 6 - Microbial Growth Increase in number of cells...

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Unformatted text preview: Microbial Growth Increase in number of cells, not size of cells Factors that influence growth: Environmental: Temperature, pH, oxygen and water availability Nutritional: Carbon, nitrogen, sulfur, and phosphorus sources, trace elements and growth factors Temperature Different microorganisms grow over different temperature ranges Min and max temp Typically ~ 30°C apart Optimum temp closer to max than min temp Temperature Psychrophiles: Psychrotrophs (moderate psychrophiles) Optimum temperature between 5- 15°C Optimum temperature between 20 - 30°C Mesophiles: Optimum temperature between 25 - 40°C Temperature Thermophiles: Optimum temperature between 50 - 70°C Hyperthermophiles: Optimum temperature 70°C or above Usually Archaea pH Most bacteria prefer range of 5 – 8 Optimum of most bacteria is 7 (neutrophiles) Some are adapted to lower pH Acidophiles optimum is lower (below 5.5) Thiobacillus ferrooxidans obtains energy from FeS Produces sulfuric acid Optimum pH ~ 2 Lactobacillus ferment sugars producing lactic acid acid creates a low pH environment Alkalophiles have optimum above 8.5 Bacillus alcalophilus ~ 10.5 Typically fungi grow over a wider pH range Usually responsible for spoilage of acidic foods Water Availability Osmotic Pressure Facultative halophiles (osmotolerant) survive high salinity Obligate halophiles require high salt concentrations Staphylococcus species Most marine microbes Certain Archaea are extreme halophiles Salt flats of Utah Dead Sea Oxygen Requirements Toxic forms of oxygen: Superoxide (O2-) free radicals: Modified by superoxide dismutase (SOD) 2 O - + 2 H+ → H O + O 2 2 2 2 Peroxide (H2O2 ): Detoxified by catalase or peroxidase Catalase: H O → H O + O 2 2 2 2 Peroxidase: H2O2 + 2 H+ → 2 H2O Obligate (Strict) Aerobes Can only grow in the presence of O2 Have SOD and catalase Can perform aerobic respiration Cannot perform anaerobic respiration or fermentation Obligate (Strict) Anaerobes Can not grow in the presence of O2 Do not have SOD or catalase Can perform anaerobic respiration and/or fermentation Cannot perform aerobic respiration Facultative Anaerobes Grow with or without O2 Have SOD and catalase Can perform aerobic respiration and anaerobic respiration and/or fermentation Grow faster in the presence of O2 Aerotolerant Anaerobes Grow with or without O2 Have SOD Can perform anaerobic respiration and/or fermentation Cannot perform aerobic respiration Grow faster in the absence of O2 Microaerophiles Grow only in low levels O2 Have small amounts of SOD and catalase Produce toxic levels of superoxide free radicals and peroxide at high levels of O2 Can perform aerobic respiration Cannot perform anaerobic respiration or fermentation Growth in liquid media Obligate Aerobes Facultative Anaerobes Obligate Anaerobes Aerotolerant Anaerobes Microaerophiles Growth of prokaryotes depends on nutritional factors as well as physical environment Main factors to be considered are: Required elements Growth factors Energy sources Nutritional diversity Carbon Found in all organic compounds One of the most important growth requirements Autotrophs utilize CO2 ½ the dry weight of a bacteria cell is carbon Carbon fixation Heterotrophs utilize organic compounds Metabolic Diversity Prokaryotes are grouped based on energy and carbon sources Energy utilize the sun’s energy Phototropic utilize chemical energy chemotropic Carbon use inorganic forms of carbon Autotropic require organic molecules Heterotropic Nitrogen Needed for amino acids, nucleic acids, and ATP Utilize amino acids from protein degradation Ammonium ions (NH4+) and nitrates (NO2-) Symbiotic bacteria in the roots of legumes nitrogen fixation - utilization of gaseous nitrogen ( N2) Sulfur Needed for building some amino acids (cysteine and methionine) and vitamins (thiamine and biotin). Sulfates (SO42-), hydrogen sulfide (H2S), and sulfur containing amino acids Phosphorus, Potassium, Calcium, and Magnesium Phosphorus is needed for building nucleic acids, phospholipids, and ATP. Phosphate ion (PO43-)is an important source Potassium, calcium, and magnesium cofactors for enzymes Trace Elements Minerals required in very small amounts Iron, copper, molybdenum, cobalt and zinc Most are enzyme cofactors Organic Growth Factors Organic compounds essential growth Cannot be synthesized by microorganisms Vitamins Amino acids Cultivation Of Microorganisms culture medium inoculation nutrient material prepared for growing microorganisms introduction of a microorganism into medium culture growth of a microorganism observed on/in a medium Types Of Culture Media Chemically defined media: Complex media: Exact composition known Exact composition varies Anaerobic growth media: Contain reducing compound (e.g.fluid thioglycolate) Removes free oxygen Selective media: Favors the growth of desired microorganisms Inhibits the growth of unwanted ones Differential media: Distinguishes between groups of microorganisms MacConkey’s Agar A selective medium: Inhibits Gram-positive bacteria growth Encourages Gram-negative bacteria growth Differential medium: Lactose fermenters produce acid and form pink colonies Non-lactose fermenters form colorless colonies MacConkey’s Agar Escherichia coli Salmonella Differential media Blood agar Streptococcus produce hemolysin to break down RBC Alpha Hemolysis Beta Hemolysis Enrichment media: Selectively enhance the growth of desired microorganisms to detectable levels Microbial Growth Refers to increase in number of cells not size of individual cells Bacteria normally reproduce by binary fission Exceptions are budding and fragmenting Generation time – the time required for a bacterial population to double Most bacteria have a generation time of 1-3 hours Generation number is expressed as a power of 2 Original cell is 20, 2nd generation (after one cell division) would be 21 and so on. Phases of The Bacterial Growth Curve Lag phase Log phase Stationary phase Decline phase Due to build up of wastes and depletion of nutrients Phases Of The Bacterial Growth Curve Growth Curve in Liquid and Solid Media Phases of the growth curve can be observed in liquid media On solid media, different parts of the colony are in different phases Continuous cultures Monitoring Bacterial Growth Direct Methods For Bacterial Counts Plate counts Filtration Direct microscopic count Indirect methods for bacterial counts Turbidity Metabolic activity Dry weight Direct Methods Plate counts 1. advantage: measures only living cells Disadvantage: incubation time, various growth requirements Plate counts are often reported as colony forming units (CFUs) It is important to limit the number of colonies that develop on a plate to a countable number • • • • • • 30-300 colonies Serial dilutions ensure colony counts are within range Count based on assumption the one cell gives rise to one colony Ideal number to count Number of colonies = number of cells in sample Between 30 and 300 colonies Sample normally diluted in 10-fold increments Plate count methods pour-plates Spread-plates methods 2. Membrane Filtration Sample (liquid) is passed through filter with pore size of 0.2-0.45 µm Filter placed on surface of solid medium Organisms retained on filter will grow after incubation Advantage: only counts live bacteria, can be used to count low cell concentrations Disadvantage: must have at least 100 ml of media Known volume of liquid passed through membrane filter Filter pore size retains organism Filter is placed on appropriate growth medium and incubated Colonies are counted 3. Direct Microscopic Count Advantage: no incubation time is required Disadvantage: dead cells may be counted, difficult to count motile bacteria, requires a high concentration of cell (10 million per ml) Number is measured in a know volume Liquid dispensed in specialized slide Counting chamber Viewed under microscope Cells counted 4. Most probable numbers (MPN) Statistical assay Multi-tube test advantage: measures only living cells Disadvantage: incubation time, estimation of cell concentration Series of dilution sets Each set inoculated with 10-fold less sample than previous set Sets incubated Results compared to MPN table gives statistical estimation of cell concentration Indirect methods 1. Turbidity Uses spectrophotometer Disadvantage Must have high number of cells Advantage May be used to generate standard growth curve Measures light transmitted through sample Measurement is inversely proportional to cell concentration Must be used in conjunction with other test once to determine cell numbers 2. Total Weight Disadvantage: Tedious and time consuming Not routinely used Advantage: Useful in measuring filamentous organisms Wet weight Cells centrifuged down and liquid growth medium removed Packed cells weighed Dry weight Packed cells dry at 100°C 8 to 12 hours Cells weighed 3. Detecting metabolic products Acid production pH indicator detects acids that result from sugar breakdown Gas production Gas production monitored using Durham tube Tube traps gas produced by bacteria ...
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This note was uploaded on 02/21/2008 for the course BIOLOGY 380 taught by Professor Littlejohn during the Spring '06 term at University Of Southern Mississippi .

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