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Microbial Growth Factors that influence growth: Physical/Environmental Chemical/Nutritional Microbial adaptations are remarkable Extremeophiles May adapt to a point of no return Temperature Microbes within different ranges (-200C – 1200C) Min and max typically ~ 30°C apart Optimum temp closer to max than min Psychrophiles: Psychrotrophs (moderate psychrophiles) Optimum temperature ~15°C Optimum temperature ~25°C Mesophiles: Optimum temperature ~37°C Thermophiles: Optimum temperature ~60°C Hyperthermophiles: Optimum temperature above 70°C Usually Archaea pH Most microbes are neutrophiles (6.5 – 7.5) Some bacteria are considered acid tolerant Optimum pH of most bacteria is 7 Helicobacter pylori Typically fungi grow over a wider pH range Usually responsible for spoilage of acidic foods Acidophiles optimum is lower (below 5.5) Sulfolobus - Archaea from acidic hot springs Lactobacillus – Bacteria produces lactic acid Thiobacillus - Bacteria produces sulfuric acid Alkalophiles have optimum above 8.0 Bacillus alcalophilus ~ 10.5 Vibrio cholerae prefers pH of 9.0 outside host Water Activity Osmotic Pressure Adaptations inclusion bodies, compatible solutes, stretch receptors Facultative halophiles Obligate halophiles Osmotolerant/Halotolerant Staphylococcus aureus Fungi tend to be more tolerant than other microbes Most marine microbes Extreme halophiles Salt flats of Utah and Dead Sea All organisms need: Macroelements CHNOPS Microelements K, Ca, Cl, Na Trace elements Growth factors Carbon One of the most important growth requirements ½ the dry weight of a bacteria cell is carbon Carbon skeleton base of organic compounds Hydrocarbons Metabolic Diversity Organisms grouped by energy, carbon and electron source Energy Source Carbon Source Phototroph or Chemotroph Autotroph or Heterotroph Electron Source Lithotroph or Organotroph Photolithoautotroph Chemoorganoheterotroph CO2, inorganic chemicals and inorganic e- donor Photoorganoheterotroph Organic carbon, organic chemicals and organic e- donor Chemolithoautotroph CO2, Light and inorganic e- donor Organic carbon, light and organic e- donor Chemolithoheterotroph Organic carbon, inorganic chemicals and inorganic e- donor Oxygen Requirements Oxygen has many toxic forms Organisms require enzyme systems to protect them Superoxide (O2-) radical Neutralized by superoxide dismutase (SOD) 2 O2- + 2 H+ → H2O2 + O2 Peroxide Detoxified by catalase or peroxidase Catalase: H2O2 → H2O + O2 Peroxidase: H2O2 + 2 H+ → 2 H2O Obligate (Strict) Aerobes only aerobic metabolism Have SOD and catalase or peroxidase Obligate (Strict) Anaerobes Destroyed by oxygen Do not have SOD, catalase or peroxidase only anaerobic metabolism Facultative Anaerobes Have SOD and catalase or peroxidase Grow with or without oxygen aerobic or anaerobic metabolism Grow faster in the presence of O2 Aerotolerant Anaerobes Have SOD Grow with or without oxygen only anaerobic metabolism Grow faster in the absence of O2 Microaerophiles Grow only in low levels O2 small amounts of SOD and catalase Produce toxic levels of superoxide free radicals and peroxide at high levels of O2 only aerobic metabolism Growth in liquid media Obligate Aerobes Facultative Anaerobes Obligate Anaerobes Aerotolerant Anaerobes Microaerophiles Nitrogen Needed for amino acids, nucleic acids, and ATP Amino acids from protein degradation Nitrogen reduction Reduce nitrate to ammonia then utilize the ammonia Nitrogen fixation assimilate gaseous nitrogen ( N2) Sulfur Needed for building some amino acids (cysteine and methionine), vitamins (thiamine and biotin) and some carbohydrates Sulfur containing amino acids from protein degradation Reduce sulfates (SO42-) or sulfides (H2S) Phosphorus Tends to be a limiting growth requirement Phosphorus is needed for building nucleic acids, phospholipids, and ATP Phosphate ion (PO43-)is an important source Microelements Several are essential for proper cell function Signal molecules, membrane potential, enzyme cofactors Trace Elements Minerals required in very small amounts Iron, copper and zinc Growth Factors Organic compounds essential growth Cannot be synthesized by microorganisms Vitamins Amino acids Nucleic acid bases Fastidious Neisseria 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: Exact composition known Complex media: Exact composition varies Selective media: Favors the growth of desired microorganisms Inhibits the growth of unwanted ones Differential media: Distinguishes between groups of microorganisms MacConkey’s Agar 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 Salmonella Differential media Blood agar Alpha Hemolysis Beta Hemolysis Gamma Hemolysis Microbial Growth Refers to increase in number of cells not size of individual cells Bacteria typically reproduce by binary fission Generation time time required for a bacterial population to double Typically 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 20= 21= 22= 23= 1 2 4 8 cell cells cells cells Phases of Bacterial Growth Curve In closed system or batch culture Lag phase Log phase Stationary phase Decline phase Phases of the growth curve can be observed in liquid media Solid media, different colonies in different phases Continuous cultures Open system Measuring Bacterial Growth Direct Methods Viable plate count, Membrane filtration, Microscopic count, Most Probable Number (MPN), Electronic Counters Indirect Methods Turbidity, Metabolic activity, Weight Direct Methods Viable Plate Count 1. Important to limit colonies to a countable number • • • • • 30-300 colonies (CFUs) Serial dilutions ensure colony counts within range Advantage: only living cells Disadvantage: incubation time, growth requirements, may underestimate count Plate count methods pour-plates Spread-plates methods 2. Membrane Filtration Sample (liquid) passed through filter Filter placed on surface of solid medium Organisms retained on filter will grow Advantage: only living cells, can be used to count low cell concentrations Disadvantage: must have at least 100 ml of media, requires incubation time, may underestimate count 3. Microscopic Count Known volume of sample placed in counting chamber Viewed under microscope Cells counted Advantage: no incubation time is required Disadvantage: dead cells may be counted, tedious, requires a high concentration of cell (10 million per ml) 4. Most probable numbers (MPN) Multi-tube statistical assay Advantage: measures only living cells, useful for culturing cells that wont grow on solid media Disadvantage: incubation time, expensive & time consuming Series of dilution sets Each set inoculated with 10X less sample than previous set Incubated and results compared to MPN table gives statistical estimation of cell concentration 5. Electronic Counter Coulter Counter – electrical current Flow Cytometry – light transmission Advantage: No incubation time Disadvantage: dead cells may be counted, not very sensitive due to clumping and debris in media Indirect methods 1. Turbidity Uses spectrophotometer Advantage: no incubation time Disadvantage: must have high concentration of cells, may count dead cells Measures light transmitted through sample Measurement is inversely proportional to cell concentration 2. Weight Wet weight Cells centrifuged and packed cells weighed Dry weight Packed cells dried at 100°C for 8 to 12 hours then weighed Disadvantage: Tedious and time consuming Advantage: Useful in measuring filamentous organisms 3. Metabolic Activity Based on enzyme activity Advantage: once metabolic rate is established provides reliable estimate of cell number Disadvantage: requires incubation time, requires metabolic rate be established in advance