Chapter 4 Lecture Outline

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Presentation transcript:

Chapter 4 Lecture Outline Bacterial Culture, Growth, and Development

Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc. Microbial Nutrition Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc.

Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc. Microbial Nutrition All life requires: Materials, to make cell parts Nutrients (C and others) Energy, to move electrons Electron flow, to drive all life processes Drives ions into, out of cells Used to create ATP Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc.

Microbial Nutrition: Nutrients Must be supplied from environment Macronutrients Major elements in cell macromolecules C, O, H, N, P, S Ions necessary for protein function Mg2+, Ca2+, Fe2+, K+ Micronutrients Trace elements necessary for enzyme function Cobalt, manganese, nickel, zinc, etc. Additional complex growth factors for fastidious organisms Amino acids, haemin, NAD, etc Some bacteria need nitrogen as N2 gas from air Some bacteria cannot be grown on artificial media Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc.

Obligate Intracellular Bacteria SEM Giemsa Stain Rickettsia Chicken fibroblast Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc.

Microbial Nutrition: Source of Carbon Heterotrophy Organic compounds Generates and releases CO2 Autotrophy CO2 from air CO2 fixation Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc.

Microbial Nutrition: Source of Energy Phototrophs Light energy Light absorption excites electrons to high energy state Perform photosynthesis Chemotrophs Chemical oxidation –reduction reactions Transfer electrons from high energy compounds to make products of lower energy Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc.

Microbial Nutrition: Electron Source Lithotrophs Inorganic molecules are electron donors Sulfur, iron, etc. Organotrophs Organic molecules are electron donors Glucose etc. Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc.

Microbial Nutrition: Summary for Prefixes for Term “-trophy” Carbon source for biomass Auto- Hetero- Energy source Photo- Chemo- Electron source Litho- Organo- Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc.

Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc.

Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc. Nutrient Up-Take Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc.

Nutrient Uptake: Diffusion Passive diffusion Some gases pass freely through membranes O2, CO2 Follows gradient of material Facilitated diffusion Transporters pass material into/out of cell Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc.

Nutrient Uptake—Active Transport Symport and Antiport Gradient of one molecule transports another Electron transport creates Proton-Motive Force PMF transports other molecules Transports material against its gradient Symport: Gradient of pumps in same direction Antiport: Gradient of pumps in opposite direction Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc.

Nutrient Uptake—Active Transport ABC Transporters ATP Binding Cassette Use ATP energy to pass material into cell Transport material against gradient Used for uptake and efflux SBP only for up-take Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc.

Siderophores and Iron Up-Take System Outer membrane protein Siderophores have high affinity for soluble ferric ion Periplasmic solute-binding protein ABC transporter Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc.

Nutrient Uptake—Active Transport Phosphotransferase System (PTS) Uses ATP energy to pass material into cell Modifies material as it enters cell Gradient is maintained, pushing material into cell glucose enters cell and is phosphorylated. As a result, gradient of pushes more glucose inside. (glucose-6-phosphate) cannot pass out of cell. Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc.

Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc. Culturing Bacteria Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc.

Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc. Culturing Bacteria Culture media has all materials necessary for growth Varies for different bacterial species Electron source Energy source If not phototrophic Carbon source If not autotrophic Nitrogen source If not N2-fixer Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc.

Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc.

Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc. Growth Media Complex media: Exact composition unknown Examples: Beef bouillon, yeast extract Enriched media Contain in addition blood components Defined synthetic media: Exact composition known Examples: Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc.

Selective and Differential Media Reveal differences in metabolism Selective Suppresses growth of unwanted microbes Differential Includes ingredients to detect certain biochemical reactions MacConkey Selective and differential Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc.

Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc. Isolating Bacteria Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc.

Obtaining Pure Cultures Dilution streaking Streak cells on plate Agar inhibits spread of microbes on plate All cells in colony derive from single cell Genetically identical Clone of that original cell Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc.

Obtaining Pure Cultures Dilution in liquid culture Reduces number of cells in each tube Spread liquid on plate to see single colonies Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc.

Determining the Concentration of Bacteria Counting Petroff-Hauser chamber Counts cells directly Gives accurate number Can’t tell if cells are alive or dead Use stain to distinguish living cells Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc.

Determining the Concentration of Bacteria Spectrophotometer Measures optical density “Shadow” of bacteria Gives rapid measurement Can’t tell if cells are alive or dead Solution must be at 107–1010 cells/ml Drawing of light bulb Photodetector Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc.

Determining the Concentration of Bacteria Viable counts Counts only cells able to reproduce Form colonies Colony forming units (CFU/ml) Assumes single cell suspension Requires time to form colonies (overnight) Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc.

Bacterial Growth Curve Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc.

Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc. The Growth Cycle Lag phase Cells synthesizing materials, not dividing Log phase = exponential growth 1  2  4  8  16 … 10 doublings increases density by ~1000 log10(N) increases linearly Stationary phase Cells no longer growing Death phase Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc.

Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc. Stationary Phase Total number of viable bacteria does not change Changes in gene regulation Quorum sensing induced Biofilm formation Up regulation of virulence factors Spore formation Cell differentiation Species specific Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc.

Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc. Cell to Communication Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc.

Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc. Biofilms Cells secrete material to hold to a surface Cells acting together Multiple species or a single species Cells signal to each other Quorum sensing Protects against dispersion Prevents antibiotics from infiltrating Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc.

Biofilm Formation in Pseudomonas aeruginosa Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc.

Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc. Endospore Formation Cells respond to changing environment Endospores Protect against bad conditions Disseminates cells Forms inside (“endo”) mother cell Bacillus and Clostridium species Exosporium Spore coat (resistant, calcium rich) Dipicolinic acid Small acid soluble proteins (DNA stabilizing) Cortex (peptidoglycan) Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc.

Click box to launch animation Cell Differentiation Animation: Endospore Formation Click box to launch animation Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc.

Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc. Cell Differentiation Cells respond to changing environment Heterocysts Different cells produce different nutrients Vegetative cells—energy Heterocysts—fixed nitrogen Myxospores Form inside fruiting body Multicellular structure Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc.

Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc. Cell Differentiation Cells respond to changing environment Actinomycetes form spores Bacteria produce aerial hyphae Specialized structures containing spores When nutrients become limited Protect genetic material Disseminate cells Spores Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc.

Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc. Concept Quiz All bacterial cells need to be supplied with a source of electrons, energy, and nutrients. carbon, nitrogen, and light. carbon, fixed nitrogen, and water. Answer: A Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc.

Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc. Concept Quiz The fastest way to measure cell density is by using a Petroff-Hauser counting chamber. spectrophotometer. petri plate. Answer: B Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc.

Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc. Concept Quiz When food supplies dwindle, cells protect themselves by forming biofilms. heterocysts. spores. Answer: C Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc.