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Microbial Nutrition and Growth
Chapter 6 Microbial Nutrition and Growth
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Result of microbial growth is discrete colony
Growth Requirements Microbial growth Increase in a population of microbes Result of microbial growth is discrete colony An aggregation of cells arising from single parent cell Reproduction results in growth © 2012 Pearson Education Inc. 2
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Colonies on a plate
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Microbes obtain nutrients from variety of sources
Growth Requirements Organisms use a variety of nutrients for their energy needs and to build organic molecules and cellular structures Most common nutrients contain necessary elements such as carbon, oxygen, nitrogen, and hydrogen Microbes obtain nutrients from variety of sources © 2012 Pearson Education Inc. 4
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Nutrients: Chemical and Energy Requirements
Growth Requirements Nutrients: Chemical and Energy Requirements Sources of carbon, energy, and electrons Two groups of organisms based on source of carbon Autotrophs Heterotrophs What is our source of carbon? What is a plant’s source of carbon? Two groups of organisms based on source of energy Chemotrophs Phototrophs © 2012 Pearson Education Inc. 5
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Nutrients: Chemical and Energy Requirements
Growth Requirements Nutrients: Chemical and Energy Requirements Oxygen requirements Obligate aerobes—organisms that cannot live without oxygen. Obligate anaerobes—organisms that cannot live in the presence of oxygen. Facultative anaerobes—can live with or without oxygen. Aerotolerant anaerobes—do not use aerobic metabolism, but can tolerate oxygen. © 2012 Pearson Education Inc. 6
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Nutrients: Chemical and Energy Requirements
Growth Requirements Nutrients: Chemical and Energy Requirements Oxygen requirements Oxygen is essential for obligate aerobes Oxygen is deadly for obligate anaerobes How can this be true? Toxic forms of oxygen are highly reactive and excellent oxidizing agents Resulting oxidation causes irreparable damage to cells © 2012 Pearson Education Inc. 7
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Nutrients: Chemical and Energy Requirements
Growth Requirements Nutrients: Chemical and Energy Requirements Oxygen requirements Two common toxic forms of oxygen Superoxide radicals (O2-) Peroxide anion (O22-) Organisms that grow in the presence of oxygen must have enzymes that can break down the toxic forms of oxygen. © 2012 Pearson Education Inc. 8
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Growth Requirements Superoxide radicals (O2-) 2O2- + 2H+ H2O2 + O2
Superoxide dismutase 2O2- + 2H+ H2O2 + O2 Peroxide anion (O22-) Catalase 2H2O2 2H2O + O2 Also peroxidase can break down H2O2 Other than these enzymes, aerobes use antioxidants such as vitamins C and E in order to protect against toxic oxygen products.
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Figure 6.2 Catalase test
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Figure 6.3 Oxygen requirements of organisms-overview
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Nutrients: Chemical and Energy Requirements
Growth Requirements Nutrients: Chemical and Energy Requirements Nitrogen requirements Anabolism often ceases because of insufficient nitrogen All cells recycle nitrogen from amino acids and nucleotides Nitrogen fixation by certain bacteria is essential to life on Earth because most organisms cannot use nitrogen gas (N2) even though it is the most makes up 79% of our atmosphere. © 2012 Pearson Education Inc. 12
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Nutrients: Chemical and Energy Requirements
Growth Requirements Nutrients: Chemical and Energy Requirements Other chemical requirements Phosphorus Sulfur Trace elements Required only in small amounts © 2012 Pearson Education Inc. 13
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Physical Requirements
Growth Requirements Physical Requirements Temperature Effect of temperature on proteins If temperature is too high, proteins can denature. If temperature is too low, proteins may not be active. Effect of temperature on membranes of cells and organelles If too low, membranes become rigid and fragile If too high, membranes become too fluid © 2012 Pearson Education Inc. 14
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Figure 6.4 Microbial growth-overview
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Temperature Requirements
Psychrophiles—grow best at temperatures below 15oC. Mesophiles—grow best in temperatures ranging from 20oC to 40oC. Thermophiles—grow at temperatures above 45oC. Hyperthemophiles—grow at temperatures above 80oC.
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Celsius to Fahrenheit oC x 9/5 + 32 37oC = 98.6oF ~30oC = skin temperature ~25oC = room temperature 65oC = will denature most enzymes
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Thermophiles Mesophiles Hyperthermophiles Growth rate Psychrophiles
Figure 6.5 Four categories of microbes based on temperature ranges for growth Thermophiles Mesophiles Hyperthermophiles Growth rate Psychrophiles Temperature (°C)
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Figure 6.6 An example of psychrophile-overview
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Quick Lab—comparison of temperature requirements among microbes.
Each group of three will receive a TSA plate and will divide it into four quadrants with a wax pencil. One quadrant will be spot-inoculated with S. epidermidis, one with S. marcescens, and one with Bacillus stearothermophilus. Some plates will be incubated overnight at 25oC, some at 37oC, and some at 65oC. Make sure your inoculum is not too heavy; in other words, don’t put a massive hunk of bacteria onto the plate.
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Physical Requirements
Growth Requirements Physical Requirements pH Organisms are sensitive to changes in acidity Neutrophiles grow best in a narrow range around neutral pH Acidophiles grow best in acidic habitats Alkalinophiles live in alkaline soils and water © 2012 Pearson Education Inc. 21
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Physical Requirements
Growth Requirements Physical Requirements Physical effects of water Microbes require water to dissolve enzymes and nutrients Water is important reactant in many metabolic reactions Most cells die in absence of water Some have cell walls that retain water Endospores cease most metabolic activity Two physical effects of water Osmotic pressure Hydrostatic pressure © 2012 Pearson Education Inc. 22
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Physical Requirements
Growth Requirements Physical Requirements Physical effects of water Osmotic pressure Pressure exerted on a semipermeable membrane by a solution containing solutes that cannot freely cross membrane Hypotonic solutions have lower solute concentrations Hypertonic solutions have greater solute concentrations Restricts organisms to certain environments Obligate and facultative halophiles—salt-loving organisms. Staphylococcus aureus can colonize the skin because it can tolerate higher salt concentrations. © 2012 Pearson Education Inc. 23
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Physical Requirements
Growth Requirements Physical Requirements Physical effects of water Hydrostatic pressure Water exerts pressure in proportion to its depth Barophiles live under extreme pressure Their membranes and enzymes depend on pressure to maintain their shape © 2012 Pearson Education Inc. 24
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Associations and Biofilms
Growth Requirements Associations and Biofilms Organisms live in association with different species Antagonistic relationships Synergistic relationships—both organisms benefit Symbiotic relationships—both organisms benefit to the extent that they do not normally live outside the relationship. We are dependent on the bacteria colonizing our bodies! © 2012 Pearson Education Inc. 25
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Associations and Biofilms
Growth Requirements Associations and Biofilms Biofilms Complex relationships among numerous microorganisms Develop an extracellular matrix Adheres cells to one another Allows attachment to a substrate Sequesters nutrients May protect individuals in the biofilm Form on surfaces often as a result of quorum sensing—microorganisms attracted to others already present Many microorganisms more harmful as part of a biofilm © 2012 Pearson Education Inc. 26
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Figure 6.7 Plaque (biofilm) on a human tooth
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Culturing Microorganisms
Inoculum introduced into medium Environmental specimens Clinical specimens Stored specimens Culture Act of cultivating microorganisms or the microorganisms that are cultivated © 2012 Pearson Education Inc. 28
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Figure 6.8 Characteristics of bacterial colonies-overview
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Culturing Microorganisms
Obtaining Pure Cultures Cultures composed of cells arising from a single progenitor Progenitor is termed a CFU Aseptic technique prevents contamination of sterile substances or objects Two common isolation techniques Streak plates Pour plates © 2012 Pearson Education Inc. 30
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Figure 6.9 Streak plate method of isolation-overview
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Streak plate Good and bad
For MICR 2909 Lecture 2, 2001 Streak plate Good and bad At all costs, there must be prevention of contamination. Often use cotton wool stoppers to flasks, tubes. Petri dish: ideal for solid medium and allows gaseous diffusion without dust Development of solid media needed for colony formation initially used surface of freshly cut vegetables eg potato gelatin (1881) used low melting point (< 37oC) protein, therefore a nutrient agar-agar (Hess, Koch’s lab) polysaccharide - nutritionally inert melts 100oC ; solidifies ~40oC Streak Plate Method of Isolation Purpose The streak plate technique is the most widely used method of obtaining isolated colonies from a mix of cultures. Principle The streak plate technique is essentially a method to dilute the number of organisms, decreasing the density. This allows for individual colonies to be isolated from other colonies. Each colony is considered "pure," since theoretically, the colony began with an individual cell. Additional Information (see also p. 53 in the lab text for diagrams.) 1. Begin with inoculating the first, or primary, quadrant of the agar plate. Use a light touch. Don't penetrate or scrape the agar surface. Cover plate with lid. 2. Flame the loop, cool by touching an uninoculated portion of the surface. 3. Now rotate the plate. Open lid and streak again, following the diagram in the exercise book. Remember: you are picking up growth from quadrant one, and using this as your inoculum for quadrant two. 4. Flame loop; rotate plate, and repeat procedure for quadrants three and four. The proper wrist action and light touch takes practice. BSc(MolBiol) Lect 2.ppt
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Figure 6.10 Pour plate method of isolation-overview
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Culturing Microorganisms
Culture Media Majority of prokaryotes have not been grown in culture medium Three types of general culture media General purpose media Selective media Differential media © 2012 Pearson Education Inc. 34
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Figure 6.12 An example of the use of a selective medium
Bacterial colonies Fungal colonies pH 7.3 pH 5.6
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Figure 6.13 The use of blood agar as a differential medium
Beta-hemolysis Alpha-hemolysis No hemolysis (gamma-hemolysis)
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Figure 6.15 Use of MacConkey agar as a selective and differential medium-overview
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Culturing Microorganisms
Special Culture Techniques Most microorganisms on earth cannot be grown in laboratories! (~99%) Techniques developed for culturing microorganisms Animal and cell culture—microorganisms grown on cell culture because artificial media is inadequate, e.g., leprosy, viruses © 2012 Pearson Education Inc. 38
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Culturing Microorganisms
Preserving Cultures Refrigeration Stores for short periods of time Deep-freezing Stores for years Lyophilization (freeze drying) Stores for decades © 2012 Pearson Education Inc. 39
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Figure 6.17 Binary fission events-overview
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Growth of Microbial Populations
Generation Time Time required for a bacterial cell to grow and divide Dependent on chemical and physical conditions © 2012 Pearson Education Inc. 41
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Four Phases of Microbial growth
Lag phase—bacteria are adjusting to environment. Log phase—rapid growth phase. Bacteria are doubling. Stationary phase—number of dying cells equals number of cells being produced. Death phase—number of dying cells exceeds number of new cells produced.
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Figure 6.20 Typical microbial growth curve
Stationary phase Death (decline) phase Log (exponential) phase Number of live cells (log) Lag phase Time
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