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Microbial Nutrition and Growth
6 Microbial Nutrition and Growth
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Growth Requirements Microbial growth Results of microbial growth
Increase in a population of microbes Due to reproduction of individual microbes Results of microbial growth Discrete colony—an aggregation of cells arising from single parent cell Biofilm—collection of microbes living on a surface in a complex community
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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
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Growth Requirements Nutrients: Chemical and Energy Requirements
Sources of Carbon, Energy, and Electrons Two groups of organisms based on source of carbon: Autotrophs Heterotrophs Two groups of organisms based on source of energy: Chemotrophs Phototrophs
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Chemical compounds (chemo-)
Figure 6.1 Four basic groups of organisms based on their carbon and energy sources. Energy source Light (photo-) Chemical compounds (chemo-) Photoautotrophs • Plants, algae, and cyanobacteria use H2O as an electron source to reduce CO2, producing O2 as a by-product • Green sulfur bacteria and purple sulfur bacteria use H2S as an electron source; they do not produce O2 Carbon dioxide (auto-) Chemoautotrophs • Hydrogen, sulfur, and nitrifying bacteria, some archaea Carbon source Organic compounds (hetero-) Photoheterotrophs • Green nonsulfur bacteria and purple nonsulfur bacteria, some archaea Chemoheterotrophs • Aerobic respiration: most animals, fungi, and protozoa, and many bacteria • Anaerobic respiration: some animals, protozoa, bacteria, and archaea • Fermentation: some bacteria, yeasts, and archaea
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Growth Requirements Nutrients: Chemical and Energy Requirements
Sources of Carbon, Energy, and Electrons Two groups of organisms based on source of electrons: Organotrophs Lithotrophs
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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
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Growth Requirements Nutrients: Chemical and Energy Requirements
Oxygen Requirements Four toxic forms of oxygen: Singlet oxygen Superoxide radicals Peroxide anion Hydroxyl radical
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Figure 6.2 Catalase test.
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Growth Requirements Nutrients: Chemical and Energy Requirements
Oxygen Requirements Many organisms can live in various oxygen concentrations Aerobes Anaerobes Facultative anaerobes Aerotolerant anaerobes Microaerophiles
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Oxygen concentration Loose- fitting cap High Low Obligate aerobes
Figure 6.3 Using a liquid thioglycolate growth medium to identify the oxygen requirements of organisms. Oxygen concentration Loose- fitting cap High Low Obligate aerobes Obligate anaerobes Facultative anaerobes Aerotolerant anaerobes
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Growth Requirements Nutrients: Chemical and Energy Requirements
Nitrogen Requirements Anabolism often ceases due to insufficient nitrogen Nitrogen acquired from organic and inorganic nutrients All cells recycle nitrogen for amino acids and nucleotides Nitrogen fixation by certain bacteria is essential to life on Earth
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Growth Requirements Nutrients: Chemical and Energy Requirements
Other Chemical Requirements Phosphorus Sulfur Trace elements Only required in small amounts Growth factors Necessary organic chemicals that cannot be synthesized by certain organisms
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Table 6.1 Some Growth Factors of Microorganisms and Their Functions
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Growth Requirements Physical Requirements Temperature
Temperature affects three-dimensional structure of proteins Lipid-containing membranes of cells and organelles are temperature sensitive If too low, membranes become rigid and fragile If too high, membranes become too fluid
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Figure 6.4 The effects of temperature on microbial growth.
Growth rate Minimum Maximum Temperature Optimum 22ºC 30ºC 37ºC
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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 –10 10 20 30 40 50 60 70 80 90 100 110 120 Temperature (ºC)
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Figure 6.6 An example of a psychrophile.
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Growth Requirements Physical Requirements pH
Organisms sensitive to changes in acidity Hydrogen ions interfere with H bonding Neutrophiles grow best in a narrow range around neutral pH Acidophiles grow best in acidic habitats Alkalinophiles live in alkaline soils and water
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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 and cysts cease most metabolic activity Two physical effects of water: Osmotic pressure Hydrostatic pressure
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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 the membrane Hypotonic solutions have lower solute concentrations Cell placed in hypotonic solution swells Hypertonic solutions have greater solute concentrations Cell placed in hypertonic solution shrivels Restricts organisms to certain environments Obligate and facultative halophiles
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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 three-dimensional, functional shape
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Growth Requirements Associations and Biofilms
Organisms live in association with different species: Antagonistic relationships Synergistic relationships Symbiotic relationships
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Growth Requirements Associations and Biofilms Biofilms
Complex relationships among numerous microorganisms Form on surfaces, medical devices, mucous membranes of digestive system Form as a result of quorum sensing Many microorganisms more harmful as part of a biofilm Scientists seeking ways to prevent biofilm formation
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Figure 6.7 Biofilm development.
1 Free-swimming microbes are vulnerable to environmental stresses. Water flow Chemical structure of one type of quorum-sensing molecule Water channel Bacteria Escaping microbes Matrix Some microbes land on a surface, such as a tooth, and attach. The cells begin producing an extracellular matrix and secrete quorum-sensing molecules. Quorum sensing triggers cells to change their biochemistry and shape. New cells arrive, possibly including new species, and water channels form in the biofilm. Some microbes escape from the biofilm to resume a free-living existence and perhaps, to form a new biofilm on another surface. 2 3 4 5 6
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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
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Figure 6.8 Characteristics of bacterial colonies.
Shape Circular Rhizoid Irregular Filamentous Spindle Margin Entire Undulate Lobate Curled Filiform Elevation Flat Raised Convex Pulvinate Umbonate Crateriform Size Colony Punctiform Small Moderate Large Texture Smooth or rough Appearance Glistening (shiny) or dull Pigmenta- tion Nonpigmented (e.g., cream, tan, white) Pigmented (e.g., purple, red, yellow) Optical property Opaque, translucent, transparent
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Table 6.2 Clinical Specimens and the Methods Used to Collect Them
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Culturing Microorganisms
Obtaining Pure Cultures Cultures composed of cells arising from a single progenitor Progenitor is termed a colony-forming unit (CFU) Aseptic technique prevents contamination of sterile substances or objects Two common isolation techniques: Streak plates Pour plates
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Figure 6.9 The streak-plate method of isolation.
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Figure 6.10 The pour-plate method of isolation.
Sequential inoculations 1.0 ml 1.0 ml 1.0 ml 9-ml broth 9-ml broth 9-ml broth Initial sample 1 ml to each Petri dish, add 9-ml warm agar, swirl gently to mix Colonies Fewer colonies
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Culturing Microorganisms
Obtaining Pure Cultures Other Isolation Techniques Some fungi isolated with streak and pour plates Protozoa and motile unicellular algae isolated through dilution of broth cultures Can individually pick single cell of some large microorganisms and use to establish a culture
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Culturing Microorganisms
Culture Media Majority of prokaryotes have not been grown in culture medium Variety of liquid and solid media used to culture microbes Nutrient broth is a common liquid medium Agar is a common addition to make media solid Used to make Petri plates and slant tubes
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Figure 6.11 Slant tubes containing solid media.
Butt
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Culturing Microorganisms
Culture Media Six types of general culture media: Defined media Complex media Selective media Differential media Anaerobic media Transport media
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Table 6.3 Ingredients of a Representative Defined (Synthetic) Medium for Culturing E. coli
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Culturing Microorganisms
Culture Media Complex Media Exact chemical composition is unknown Nutrients commonly derived from breakdown of yeast, beef, soy, and proteins Supports growth of a wide variety of microorganisms Useful when nutritional needs of an organism are unknown
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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.
Alpha-hemolysis Beta-hemolysis No hemolysis (gamma-hemolysis)
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Acid fermentation with gas
Figure The use of carbohydrate utilization tubes as differential media. Durham tube (inverted tube to trap gas) No fermentation Acid fermentation with gas
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Table 6.4 Representative Differential Complex Media
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serotype Choleraesuis
Figure The use of MacConkey agar as a selective and differential medium. Escherichia coli Escherichia coli Escherichia coli Staphylococcus aureus Staphylococcus aureus (no growth) Salmonella enterica serotype Choleraesuis Nutrient agar MacConkey agar MacConkey agar
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Figure 6.16 An anaerobic culture system.
Clamp Airtight lid Chamber Palladium pellets to catalyze reaction removing O2 Envelope containing chemicals to release CO2 and H2 Methylene blue (anaerobic indicator) Petri plates
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Culturing Microorganisms
Culture Media Transport Media Used by health care personnel to ensure clinical specimens are not contaminated and to protect people from infection Rapid transport of samples is important
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Culturing Microorganisms
Special Culture Techniques Techniques developed for culturing microorganisms Animal and Cell Culture Used when artificial media is inadequate Viruses only grow within living cells Low-Oxygen Culture Carbon dioxide incubators Candle jars
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Culturing Microorganisms
Preserving Cultures Refrigeration Stores for short periods of time Deep-freezing Stores for years Lyophilization Stores for decades
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Bacterial Growth: Overview
PLAY Bacterial Growth: Overview
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Figure 6.17 Binary fission. Cytoplasmic membrane Chromosome 1
Cell wall Replicated chromosome Septum 30 minutes Completed septum 60 minutes Septum 90 minutes 120 minutes
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Binary Fission PLAY Binary Fission
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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
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Figure 6.18 A comparison of arithmetic and logarithmic growth.
70 70 60 60 50 Species growing arithmetically 50 Species growing logarithmically 40 40 Number of cells Number of cells 30 30 20 20 10 10 1 2 1 2 Time (hours) Time (hours)
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Figure 6.19 Two growth curves of logarithmic growth.
5000 1015 4,096 4000 1010 3000 Number of cells Number of cells (log scale) 2000 105 104 1000 103 512 102 8 64 101 5 10 5 10 Time (hours) Time (hours)
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Figure 6.20 A typical microbial growth curve.
Stationary phase Death (decline) phase Log (exponential) phase Number of live cells (log) Lag phase Time
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Bacterial Growth Curve
PLAY Bacterial Growth Curve
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Growth of Microbial Populations
Continuous Culture in a Chemostat Chemostat used to maintain a microbial population in a particular phase of growth Open system Requires addition of fresh medium and removal of old medium Used in several industrial settings
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Figure 6.21 Schematic of chemostat.
Fresh medium with a limiting amount of a nutrient Flow-rate regulator Sterile air or other gas Culture vessel Culture Overflow tube
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Growth of Microbial Populations
Measuring Microbial Reproduction Direct Methods Not Requiring Incubation Microscopic counts
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Figure 6.22 The use of a cell counter for estimating microbial numbers.
Cover slip Pipette Bacterial suspension Location of grid Overflow troughs Place under oil immersion Bacterial suspension 1 mm 1 mm
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Growth of Microbial Populations
Measuring Microbial Reproduction Direct Methods Not Requiring Incubation Electronic counters Coulter counters Counts cells as they interrupt an electrical current Flow cytometry Detects changes in light transmission as cells pass a detector
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Growth of Microbial Populations
Measuring Microbial Reproduction Direct Methods Requiring Incubation Serial dilution and viable plate counts Membrane filtration Most probable number
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Figure 6.23 A serial dilution and viable plate count for estimating microbial population size.
1 ml of original culture 1 ml 1 ml 1 ml 1 ml 9 ml of broth + 1 ml of original culture 1:10 dilution (10–1) 1:100 dilution (10–2) 1:1000 dilution (10–3) 1:10,000 dilution (10–4) 1:100,000 dilution (10–5) 0.1 ml of each transferred to a plate 0.1 ml 0.1 ml 0.1 ml 0.1 ml Incubation period Too numerous to count (TNTC) TNTC 65 colonies 6 colonies 0 colonies
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Figure 6.24 The use of membrane filtration to estimate microbial population size.
Membrane transferred to culture medium Sample to be filtered Membrane filter retains cells To vacuum Incubation Colonies
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1 ml 1 ml Undiluted 1:10 1:100 Inoculate 1 ml into each of 5 tubes
Figure The most probable number (MPN) method for estimating microbial numbers. 1 ml 1 ml Undiluted 1:10 1:100 Inoculate 1 ml into each of 5 tubes Phenol red, pH color indicator, added Incubate Results 4 tubes positive 2 tubes positive 1 tube positive
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Table 6.5 Most Probable Number Table (Partial)
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Growth of Microbial Populations
Measuring Microbial Growth Indirect Methods Turbidity
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Figure 6.26 Turbidity and the use of spectrophotometry in indirectly measuring population size.
Direct light Light source Uninoculated tube Light-sensitive detector Light source Inoculated broth culture Scattered light that does not reach reflector
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Growth of Microbial Populations
Measuring Microbial Growth Indirect Methods Metabolic activity Dry weight Genetic methods Isolate DNA sequences of unculturable prokaryotes
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Micro Matters In the Micro Matters video in chapter 6, Jen and Mike learn about the cause of listeriosis, Listeria monocytogenes. Listeria monocytogenes survives in a variety of environmental conditions. Listeria monocytogenes can cause central nervous system (CNS) infections in individuals with a weakened immune system. Listeria monocytogenes can be identified using selective and differential media. Some antibiotics used to treat listeriosis are inhibitors of bacterial enzymes.
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