Post on 18-Jan-2016
PowerPoint® Lecture Presentations prepared byMindy Miller-Kittrell,North Carolina State University
C H A P T E R
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6
Microbial Nutrition and Growth
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Growth Requirements
• Microbial growth
• Increase in a population of microbes
• Due to reproduction of individual microbes
• Result of microbial growth is discrete colony
• An aggregation of cells arising from single parent cell
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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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Figure 6.1 Four basic groups of organisms based on their carbon and energy 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 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
• Aerobes
• Anaerobes
• Facultative anaerobes
• Aerotolerant anaerobes
• Microaerophiles
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Loose-fittingcap
OxygenconcentrationHigh
LowObligateaerobes
Obligateanaerobes
Facultativeanaerobes
Aerotolerantanaerobes
Figure 6.3 Using a liquid thioglycollate growth medium to identify the oxygen requirements of organisms.
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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 from 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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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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Maximum
Optimum
Minimum
37ºC30ºC22ºC
Figure 6.4 The effects of temperature on microbial growth.
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Figure 6.5 Four categories of microbes based on temperature ranges for growth.
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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
• H+ and OH– 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
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.
Free-swimming microbes are vulnerable to environmentalstresses.
Bacteria
Some microbes land on a surface, such as a tooth, and attach.
The cells begin producing an intracellular 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, form a new biofilm on another surface.
Chemical structure of one type of quorum- sensing molecule
Matrix
Water flow
Water channel
Escapingmicrobes
1
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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Shape
Circular Rhizoid Irregular Filamentous Spindle
Margin
Elevation
Size
Texture
Appearance
Pigmenta-tion
Opticalproperty
Entire Undulate Lobate Curled Filiform
Flat Raised Convex Pulvinate Umbonate
Punctiform Small Moderate Large
Glistening (shiny) or dull
Nonpigmented (e.g., cream, tan, white)Pigmented (e.g., purple, red, yellow)
Opaque, translucent, transparent
Smooth or rough
Colony
Figure 6.8 Characteristics of bacterial colonies.
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© 2014 Pearson Education, Inc.
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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Sequential inoculations
1.0 ml
Initialsample
9 mlbroth
Colonies Fewer colonies
1.0 ml to eachPetri dish, add9 ml warm agar,swirl gently to mix
1.0 ml 1.0 ml
9 mlbroth
9 mlbroth
Figure 6.10 The pour-plate method of isolation.
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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
• Agar is a common addition to many media
• Six types of general culture media
• Defined media
• Complex media
• Selective media
• Differential media
• Anaerobic media
• Transport media
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Slant
Butt
Figure 6.11 Slant tubes containing solid media.
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© 2014 Pearson Education, Inc.
Bacterial colonies Fungal colonies
pH 7.3 pH 5.6
Figure 6.12 An example of the use of a selective medium.
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Beta-hemolysis
Alpha-hemolysis
No hemolysis(gamma-hemolysis)
Figure 6.13 The use of blood agar as a differential medium.
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Durham tube(inverted tubeto trap gas)
Acid fermentationwith gas
No fermentation
Figure 6.14 The use of carbohydrate utilization tubes as differential media.
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Escherichia coli Escherichia coli Escherichia coli
Staphylococcusaureus
Staphylococcusaureus (no growth)
Salmonella entericaserotype Choleraesuis
MacConkey agar MacConkey agarNutrient agar
Figure 6.15 The use of MacConkey agar as a selective and differential medium.
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Airtight lid
Palladium pelletsto catalyze reactionremoving O2
Methylene blue(anaerobicindicator)
Envelopecontainingchemicals torelease CO2
and H2
Petri plates
Chamber
Clamp
Figure 6.16 An anaerobic culture system.
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Culturing Microorganisms
• Culture Media
• Transport media
• Used by hospital 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
• Low-oxygen culture
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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
Bacterial Growth: OverviewPLAYPLAY
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Binary Fission
Binary FissionPLAYPLAY
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Cytoplasmic membraneChromosome
Cell wall
Replicatedchromosome
Septum
Completedseptum
30 minutes
60 minutes
90 minutes
120 minutes
Septum
1
2
3
4
5
Figure 6.17 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.
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Figure 6.19 Two growth curves of logarithmic growth.
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Figure 6.20 A typical microbial growth curve.
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Bacterial Growth Curve
Bacterial Growth CurvePLAYPLAY
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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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Fresh medium witha limiting amountof a nutrient
Sterile airor othergas
Flow-rateregulator
Culturevessel
Overflowtube
Culture
Figure 6.21 Schematic of chemostat.
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Growth of Microbial Populations
• Measuring Microbial Reproduction
• Direct methods not requiring incubation
• Microscopic counts
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Cover slip
Location ofgrid
Bacterialsuspension
Overflow troughs
Place underoil immersion
Bacterialsuspension
Pipette
Figure 6.22 The use of a cell counter for estimating microbial numbers.
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Growth of Microbial Populations
• Measuring Microbial Reproduction
• Direct methods not requiring incubation
• Electronic counters
• Coulter counters
• Flow cytometry
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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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1 ml of originalculture
9 ml of broth +1 ml of originalculture
0.1 ml of eachtransferred toa plate
Incubation period
1.0 ml
1:10dilution(10-1)
Too numerous to count(TNTC)
TNTC 65 colonies 6 colonies 0 colonies
1:100dilution(10-2)
1:1000dilution(10-3)
1:10,000dilution(10-4)
1:100,000dilution(10-5)
1.0 ml 1.0 ml 1.0 ml
0.1 ml0.1 ml0.1 ml0.1 ml
Figure 6.23 A serial dilution and viable plate count for estimating microbial population size.
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Membrane transferredto culture medium
Sample to be filtered
Membrane filterretains cells
To vacuum
Colonies
Incubation
Figure 6.24 The use of membrane filtration to estimate microbial population size.
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1:1001:10Undiluted
1.0 ml1.0 ml
Inoculate 1.0 ml intoeach of 5 tubes
Phenol red, pHcolor indicator,added
Incubate
Results
4 tubes positive 2 tubes positive 1 tube positive
Figure 6.25 The most probable number (MPN) method for estimating microbial numbers.
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Growth of Microbial Populations
• Measuring Microbial Growth
• Indirect methods
• Turbidity
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Direct light
Light source Uninoculatedtube
Light-sensitivedetector
Light source Inoculatedbroth culture
Scattered lightthat does notreach reflector
Figure 6.26 Turbidity and the use of spectrophotometry in indirectly measuring population size.
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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