Download Chapter 6 Microbial Nutrition and Growth

MICROBIOLOGY
WITH DISEASES BY TAXONOMY, THIRD EDITION
Chapter 6
Microbial Nutrition and Growth
Lecture prepared by Mindy Miller-Kittrell, University of Tennessee, Knoxville
Copyright © 2011 Pearson Education Inc.
Microbial Growth
• Metabolism Results in Reproduction
• Reproduction results in Growth
• What is microbial growth?
– an increase in a population of microbes (rather
than an increase in size of an individual)
• Result of microbial growth?
– a discrete colony – an aggregation of cells
arising from single parent cell
• Animations: Bacterial Growth Overview
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Factors affecting growth:
Nutritional Factors
Nutrients – chemicals taken in and used by organisms for
energy, metabolism and growth
 Water (Hydrogen and Oxygen)
 Carbon
 Nitrogen
 Sulfur
 Phosphorus
 Trace Elements
 Growth Factors
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Factors affecting growth:
Nutritional Factors
Macronutrients - Required in large amounts
 Carbon
 Needed for synthesis of cellular material and energy source
 Nitrogen
 Needed for protein synthesis, nucleic acids, ATP
 Sulfur
 Needed to synthesize amino acids and vitamins (thiamine, biotin)
 Phosphorus
 Needed to synthesize nucleic acids, ATP, phospholipids
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Factors affecting growth:
Nutritional Factors
Trace Elements
 required in trace amounts
 involved in enzyme function and protein structure
 Examples: Zn, Cu, Fe
 Present in tap water and distilled
Growth factors
 Organic compounds that cannot be synthesized by bacteria
 bacteria are “fastidious”
 examples: amino acids, purines, pyrimidines, vitamins
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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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Sources of Carbon, Energy, and Electrons
Carbon sources
Organisms are categorized into two groups:
1. Autotrophs
 Those using an inorganic carbon source (carbon
dioxide)
2. Heterotrophs
 Those catabolizing organic molecules (proteins,
carbohydrates, amino acids, and fatty acids)
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Sources of Carbon, Energy, and Electrons
Energy sources
Organisms are categorized into two groups:
1. Chemotrophs
 Acquire energy from redox reactions involving
inorganic and organic chemicals
2. Phototrophs
 use light as their energy source
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Groups of organisms based on carbon and energy source
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Figure 6.1
Oxygen Requirements
Oxygen sources
 Found as gaseous O2 or covalently bound in compounds
 Essential for aerobic respiration
 Oxygen is the final electron acceptor
• Deadly for some types of bacteria (anaerobes)
 Toxic forms of oxygen are highly reactive
 are excellent oxidizing agents
 Results in irreparable damage to cells by oxidizing compounds
such as proteins and lipids
http://www.exrx.net/Nutrition/Antioxidants/Introduction.html
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Toxic Forms of Oxygen
Singlet oxygen: 1O2
Oxygen boosted to a higher-energy state; extremely reactive
Superoxide free radicals: O2
O2 + 2H+
Superoxide Dismutase
H2O2 +O2
Peroxide anion: O22
2H2O2
Catalase
H2O2 + NADH+H+
Peroxidase
2 H2O + O2
2 H 2O
Hydroxyl radical (OH)
• Result of ionizing radiation & incomplete reduction of hydrogen
peroxide;
• extremely reactive but danger averted in aerobes because of
catalase & peroxidase
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Oxygen and Carbon Dioxide Requirements
– Aerobes – must use oxygen and can detoxify it
– Anaerobes- can not use oxygen nor detoxify it
– Facultative anaerobes- do not require oxygen
but can use and detoxify it
– Aerotolerant anaerobes – can not use aerobic
metabolism but have some enzymes to detoxify
oxygen’s poisonous forms
– Microaerophile – requires a small amount of
oxygen for growth
– Capnophile – requires higher CO2
than normally found in the atmosphere
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tension (3-10%)
Classification of Organisms Based on
Oxygen Requirements
 Microbial Growth is affected by Oxygen Concentration
Obligate
aerobes
Facultative
anaerobes
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Obligate
anaerobes
Aerotolerant
anaerobes
Microaerophiles
Nitrogen Requirements
Nitrogen Sources
 Acquired from organic and inorganic nutrients
 Recycled from amino acids and nucleotides to make other
proteins and nucleotides
 Nitrogen fixation Nitrogen gas reduced to ammonia
 Essential to life on Earth because nitrogen is made available in
a usable form
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Factors that Affect Microbial Growth
Temperature –
– Affects proteins and lipid membranes
– If too low, membranes become rigid and fragile
– If too high, membranes become too fluid
•
Categories based on Optimum Temperature
–
–
–
–
Psychrophile – optimum below 15oC
Mesophile – optimum between 20oC – 40oC
Thermophile – optimum higher than 45oC
Hyperthermophiles – optimum above 80oC
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Effects of temperature on microbial growth
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Figure 6.4
Catagories of Microbes Based on Temperature Range
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Figure 6.5
Temperature
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Use of Temperature to Preserve Microbes
Preserving Bacteria Cultures:
• Refrigeration:
– Storage for short periods of time
• Deep-freezing:
– -50° to -95°C
– Preserves cultures for years
• Lyophilization (freeze-drying):
– Frozen (-54° to -72°C) and dehydrated
in a vacuum
– Can last decades
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Effects of pH
• Classification of Microbes based on pH
– Organisms sensitive to changes in acidity
– H+ and OH– interfere with H bonding
– Acidophiles – prefer below 7
– Neutrophiles – prefer 7
– Alkalinophiles – prefer above 7
– Most bacteria grow between pH 6.5 and 7.5
– Molds and yeasts grow between pH 5 and 6
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Physical Effects of Water
Microbes require water
 to dissolve enzymes and nutrients required in metabolism; to react
in many metabolic reactions
 Some microbes have cell walls that retain water
 Endospores and cysts stop most metabolic activity to survive in a
dry environment for years
 Two physical effects of water
 Osmotic pressure
 Hydrostatic pressure
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Osmotic Pressure
Osmotic pressure
The pressure exerted on the semipermeable membrane by a
solution containing solutes, which cannot move across the
membrane.
Osmosis
Diffusion of water across a semipermeable membrane driven by
unequal concentration of solutes across the membrane.
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Osmotic Variations in the Environment
– Isotonic
– External concentration of solutes is equal to cell’s
internal environment
– Diffusion of water equal in both directions
– No net change in cell volume
– Hypotonic
– External concentration of solutes is lower than cell’s
internal environment
– Cells swell and burst
– Hypertonic
– Environment has higher solute concentration than
cell’s internal environment
– Cells shrivel (crenate)
– Halophiles tolerate higher salt concentrations
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Osmotic Pressure
ISOTONIC
Physiologic Saline
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HYPERTONIC
Hydrostatic Pressure
 Water exerts pressure in proportion to its depth
 For every addition of depth, water pressure increases 1 atm
 Organisms that live under extreme pressure are barophiles
 Their membranes and enzymes depend on this pressure to
maintain their three-dimensional, functional shape
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Microbial Growth
 Binary fission
 Splitting parent cell to form two similar-sized daughter cells to
increase number of cells
 Generation time
 Duration of each division
 Determined by type of bacteria
 Example: E. coli (20 min)
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Exponential Growth by Binary Fission
1.
2.
3.
4.
5.
DNA replication
Cell elongation
Septum formation
Septum completion
leads to separation or
further division
Process repeats
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Figure 6.17a
Bacterial Growth Curve
Animation: Bacterial Growth Curve
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Figure 6.20
Bacterial Growth Curve
 Graph of a closed bacterial population over time
 Lag phase
 Acquire cell mass, no reproduction
 Log (Exponential growth) phase
 Cells dividing
 Stationary phase
 Cells stop growing, cells dividing and dying at same rate
 Death phase
 Cells dying due to lack of nutrients and increased waste
products
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Methods of Culturing Microbes
 Specimen Collection
 Taking a sample of infected material
 Sterile (aseptic) technique required to avoid introducing
unwanted microbes into the sample
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Clinical Sampling
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Table 6.3
Culturing Microorganisms
Inoculate
 Implant microbes onto medium (broth or solid)
Inoculum
 Sample of microbes from specimen
Culture
 act of cultivating microorganisms
 or the microorganisms that are cultivated
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Streak Plate Method
Pure Culture
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Figure 6.8 - Overview
Mixed Culture
Can you see 12 different bacterial colonies?
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Figure 6.9 - Overview
Culture Media
MEDIA
• Nutrient preparation for microbial growth
• Must provide all chemical requirements
• Physical state depends on amount of AGAR
– Used as solidifying agent for culture media
– Composed of complex polysaccharides
– Advantages of agar vs gelatin:
– Generally not metabolized by microbes
– Liquefies at 100°C
– Solidifies ~40°C
– Fanny Hesse used agar from seaweed in her jams and jellies, which
she learned from a neighbor who had lived in Java (Indonesia).
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Chemically Defined vs Complex Media
Comparison of Media
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Types of Media Used in the Clinical Lab
 Basic Nutrient
 Designed to grow broad-spectrum microbes
 Enriched
 Add enrichment to encourage growth of microbes
 Blood, growth factors, serum
 Selective
 Suppress unwanted microbes and encourage desired
microbes to grow
 Salt, dyes, alcohol
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An example of the use of a selective medium
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Figure 6.12
Types of Media Used in the Clinical Lab
 Differential
 To distinguish colonies of different microbes from one another
 Dyes, pH indicators
 Reduced (anaerobic) media
 Contain chemicals (thioglycollate) that combine O2
 Used for anaerobic cultures
 Transport
 Maintain and preserve microbes
 Include atmospheric buffers
 Prevent drying
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MacConkey agar as a selective and differential medium
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Figure 6.15
Anaerobic Culture Methods
Gas Pak Jar
Glove Box
Figure 6.15 - Overview
Capnophiles require high CO2
• Candle jar
(3-10% CO2)
• CO2-packet
Environment to Culture Microbes
• Incubation
– Temperature
– 35-37oC – body temperature
– 25-30oC – room temperature
– 4-8oC – refrigerator temperature
– Atmosphere
– Aerobic - free oxygen present
– Microaerophilic – free O2 present; increased
CO2
– Anaerobic – NO free O2 present
– Time
– 18 – 24 hours
– Longer for slow-growing microbes
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Planktonic vs Sessile Bacteria
•All lab tests use “pure cultures” of
suspended cells called planktonic
bacteria since they float around in
liquid.
•In fact, pure cultures are virtually
absent in nature.
Robert Koch
Courtesy of the
National Library of Medicine
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•Most microbes exist as sessile
bacteria– attached to a surface –
and they live in communities called
biofilms.
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
– Many microorganisms more harmful as part of a biofilm
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What is a biofilm ?
An organized, layered system of microbes
attached to a surface
Biofilms form when microbes adhere to a surface that
is moist and contains organic matter
Ingredients needed for a biofilm :
 Surface
 Bacteria
 Aqueous environment
 Nutrients
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How does a biofilm develop?
1. Planktonic cells attach to surface
2. Cells multiply ;Produce glycocalyx
3. Slime layer entraps nutrients, cells, microbes
4. Dynamic pillar-like layers form
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How do biofilms communicate?
•Cell to cell
communication
- send and receive
chemical signaling
molecules
•Quorum sensing
- accumulation of
signaling molecules
Center for Biofilm Engineering Montana State University–Bozeman
- enables a cell to
sense the cell density
http://www.ted.com/talks/lang/eng/bonnie_bassler_on_how_bacteria_communicate.html
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Biofilm Behavior
• Biofilm bacteria “turn on” a different set of genes
than planktonic bacteria
Examples:
– Turn off flagellar protein and turn on pili genes
– Turns on genes for antibiotic resistance
• Form a "division of labor" by nutrient cycling
– Some cells turn on metabolic pathways that degrade
particulate matter, while other adjacent cells of the same
population use the degradation products to produce new
cells that are dispersed in the environment
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Where are Biofilms Found?
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Biofilms Found in Health Care
 Dental caries
 Contact lenses
 Lungs of Cystic Fibrosis patients
Biofilm on a contact lens
 Indwelling medical devices
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



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Endotracheal tube
Mechanical heart valves
Pacemakers
Urinary catheters
IV connectors
Prosthetic joints
Staphylococcus biofilm
on inner surface of
IV connector
Rodney M. Donlan, CDC
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Where are Biofilms Found?
Biofilm on soft, daily-wear,
contact lens
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Biofilm on urinary catheter
Biofilms and Chronic Wounds
 60 % of chronic wounds
had biofilms
 Only 6% of acute wounds
had biofilms
 Found normal flora and
pathogens produced
different chemicals for their
communication.
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Medical Importance of Biofilms
 Are 1000X more resistant to antimicrobial agents than
planktonic cells
 Easily transfer genes to express new and sometimes more
virulent phenotypes
 Are more resistant to host defense mechanisms
 80% of nosocomial infections are biofilm associated (NIH)
 20% of patients with biofilm-related septicemia die
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