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									BACTERIAL UBIQUITY
Prokaryotes are found almost everywhere
   From deep oceans to volcanoes
   From polar regions to equator
   From the Great Salt Lake to freshwater streams
Individual species differ, and grow in a limited
set of environments
   May grow at temperatures near boiling, but not at
    moderate temperatures
   May grow in the Great Salt Lake but not in
    freshwater streams
BACTERIAL UBIQUITY
Growth is dependent upon many factors
   Host organism
   Temperature
   pH
   Available nutrients
   Etc.
CULTURING BACTERIA
Microorganisms grow in mixed populations in
nature
   Joint contributions to numerous processes
Microorganisms are grown in pure culture in the
laboratory
   Requires isolation
    from a mixed culture
   Facilitates study
    of functions of a
    particular species
CULTURING BACTERIA
Only ~0.1% of prokaryotes can be grown in pure
culture
   Most environmental microbes are difficult to study
      Growth requirements difficult to determine
   Most medically important bacteria can be grown in
    pure culture
      Why do you suppose this is the case?
CULTURING BACTERIA
Various techniques of culturing microorganisms
   Equipment must be sterile
      Free of microorganisms
   Aseptic technique employed
      Minimize chance of inadvertent introduction of
      microorganisms
   Various types of media
      Contain necessary
      nutrients
      Solid or liquid
                       MEDIA
Liquid media
   Nutrients required for growth
       Different requirements for different species
   Cannot be used to isolate pure cultures
Solid media
   Liquid media plus solidifying agent agar
   Allows isolation of pure cultures
Containers
   Petri dish (“plates”), tubes (“deeps”,
    “slants”)
       Not airtight (allows gas exchange)
       Excludes airborne microorganisms
                      AGAR
Resistant to bacterial degradation
Survives high-temperature treatment
   Can be sterilized in autoclave
Liquefies at high temperatures
   Can be poured into convenient containers
Remains liquid until below 45oC
Once solidified, remains solid
over a wide range of temperatures
   Melts at 95oC
Translucent
   Colonies are visibly apparent
    PURE CULTURE
Bacteria are separated and placed on solid medium
   Various types of dilutions and plating techniques employed
Individual bacteria divide to form visible colonies
   Visible at > 1 million cells
   Genetically identical
   “Clone”
   Each clone is a pure
    culture
    STOCK CULTURES
After obtaining a pure culture, it is maintained as
a stock culture
   Maintained as an inoculum for later study
Various means of storage
   “Agar slant”
   Frozen in glycerol solution
      Glycerol prevents damage to cells from ice crystals
   Lyophilized (freeze-dried)
BACTERIAL GROWTH
Defines as an increase in cell
number
Division by binary fission
   Simpler than mitotic division
   Cell size doubles
       Cell components doubled
   Cell divides
   Repeat
   Growth is exponential
   1248, etc.
BACTERIAL GROWTH
Doubling time / generation time
   Time required for population to double in
    number
   Variable, dependent upon
       Species
       Growth conditions (nutrients, temp, etc.)
   Escherichia coli: optimally 20 minutes
   Mycobacterium tuberculosis: ~ 12 – 24
    hours+
   Longer under suboptimal conditions
EXPONENTIAL GROWTH
How to get rich quick
 On the first day of the    Day 1            $0.01
 month, put a penny in a    Day 5            $0.16
 jar
                            Day 10           $5.12
 On the second day,
                            Day 15         $163.84
 double this amount
 (2 cents)                  Day 20       $5,242.88
 On the third day, double   Day 25    $167,772.16
 this amount (4 cents)      Day 28   $1,342,177.28
 Etc.                       Day 30   $5,368,709.12
EXPONENTIAL GROWTH
Bacteria have the ability to increase
exponentially
   Exponential growth increases numbers incredibly
    quickly
Exponential growth is not
always possible
   Growth can be limited by
    various factors
      Environmental factors
      Nutrient availability
ENVIRONMENTAL FACTORS
Various environmental factors affect prokaryotic
growth
   Temperature requirements
   O2 requirements
   pH requirements
   Water availability
   Interactions with other organisms
TEMPERATURE REQUIREMENTS
All species can grow within a particular range of
temperatures
    Enzymes denatured above this range
    Optimal temperature
     within this range
        Divided into groups
        based on temperature
        optimum
        How does temperature affect
        the rate of chemical reactions?
        How would the enzyme
        DNA polymerase differ
        between mesophiles and
        thermophiles, etc.?
TEMPERATURE REQUIREMENTS
Psychrophiles (-5ºC – +15ºC)
    Psychrotrophs prefer > 15ºC, but tolerate lower
        e.g., Listeria monocytogenes (food poisoning)
Mesophiles (25ºC – 45ºC)
    e.g., Escherichia coli
    Most other common bacteria, most human pathogens
Thermophiles (45ºC – 70ºC)
    Thermus aquaticus from thermal springs
Hyperthermophiles (70ºC – 110ºC)
    Members of Archaea
        Many members of Archaea are “extremophiles”
    Pyrolobus fumarii isolated from a hydrothermal vent has max
     growth temp of 113ºC
   TEMPERATURE AND DISEASE
Leprosy (Hansen’s disease)
  Caused by Mycobacterium leprae
  Typically affects body extremities
      Ears, hands, feet, fingers
Syphilis
  Caused by Treponema pallidum
  Lesions generally appear on genitalia,
  lips, tongue, & throat
      Early treatments involved induction of
       fever by deliberate infection with malaria
       parasite
      Antibiotics are a kinder, gentler treatment

  What can you determine regarding
  the temperature requirements of these
  bacteria?
OXYGEN REQUIREMENTS
O2 can be useful
   e.g., aerobic cellular respiration
O2 can be damaging
   Readily converted into toxic compounds
       esp. superoxide (O2-) and hydrogen peroxide (H2O2)
   Enzymes detoxify these toxic compounds
       Superoxide dismutase: O2-  H2O2
       Catalase: H2O2  H2O & O2
       Not all bacteria possess these enzymes
       Other enzymes exist that detoxify these compounds
OXYGEN REQUIREMENTS
O2 levels vary widely in different environments
   Earth’s atmosphere is ~20% O2
   O2 is absent from some environments
       e.g., swamps, beneath soil surface, human intestines, etc.
Bacteria differ in their requirements for O2
   e.g., Some bacteria absolutely require O2 for cellular
    respiration
       Obligate aerobes
       Typically possess enzymes detoxifying O2- and H2O2
Bacteria differ in their tolerance of O2
   e.g., Some bacteria cannot tolerate O2
       Obligate anaerobes
       Typically lack enzymes detoxifying O2- and H2O2
OXYGEN REQUIREMENTS
O2 requirements determined using a “shake tube”
   (Could also use thioglycollate broth)
   Sterile tube of nutrient agar boiled
       Agar melted, O2 driven off
   Cooled to 50oC, bacteria added
   Agar hardens
   Incubate, note areas of growth




         What enzymes do you think each of these bacteria possess?
        Why do facultative anaerobes grow better when O2 is present?
pH REQUIREMENTS
All species can grow within a particular
range of pH values
   Optimal pH within this range
      Divided into groups based on pH optimum
          Acidophiles (optimal pH < 5.5)
          Neutrophiles (optimal pH near neutral)
          Alkalophiles (optimal pH > 8.5)
   Internal pH constant and typically near neutral
      Often maintained by ion pumps
WATER AVAILABILITY
Water is absolutely
required for growth
   Constitutes ~70% of cell
May be present but
osmotically unavailable
   High solute environment
    removes H2O from cell
WATER AVAILABILITY
Microbes differ in their ability to live in high salt
environments
Two main ways to deal with high osmolarity in the
environment
   Actively pump ions into cell
       e.g., K+
   Produce small solute molecules to increase internal
    osmolarity
       e.g., proline
WATER AVAILABILITY
Osmotolerant
   Organisms able to tolerate salt concentrations up to 10%, but
    not requiring high salt concentrations
   a.k.a. “facultative halophiles”
Halophiles
   a.k.a. “obligate halophiles”
   Organisms requiring high levels of NaCl to grow
       3 – 9% minimum NaCl required
   Frequently spoilage organisms in high-salt or high-sugar
    preserved foods
       Examples of such foods?
   Many extreme halophiles belong to domain Archaea
MICROBIAL ASSOCIATIONS
 Microbes do not grow in pure culture in nature
    Live in shared habitats and interact with other organisms
 Symbiosis
    Close partnership between organisms
    Three main types
        Mutualism
        Commensalism
        Parasitism
 Non-symbiotic relationships
    Synergism
    Antagonism
             MUTUALISM
Association between organisms in which both benefit
   Lichen consist of a fungus and an alga (or cyanobacterium)
       What does the fungus gain?
       What does the photosynthetic partner gain?
   Protozoa in a termite’s gut hydrolyze cellulose
       What does the termite gain?
       What does the protozoan gain?
     COMMENSALISM
Association between two organisms in which
one partner benefits and the other is unaffected
   e.g., Satellitism
       One bacterium produces a growth factor required by the
       second bacterial species
       Small colonies of the second species are able to grow near
       colonies of the first
       See figure 7.11, p. 206 Talaro & Talaro
         PARASITISM
Association between two organisms in which one
partner benefits and the other is harmed
NON-SYMBIOTIC ASSOCIATIONS
Synergism
    Interrelationship between two or more free-living
     organisms of benefit to all
    Relationship not necessary for survival
    Similar to mutualism
Antagonism
    Association between free-living species arising from
     competition
    One organism secretes a substance that inhibits or
     destroys other organisms
       e.g., antibiotics
MICROBIAL NUTRITION
Process by which chemical substances
(nutrients) are acquired from the environment
and used in cellular activities
Nutritional requirements
   Source of elements
   Source of energy
Substances required by an organisms are termed
“essential nutrients”
 MICROBIAL NUTRITION
Energy source               Carbon source
 Phototroph                  Autotroph
     Derives energy from        Obtains carbon as CO2
      sunlight               Heterotroph
 Chemotroph                      Obtains carbon in
     Derives energy from         organic forms
      chemicals
                 NUTRIENTS
Macronutrients                          Organic nutrients
   Required in relatively large           Contain both C and H
    amounts                                e.g., carbohydrates, lipids,
   Principle roles in cell structure       nucleic acids, proteins, CH4,
    and metabolism                          etc.
        e.g., energy source                Not required by all
   e.g., carbohydrates, proteins,          microorganisms
    etc.                                Inorganic nutrients
Micronutrients                             Lack either C or H
   a.k.a. “trace elements”                e.g., metals, salts, gases, water
   Enzyme cofactors, etc.                 Natural reservoirs are mineral
   e.g., Mg, Zn, Ni, etc.                  deposits, water, air
   Requirements differ between            Required by all microorganisms
    species
 CELLULAR CHEMISTRY
Cytoplasmic composition of Escherichia coli
 Aside from the ~70% H2O:
     97% organic molecules (mainly proteins)
        What are the four major classes of biological
        macromolecules?
     96% composed of six elements (CHNOPS)
     ~5,000 different compounds

     Nutritional requirements very minimal
        Water, glucose, and a few salts
        No growth factors required (more on these later)
REQUIRED ELEMENTS
Major elements
   Elements comprising cell constituents
   e.g., C, H, N, O, P, S, etc.
   Must be supplied in a usable form
      e.g., CO2 is usable by some (not all) organisms
      e.g., N2 is usable by very few organisms
Trace elements
   Required in minute amounts
      e.g., Co, Zn, Cu, Mb, Mn, etc.
      Typically enzyme cofactors
GROWTH FACTORS
Some bacteria cannot             Not all bacteria require
synthesize some of               growth factors
their cell constituents          Growth factor requirements
   e.g., certain amino acids,   differ for different bacteria
    vitamins, etc.                  e.g., E. coli requires no
   A supply of these                growth factors
    compounds is required           e.g., species of Neisseria
    for growth                       require 40+ growth factors
   These required                      7 vitamins, 20 amino acids, etc.
    compounds are termed                Bacteria requiring many
    “growth factors”                    growth factors are termed
                                        “fastidious”
NUTRIENT SOURCES
Ultimately derived from an inorganic reservoir
   Source of nutrients
   Replenished by organisms
      Nutrient cycling is a critically important ability of
      microorganisms
                     CARBON
Heterotroph
   Requires carbon in an organic
    form
       e.g., proteins, carbohydrates, etc.
   Nutritionally dependent upon
    other life forms
   Not all organic carbon us usable
    by all organisms
       Did you ever eat a pine tree?
Autotroph
   Uses CO2 as its carbon source
       Converts CO2 into organic carbon
   Not nutritionally dependent upon
    other life forms
                NITROGEN
Important component of proteins, DNA, & RNA
   Primary nitrogen source for heterotrophs
Main inorganic reservoir is atmospheric gas (N2)
   ~79% of Earth’s
    atmosphere
   N2 can be used by a
    small number of
    microorganisms
       Nitrogen fixation”
       N2  NH3
             NITROGEN
Decomposers convert organic nitrogen into NH3
   “Ammonification”
NH3 can be converted to other inorganic forms of
nitrogen by various
microorganisms
   NO3-, NO2-, NH3
Many organisms
can use one or
more of these
inorganic forms of
nitrogen
                  OXYGEN
Required for aerobic cellular respiration
Major component of various macromolecules
   Even anaerobes require oxygen in some form
Main inorganic reservoirs
   Free oxygen (O2) constitutes ~20% of Earth’s
    atmosphere
   Oxygen is also present in many inorganic salts
      e.g., sulfates, phosphates, nitrates, etc.
            HYDROGEN
Major element in all organic molecules
   esp. carbohydrates, proteins, etc.
Also important element in many inorganic
compounds
   e.g. H2O, many salts
Important element in certain gases
   H2S, CH4, H2
   These gases both used and produced by microbes
         PHOSPHORUS
Important component of certain molecules
   DNA, RNA, ATP, phospholipids, etc.
Main inorganic source is phosphate (PO43-)
   Present in rocks ad other mineral deposits
Generally very scarce in
environment
   Scarcity of phosphorus is
    typically limiting factor for
    growth
   Overabundance in water
    leads to eutrophication
                   SULFUR
Essential component of some vitamins and amino acids
Mineral form widely
distributed in
environment
   Hydrogen sulfide
    gas (H2S)
   Elemental sulfur (S)
   Sulfides (e.g., FeS)
   Sulfate (SO42-)
Various forms
usable by various           Acid drainage from a mine
microorganisms              Bacteria oxidize metal sulfides,
                           producing sulfuric acid (low pH)
MEASURING GROWTH
Direct cell counts
   Counts all cells – alive or dead
Viable cell counts
   Counts only cells able to multiply
Measuring biomass
   Measures turbidity, total weight, or nitrogen
Measuring cell products
   Byproducts of metabolism
DIRECT CELL COUNTS
Direct microscopic count
   Bacteria placed in counting chamber
       Holds known volume of liquid
   Counted under microscope
   Requires >107 cells/ml
Cell-counting instruments
   Coulter counter
       Counts cells as they pass through a small
       hole single file
            Cells interrupt an electrical current
   Flow cytometer
       Similar to Coulter counter
       Measures passing cells by scattering of
       light from a laser
VIABLE CELL COUNTS
Plate counts
   Prepare serial dilutions
   Known volume added to plate
       Pour plate or spread plate
   Incubate, count colonies
       Isolated cell  colony
            “Colony-forming unit”
   Requires >100 cells/ml
Membrane filtration
   Used when cell concentration is low
   Known volume of liquid passed
    through filter
   Bacteria retained by filter, plated
VIABLE CELL COUNTS
Most Probable Number (MPN)
   Prepare serial dilutions
   Incubate tubes
   Note growth
   Compare results
    to MPN table
   Gives statistical
    estimate of cell
    concentration
   Frequently used
    to quantify fecal
    coliforms
MEASURING BIOMASS
Measuring turbidity          Measuring total weight
   Spectrophotometer           Wet weight
    measures light              Dry weight
    transmittance through
    specimen
                             Measuring N content
   Requires >107 cells/ml      Treat cells with H2SO4
                                    Nitrogen converted to NH3
                                Assay NH3
                                Compute biomass
                                    Cells are typically 14% NH3
                             Cannot differentiate
                             between living and dead
                             cells
MEASURING CELL PRODUCTS
 Acid production
    Acids often produced as a result of the breakdown of sugars
        Detected by pH indicators
 Gas production
    Visually detected in Durham tubes
    Detected by pH indicators
        CO2 slightly reduces pH
             CO2 + H2O  H2CO3  H+ + HCO3-

 ATP production
    Add firefly enzyme luciferase
        Fluorescence requires ATP hydrolysis
        Fluorescence quantified
 BACTERIAL GROWTH
Bacteria have the ability to increase
exponentially
   Exponential growth increases numbers incredibly
    quickly
Exponential growth is not
always possible
   Growth can be limited by
    various factors
      Environmental factors
      Nutrient availability
    GROWTH CURVES
Populations typically a predictable pattern of
growth over time
   “Growth curve”
Four distinct phases
   Lag phase
   Log (exponential)
    phase
   Stationary phase
   Death phase
             LAG PHASE
No cell division
Preparation for cell division
   Synthesis of macromolecules
Length of phase
depends on conditions
   e.g., age of cells,
    richness of medium,
    storage temperature,
    etc.
             LOG PHASE
Exponential growth
   Cell division exceeds cell death
   Shortest generation time measured here
   More susceptible to antibiotics and other chemicals
STATIONARY PHASE
Resources depleted or toxins accumulate
Cell division = cell death
   Total number of viable cells remains relatively constant
Cells more resistant to antibiotics and other chemicals
       DEATH PHASE
Cell death exceeds cell division
Cells die at a fairly constant rate
Slope generally less steep than growth phase
    COLONY GROWTH
Growth in colonies follows a growth curve
Different parts of the colony are at different
points in the growth curve
   Resources depleted sooner in center of colony
      Reach stationary phase
      sooner
   Exponential growth
    continues at edges
MIXED MICROBIAL COMMUNITIES
 In nature, species live in close association with
 other species
    e.g. mouth, intestines, soil, water
 Interactions between species can optimize
 environment for other species
    e.g. aerobes use up O2, reducing O2 levels for
     anaerobes
    e.g. metabolic wastes may provide nutrients for
     another species
 Difficult to reproduce in laboratory
    Generally hard to grow these species
                BIOFILMS
Bacteria attached to surfaces encased in polysaccharide
   Slippery rocks in streams
   Slime in kitchen drain
   Scum in toilet bowls
   Plaque on teeth
   Etc.
BIOFILM FORMATION
Bacteria adhere to surface
Loose glycocalyx produced
Other species may attach to glycocalyx and grow
Cells form characteristic
architecture with open
channels for nutrients
and waste
Cell-cell communication
important in structure

								
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