Prokaryotic Growth

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ProkaryoticGrowth

• Kathy Huschle
• Northland Community Technical College

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Pure Cultures

• pure culture population of organisms descended
  from one organism
• only approximately 1 of all bacteria can be
  cultured successfully in the lab

Vibro chlorae
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Pure Culture

• colony, clone
• begins with a single bacterial cell placed on a
  solid medium such as agar
• agar
• provides specific nutrition for bacteria and a
  medium to grow on

Colonies on agar
Nutritional Agar
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Binary Fission

• method of bacterial reproduction
• cell divides exactly in half
• single cell division
• reproduction of the entire organism

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Binary Fission

• asexual
• no genetic recombination
• the DNA molecule replicates itself when bacterial
  reproduction takes place

E. coli undergoing cell division
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Bacterial Growth

• bacterial growth bacterial cell reproduction
• the process of binary fission doubles the
  population each time binary fission takes place
• this doubling time demonstrates exponential
  growth
• each generation results in a doubling of the
  population generation time is to doubling time
• measure of microbial growth rate

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Bacterial Growth Curvelaboratory conditions

• bacterial growth generally follows a
  characteristic pattern
• 5 phases
• normal growth curve, with optimum environmental
  and nutritional conditions

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Bacterial Growth Curvelaboratory conditions

• lag phase
• no increase in cell numbers
• cells are adapting to the environment
• cells are preparing for reproduction
• synthesizing new DNA, etc.

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Bacterial Growth Curvelaboratory conditions

• log phase
• exponential phase
• maximal rate for reproduction
• this happens with a specific set of growth
  conditions
• those resources for growth are abundantly
  available

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Bacterial Growth Curvelaboratory conditions

• stationary growth phase
• maximum population for the resources available
• required nutrients become depleted
• inhibitory end products from cell metabolism
  accumulate
• cell growth cell death

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Bacterial Growth Curvelaboratory conditions

• death phase
• cell death gt new cell formation

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Bacterial Growth Curvelaboratory conditions

• phase of prolonged decline
• can last from months to years
• survival of the fittest

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Solid Media

• on solid media
• cells do not disperse readily
• nutrients become limited in center
• death phase occurs in the center with exponential
  phase at periphery of the bacterial colony

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Bacterial Growth

• most lab organisms are grown in a batch culture
• closed system
• new materials are not added
• waste products are not removed
• under these conditions bacteria populations
  follow distinct patterns of growth

Algae batch cultures
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Bacterial Growth

• continuous culture maintained
• nutrients must be continually supplied
• end products must be removed
• exponential growth phase maintained

Continuous culture in lab
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Natural Chemostat

• chemostat
• continuous culture device

A cow, with its four stomachs, is natures
perfect chemostat constantly grazing to add
nutrients and continually belching and other such
mechanics to remove bacterial metabolic end
products
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Environmental Parametersinfluencing bacterial
growth

• not all bacteria favor the same environmental
  conditions
• the effects of varying conditions are seen as
  differences in reproduction (bacterial growth)
• some environmental conditions that can affect
  bacterial growth include
• temperature
• oxygen
• salinity
• pH

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Environmental Influencing Factorstemperature

• temperature
• ideal temperature for growth varies between
  organisms
• specified by the bacterial genome

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Environmental Influencing Factorstemperature

• temperature growth range
• minimum to maximum temperatures for bacterial
  growth
• optimal growth temperature
• temperature at which the highest rate of
  reproduction occurs

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Environmental Influencing Factorstemperature

• 5 divisions of prokaryotes, based on optimal
  growth temperature
• psychrophiles
• psychrotrophs
• mesophiles
• thermophiles
• hyperthemophiles

Psychrophile Desulfofaba gelida
Thermophile Pyrococcus sp.
Hyperthermophile Thermococcus barophilus
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Environmental Influencing Factorstemperature

• psychrophiles
• optimum growth temperature -50C 150C
• found in the Arctic and Antarctic regions of the
  world

Bacteria found in melt from a Russian outpost on
Lake Vostok
Desulfofrigus oceanense
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Environmental Influencing Factorstemperature

• psychotrophs
• optimum growth temperature 200C 300C
• will grow at lower temperatures
• most commonly found in refrigerated food spoilage

Stemphlium sarcinaeforme
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Environmental Influencing Factorstemperature

• mesophiles
• optimum growth temperature 250C 450C
• most human pathogens are mesophiles
• adapted well to growth in the human body, whose
  normal temperature is around 370C

Salmonella
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Environmental Influencing Factorstemperature

• thermophiles
• optimum temperature 450C 700C
• commonly found in compost heaps and hot springs,
  water heaters

Sulfolobus
Thermophile in a hot spring
Sulfur pots in Yellowstone
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Environmental Influencing Factorstemperature

• hyperthermophiles
• optimum growth temperature 700C 1100C
• usually member of the Archae domain
• found in hydrothermal vents in the depths of the
  ocean

Deep Sea Vent
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Temperature Ranges

• psychrophiles
• -50 C to 150 C
• psychotrophs
• 200 C to 300 C
• mesophiles
• 250 C to 450 C
• thermophile
• 450 C to 700 C
• hyperthermophiles
• 700 C to 1100 C

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Temperature Considerations

• food preservation
• refrigeration
• inhibits fast growing mesophiles
• psychrophiles can still grow in refrigeration,
  but at a diminished rate
• freezing destroys microorganisms that require
  water to grow

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Temperature Considerations

• disease
• body temperature varies extremities are usually
  cooler than 370C
• some microorganisms can cause disease in certain
  body parts but not in others due to variations in
  body temperatures

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Environmental Influencing Factors oxygen

• oxygen levels vary between environments and
  within the same environment
• based on O2 requirements, prokaryotes are
  separated into the following groups
• obligate aerobes
• obligate anaerobes
• facultative anaerobes
• microaerophiles
• aerotolerant anaerobes

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Environmental Influencing Factors oxygen

• obligate aerobes
• need oxygen present to multiply

Giardia
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Environmental Influencing Factors oxygen

• obligate anaerobes
• cannot multiply in the presence of oxygen
• often killed by traces of oxygen in their
  environment

C. perfringens
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Environmental Influencing Factors oxygen

• facultative anaerobes
• grow best with oxygen, but can grow without
  oxygen
• respiration if oxygen is available
• fermentation if no oxygen is present
• growth is greater in the presence of oxygen due
  to the production of more ATP (energy source of
  the cell)

Aeromonas hydrophilia on intestinal cells
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Environmental Influencing Factors oxygen

• microaerophiles
• require oxygen but have maximal growth at reduced
  oxygen concentration
• high concentration of oxygen inhibit growth

Helicobacter sp.
Helicobacter sp.
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Environmental Influencing Factors oxygen

• aerotolerant anaerobes
• indifferent to oxygen

S. mutans
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Environmental Influencing Factors pH

• based on pH of the environment, microorganisms
  are separated into the following groups
• neutrophiles
• acidophiles
• alkalophiles

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Environmental Influencing Factors pH

• neutrophiles
• optimum pH of 7 (neutral)
• most microorganisms grow best between pH of 5
  (acidic) and pH of 8 (alkaline)
• acidophiles
• optimal growth, pH of less than 5.5
• alkalophiles
• optimum pH of 8.5 or greater

Copper
Urinary bacterial infection caused by alkaline
urine
Copper tolerant acidophile
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Environmental Influencing Factors salinity

• H2O is required by all microorganisms for growth
• in some places H2O is hard to come by such as in
  salt concentrations
• if a cell is in an environment that has a greater
  solute concentration than the interior of the
  cell, then by osmosis the water will leave the
  cell causing plasmolysis (shrinking of the cell)

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Environmental Influencing Factors salinity

• halophiles are microorganisms that have adapted
  to this kind of environment
• halophiles
• require high levels of sodium chloride
• moderate halophiles
• 3 salt concentration
• extreme halophiles Archaea
• require at least 9 salt solution
• found in the Dead Sea

Dunaliella salina cell, near a salt crystal. 40X

Dead Sea
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Nutritional Influencing Factors

• major elements
• C, O, H, N, S, P, K, MG, Ca Fe
• essential components of protein, carbohydrates,
  lipids and nucleic acid
• needed to synthesize cell components

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Nutritional/Energy Influencing Factors

• heterotrophs
• utilize organic carbon
• autotroph
• utilize inorganic carbon
• phototrophs
• harvest the energy of sunlight
• chemotroph
• obtain energy by metabolizing chemical compounds

Dinoflagellates
Myxobacteria
Purple Sulfur Bacteria a chemotroph
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Nutritional Diversity

• prokaryotes are able to use diverse sources of
  carbon (an essential element) and energy
• this ability allows them to thrive in virtually
  and environment

Forms of Carbon
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Nutritional Diversity

• photoautotrophs
• utilize the energy of sunlight
• obtain carbon from CO2
• primary producers of the microbial world
• 6CO2 12H2O C6H12O6 6H2O 6O2
• photoheterotrophs
• utilize the energy of sunlight
• obtain carbon from organic compounds

Cyanobacteria
Rhodobacter sphaeroides
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Nutritional Diversity

• chemolithoautotrophs
• AKA as
• chemoautotrophs or chemolithotrophs
• energy from inorganic compounds such as hydrogen
  sulfide
• carbon from CO2

Thiobacillus denitrificans
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Nutritional Diversity

• chemoorganoheterotrophs
• AKA
• chemoheterotrophs or chemoorganotrophs
• utilize organic compounds for energy and as a
  carbon source
• most common group of microorganisms associated
  with humans and animals
• important organic degraders

Brachionus calyciflorus
B. vietnamiensis
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Prokaryotes in the Lab

• studying microorganisms in their environment,
  enhances our ability to grow them in the lab
• lab growth is important for the study of the
  microbial world and its effect on human life

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Lab Cultivation of Microbes

• complex media
• used for routine purposes
• variety of ingredients needed by the
  microorganism are included in the media
• nutrient agar, blood agar, PEA agar, Mannitol
  Salt agar are some examples

S. aureus on blood agar
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Lab Cultivation of Microbes

• selective media
• formulated with ingredients that inhibit the
  growth of some bacteria, such as an antibiotic,
  but enhance growth of the target organism
• ie MacConkey agar can be used to isolate
  Gram-negative rods

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Lab Cultivation of Microbes

• differential media
• includes ingredients, such as chemical
  indicators, that produce observable differences
  between species of bacteria
• ie ph indicator may be incorporated with the
  agar medium allowing for the detection of acid
  producing microorganisms

mannitol salt agar pH indicator turns the agar
yellow in the presence of a salt tolerant organism
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Creating Appropriate Environmental Conditions

• to enhance microbial growth in a lab, certain
  environmental conditions need to be created
• atmospheric pressure
• temperature
• oxygen availability

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Creating Appropriate Environmental Conditions

• atmosphere
• increase CO2 for some species of microbes

Candle jar used in lab to increase CO2
concentration
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Creating Appropriate Environmental Conditions

• anaerobic microorganisms require anaerobic
  conditions required growth
• these are some of the most difficult types of
  microorganisms to culture in the lab, due to the
  fact that even a brief exposure to oxygen
  generally results in the death of the organism

Anaerobic jars used in labs
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Creating Appropriate Environmental Conditions

• temperature
• controlled with the use of an incubator
• allows for setting the optimum temperature for
  individual microorganisms

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Bacteria Enumeration

• lab techniques that monitor bacterial growth
• viable plate count
• direct count
• most probable number
• membrane filtration
• measuring biomass
• turbidity
• total weight
• chemical constituents

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Bacteria Enumeration

• viable plate count
• measure the number of cells in a sample based on
  the fact that one cell gives rise to one colony
• utilizes a series of dilutions in order to
  calculate the number of viable bacteria in the
  original sample

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Bacteria Enumeration

• direct count
• using special equipment capable of making
• a direct microscopic count
• a count of cells suspended in a suspension
• a count by analyzing the scattering of light as
  cells pass by a laser

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Bacteria Enumeration

• most probable number
• a statistical analysis of cell numbers based on
  the theory of probability

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Bacteria Enumeration

• membrane filtration
• used when cell numbers are low
• allows for a concentration of the microbes by
  filtering before plating

Membrane filtration on mEnterococcus agar. The
plate at the bottom is uninoculated. The red
colonies typical of the Enterococci are clearly
visible on the white membrane filters.
Membrane filtration equipment
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Bacteria Enumeration

• measuring biomass
• turbidity
• total weight
• chemical constituents

turbidity
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Bacteria Enumeration

• turbidity
• cloudiness, which indicates the presence of
  microbial growth
• cell numbers can be measured with a
  spectrophotometer

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Bacteria Enumeration

• total weight
• tedious work
• measure the wet weight, centrifuge and then
  measure dry weight

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Bacteria Enumeration

• chemical constituents
• analyzing the quantity of chemical (metabolic
  byproduct) in a bacterial sample and using that
  information to calculate biomass

Spectroanalysis
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Bacterial Growth in Nature

• similar to a continuous culture
• nutrients are continually added and byproducts
  are removed
• generally multiply more slowly than under lab
  conditions
• often the waste of one microorganism is the
  nutrient of another

Microbial mat in Yellowstone
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Bacterial Growth in Nature

• biofilms
• polysaccharide-encased community
• slippery rocks, gunk in drains, plaque on teeth,
  IVs are all examples of biofilms
• begins with adherence of a bacterium to a surface
• bacteria multiplies
• synthesizes a loose glycocalyx allowing unrelated
  cell to attach and grow

Methanogen biofilm
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Bacterial Growth in Nature

• biofilms
• medical problems
• resist antibiotics
• 65 of human infections involve biofilms
• often times 100X more resistant to disinfectants

Biofilm on endotrachial tube
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Bacterial Growth in Nature

• bioremediation
• bacteria used to degrade chemicals are enhanced
  by organisms present in biofilm

Acid from an abandoned mine. Microorganisms are
introduced to this environment and are
successfully able to clean up the problem.