Microbial Growth

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Chapter 6

• Microbial Growth

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Bacterial Cell Division

• Cell growth is defined as an increase in the
  number of cells, requires continued growth to
  maintain species
• 2000 chemical reactions with a wide variety of
  types
• main rxn is polymerization reaction
• monomer to polymer

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Binary Fission

• Cell growth continues until divides into 2 new
  cells
• Cells create a septum between new cells
• starts as cytoplasmic membrane and eventually
  becomes cell wall
• Each batch of new cells is a generation
• Cellular components increase proportionally so
  each cell gets enough of everything to the new
  cell
• Time to generate new cells is dependent on
  nutritional and genetic factors
• division is tied to chromosomal replication

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Fts Proteins

• Filamentous temperature sensitive proteins
  mutation in genes that encode the Fts proteins
• bacteria without FtsZ have difficulty dividing
• FtsZ is universally distributed in all
  prokaryotes
• see FtsZ-like proteins in mitochondria and
  chloroplasts, also similar to tubulin in eukarotes

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Fts Complexes Division Apparatus

• Fts interact to form the division apparatus
  called the divisome
• FtsZ attach in a ring to the cell at the membrane
  and then attracts FtsA and Zip A
• FtsA ATP hydrolyzing enzymes for proteins in
  divisome
• ZipA anchor attachment of FtsZ to membrane
• Also contain Fts proteins involved in
  peptidoglycan synthesis FtsI is a
  penicillin-binding protein (activity site for
  penicillin)
• Divisome makes new cytoplasmic membrane and cell
  wall in both directions until large enough to
  divide

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DNA Replication

• Occurs prior to FtsZ ring formation and when done
  get the ring formation between the 2 nucleoid
  regions using min proteins
• min C inhibits cell division until exact center
  of the cell is found
• min E inhibits min C activity and attached at
  center of cell, recruits the FtsZ and ring
  formation

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Cell Shape

• Morphology cell shape
• Peptidoglycans thought to dictate shape but now
  know only a minor role
• Protein for shape is homologous to actin
• Major protein MreB forms an actin-like
  cytoskeleton
• filamentous, spiral-shaped bands in cell under
  cytoplasm membrane
• cocci lack MreB and its gene, default shape
  sphere
• Bacteria make FtsZ and MreB tubulin- and
  actin-like proteins
• evolutionary similarities between eukaryotes and
  prokaryotes

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Peptidoglycan Snthesis

• Must make the cell wall before cell division
  add new cell wall to existing cell wall
• At FtsZ autolysins make openings in wall
• enzyme similar to lysozyme
• present in the divisome
• Cell wall material added thru the holes
• Between new and old cell wall, a ridge forms
  like a scar

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Cell Wall Formation

• Precursors to the cell wall are spliced into
  existing peptidoglycan
• If the precursors arent coordinated with the
  old, the cell goes through spontaneous autolysis
  cell ruptures

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Biosynthesis of Peptidoglycan

• Cut pre-existing peptidoglycans by autolysins
  with simultaneous insertion of precursors
  bactoprenol lipid carrier molecule, hydrophobic
  C55 alcohol
• Bactoprenol makes the peptidoglycan precursors
  hydrophobic so they can cross membrane to be
  inserted, spend time in the periplasm to build
  cell wall and make glycosidic bonds

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Transpeptidation and Penicillin

• Final step need to insert the peptide
  components of the cell wall between the muramic
  acid (refer to cell wall structure from before)
• This reaction is inhibited by penicillins
  prevent cell wall formation by binding to FtsI,
  autolysins continue to weaken the cell wall and
  leads to lysis
• used in humans
• since we do not have cell walls, can use drug at
  high levels
• virtually all bacterial pathogens have
  peptidoglycan so works on most bugs

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Final Interactions

• Interaction with several amino acids based on the
  organism
• E coli between diaminopimelic acids and D-Ala
  on adjacent peptides
• Removal of the 2nd D-Ala drives the rxn as there
  is not ATP (outside the cell)
• In gram , glycine interbridge is usaully
  present, cross-link accur across the interbridge
  on L-Lys and D-Ala

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Growth of Bacterial Populations

• Increase in the number of organisms in a
  population
• Terminology
• 1 cell to 2 cells is a generation
• time for the new cell to form is the generation
  time, mass also doubles so also called doubling
  time
• These vary between organisms and are based on
  growth medium, growth conditions
• usually differ out in nature vs. the test tube

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Exponential Growth

• Where number of cells double during a regular
  tine interval
• Graph on linear scale, see a dramatic increase in
  the numbers over time
• Graph on semi-log paper, you get a straight line,
  meaning exponentially growing
• use to estimate growing time

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Estimating Growth Rate
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Growth

• In exponential growth rate increase is slow
  initially but increases in cell number
• in non-sterile, nutrient rich environments, such
  as milk slow growth is good, leave milk out an
  hour, not to many bacteria, but if leave out
  several hours, the level of bacteria will be much
  higher

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Growth Cycle

• Exponential growth cannot continue forever
• Cycle has 4 distinct areas lag, exponential,
  stationary and death phases

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Lag Phase

• Delay in growth of bacteria
• Interval may be different
• based on organism and growth conditions
• See when using old or stationary phase cultures
  to start your growth curve
• Lag is caused by cells being depleted in
  essential constituents, must also repair if
  damaged by heat or radiation, etc
• also see if moving from a rich medium to a poorer
  medium

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Exponential Phase

• Cells divide for a brief time based on
  resources and other factors
• Rate of growth vary greatly
• influenced by environmental conditions and
  genetic characteristics of the organism

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Stationary Phase

• Limitation on growth caused by 2 factors
• essential nutrients of culture medium is used up
• some waste products of the organism build up in
  medium and inhibit growth
• can be a combination of both
• Exponential growth stops and there is no net
  increase or decrease in the cell number may be
  slow growth
• Cellular functions continue energy metabolism
  and biosynthetic processes
• some divide and some die cryptic growth

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Death Phase

• Cells will die eventually
• Death accompanied by cell lysis
• Exponential death but slower than growth
• Figure 6.8 is of a POPULATION and not a single
  cell, this process does NOT apply to them

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Measuring Microbial Growth

• 2 methods of direct measurement
• total counts
• viable count
• Important to know the number of bacteria for some
  tests

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Total Counts

• Total count using a microscope and hemocytometer
  a special counting chamber with a square on
  surface of glass with a known volume under a
  cover slip
• count the number of cells on the grid and then
  calculate the number of cells based on the volume
  on the chamber
• also count dead cells, miss small cells,
  precision is hard to achieve, requires phase
  contrast microscopy when not stained, not good a
  low density and motile cells must be immobilized

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Viable Counts

• Viable cells can divide and make offspring
• Determine whether capable of forming colonies on
  suitable agar
• plate count or colony count, assume each cell can
  yield a colony
• 2 methods spread plate and pour plate
• spread plate use small volume of diluted cells
  and spread over surface of agar, count colonies
  and calculate number using dilution
• pour plate add volume of culture into Petri
  dish, add melted agar, mix by swirling colonies
  form throughout the agar, not just on top like
  above method, examine carefully

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Dilutions

• Use dilutions to determine the number of colonies
  in a countable range count/plate should be
  between 30 and 300 colonies
• Must determine optimum conditions to grow the
  bacteria temp, medium, time, etc
• Perform serial dilutions to get into the
  countable range
• Sources of error not using correct growing
  conditions, errors also in technique pipeting,
  mixing, etc.

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Serial Dilutions

• Usually do serial 10-fold dilutions by mixing 0.5
  ml of sample with 4.5 ml of fresh medium (1 part
  in 9 parts 10)
• Do consecutive dilutions in the same manner and
  plate a volume on the agar
• After growth, count the colony forming units
  (cfu) and calculate the number of bacteria using
  dilution and volume placed on plates

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Colony Forming Units

• Use colony forming units because occasionally you
  may have 2 bacteria in the same area that make a
  single colony you cant tell apart
• Can use selective media to count only a
  particular organism
• Great plate anomaly may be unreliable to assess
  total number of cells in natural samples soil,
  water plate count is usually lower than direct
  count
• organisms may have really different nutrient
  needs
• may need selective media to get a better count

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Indirect Measurements

• Use turbidity as an indirect measurement more
  cells mean it is more cloudy
• Use a photometer or spectrophotometer
• similar in use light scattered by cells and all
  light that passes thru will be collected
• differences is with the light source photometer
  is a broad pass filter and spec is prism or
  diffraction grating
• both measure only unscattered light but report in
  Klett units or optical density

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Generation of a Standard Curve

• Can substitute turbidity for direct counting
  methods but need to make a standard curve
• Relate direct count to indirect method
• may use cell number or cell mass
• Must use within limits really dense samples
  may deflect light and then another cell
  re-deflects them to the detector
• makes things non-linear
• can check sample repeatedly without altering test
  outcome

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Continuous Culture - Chemistat

• Previous growth was based on batch cultures
  fixed volume of medium altered by metabolism in
  culture closed system
• Constant environment needed over long periods of
  time to generate a continuous supply of
  exponential phase bacteria continuous culture
  open system
• add fresh medium and remove the old medium in a
  chemistat
• volume, cell number and nutrient state are
  constant steady state

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Chemistat

• Constant growth rate and population density
• 2 important factors
• dilution rate
• concentration of limiting nutrient, usually N or
  C

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Affect of Nutrient on Growth and Yield

• In batch cultures nutrient can affect growth
  rate and growth yield
• At low concentrations only the rate is reduced
• cannot meet the needs of organisms
• Moderate to high may not change the rate but
  the yield will increase

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Chemistat Control of Rate and Yield

• Both rate and yield can be controlled
  independently by altering the dilution rate which
  effects the of nutrients present
• Dilution rate at high and low rates the steady
  state breaks down, at high bacteria arent
  growing fast enough and at low not feeding
  fast enough so cells are dying
• Cell density (cells/ml) controlled by level of
  limiting nutrient

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Environmental Effects - Temperature

• Most important as alter the temperature to
  drastically the bacteria will die
• Raising the temperature may speed up growth rates
  but over a limited range may be detrimental if
  too high maximum temperature or too low
  minimum temperature
• Optimum temperature temperature that growth
  occurs most rapidly usually nearer to the
  maximum than the minimum

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Cardinal Temperatures

• Maximum, optimum and minimum are the cardinal
  temperatures
• Cardinal temps are not fixed and may fluctuate
  depending on growth medium
• Maximal temperatures reflects denaturation of 1
  or more proteins
• Not sure what causes minimal temperature but may
  be the composition of the cytoplasmic membrane
• alter composition resulted in a change in the
  maximum and minimum temperatures

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Temperature Classes of Organisms

• Psychrophile very low temperatures
• Mesophiles moderate temperatures
• Thermophiles high temperatures
• Hyperthermophiles very high temperatures
• All but mesophiles can also be classified as
  extremophiles

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Important Thermophile

• Thermus aquaticus DNA polymerase that works in
  artificial or in vitro DNA replication
• Enzyme is taq polymerase and is used in PCR

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Effect of pH

• pH scale logarithmic scale that measure the
  H in a solution
• 10-fold difference between numbers
• Bacteria grow in media with various pHs
• 0-6.9 are acidophiles
• 7.1-14 are alkaliphiles

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pH

• Each organism has a range that it can grow in
  (external pH) usually 2-3 pH units and between
  pH 5-9
• Acidophiles usually live at lt pH 2, fungi are
  more tolerant of low pH, some obligate
  acidophiles as they need a large amount of H to
  maintain membrane structure
• Alkaliphiles usually gt pH 10, some are also
  halophilic (love salt) use the Na to
• proteases and lipases from alkaliphile bacteria
  seen in household cleaners
• Neutrophiles live between pH 6-8
• Internal pH must remain close to neutral

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Buffers

• We add buffering chemicals to the media to insure
  to proper pH for the organisms
• Metabolic reactions will increase or decrease the
  pH depending on what is happening in the cell
• Potassium phosphate is used quite frequently, use
  others depending on the pH range needed for the
  bacteria

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Osmotic Effects

• Water availability is expressed as water activity
• Water diffuses from high to low thru a
  membrane osmosis
• Solute usually higher outside the organism so
  water moves into the cell
• cell in a positive water balance, in an area of
  low water activity, then water leaves the cells
• causes many problems

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Halophiles

• Osmotic effects seen in habitats with high salt
  
• Mild halophile 1-6, moderate halophile 7-15
  NaCl
• Halotolerant can adjust to increase in solute
  by decreasing water in the cell
• Extreme halophiles 15-30 NaCl

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Other Types of Organisms

• Osmophiles grow in environments with a high
  sugar
• Xerophiles grow in very dry environments

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Compatible Solutes

• Organisms grown in an area of low water activity
  need to adjust to this
• Gain water by increasing the concentration of
  internal solutes
• Accomplish this by
• pumping inorganic ions into cell from environment
• synthesizing or concentrating and organic solute
• Solute must not inhibit the biochemical processes
  in the organism

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Solutes
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Where do the Solutes Come From?

• Synthesize or take up solute genetically
  determined by the organism
• Staphylococcus species are salt tolerant use to
  select over salt intolerant organism and use
  proline as a compatible solute
• See glycine betaine in halophilic bacteria and
  cyanobacteria
• Extreme halophiles produce ectoine (cyclic
  derivative of aspartate)

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Oxygen and Microbial Growth

• Anoxic organisms grow without oxygen
• Classes of microorganisms vary in use of oxygen
  and tolerance
• Aerobe grow in 21 O2 and respire O2 in
  metabolism
• Microphiles require less than 21 O2 may
  contain an O2 labile protein, limited capacity to
  respire
• Facultative aerobe under appropriate nutrient
  and culture conditions either grow anoxic or oxic
  condition
• Anaerobes cannot respire in O2
• 2 kinds aerotolerant anaerobes can tolerate O2
  and grow in the presence of O2 but do not use it
  and obligate or strict anaerobes inhibited or
  killed by O2

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3 Types of Obligate Anaerobes

• Prokayotes important one is clostridium family
  that is gram positive spore forming rob that
  causes food poisoning
• Some fungi
• Few protozoans
• Sensitivity to O2 varies in all these groups

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Culture Techniques

• Anaerobes need the O2 removed form the culture
  use a reducing agent such as thioglycolate in
  broth to determine oxygen requirements
• Growth at the top is obligate aerobes,
  facultative organisms grow throughout the medium
  and anaerobes grow only at the bottom of the
  tubes
• Also use resazurin in the medium to indicate if
  O2 is present should see only near the top

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Anoxic Jar or Anaerobic Hood

• Use a tightly sealed jar or bag that you use a
  chemical reaction to remove all the O2 from it to
  grow anaerobes
• Hood uses a series of vacuum pumps to remove O2
  and replace usually with N2

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Toxic Forms of O2 and Enzymes
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Enzymes

• Catalase is the most common enzyme to remove H2O2
  
• Used in conjunction with superoxide dismutase
  which generates H2O2 when combining 2 superoxide
  ions, also makes O2
• Peroxidase removes H2O2 but requires NADH to make
  water
• Superoxide reductase in Archaea reduce
  superoxide to H2O2 without the production of O2,
  remove H2O2 with peroxidase-like enzyme