Microbial growth

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Microbial growth

• Dependent on both physical and chemical factors

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Environmental factors which exert the greatest
influence on the survival of microorganisms

• 1.
• 2.
• 3.
• 4.
• 5.
• 6.
• 7.
• 8.
• The ability to survive in environmental
  conditions and reproduce is called________________
  _________

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Temperature

• There are no organisms that can grow at all
  temperatures
• Survival in very hot environments is difficult
  due to the fact that even a slight increase in
  temp speeds up enzyme reactions and may cause
  denaturation of the protein enzymes
  (denaturation means ____________________)

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Temperature

• Each species has a
• Minimal
• Maximal
• Optimal
• Most bacteria will grow in the range of 30-40
  degrees
• Temperature has an effect on growth rate,
  metabolism, reproduction rate, metabolism and
  morphology

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Psychrophiles

• Group I- can grow at 0oC with an optimum of 15o.
• Grow in oceans, glaciers and other constantly
  cold environments
• Can exist in cold environments because they have
  high concentrations of _________ fatty acids in
  the phospholipids of their cell membranes which
  helps maintain fluidity
• Seldom cause problems in food preparation
• No bacterial growth occurs in frozen solid
  conditions but remain dormant or in a state of
  suspended animation

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Psychrophiles

• Group II optimal temperature range is 20-30o
  and cannot grow above 40oC.
• Spoilage microbes in this group
• Can be termed
• Psychotrophs
• Moderate psychrophiles
• Facultative psychrophiles
• Some mesophilic bacteria that continue to grow in
  cold environments (refrigerator) are termed
  _______________ mesophiles

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Refrigeration

• Most common method of preserving food supplies
• Will prevent the growth of all but a few
  pathogenic bacteria
• Freezing- organisms become dormant
• Spoilage in refrigerator due to psychotrophs are
  mold mycelium, slime on food surfaces, off
  colors, off tastes

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Mesophiles

• Most common type of bacteria
• Optimum temperature range 25- 40 oC
• Saprophytes also known as __________ grow best at
  _____ oC
• Human pathogens grow best at _____ oC. This group
  includes most of the spoilage and disease-causing
  organisms

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Thermophiles

• Optimum growth temperature is between 50-60 oC,
  the temperature of hot tap water
• Some members of Archaea grow optimally at tempgt
  80 oC and are considered _________________________
  __and grow near hydrothermal vents in the ocean,
  sulfur is important in their metabolic activity
  (record is 110oC near vent, kept from boiling due
  to ocean water pressure)

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Thermophiles

• Grow on soil surfaces that get to 50-70oC in
  deserts, power plants, compost heaps, hot springs
• To survive denaturation of proteins, bacteria
  rapidly regenerate enzymes lost to high
  temperature and use more heat- stable amino
  acids. Cell membranes have high concentrations
  of _________ fatty acids
• Many cannot grow at temperatures lt 45 oC
• Thermophilic bacteria are not considered a public
  health problem

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pH

• In microbial growth media, ___________ are often
  used to maintain the relative constancy of the
  media (usually phosphate because it also
  provides _____, an essential nutrient.
• _____________ media employs pH indicators to
  distinguish between bacterial species and the
  enzymes they contain

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pH

• Most bacteria grow best in a narrow range near
  neutrality (Enterics are neutrophiles)
• Foods such as sauerkraut, pickles and cheeses are
  preserved from spoilage by acids produced in
  bacterial _______________
• Acidophiles are tolerant of acid pH
• Thiobacillus a true acidophile (pH5)
• Bacteria that produce acids and are obligate
  acidophiles- love pH levels lt 6.5
• Fungi appear to be more tolerant of acid
  conditions (ph 5-6) than bacteria
• Alkalophiles like pH levels above 7.5
• Ex Bacillus pH gt 9

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Water

• All organisms need water to survive
• Osmosis- movement of water from an area of higher
  concentration to lower (or lower solute
  concentration to higher)
• High osmotic pressure will remove necessary water
  from a cell
• Plasmolysis- cells placed in a hypertonic
  solution will cause the __________________ to
  shrink away from the cell wall and can result in
  cell death
• Therefore, addition of salts are used to preserve
  foods such as ___________________

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Water

• Halophiles- like ______________
• Staphylococcus __________can be differentiated by
  its ability to transform energy on MSA. It is a
  ___________ halophile
• Haloanaerobium likes 30 NaCl
• Osmophiles- high ___________ environment Ex
  bacillus
• Xerophile- can live in ________ environments Ex
  mycobacterium tuberculosis
• Use intracellular solutes to absorb any available
  water from the environment

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Oxygen needs

• Our environment contains approximately 21 oxygen
  and 78 nitrogen. Both exist as diatomic
  molecules (_____________)
• Thioglycolate media is used to test the oxygen
  requirements for each species
• Contains the pink dye, resazurin to detect O2 gas
• Lower levels of media have _____ oxygen than
  higher levels

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Strict aerobes

• These organisms produce H2O2 intracellularly as a
  result of cell respiration
• Have enzymes to remove this toxic peroxide from
  their cells
• Catalase
• Commonly used to identify bacteria
• Human cells also have catalase (when peroxide is
  added to a wound.)
• H2O2 ? H2O O2 (bubbles) this is the catalase
  test
• Easy way to distinguish Staphylococcus spp from
  Streptococcus spp
• Peroxidase
• does not produce oxygen bubbles
• H2O2 2 H ? 2 H2O

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Facultative anaerobes

• Grow better in oxygen environments but can
  survive in the absence of oxygen gas
• Use fermentation or anaerobic respiration when
  oxygen is not available (ability to produce
  energy decreases)
• Examples E coli found in the human intestinal
  tract and many yeasts

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Microaerophiles

• Aerobic, do require oxygen
• Grow only in oxygen concentrations lower than
  air.
• In solid nutrient media, grow only at a depth
  where small amounts of oxygen has diffused into
  the media
• Optimum O2 concentration is 1-15

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Strict anaerobes

• Killed in the presence of O2 gas
• Best examples are members of the genus,
  Clostidium
• C.__________
• C. __________
• C. __________
• In the presence of oxygen, obligate anaerobes
  appear to form some superoxide free radicals
  which are so toxic that those organisms
  attempting to grow in oxygen atmosphere must
  produce SOD to neutralize them (SOD is
  _______________)

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Microbial nutrition and energy sources

• How do microbes obtain their nutrients
  (substances necessary for metabolism)?
• Nutrients are needed in either macro or micro
  quantities
• Macronutrients C, H, O, P, S (are 96 of cell) K,
  Na, Ca, Mg, Cl, Fe are other macronutrients

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Carbon

• One of the most important requirements for growth
• Structural backbone of all living matter
• Needed for all organic compounds that make up the
  cell
• Chemoheterotrophs get most of their carbon from
  the source of their energy, the organic
  materials, __________, ___________ and lipids
  from other organisms

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Carbon

• Autotrophs- get their carbon from carbon dioxide
• Chemoautotrophs- get their energy form inorganic
  compounds such as sulfur, nitrogen or hydrogen
  gas or minerals (lithotrophs)
• Photoautotrophs get their energy from light

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Hydrogen

• Maintains pH levels
• Forms hydrogen bonds in macromolecules
• Needed for chemiosmosis- to form a gradient
  across membranes to produce ATP

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Nitrogen

• Plants and animals are unable to use the 78
  nitrogen gas available in the air
• Some bacteria are nitrogen fixing bacteria which
  can convert N2 to a useable form for plants (NO3)
  to use as fertilizer
• Macromolecules DNA, RNA, ATP, proteins and
  peptidoglycan all contain nitrogen
• Makes up 14 of the dry weight of a bacterial cell

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Oxygen

• Needed for cell respiration (to make ATP, it is
  the final hydrogen receptor)
• Part of the water molecule which is the solvent
  found in cells

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Phosphorus

• Required for ATP, DNA, RNA and phospholipids
• One of the main ingredients in commercial
  fertilizers along with nitrogen and potassium

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Sulfur

• Used to synthesize sulfur-containing amino acids
  and vitamins such as thiamine and biotin
• Sulfur contributes to the tertiary configuration
  of protein structure

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Other macromolecules

• K, Na, Ca, Mg, Cl, Fe
• Potassium, magnesium and calcium are element that
  microorganisms also require
• Are often cofactors for enzyme reactions
• May help catalyze a reaction by forming a bridge
  between an enzyme and a substrate

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Micronutrients

• Trace elements- microbes require some small
  amounts of certain mineral elements such as Fe,
  Cu, Mb, Zn, Co, Ni, W, Se
• Usually cofactors

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Culture media

• Nutrient material prepared for the growth of
  microorganisms in the lab
• Initially must be sterile
• Inoculate the culture media with the
  microorganism, referred to as a culture when
  grows and multiplies
• Synthetic media- chemically defined in the
  identity and amount of each component of the
  media
• Complex- chemically undefined and non-synthetic,
  made up of nutrients such as extracts from yeast,
  meat or plants or digests of proteins from these
  or other sources

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Media

• Can be solid or broth
• Agar (red algae) is used because of its chemical
  properties and is not a source of nutrient (that
  is why gelatin is not used)for microorganism
  (inert gel)
• Liquifies at 100o, remains a liquid until 40oC
  and once solidified does not liquify until the
  temperature nears 100oC (good for culturing
  thermophilic bacteria)
• Nutrient broth- complex media in liquid form
  (- the agar)

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Basic types of media

• General purpose- non selective used to grow as
  many types of bacteria as possible
• Nutrient agar- N.A.
• Trypticase soy agar T.S.A.
• Enriched media- general purpose with added growth
  factors
• Compounds such as amino acids or vitamins which a
  particular species of bacteria are unable to
  produce metabolically
• Those which require growth factors are known as
  fastidious
• Chocolate agar
• Blood agar (uses sheep blood)

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Enriched media

• Those which require growth factors are known as
  fastidious
• Chocolate agar
• Defibrinated sheep blood added to sterile molten
  agar heated at 80oC until chocolate color
  develops
• Blood agar (uses sheep blood)- cool agar base to
  45-50oC, aseptically add defibrinated blood

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Selective media

• Media which contains agents that suppress the
  growth of some microbes while encouraging others
  to grow
• Mannitol salt agar- (M.S.A.)- contains a high
  salt concentration of 7.5 which is inhibitory to
  most bacteria other than Staphylococci
• Medium also has a differential function it
  contains the sugar, mannitol which some
  Staphylococci are capable of fermenting and a pH
  indicator, phenol red which changes color when
  acid is produced

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Selective media

• Eosin methylene blue agar- (Levine) lactose and
  the dyes, eosin and methylene blue permit
  differentiation between enteric lactose
  fermenters and non-fermenters as well as the
  identification of the colon bacillus, E. coli
• E. coli are blue-black with a metallic green
  sheen caused by the large quantity of acid that
  is produced and that precipitates the dyes onto
  the growths surface

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Selective media

• MacConkey agar- (Mac) the inhibitory action of
  crystal violet on the growth of gram positive
  bacteria allows the isolation of gram negative
  bacteria
• Incorporation of lactose, bile salts and the pH
  indicator, neutral red, allows differentiation of
  enteric bacteria on the basis their ability to
  ferment lactose

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Differential media

• Make it easier to distinguish colonies of a
  desired organism from other colonies growing on
  the same plate
• Can support the growth of several types of
  bacteria
• Pure cultures have identifiable reactions with
  differential media
• These media include M.S.A., E.M.B., B.A.,Mac,
  T.S. I. (triple sugar iron)

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Differential media

• Blood agar- used to identify species that destroy
  RBCs
• Streptococcus pyogenes shows beta hemolysis or a
  clear ring around their colonies
• M.S.A.- can be used to distinguish Staphylococcus
  aureus which is a mannitol fermenting organism
  from other organisms which are unable to (pH
  indicator phenol red from red to yellow)

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Differential media

• MacConkey- lactose containing media which gram
  neg bacteria can metabolize (pink or red color)
  can be differentiated from the non-fermenters
  (colorless)

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Reducing media

• Encourages growth of obligate anaerobes
• Media includes sodium thiogylcolate that
  chemically combines with dissolved oxygen to
  deplete the oxygen levels in the media
• media is kept in tightly capped tubes which are
  heated prior inoculation to drive off any
  absorbed oxygen

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Microbial growth

• Refers to the number of cells, not the growth of
  individual cells
• Based on the availability of nutrients but may be
  influenced by other factors
• Due to the asexual reproductive method, binary
  fission
• Growth in a closed system typically demonstrates
  the following growth curve

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Bacterial growth curve- closed system

• Lag phase
• Log phase (exponential growth)
• Stationary phase
• Death phase

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Generation time

• The time required for a cell to divide and its
  population to double is called the generation
  time
• If doubling occurred every twenty minutes, (which
  it does with E. coli) in less than seven hours,
  there would be over one million cells from just
  one cell initially

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Generation time problem

• If you had one bacterium that divided every 30
  minutes, after four hours, how many bacteria
  would be present?
• Nf final number of bacteria
• Ni initial number of bacteria
• N the number of generations
• Generation rate (expressed as generations per
  hour 1/ generation time (in hours)

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Maintaining a bacterial culture

• What are ways to maintain the viability of a
  bacterial culture?
• Temperature
• Subculturing
• Continuous culturing using a CHEMOSTAT)-specialize
  d apparatus keeps the population in exponential
  growth phase by draining off spent media and
  adding fresh media (used in industrial
  fermentation)

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