Microbiology - Chapter 7

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Microbiology - Chapter 7 8

• Microbial Growth Bacteria reproduce by binary
  fission, a cell divides into two, two to four,
  four to eight, etc.
• Cell division can occur quite rapidly depending
  on nutrient levels, temperature, etc. E. coli
  can divide every 20 minutes (time to double
  generation time) in nutrient media at 37 degrees
  C. The numbers get so large we express them as
  the log of the number of cells.

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Microbiology - Chapter 7 8

• Microbial Growth is affected by two major
  factors
• Environmental temperature, pH, Osmotic
  conditions
• Chemical Proper concentrations of C, H, O, N,
  P, S, some trace elements, and some organic
  cofactors

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Microbiology - Chapter 7 8

• Bacterial Growth Curve
• A Lag phase C Stationary phase
• B Log phase D Death Phase
• Know this and be able to explain what is
  occurring during each phase.
• Bacteria that produce endospores may not have a
  death phase. Why?

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Microbiology - Chapter 7 8

• Why study such a growth curve?
• Helps us understand how microbes grow under
  different conditions
• Helps us understand how pathogen grow in our body
  
• Helps us study the effect of different chemicals,
  osmotic conditions, even the effect of
  temperature on bacterial growth.
• Ex. What would the growth curve for E.coli look
  like if we incubated at 4 degrees Celsius? At 65?

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Microbiology - Chapter 7 8

• How do we measure growth of bacteria in a growth
  curve?
• Direct cell count using stained slides that have
  a grid for counting. (Tedious, a real pain)
• Indirect Serial dilution, plates are
  innocculated - incubated and colonies counted.
  Number of colonies X dilution factor gives the
  number of bacteria.

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Microbiology. Chapter 7 -8 How do we measure
growth of bacteria in a growth curve? Direct
cell count. Tedious and time consuming
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Microbiology - Chapter 7 8

• How do we measure growth of bacteria in a growth
  curve?

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Microbiology - Chapter 7 8

• How do we measure growth of bacteria in a growth
  curve? Measure cloudiness in a test tube as the
  number of cells increase (turbidity) using a
  spectrophotometer. Correlate this data with a
  standard plate count and now just use the
  turbidity measurement look up number from a
  chart from then on. Saves time and money.

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Microbiology - Chapter 7 8

• How do we measure growth of bacteria in a growth
  curve?
• Coulter counter. Electronically counts number of
  bacteria as bacteria pass through a tiny tube.
  Expensive.

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Microbiology - Chapter 7 8

• Physical factors that affect bacterial growth
• Mesophiles grow best moderate temp. 25 40
  degrees
• most of our lab microbes
• Psychrophiles adapted to survive and grow at
  cooler temp., even in the frig (below 25
  degrees)
• Listeria (in cheeses and meat)
• Thermophiles adapted to and grow at much higher
  temp.
• Thermus aquaticus, from oceanic vents, survives
  at 60 degrees C
• Leprosy bacilli prefer 30 degrees, most pathogens
  prefer 37 degrees.

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Microbiology - Chapter 7 8

• Physical/Chemical factors that affect bacterial
  growth pH measure acidity and alkalinity of
  media
• Bacteria grow best at pH range of 6.5 to 7.5
• Fungi grow better at slightly acid condition (5.0
  to 5.5)
• Sabaraud dextrose and Potato dextrose agars
• One pathogen, Helicobacter pylori, is adapted to
  and survives in stomach acid (cause of ulcers)
• Hydrostatic pressure some bacteria grow really
  well deep in the ocean at pressures that crush
  submarines like and egg

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Microbiology - Chapter 7 8

• Physical/Chemical factors that affect bacterial
  growth pH measure acidity and alkalinity of
  media
• Osmotic pressure relative salt concentrations in
  water solutions
• Hypertonic higher salt concentrations, slows or
  stops bacterial growth salt preservative in meat
• some prefer higher salt Halophiles
• some survive and thrive, Vibrio bacteria, V.
  cholera
• Hypotonic lower salt, fresh water, net flow
  water into cells, bacteria have rigid cell wall
  resist rupture
• Isotonic equal solute (salt) no net flow,
  preferable

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Microbiology - Chapter 7 8

• Chemical factors that affect bacterial growth
  Nutrition
• How microbe acquire nutrients. C, H, O, N, S, P,
  Ca, Mg, etc
• Carbon
• Autotroph producers, photosynthetic, use CO2
  and H2O, sunlight as energy, make their own
  food
• Heterotroph require preformed food, digestive
  and absorptive, most microbes
• Chemoautotroph unique metabolism, use chemical
  energy from inorganic molecules, Sulfur and
  Iron

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Microbiology - Chapter 7 8

• Chemical factors that affect bacterial growth
  Nutrition
• How microbe acquire nutrients. C, H, O, N, S, P,
  Ca, Mg, etc
• Oxygen
• Obligate aerobes require molecular oxygen (as
  final electron acceptor in catabolism)
• Pseudomonas spp.
• Obligate anaerobes require atmosphere with no
  O2 an organic molecule is final electron
  acceptor in catabolism (like a fermentation
  pathway)
• Clostrida - grow in Brewer Jar
• Facultative anaerobes grow with or without O2,
  usually are also fermenters, like E. coli
• Microaerophile grow best in lower oxygen and
  higher carbon dioxide, Strep., candle jar

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Microbiology - Chapter 7 8

• Problems with oxygen oxygen can be toxic, it
  oxidizes and destroys vital cell chemicals
  aerobic organisms have enzymes and systems to
  handle it
• SOD superoxide dismutase, enzyme that
  chemically alters toxic oxygen free radicals and
  toxic high energy singlet oxygen to less toxic
  hydrogen peroxide
• Catalase Converts hydrogen peroxide to oxygen
  and water

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Microbiology - Chapter 7 8

• Nitrogen Found in all the amino acids,
  nitrogenous bases of nucleic acids, etc.
• Hydrogen found in all biological molecules,
  Carbs, fats, proteins, nucleic acids, etc
• Phosphorous found in nucleic acids, ATP, and
  phospholipdids of membranes
• Sulfur found in 2 or 3 amino acids of microbes
• Trace elements inorganic elements needed in very
  tiny concentrations (manganese, cobalt, Zn, Cr)

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Microbiology - Chapter 7 8

• Organic cofactors
• Vitamins
• Required by certain bacteria, fastidious hard
  to grow
• Coenzymes Many microbes produce their own from
  scratch, source of our supplements (one a day,
  GNC)
• Fastidious organisms may require enriched media
  to get them to grow (blood, eggs, etc)
• Some organisms are almost impossible to culture
  because of their strict parasitic-fastidious
  nature (syphilis, leprosy)

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Microbiology chapters 7 - 8 part 2

• Metabolism
• Catabolism
• Anabolism Both occur simultaneously in
  cells
• Catabolism eventually produces the chemical
  energy (ATP) required for all cellular functions
  such as anabolism (synthesis), membrane
  transport, etc.

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Microbiology chapters 7 - 8 part 2

• ATP Adenosine triphosphate, universal cellular
  energy
• Cyclically made and energy is stored and then
  broken down and the energy is released

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Microbiology chapters 7 - 8 part 2

• Note ATP is a ribonucleotide, it has ribose, a
  nitogenous base (adenine), and phosphate. The
  high energy bond of the terminal of the three
  phosphates is the one cyclically broken and
  regenerated.
• Sugars like glucose can be broken down in a
  catabolic pathway controlled by many cellular
  enzymes. Some of the energy released by the
  breaking of covalent bonds is harvested and
  stored in the energy bonds of ATP.
• Most any biomolecule can be used for energy we
  will focus on the catabolism of glucose (a
  monosaccharide) and later show how the others are
  involved (lipids, AA, etc)

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Microbiology chapters 7 - 8 part 2

• Quick review on enzymes
• Organic catalyst (made of carbon, speed up rate
  of chemical reactions)
• Made of protein chains of Amino acids in a
  specific sequence that fold and coil into
  specific shapes. Their shape is key to
  understanding their function. (remember shape
  determines function) Also, shape is easily
  affected by changes in temperature. So, heat or
  cold can cause enzymes to slow down or even stop.
  
• An enzyme lowers activation energy energy
  required for a reaction to begin

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Microbiology chapters 7 - 8 part 2

• Quick review on enzymes
• Substrates are the material that are acted on by
  the enzyme
• Enzymes are often named using the name of the
  substrate and adding ase. Sucrase breaks down
  sucrose to glucose and galactose. Enzyme driven
  reactions are often reversible.

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Microbiology chapters 7 - 8 part 2

• Aerobic metabolism specifically glucose
  catabolism
• This stuff is hard Just do It
• Goal
• 1. List the three stages of glucose catabolism
• 2. Know the basic steps of each stage
• 3. Know how much ATP is made at each stage per
  molecule of glucose
• 4. Starting products and end products, other
  important carriers (NAD)
• 5. The difference between substrate level
  phosphorylation and oxidative phosphorylation
• 6. Theory of chemiosmosis and ATP production at
  the membrane of the mitochondria

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Microbiology chapters 7 - 8 part 2

• Glucose is a hexose, monosaccharide, C6H12O6
• It is systematically broken down through three
  related pathways to Carbon dioxide (CO2) and
  Water (H2O)
• Overall FormulaC6H12O6 ___ O2 ? CO2 ___
  H2O
• The three stages
• Glycolysis (anaerobic) (in cytoplasm)
• Krebs cycle (aerobic)
• (in mitochondria)
• Electron transport (with chemiosmosis) (aerobic)

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Microbiology chapters 7 - 8 part 2

• Glycolysis Anaerobic, no oxygen required,
  linear NZ pathway
• Begins with ______
• End products _________
• How much ATP? _______
• How many carrier molecules? ____
• Name the carrier molecule. ____
• Where in the cell? _______
• What happens after if the organism
• Is an aerobe? _____
• Facultative? ______
• Strict anaerobe? ______
• Aerobe deprived of oxygen? ____

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Microbiology chapters 7 - 8 part 2

• Krebs cycle (TCA, Citric acid cycle) Aerobic
  stage, Occurs in the fluid of the Matrix

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Microbiology chapters 7 - 8 part 2

• This is a cyclic pathway
• Pyruvic acid is first acted on by an NZ and a
  coenzyme (COA). The end product is Acetyl-Coa
  and a CO2 molecule.
• Remember this occurs twice for each glucose
  molecule. (One glucose is split into two
  pyruvic acid molecules.)

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Microbiology chapters 7 - 8 part 2

• The acetyl-COA reacts with an enzyme and another
  substrate (a 4-C molecule called oxaloacetic
  acid) to produce Citric Acid, a 6 carbon
  tri-carboxylic acid 3 carboxyl groups
• Several enzymes systematically oxidize the citric
  acid into a 5-C acid, then a 4-C acid and
  eventually back to the original oxaloacetic acid
  thus a cycle. Each time the terminal carboxyl
  group is removed a CO2 molecule is produced.
• Thus, one glucose, causes the cycle to turn
  twice, each turn produces 3 CO2 (one at Acetyl
  COA step and two in the cycle)
• Now for the hard part. Understanding that an
  oxidation reduction reaction is going on at each
  step. (Here Krebs), at glycolysis, and even
  electron transport)
• Lets first review oxidation- reduction (aka
  redox)

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Microbiology chapters 7 - 8 part 2

• Oxidation Reduction
• Organic molecules like glucose have covalent
  bonds between C-C, C-H, C-O, O-H
  C6H12O6
• When the molecule is broken down -, the covalent
  bonds are broken electrons are removed and
  transferred to carrier molecules.
• Oxidation is the removal of electrons and/or
  adding Oxygen
• In Glycolysis the glucose is broken into two
  Pyruvates,
• The electrons and a H are transferred to a
  carrier, NAD.
• NAD gains the electron (and Hydrogen too), it is
  reduced
• to NADH, thus oxidation and reduction go
  together.

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Microbiology chapters 7 - 8 part 2

• Oxidation Reduction
• Look again at glycolysis.
• Glucose is oxidized and the carrier NAD is
  reduced.
• For every glucose, two Carriers are produced
• 2 NADH (what happens to them, they have to
• be regenerated oxidized back to NAD)
• Aerobes eventually produce CO2 and H2O
• Thus oxygen is the final electron acceptor(
  producing
• Water).
• Anaerobes use a different set of enzymes, a
• Fermentative pathway that generates other acids,
• alcohols or gasses (lactic acid, ethanol, CO2)
• electron acceptor is an organic molecule
• If no regeneration of NAD, the glycolysis pathway
  
• shuts down and the organism dies no ATP

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Microbiology chapters 7 - 8 part 2

• Glycolysis, no oxygen, fermentation, only 2 ATP
  per molecule of glucose
• Glycolysis, with oxygen, followed by Krebs and
  electron transport, can generate much more ATP
  (sometimes as much as 36). Aerobic mechanisms
  are much more energy efficient.
• In the Krebs cycle many more carrier molecules
  like NADH are generated and thus lead to more
  ATP. (Other carriers FAD, NADP we just use NAD
  as a representative type of carrier).
• The constantly turning of the cycle produces a
  steady stream of reduced carriers (NADH) which
  pass the electrons to a set of carrier-processor
  molecules imbedded in the membrane of the
  Mitochondria. These carriers are called the
  electron transport chain.

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Microbiology chapters 7 - 8 part 2

• Return to Krebs and show how it works with
  electron transport chain. Note how glycolysis
  and Krebs are working together. Note that each
  produces reduced carriers that need to be
  processed.

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Microbiology chapters 7 - 8 part 2

• The electrons are passed down the chain and end
  up being added to oxygen. The Hydrogen ion (H)
  is pumped out (proton pump) and flows back in at
  special sites to be added to the Oxygen and
  electron to form Water. Energy is conserved
  (harvested stored) in the bonds of ATP

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Microbiology chapters 7 - 8 part 2

• Theory of Chemiosmosis Proton pump, increased
  H ion concentration, flow through ATP synthase
  related channel, energy is harvested in high
  energy bonds of ATP. Enough to generate 34 more
  ATP.

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Microbiology chapters 7 - 8 part 2

• Fermentation Many microbes ferment sugars and
  other substrates to make ATP without oxygen
• See pg 234 in text NADH reduces pyruvate and
  ethanol and carbon dioxide are produced
• Other end products are seen lactic acid,
  acetic, acid
• We use biochemical tests and the end products of
  sugar fermentation to ID bacteria (charts in
  Bergeys) later in lab, particularly with
  unknown 2
• Some bacteria, like E. coli and Bacillus use
  nitrogen electron acceptors to regenerate NAD.
  Nitrate and Nitrite reduction are examples. Pg
  233 in text. The enzyme system is called Nitrate
  reductase

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Microbiology chapters 7 - 8 part 2

• Other fuels
• Proteins digested to amino acids
• Amino acids are
• deaminated amino group removed, the
  reulting acid can be further metabolized,
  more ATP
• decarboxylated carboxyl group removed, the
  end products then enter glycolysis or Krebs to
  make ATP

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Microbiology chapters 7 - 8 part 2

• Lipids are catabolized to Glyerol and Fatty acids
• Glycerol easily enters glycolysis and Krebs just
  like pyruvate
• Fatty acids are chopped into 2 or 3 acid
  fragments that are broken downt to carbondioxide
• Even nucleic acids OH SO MUCH MORE!!! Take
  biochem.