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Microbiology - Chapter 7 _ 8

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

How do we measure growth of bacteria in a
growth curve?
             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.
             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.
             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.
              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”
              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
             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
             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
             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
             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)
                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)
  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.
  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
  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)
  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
  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.
  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
   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 Formula:C6H12O6 + ___ O2  CO2 + ___ H2O

The three stages:

Glycolysis (anaerobic)                             (in cytoplasm)

Krebs cycle (aerobic)
                                                   (in mitochondria)
Electron transport (with chemiosmosis) (aerobic)
   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? ____
  Microbiology chapters 7 - 8 part 2

Krebs cycle (TCA, Citric acid cycle) Aerobic stage, Occurs in
the fluid of the Matrix
   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.)
  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)
Let‟s first review oxidation- reduction (aka: redox)
    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.
   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
   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.
   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.
    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
   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.
   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 Bergey‟s) ** 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
   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
         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.

				
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