LECTURE 5. CHAPTER 6 - Microbial Growth

• pump in protons
• generate ammonia (+) by ammino acid degradation

3. Classification based on water activity/osmotic pressure (remember, low water activity = high osmotic pressure)

• halophiles- require sodium >9%; extremes may live in 30%
• marine salt about 3%
• cell wall and membrane fall apart w/o sodium
• nonhalophile - 0 to 1.5%

What happens when water activity is low (cell hypotonic to environment)? Water leaves cell; dehyration 

How do microbes adapt to low water activity?

• Microbes can change their internal osmotic environment
• E. coli in GI tract
• produce or import compounds that increase internal solutes
• compatible solutes - do not harm host
• ions, sugars, amino acids

4. Oxygen:

How does oxygen affect optimal growth?

• aerobes - require oxygen
• obligate/strict anaerobes - killed by oxygen
• aerotolerant anaerobes - does not require oxygen but not killed
• facultative anaerobe - grows in presence or absence of oxygen with different metabolic strategies
• microaerophiles - require reduced levels of oxygen

Why is oxygen toxic to some microbes?

• metabolic byproducts all organisms produce are toxic to cells
• hydrogen peroxide
• superoxide
• present in WBC to kill bacteria
• aerobes have catalase and/or peroxidase and superoxide dismutase (SOD)
• catalase: 2H₂0₂ -> 2H₂0 + 0₂
• peroxidase: H₂0₂ + 2H+ -> 2H₂0
• SOD: 20₂- + 2H+ -> 0₂ + H₂0₂
• strict anaerobes lack these enzymes
• microaerophiles either reduced amounts of these enzymes or oxygen-sensitive forms of enzymes
• aerotolerant have these enzymes but not those used in aerobic metabolism

• Toxic forms of oxygen are broken down by several enzymes; one of these is catalase. This enzyme breaks down hydrogen peroxide to form water and molecular oxygen. Our tissues as well as many microorganisms are catalase positive - thats why the bubbles when you pour peroxide on a cut.

Why would our tissues have peroxide in them?
Some theories of cancer hypothesize that oxidants (like toxic forms of oxygen) damage DNA and thus cause the mutations which in turn cause cancer. These theories predict that antioxidants such as beta-carotene, vitamin E and vitamin C would thus have anti-mutation activity. 

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commonly called vitamins, these are organic substances required for the growth of an organism but which the organism can not synthesize.

What is the difference between a defined and an undefined medium?

WHAT FACTORS AFFECT GROWTH?

• Nutrients - Cell constituents and energy sources
• Physical factors - temp, pH, water, oxygen, pressure

I.  Nutrients:
 

ELEMENT		CELL FUNCTION
C		backbone of organic cell components, energy
H		water, organic components, pH, hydrogen bonds, re-dox
O		water, organic components, respiration
N		amino acids, nucleotides, coenzymes, ATP
S		amino acids, coenzymes, enzymes
P		nucleic acids, phospholipids, coenzymes, ATP
Fe		cytochromes, enzymes
Na, K, Ca, Cl, Mg, Mn		Trace elements: transport, ionic balance, cofactors (e- donor/acceptors)

II. CLASSIFICATION OF ORGANISMS BASED ON carbon, energy, and electron sources

• Chemotrophs - Derives energy from chemicals
• Phototrophs - Derives energy from sunlight
• Autotrophs - Use carbon dioxide for carbon
• Heterotrophs - Use organic substrates for carbon
• Lithotrophs - Inorganic compounds for electrons

Bacterial Growth

• Bacteria grow by binary fission. Starting with one organism how many organisms would you have after 1,2,3,4,5,6,7,8,9,10,11,12 generations.

If you started with 10 organisms, how many would you have after each of the above generations?

What is microbial growth?

• Increase in cell numbers
HOW DO BACTERIA REPRODUCE?
• Binary fission
• Budding
• Fragmentation
What occurs during binary fission?
• DNA duplication
• DNA repication
• single origin of replication, bidirectional
• theta intermediate
• Separation of DNA and cytoplasmic contents
• Cross wall formation
Generation time = doubling time
• time required for a cell to divide or a population to double
• 1 to 2 or 100 to 200 or 1 million to 2 million
• Most common bacteria have a generation time 30-60 min under opt. conditions.
• Most common pathogens in the body, about 5-10 hours.

Mycobacterium tuberculosis (in lab)	12 hrs
Clostridium botulinum (in lab)	0.58
E. coli (in lab)	0.30
E. coli (in mouse)	20 hrs

What happens when you inoculate a single bacterium from slant stored in refrigerator into nutritional medium and incubate?



• Why doesn't it grow immediately?
• What determines how fast it grows?
• Why does it stop growing?


Phases in bacterial growth in batch culture (see above picture)

• Lag phase - synthesis of new components or repair
• Log phase - reproduction at maximum rate (shortest generation time)
• exponential growth 1->2->4->8->16->32->64    Bf = Bi x 2n    Log Bf = log Bi + .3t/dt    
• Stationary phase - no net increase, balance between cell division, cell "death", maintenance    Nutrients vs toxins    synthesis of storage materials    sporulation, survival genes    
• Death phase - do they really die?    some autolysis    small subpopulation persists;    accumulation of toxins    

GROWTH EQUATION: Log Bf = LogBi + .3t/dt

PRACTICE PROBLEM: A culture is inoculated at noon with 10³ cells/ml. At 10 pm the population is determined to be 10⁹ cells/ml. What is the doubling time? Describe the phases of bacterial growth.

PRACTICE PROBLEM: A culture is inoculated at noon with 10³ cells/ml. At 10 pm the population is determined to be 10⁹ cells/ml. What is the number of generations? What is the doubling time?

Write out the growth equation
n = Log₁₀ f - Log₁₀ i/0.3

Indicate the known and unknown values
Log₁₀ f  = 9, Log₁₀i = 3, t = 10, n = ?

Substitute the values and do the math

n = 9 - 3 = 6/0.3 = 20 generations

Generation time equation = (g = t/n)

Substitute values: g = 10 hr/20 = 0.5 hr = 30 min/doubling