4 - Bacterial Growth & Control

What is bacterial growth and generation time?

Bacterial growth - increase in # of cells in population

• exponential - population x2 w/ ea. generation
  Generation time - time needed for population to x2 (unique to each species)

Equation to calculate bacterial population growth

N_t = N₀ × 2ⁿ

N_t: # of cells in population at time, t

N₀: initial # of cells in population

n: # of divisions

What happens during binary fission?

Binary fission - bacteria division process

• growth → more ribosomes, macromolecules, ions

• genome copied & separated to opp. sides

• cell elongated due to new peptidoglycan & membrane synthesis (penicillin blocks this step)

• FtsZ ring forms a septum in middle → pinches

Transpeptidases & transglycosylases in peptidoglycan expansion (AKA penicillin binding proteins / PBPs)

• Cell wall expanding, peptidoglycan is cut, new NAG-NAM dimers inserted

• Proteins transpeptidases & transglycosylases help ligate sugars & protein crosslinks

• Penicillin blocks PBPs - peptidoglycan is cut but not reconnected

Planktonic vs biofilm growth

Planktonic growth -

1. bacteria in liquid

2. division of free-floating individual cells

Biofilm growth -

1. multicellular communities

2. attached to inanimate surface or tissue

3. encased in polysaccharide

4. protected from antibiotics, disinfectants, physical stress

5. causes >75% of human bacterial infections

Quorum sensing - events, purpose & autoinducers

• Bacteria secrete small chemicals called autoinducers (AI)

• increased population size → higher AI concentration

• bind to receptors, change gene expression

• allow population coordination

  • toxin production, antibiotic resistance, growth rate, planktonic → biofilm

Stages of bacterial growth in closed systems

1. Lag phase

2. Log phase

3. Stationary phase

4. Death/prolonged decline phase

Lag phase

• 1st stage in bacterial growth

• no NET change in popl. size

  • rate of division = rate of death

• bacteria make enzymes to prep for rapid growth

• length can vary depending on cell health

Log phase

• 2nd stage in bacterial growth

• maximal rate of growth & use of nutrients

• exponential growth

• early: lots of primary metabolites

• late: prepares for impending starvation

Stationary phase

• 3rd stage in bacterial growth

• environment becomes harsh

• cell death = cell division

• dying cells release nutrients → used by survivors

• many species produce toxins

Death/prolonged phase

• 4th stage in bacterial growth

• very unfavorable life conditions

• exponential death rate, then slow decline in #s

• total popl. loss may take years

Effect of temperature on bacterial growth

• All bacteria have their own livable range of temps

• Optimum temperature → best growth

Cold temps -
Psychrophiles: -5 - 15^oC (Arctic/Antarctic)
Psychrotrophs: 20 - 30^oC (soil/water of temperate climates; fridge spoilage of food)

Moderate -
Mesophiles: 25 - 45^oC (harmful to humans prefer 35 - 40^oC)

Hot -

Thermophiles: 45 - 70^oC (hot springs, water heaters, hot tubs)
Hyperthermophiles: 70 - 110^oC (hot springs, hydrothermal vents, boiling water)

Effect of oxygen on bacterial growth

• O₂: final e^- acceptor in aerobic respiration & extremely reactive & toxic (converted to free radicals & hydrogen peroxide; enzymes needed to detoxify

1. Obligate aerobe - growth at top of liquid

2. Facultative anaerobe - concentration gradient growth; most at top → some at bottom

3. Obligate anaerobe - even growth between middle down

4. Microaerophile - growth near top with some space below surface

5. Aerotolerant anaerobe - even growth throughout

Effect of pH on bacterial growth

Neutrophiles - most bacteria grow best in ~neutral pH 7

Acidophiles - acidic pHs; must ship out H⁺ or neutralize nearby

Alkalophiles - alkaline pHs

Effect of water availability / solute concentration on bacteria growth

All bacteria require water, varies in amount; most prefer isotonic (to own cytosol) environments

Plasmolysis - high salt, low water → shrinking in hypertonic environment

Halophiles - like salt & require higher solute conc.

Extreme halophiles - require very high solute

Halotolerant - can tolerate higher solute

Direct bacterial growth measurement

Serial dilutions - dilutions of dilutions…

• lowers count of bacteria; easier

Plate counting - small sample of ea. dilution & spread on petri dishes

• count CFU when it’s between ~30-300

  • calc titer: (CFU * 1/dilution) / vol. plated

• Pro: measures live bacteria

• Con: can’t tell 1 single cell vs 1 clump

Mean probably number (MPN) - good if bacteria can’t grow on plates

• dilutions of OG sample & add to sterile tubes of broth

  • look for growth

• patterns of growth → statistical probability of counts

• MPN index/100 mL from combo of +’s

Microscopy counting -

• bacteria → specialized microscope slide Petroff-Hausser cell counter

• count # w/i square grid → multiple # cells by 1,250,000 → # cells/mL

• Pro: can differentiate clumps

• Con: can’t distinguish live vs. dead

Filtration & plating -

• pass large vol. of water through paper filter (traps bacteria) → touch filter onto Petri dish → count colonies

Indirect bacterial growth measurement

Turbidity / spectrometry

• growth in broth → cloudy

• spectrophotometer - shine light through tube at specific wavelength

Enzymes

• measure specific enzyme activity / metabolic pathway; more activity = more cells

Dry weight

• centrifuge cells & discard liquid → desiccate (dry) overnight; more weight = more cells

Sterilization vs disinfection vs sanitization

Sterilization - complete removal of all microbes (+viruses & endospores)

Disinfection - removal of most microbes

Sanitization - removal of enough microbes for health standards

Physical control of bacterial growth

1. Heat - denatures proteins & oxidizing other cellular components

• dry heat - very high temps, sterilization

• boiling - destroys most pathogens

• pasteurization - high heat, short time; not total sterilization

• autoclaving - pressurized steam; sterilize equipment/canning

1. Radiation -

• UV light - dmgs DNA of microbes on surfaces/air/water; doesn’t penetrate thick specimen

• ionizing radiation - very high energy; dmgs DNA & membranes; can penetrate products after packaging

1. Filtration -