Lectures 3+4: Bacterial Growth and Culture, Environmental Influences/Control of Growth

Essential Nutrients

must be supplied from the environment (i.e. microbes cannot make).

Macronutrients

• needed in large amounts

• major elements CHNOPS in cell macromolecules (carbohydrates, lipids, nucleic acids, proteins)

• cations necessary for protein fxn (enzyme cofactors, regulatory molecules)

Micronutrients

• needed in small amounts

• Trace elements necessary for enzyme function (components of cofactors or enzymes)

Autotrophy vs Heterotrophy

A: fix CO2 to assemble organic molecules (mostly sugars).

H: use pre-formed organic molecules directly or break them down to assemble other organic molecules.

Phototrophs

obtain energy from light (photons converted into chemical energy via photosynthesis)

Chemotrophs

obtain energy from inorganic (e.g. H2, S, Fe) or organic (e.g. sugars, lipids, proteins) molecules.

Lithotrophs

obtain electrons (i.e. transfer of energy; oxidation) from inorganic molecules

Organotrophs

obtain electrons from organic molecules

Most phototrophs and chemolithotrophs are strict autotrophs/hemotrophs?

strict autotrophs

Nutrient are taken up by __.

Porins: Membrane-spanning protein channels discriminate between substrates of certain sizes or chemical composition, then permit those substances to enter into the cell.

Permeases

Substrate-specific membrane proteins transport the nutrient to the cytoplasm (ATP energizes)

Complex media

nutrient rich, poorly defined

Minimal defined media

contain only those nutrients that are essential for growth of a given microbe.

Enriched media, potential reason for use

complex media to which extra nutrients (e.g. blood, serum) are added

• some organisms are fastidious, meaning they have complex/specific/demanding needs, and will only grow if those specific needs are met

Selective media

favor growth of one organism over another

Differential

exploit differences between two species that grow equally well

Growth factors

Specific nutrients required by a species that must be provided in lab media for growth

T/F: Microbes can evolve to lose various biosynthesis genes

True: (e.g. amino acids) due to the nutrient richness of their natural ecological niche.

Batch culture

liquid medium within a closed system (e.g. flask).

Lag phase

Bacteria are preparing cell machinery for growth

Log phase

growth approximates an exponential curve

Stationary phase

Cells stop growing and shut down growth machinery while turning on stress responses to help retain viability

Death phase

Cells die with a “half-life” similar to that of radioactive decay

Exponential growth (log phase) only achieved when…

Nutrients are not limited and metabolic wastes (bacterial poo) are not at a toxic level

Continuous culture

All cells in a population achieve a steady state of exponential growth, which allows detailed study of bacterial physiology.

Chemostat

ensures logarithmic growth by constantly adding and removing equal amounts of culture medium - the human gastrointestinal tract is engineered much like a chemostat.

Growth rate

measured as the number of cell divisions over a given time during the log phase.

Generation time

the time it takes for a population to double during the log phase

Biofilms

specialized, surface-attached communities

• can be constructed by one or multiple species and can form on a

  range of organic or inorganic surfaces

• extremely resistant to dehydration, antibiotics, immune system, and

  predation

Quorum sensing

Chemical signals enable bacteria to communicate, and sometimes form biofilms

Which of the following uses pre-formed organic molecules for both

carbon and energy?

a) Photoautotroph

b) Chemoautotroph

c) Photoheterotroph

d) Chemoheterotroph

D

A medium that exposes biochemical differences between two

species that grow equally well is best described as a(n):

a) synthetic medium

b) selective medium

c) complex medium

d) differential medium

e) enriched medium

D

What is the correct order of phases in a bacterial growth curve?

a) lag → log → stationary→ death

b) log → lag → stationary→ death

c) lag → stationary → log→ death

d) stationary → log → lag→ death

e) lag → log → death → stationary

A

“Normal” growth conditions, defined by humans

• sea level, 1atm pressure

• temperatures 20-40C

• near-neutral pH

• 0.9% salt and ample nutrients

Extremophiles

microbes (bacteria, archaea, and a few eukaryotes) that are able to grow in conditions very different from those for humans (e.g. temperature > 100°C; pH 2).

Temperature classifications

• Hyperthermophile: above 80 C

• Thermophile: 50-80 C

• Mesophile: 15-45 C

• Psychrophile: below 15 C

Osmolarity classifications

• Halophile: high salt, >2M

• Halotolerant: high salt not required, but able to grow up to 2M

Pressure classifications

• Barophile: high pressure, >380atm

• Barotolerant: btwn 10-500atm

Heat-shock response, products

Caused by rapid temperature changes during growth that activate groups of stress response genes

• chaperones: maintain protein shape

• enzymes: change membrane lipid composition.

Increased hydrostatic pressure and cold temperatures __ membrane fluidity. Therefore…

Reduce. Therefore, specifically-designed membranes and protein structures are needed

Aquaporins

Membrane-channel proteins that allow water to traverse the membrane much faster than by diffusion- they help protect the cell from osmotic stress.

Mechanisms used by microbes to minimize osmotic stress (2)

1. hypertonic: protect their internal water by synthesizing or importing compatible solutes (e.g., proline, that are not charged and not toxic to the cells)

2. hypotonic: pressure-sensitive or mechanosensitive channels can be used to leak solutes out of the cell.

These strategies become ineffective under extreme osmotic shock

Temperature effects on phys

• cell temp matches environment

• optimum, minimum, and maximum temp normally span 30-40C

• species grows most quickly at temp where all cell proteins work most efficiently, growth stops when rising temperatures cause structures to fail

Ways bacteria can regulate pH

1. uncharged organic acids can cross membrane then dissociate inside the cell, releasing a proton and disrupting internal pH

2. exchange extracellular K+ for intracellular H+ when the internal pH becomes too low

3. use sodium motive force in addition to PMF to generate energy. Rely on Na+/H+ antiporters (out/in) to bring H+ into the cell – since their environment is very high in pH (low levels of H+)

4. altered membranes keep fragile cytoplasmic enzymes away from harsh extracellular pH

Strict aerobes

can only grow in o2

microaerophiles

grow only at lower O2 levels

strict anaerobes

die with the slightest amount of oxygen

facultative anaerobes

can live with or without O2

Techniques to culture anaerobes in lab (3)

1. Reducing agents (thioglycolate) eliminate dO2 from liquid culture

2. Palladium catalyst within sealed jar removes O2 by reacting with H gas and forming water

3. Anaerobic glove box: vacuum system removes all atmospheric gases

Depletion of nutrients elicits a __ response…

“Starvation response” where enzymes are produced to increase the efficiency of nutrient gathering and protect cell macromolecules from damage

Programmed cell death

Dying cells release nutrients that neighboring cells use to survive when severely stressed by starvation

Oligotrophic

natural ecosystems low in nutrients

Eutrophication

Sudden infusion of large quantities of a formerly limiting nutrient.

• can lead to “bloom” or microbes, which can threaten competing species

Sterilization

killing of all living cells, spores, and viruses

Disinfection

killing or removal of pathogens from inanimate objects; does not necessarily result in sterilization

Antisepsis

killing or removal of pathogens from the surface of living tissues

Sanitization

reducing the microbial population to safe levels

Physical control of microbes

1. high temp, high pressure: autoclave

2. pasteurization: ultra high temp for short period of time

3. cold: low temp slows growth but doesn’t kill

4. filtration: micropore filters can remove microbial cells but not viruses

5. irradiation: rays, beams, xrays to penetrate

T/F: Antibiotics are extremely effective against biofilms and microbes

False. They can quickly evolve to resist the antibiotics

Biological control and examples

The use of one microbe to control the growth of another

1. Probiotics: contain certain microbes that, when ingested, aim to restore balance to intestinal flora

2. Phage therapy: aims to treat infectious diseases with a virus targeted to the pathogen – phages only target specific bacteria

3. Predatory bacteria: hunt and consume other bacteria (not yet approved)

Barophiles

grow at high pressure

halophile

require high salt concentration

Which of the following environments is MOST likely to harbor a

microbe that could function as a human pathogen?

a) Glacier on Mount Everest

b) Seafloor vent at the bottom of the Pacific Ocean

c) Human colon (large intestine)

d) Soil beneath the Bonneville Salt Flats

e) Table top in a school cafeteria

C

Which of the following statements about microbes and temperature is FALSE?

a) Within a specific range, the microbial growth rate roughly doubles for every 10°C rise in temperature.

b) Changes in temperature affect membrane fluidity, and thus influence nutrient transport.

c) Thermophiles are microbes that grow at temperatures between 40°C and 80°C

d) Microbes have mechanisms to control their temperature

D

What are microbes that grow at very high pressures called?

a) Osmophiles

b) Mesophiles

c) Xerophiles

d) Halophiles

e) Barophiles

E

Why can’t bacteria grow in solutions with very high concentrations of sugar?

a) Bacteria cannot digest pure sugar.

b) Sugar raises the solution’s osmolarity.

c) Sugar lowers the solution’s osmolarity.

d) Sugar raises the solution’s pH.

e) Sugar lowers the solution’s pH.

B

Jerky recipes typically include a vinegar-based marinade, copious

quantities of salt, and dehydration in a food smoker. The finished

product is stored at cool temperatures. Which of the following

physical parameters for growth is NOT addressed by this recipe?

a) Barometric pressure

b) Temperature

c) Osmolarity

d) Hydronium ion concentration (pH)

A

You work for an engineering firm attempting to improve upon the

sterilization capabilities of a standard autoclave. Which one of the

following design changes is the MOST promising area to research?

a) Using dry heat instead of steam

b) Increasing the chamber pressure

c) Decreasing the treatment time

d) Decreasing the treatment temperature

B