lecture 5

how do we grow microbes in the lab?

• culture media: solutions tailored to the nutritional needs of an organism of interest

• media is always sterilized before use to eliminate all microorganisms other than the one you will inoculate the medium with

• culture media can be liquid or solid

defined media

uses known concentrations of specific chemicals: consistent and reliable environment for precise control

complex media

uses undefined natural components (blood, yeast extract, brains, etc.): supports growth of a wider range of organisms inc. those w/ unknown needs

why measure population size or growth rate?

• learn about the ecological role of a microbe

  • determine how a microbe is growing under known environmental and nutritional conditions

how can you obtain a microscopic total cell count?

using a counting chamber — slide with a grid

viable cell count (VCC)

obtains a more representative measure of the population by counting ‘viable‘ cells, those that are alive and able to grow

• spread a liquid culture on a plate

• done with diluted cultures to allow accurate counting

turbidimetric measurement of growth

OD = -log₁₀(intensity_blank/intensity_sample)

OD is roughly proportional to the number of cells per unit volume

growth phases in batch culture

• when microbial populations are growth in batch culture they are grown in a fixed volume of liquid with finite nutrients and other resources

• leads to a growth curve with different growth phases relative to resource consumption

lag phase

microbes pause to acclimate to new environmental conditions after transfer to a new batch culture

what affects lag phase duration?

• nutrient conditions in the growth medium

• the history and age of cells

exponential phase

• the cell population doubles at regular intervals

• cells are as close to being metabolically identical as possible

what is the absolute rate of exponential growth dependent on?

environmental conditions, media, and inherent growth properties of the organism

stationary phase

exponential growth needs to end because the fixed number of resources will become limiting to growth of new cells (carrying capacity)

• during the stationary phase, there is no net change in cell number, so the growth rate is 0

• cellular metabolism will change to prepare for maintenance and survival

decline phase

the total number of cells decrease as cells die

• cell division may still continue at a slow rate, but it is overshadowed by the death rate

• some cells may evolve to grow using recycled resources or simply survive during this time

microbe growth rates?

have both the fastest and the slowest growth rates of any known organisms

• some hot-springs bacteria can double in as little as 10 minutes, whereas deep-sea-sediment microbes may take as long as 100 years

microbial growth is often plotted on what kind of scale?

logarithmic

binary fission

cells elongate and then form a partition to separate two identical daughter cells

generation time

refers to the time required for binary fission to occur (= doubling time)

environmental factors that influence microbial growth

• temperature

• osmolarity (osmotic concentration)

• pH: hydronium and hydroxide ion concentration

• oxygen

the environmental habitat that a species inhabits is based on what one main criterion?

the tolerance of that organism’s proteins and other macromolecular structures to the local physical and chemical conditions

what typically occurs at extreme low temps?

growth stops because enzymatic processes become too slow and the cell membrane becomes too stiff

what typically occurs at extreme high temps?

critical proteins denature (e.g. transport proteins) and cell structures fail

how are microbes classified by temperature?

• growth rate roughly doubles for every 10°C rise in temperature

• the typical span between its minimum and maximum growth temperatures is around 30°C

• microbes are adapted to different environments

thermophilic environments and thermophiles exist how?

Taq DNA polymerase is able to withstand the protein-denaturing conditions required during PCR because it is heat stable

• the enzyme stays active even after repeated exposure to high temps needed to denature DNA and make PCR possible

characteristics of thermophiles?

have specially adapted membranes and protein sequences

• they have special membranes with more saturated (as opposed to unsaturated) lipids

• enzymes do not unfold as easily as would be found in a mesophile and so they hold their shape at higher temps

• they have DNA binding proteins that stabilize their DNA

characteristics of psychrophilic microbes?

psychrophilic lipases are “looser“ and work in 10-20°C temps, allowing them to be used in products like cold-water laundry detergents

• have low growth rates—all chemical reactions slow as temp drops; this includes enzyme activity, and just the underlying physics of things like DNA replication, RNA folding, etc.

Why isn’t growth rate for hyperthermophiles higher than thermophiles?

Heat does speed up chemistry, to an extent. However growth rate is a property of the whole organism, not just temperature.

• Some hyperthermophiles run low-yield metabolisms

• there’s also a cost to temperature adaption—energy is diverted away from division towards surviving high temps

what typically occurs at extreme low and high pH’s in the environment?

cells become unable to maintain pH

characteristic of neutralophiles, acidophiles, and alkaliphiles?

cells move protons across the membrane (different than ETC) to hold cyctoplasmic pH steady (e.g. H+/cation antiporters that use ions to buffer against acid or alkaline stress)

what typically occurs at low osmolarity?

water movement in or out of the cell can create pressure on the membrane

what typically occurs at high osmolarity?

‘salting out‘ — proteins fall out solution

osmolarity is important for a cell because….

of the cell’s selectively permeable plasma membrane

what polar molecule is small enough to diffuse directly through the membrane?

water is small and can diffuse directly through the membrane at a slow rate; also, microbes encode membrane channels called aquaporins that allow the passage of water

what occurs to a cell in a hypertonic (salty) medium?

water will leave the cell in an attemept to equalize osmolarity across the membrane which causes concentration of molecules in the cytoplasm to become too high

what is a result of concentration of molecules increasing in the cytoplasm due to hypertonic medium?

microbes change the solute balance inside the cell so that the cytoplasm is similar to the environment

• increase active transport of ions; they produce small organic molecules called compatible solutes (synthesize amino acids or sugars) that help survive against extreme osmotic stress

How are microbes classified by preferred temperature?

• Psychrophile = cold (<15°C)

• Mesophile = moderate (15–45°C)

• Thermophile = hot (50–80°C)

• Hyperthermophile = extremely hot (>80°C)

How are microbes classified by preferred pH?

• Acidophile = acidic (<pH 3)

• Neutralophile = neutral-ish (pH 5–8)

• Alkaliphile = basic (>pH 9)

What is a halophile?

A microbe that grows in high-salt environments. (>2M NaCl)

How are microbes classified by their O₂ requirements?

• Aerobe → needs O₂

• Facultative anaerobe → grows with OR without O₂

• Microaerophile → needs small amounts of O₂

• Anaerobe → grows without O₂; strict anaerobes cannot tolerate O₂