Microbio EX1: Chapter 4; Microbial Growth and Its Control

Chemical Makeup of a cell

-Handful of elements dominate living systems.

-C, O, N, H, P, S are ~96% of dry weight of bacterial cell and required by all life (macronutrients).

-K, Na, Ca, Mg, Cl, Fe ~3.7% of dry weight, 62 total elements can be metabolized.

-Marcomolecules make up ~96% of dry weight, mostly proteins and RNA… not DNA.

Heterotrophs

Carbon, breakdown of organic polymers, uptake of monomers

Autotrophs

synthesize organics from CO2.

Nitrogen

nearly all microbes can use ammonia (NH3), many use nitrate (NO3-), some use organics (ex: amino acids), and nitrogen fixation (N2) done by some.

Phosphorous

Usually inorganic phosphate (PO4^3-)

Sulfur

vitamins (Ex: thiamine, biotin, lipoic acid), microbes assimilate sulfate (SO4^-2), sulfide (H2S), or organics. Sulfur-containing amino acids (cysteine and methionine)

Potassium

required by several enzymes

Magnesium

stabilizes ribosomes, membranes, and nucleic acids. Required by many enzymes.

Calcium and Sodium

are only essential for only a few microbes (Ex: sodium). Halophiles (marine microbes) live in salty (NaCl) environments.

Micronutrients

many enzymes require metal ion or small organic as a cofactor for catalysis. Trace metals = required in small amounts.

Iron (Fe)

cellular respiration (cytochromes), related oxidation-reduction reactions.

Growth Factors

organic micronutrients. Vitamins most function as coenzymes, most frequently required growth factors. Others: amino acids, purines, pyrimidines, other organics.

Culture Media

Nutrient solutions used to grow microbes in the laboratory, typically sterilized in an autoclave.

Defined media

exact chemical composition known (ex: EZ medium/culture medium for enterobacteria)

Complex media

Composed of digests of microbial, animal, or plant products (ex: yeast and meat extracts), examples are tryptic soy broth.

Selective media

contains compounds that selectively inhibit growth of some microbes but not others.

Differential medium

contains an indicator, usually a dye, that detects particular metabolic reactions during growth.

Colony

genetically identical clones derived from a single mother cell, contains millions or billions of cells. Can be used to indicate if the culture is pure; all colonies should look the same.

Total cell count

Uses microscope cell count.
Total cell count is quick and easy to estimate microbial cell #s, several limitations = difficult to see small cells, can’t distinguish live from dead cells, and dependent on high cell density.

Microscopic cell count

observing and enumerating cells present, dried on slides or on liquid samples, counting chambers with squares etched on a slide used for liquid samples, several limitations.

Binary Fission

cell division following enlargement of a cell to twice original size.

Septum

partition between dividing cells, pinches off between two daughter cells.

Steps of binary fission

DNA is copied, cell grows, copied chromosome is drawn to opposite ends of the cell, septum (partition) begins to form at the midpoint, and septum eventually walls off the resulting two daughter cells from one another.

Generation (doubling) time

time required for microbial cells to double in number. Differs for microbes and varies depending on conditions.

Batch culture

a closed-system microbial culture with a fixed volume, such as a test tube or flask.

Lag phase

Interval between inoculation of a culture and the beginning of growth. New conditions require altering metabolic state. Time needed for biosynthesis of new enzymes and to produce require metabolites before growth can begin.

Exponential Phase

Doubling at regular intervals. Close to metabolically identical, rates vary greatly, influenced by media, conditions, organism itself. Continues until conditions can no longer sustain growth.

Stationary and Death Phases

-Growth limited by nutrient depletion or waste accumulation

Stationary phase

growth rate of population is zero. Metabolism continues at greatly reduced rate.

Decline phase

total number decreases due to cell death.

Cryptic growth

Some subpopulations may adapt by cannibalizing and reusing resources from dead cells.

Generation time

g = t/n.

t = duration of exponential growth. N = number of generations during the period of exponential growth.

Relationship between initial number of cells present X the number present in an exponential growing culture at any point in time

N_t = N₀2ⁿ

-N_t = cell number at time t

-N₀ = initial cell number (time 0)

-n = number of generations during the period of exponential growth.

Continuous Culture

An open system

Chemostat

Most common type of continuous culture device, known volume added while spent medium is removed at the same rate.
-can maintain exponential growth phase for weeks/months, used to study physiology, microbial ecology and evolution, enrichment and isolation of bacteria from nature.

Steady state

cell density and substrate concentration do not change over time.

Planktonic Growth

Growth in suspension of free-floating/free-swimming cells.

Sessile Growth

attached to surface. Can develop into biofilms: important in medical and industrial applications, and different properties than planktonic cells.

Biofilm

-population of cells enmeshed in a polysaccharide matrix attached to a surface. Form in stages: planktonic cells attach (flagella, fimbriae, pili), colonization (growth and extracellular polysaccharide (EPS) production), development (metabolic changes), and dispersal (colonize new sites). Can be visualized using a flow chamber.

Microbial Mats

Multilayered sheets with different organisms in each layer (ex: hot springs, intertidal regions)

Biofilm and humans

implicated in joint infections, implanted medical devices, responsible for cavities and cause gum disease. Fouling, plugging, and corroding pipes. Form in fuel tanks and on ship hulls, slowing down ships.

Alternatives to binary fission

-Balanced growth, budding cell division, and hyphal growth and multiple fission.

Balanced growth

result of binary fission, producing nearly identical cells.

Budding cell division

unequal cell growth forming different daughter cells.

Hyphal Growth

Hyphae = long, thin filaments of actinomycetes. Hyphal growth occurs only at filament tip, cell growth isn’t linked directly to division (no septa).

Multiple fission

hyphal filament forms many septa simultaneously

Cardinal temperatures

minimum temperature to grow, microbes don’t grow below this temperature. Optimum temperature to grow = growth rate is the fastest, all/most cellular components are functioning at a maximum rate.

Psychrophile

low, found in cold environments… permanent snowfields, glaciers

Mesophile

midrange, most commonly studied.

Thermophile

high, found in hot environments.   

Hyperthermophile

very high, found in extremely hot habitats such as hot springs and deep-sea hydrothermal vents.

Extremophiles

organisms that grow under very hot or very cold conditions.

Psychrotolerant

More widely distributed in nature than psychrophiles. Isolated from soils and water in temperate climates and food at 4 degrees C.

Molecular adaptations to life in the cold

Production of enzymes that function optimally in the cold. Having more a-sheets than B-sheets provides greater flexibility for catalyzing reactions at cold temperatures.

Neutrophiles

grow optimally at pH range termed circumneutral = pH 5.5-7.9

Acidophiles

grow best at low pH (<5.5). Governed by stability of cytoplasmic membrane. High concentrations of protons are required. Neutral pH causes membranes to be destroyed and cell lysis.

Alkaliphiles

pH > 8. Found in highly alkaline habitats: soda lakes and high-carbonate soils. Some used commercially (ex: secreted proteases and lipases that are added to laundry detergents to remove proteins and fats)

Microbial culture media typically contain buffers to…?

maintain constant pH.

Water activity (a_w)

varies from zero (no free water) to one (pure water).

Osmosis

Water diffuses from high to low solute (dissolved substance) concentrations.

Halophiles

grow best at aw = 0.98 (seawater); have a specific requirement for NaCl.

Halotolerant

tolerate some dissolved solutes but generally grow best in the absence of added solute.

Extreme halophiles

require very high levels (15-30%) of NaCl; often unable to grow at lower concentrations.

Osmophiles

live in environments high in sugar

Xerophiles

able to grow in very dry environments

Compatible Solutes

Solutes acquired from the environment or synthesized in cytoplasmic. Highly water-soluble (ex: sugars, alcohols, glycine betaine, KCl). Do not inhibit biochemical processes.

Aerobes

Grow at full O2 tension (~21%) and respire O2.

Microaerophiles

Can use O2 only at levels reduced from that in air (microxic) due to limited respiration or oxygen sensitivity.

Facultative organisms

can live with or without oxygen

Anaerobes

Cannot respire oxygen

Aerotolerant anaerobes

tolerate oxygen and grow in its presence even though they cannot respire.

Obligate anaerobes

inhibited or killed by oxygen. (ex: Bacteria and Archaea, few fungi, and few protozoa)

Why is oxygen toxic?

Molecular oxygen (O2) isn’t toxic. Exposure to oxygen yields toxic byproducts.
Superoxide (O₂^-), hydrogen peroxide (H₂O₂), and hydroxyl radical (OH-)

What enzymes eliminate toxic oxygen?

catalase, peroxidase, superoxide dismutase, and superoxide reductase.

Decontamination

the treatment of an object to make it safe to handle.

Disinfection

directly targets pathogens, not necessarily all microorganisms, and kills or severely inhibits growth.

Sterilization

kills all microbial cells

thermal death time

time to kill all cells at a given temperature; affected by population size. Endospores may survive.

Autoclave

Sealed heating device that uses steam under pressure. Steam under pressure yields temp. of 121 degrees C. Kills endospores. Not the pressure but the high temperature that kills the microbes.

Pasteurization

Uses heat to significantly reduce the microbial load in heat-sensitive liquids. Doesn’t kill all organisms, so it is different from sterilization. All known pathogenic bacteria are killed.

Ultraviolet Radiation

affects DNA leading to death. Useful for decontaminating surfaces, poor penetration… can be dangerous to humans.

Ionizing Radiation

electromagnetic radiation that produces ions and other reactive molecules with which radiation particles collide. Some microorganisms more resistant to radiation than others (ex: endospores v. vegetative cells, viruses v. bacteria).

Filter Sterilization

used on heat-sensitive liquids and gases. Pores of filter (0.45 and 0.2 um) are too small for living organisms to pass through but do not trap most viruses.

Depth filter

fibrous sheet made of overlapping paper or glass fibers that traps particles. (ex: HEPA/High efficiency particulate air) filters remove 0.3. um+ particles from air.

Membrane filters

most common for liquid sterilization, syringe or pump forces liquid into sterile vessel.

Nucleopore filters

thin irradiated film etched to make holes, used for scanning electron microscopy, microorganisms observed directly on filter.

Antimicrobial agent

chemical that kills or inhibits growth

-cidal does what?

kills microorganisms (ex: bactericidal, fungicidal, viricidal)

-static does what?

inhibits growth (ex: bacteriostatic, fungistatic, viristatic)

Bacteriostatic

agents inhibit important biochemical processes and bind weakly.

Bactericidal

agents bind tightly and kill the cell without lysis

Bacteriolytic

agents kill by lysis (ex: detergents)

Minimum Inhibitory Concentration (MIC)

smallest amount of an agent needed to inhibit growth of a microorganism. Can be determined with liquid medium and agent dilutions.

Zone of growth inhibition

area of no growth around disk

Sterilants

destroy all microorganisms, including endospores.

Disinfectants

kill microorganisms but not necessarily endospores, used on surfaces.

Sanitizers

less harsh, reduce microbial numbers, do not sterilize.

Antiseptics (germicides)

kill or inhibit microbial growth and are nontoxic enough to be applied to living tissues.