Microbial Reproduction, Growth, and Control Methods: Prokaryotes, Environments, and Techniques

What is the primary method of reproduction in prokaryotes?

Binary fission, which involves elongation, replication, distribution, and septum formation.

What are some alternative reproductive strategies in prokaryotes?

Budding (e.g., Listeria monocytogenes), baeocyte formation (e.g., Cyanobacteria), and spore formation (e.g., Streptomyces coelocolor).

What are the two pathways in the bacterial cell cycle?

Chromosome replication/partitioning and cytokinesis.

What is the role of the Z ring in cytokinesis?

It assembles at the site of division, linking to the membrane and forming the divisome for cell wall synthesis.

What is water activity (aw) and its significance for microorganisms?

A measure of water availability; most microorganisms require an aw ≥ 0.98 for growth.

What are halophiles and their osmotic requirements?

Salt-tolerant prokaryotes that grow optimally at Na+ concentrations greater than 0.2 M.

How do acidophiles, neutrophiles, and alkalophiles differ in pH preference?

Acidophiles thrive at pH 0-5.5, neutrophiles at pH 5.5-8.0, and alkalophiles at pH 8.0-11.5.

What adaptations do psychrophiles and thermophiles have?

Psychrophiles have membranes with unsaturated fatty acids, while thermophiles have proteins stabilized by more hydrogen bonds and proline.

What are the toxic products generated from oxygen reduction?

Superoxide radical (O2-•), hydrogen peroxide (H2O2), and hydroxyl radical (OH-•).

What protective enzymes do some microorganisms produce against toxic oxygen products?

Superoxide dismutase (SOD), catalase, and peroxidase.

What is the significance of Liebig's law of the minimum?

It states that the total biomass of an organism is determined by the nutrient present at the lowest concentration.

What is quorum sensing in microbial communities?

A mechanism of microbial communication that involves the secretion and detection of chemical signals to coordinate behavior.

What are some processes sensitive to quorum sensing in Gram-negative bacteria?

Bioluminescence, synthesis of virulence factors, conjugation, and biofilm production.

What are biofilms?

Complex microbial communities that are ubiquitous and can pose industrial and medical challenges.

What is the purpose of culture media in microbiology?

Preparations that support the growth of microorganisms, which can be either liquid or solid (solidified with agar).

What are the cardinal growth temperatures for microorganisms?

Each organism has minimal, maximal, and optimal temperatures for growth.

What is the role of compatible solutes in microorganisms?

They help maintain osmotic pressure and are compatible with metabolism, such as choline and proline.

What is the impact of pH on microbial growth?

Cytoplasmic pH extremes can be detrimental; microorganisms maintain near-neutral internal pH through proton/ion exchange mechanisms.

What adaptations do barophilic organisms have?

They require or grow better under high pressure and may have increased unsaturated fatty acids in phospholipids.

What is the significance of the E. coli divisome?

It is crucial for the process of cytokinesis, involving the assembly of the Z ring and cell wall synthesis machinery.

What is the function of autolysins in bacterial cell growth?

They are involved in peptidoglycan synthesis, aiding in the formation of the cell wall.

What environmental factors influence microbial growth?

Factors include osmotic pressure, pH, temperature, oxygen concentration, and pressure.

What are the effects of ionizing radiation on microorganisms?

It can cause mutations and death by disrupting chemical structures, including DNA.

What is the role of carotenoids in microbial protection?

They protect organisms from photooxidation caused by high-intensity visible light.

What is defined (synthetic) media?

Defined media contain ingredients of known composition and concentration, allowing controlled growth environments for specific bacteria.

What are complex media?

Complex media contain some ingredients of unknown composition and/or concentration, making them suitable for a wider range of bacteria.

What is selective media?

Selective media favor the growth of some microorganisms while inhibiting others, such as MacConkey agar, which selects for gram-negative bacteria.

What is differential media?

Differential media distinguish between different groups of microorganisms based on biological characteristics, like blood agar differentiating hemolytic from nonhemolytic bacteria.

What is the significance of colony morphology?

Colony morphology helps in identifying individual species, as each species forms characteristic colonies.

What are the common techniques for isolating pure cultures?

Common techniques include spread plate, streak plate, and pour plate methods.

What occurs during the lag phase of microbial growth?

In the lag phase, cells synthesize new components to adapt to new conditions, and growth varies in length.

What characterizes the exponential (log) phase of microbial growth?

In the exponential phase, the growth rate is constant, and cells exhibit balanced growth with uniform chemical and physical properties.

What happens during the stationary phase of microbial growth?

In the stationary phase, the total number of viable cells remains constant due to nutrient limitation and waste accumulation.

What is the death phase in microbial growth?

In the death phase, there is a decline in viable cells due to lack of nutrients, leading to cell death or viable but nonculturable states.

What is the long-term stationary phase?

In the long-term stationary phase, the population size remains the same, and genetically distinct variants may arise through natural selection.

What is the mean growth rate (µ) in microbial growth?

The mean growth rate (µ) is the number of generations per unit time, usually expressed in generations per hour.

How is the doubling time (g) calculated in microbial growth?

Doubling time (g) is calculated as the time required for the population to double in size, often derived from the mean growth rate.

What are direct cell counts?

Direct cell counts involve methods like counting chambers and electronic counters to quantify total cell numbers.

What are viable cell counts?

Viable cell counts estimate the number of living cells using techniques like spread plating and membrane filtration.

What is a Petroff-Hausser chamber?

A Petroff-Hausser chamber, also known as a hemacytometer, is used for direct cell counting and is quick and inexpensive.

What is the purpose of a Coulter counter?

A Coulter counter counts cells in a suspension by disrupting an electric current as cells pass through a small orifice.

What is flow cytometry?

Flow cytometry uses lasers and scattered light to quantify and analyze cells in a fluid stream.

What is the purpose of turbidometric measurement?

Turbidometric measurement assesses cell mass by measuring light scattering, providing a quick and sensitive method.

What is a chemostat?

A chemostat is a continuous culture system where the rate of incoming culture medium equals the rate of medium and cell removal.

What is a turbidostat?

A turbidostat maintains a constant biomass by continuously supplying nutrients and removing waste, keeping cells in log phase.

Objective

To become acquainted with methods used to kill or control the growth of pathogens.

Sterilization

Elimination of all microorganisms / spores.

Sterilant

A substance used to achieve sterilization.

Disinfection

Killing, inhibition, or removal of pathogens; not necessarily sterilizing.

Disinfectants

Agents used to disinfect inanimate objects.

Sanitization

Reduction to 'safe' levels according to public health standards.

Antisepsis

Prevention of infection of living tissue.

Antiseptics

Agents applied to tissue that are less toxic.

-cidal agents

Agents that kill microorganisms.

Germicide

A type of -cidal agent that kills pathogens and many nonpathogens.

Bactericides

Agents that kill bacteria.

Fungicides

Agents that kill fungi.

Algicides

Agents that kill algae.

Viricides

Agents that kill viruses.

-static agents

Agents that inhibit the growth of microorganisms.

Bacteriostatic

An agent that inhibits the growth of bacteria.

Fungistatic

An agent that inhibits the growth of fungi.

Filtration

A mechanical method that physically removes microorganisms from liquids and air.

Membrane filters

Porous membranes with defined pore sizes, such as 0.2µm and 0.45µm.

N-95 hospital masks

Masks that filter particles down to 0.3µm.

HEPA filters

High-efficiency particulate air filters used in laminar flow biological safety cabinets.

Moist heat

A physical method effective against all types of microorganisms, such as autoclaving.

Autoclave

A device that uses high temperatures (>121°C) and pressurized steam for sterilization.

Pasteurization

Controlled heating that reduces total microbial population and increases shelf life.

Pasteurization of milk

Historically done at 63°C for 30 minutes.

flash pasteurization

high temperature short-term; HTST - 72°C / 15 sec; rapid cooling

ultrahigh-temperature (UHT) sterilization

140-150°C; 1-3 sec; can be stored @RT for 2 mo. - e.g. Parmalat, creamers

decimal reduction time (D value)

time to kill 90% of microorganisms / spores at specific temp

z value

increase in temperature required to reduce D by 1/10

D121

0.204 min (1012 => 1011); therefore time = 12(D) = 2.5 min @121°C

D value calculations

Given the D value at one temperature and the z value, we can derive an equation to predict the D value at a different temperature

freezing

<-20°C; stops microbial reproduction due to lack of liquid water and some killing by ice crystal disruption of cell membranes

refrigeration

slows microbial growth and reproduction; short-term

UV radiation

260nm; surface sterilization (doesn't penetrate); kills microorganisms; inactivates viruses

ionizing radiation

usually gamma rays; penetrate deep; destroys cells and endospores but not always viruses

phenolics

first antiseptic; mechanism: denature proteins; disrupt cell membranes; benefits: tuberculocidal, effective in presence of organic material, long lasting; drawbacks: odor; skin irritation

alcohols

bactericidal, fungicidal, not sporicidal; inactivate some viruses; mechanism: denature proteins; possibly dissolve membrane lipids

iodine

skin antiseptic; mechanism: oxidizes cell constituents; iodinates proteins; may kill spores; drawbacks: skin damage, staining, and allergies can be a problem

iodophor

iodine + organic carrier; water-sol., non-staining; used in pre-op. - e.g. Wescodyne, Betadine

classes of chemical agents

A. phenolics B. alcohols C. halogens D. heavy metals E. quaternary ammonium compounds F. aldehydes G. sterilizing gases

overuse problematic

e.g. Triclosan; resistant Pseudomonas aeruginosa

Chlorine

Oxidizes cell constituents; important in water supply, swimming pool disinfection, dairy and food industries, and as a household disinfectant; destroys vegetative bacteria and fungi but not spores; can react with organic matter to form carcinogenic compounds.

Heavy metals

Includes ions such as mercury, silver, arsenic, zinc, and copper; effective but usually toxic; inactivate proteins by binding sulfhydrils and may also precipitate proteins.

Quaternary ammonium compounds

Amphipathic organic molecules that solubilize things and are effective cleansers; cationic detergents are effective disinfectants that kill most bacteria by disrupting membranes and denaturing proteins, but not effective against Mycobacterium tuberculosis or endospores; safe and easy to use but inactivated by hard water and soap.

Aldehydes

Highly reactive molecules that act as crosslinkers and inactivate nucleic acids and proteins; serve as chemical sterilants and are sporicidal.

Sterilizing gases

Used to sterilize heat-sensitive materials such as sutures and catheters; microbicidal and sporicidal; combine with and inactivate proteins and penetrate packing materials.

Evaluation of antimicrobial effectiveness

A complex process regulated by US federal agencies such as the Environmental Protection Agency and the Food and Drug Administration; includes determining the potency of a disinfectant compared to phenol.

Use dilution test

Determines the rate at which selected bacteria are destroyed by various chemical agents.

In-use testing

Tests conditions that approximate normal use of disinfectant.

Population size

Larger populations take longer to kill than smaller ones.

Population composition

Differences in sensitivity among microorganisms, such as Mycobacterium, spores, and young vs. old cells.

Concentration / intensity of antimicrobial

Usually, higher concentrations kill faster, but this is not always the case; for example, EtOH 70% vs. 95% shows a non-linear relationship.

Exposure duration

Longer exposure leads to more killing.

Temperature

Higher temperatures usually increase the killing effect of antimicrobials.

Local environment

Factors such as pH, viscosity, organic material, and the presence of biofilms can influence antimicrobial effectiveness.