Microbiology: Bacterial Growth and Biofilms

• These notes provide a summary of key concepts in microbiology, suitable for study purposes.

Binary Fission and Exponential Growth

• Binary fission is an asexual bacterial reproduction resulting in exponential growth.
• During binary fission, one cell divides into two daughter cells.
• Generation time is the time required for a population to double and varies among species, impacted by the environment.
• Microbial growth can be calculated using the formula: Nt = N0 x 2^N.

Biofilms

• Biofilms are polymer-encased communities of microorganisms.
• Biofilms degrade harmful chemicals, offering both beneficial and detrimental effects.
• They enhances microbial resistance to antibiotics protecting them.

Microbial Growth in Mixed Communities

• Only a small fraction (about 1%) of microorganisms can be cultured.
• Microbes in natural environments often compete with each other.

Streak Plate Method

• It is used to obtain a pure culture by streaking in four sectors to isolate single colonies.
• The resulting pure culture can be stored as an inoculum in a refrigerator for later use.

Stages of a Growth Curve

• Lag phase: Bacteria prepare to reproduce.
• Log phase: Characterized by growth, including primary metabolites (essential for growth, made early) and secondary metabolites (natural products not directly contributing to growth).
• Stationary phase: Wastes accumulate, the number of cells dying equals the number of new cells, leading to a population plateau.
• Death phase: More cells die than are created.
• Phase of prolonged decline: Most cells die, with very few mutants continuing to grow.

Microorganism Requirements for Temperature, O2, pH, and Water

• Essential for studying and understanding the roles of microbes in ecosystems.
• Temperature.
  • Psychrophiles thrive at -5°C to 15°C.
  • Psychrotrophs grow at 15°C to 30°C, common in refrigerated environments.
  • Mesophiles prefer 25°C to 45°C.
  • Thermophiles grow at 45°C to 70°C.
  • Hyperthermophiles require temperatures of 70°C or greater.
• O2.
  • Obligate aerobes require oxygen.
  • Facultative anaerobes grow best with oxygen, but can grow without it.
  • Obligate anaerobes require no oxygen.
  • Microaerophiles require small amounts of oxygen but are inhibited by high concentrations.
  • Aerotolerant anaerobes are indifferent to oxygen.
• pH.
  • Neutrophiles prefer a pH range of 5-8.
  • Acidophiles prefer a pH below 5.5.
  • Alkaliphiles prefer a pH above 8.5.
• Water availability.
  • Halotolerant organisms can withstand up to 10% salt.
  • Halophiles require high salt concentrations to grow (~3% for marine bacteria, some >9% for extreme halophiles).
  • Plasmolysis occurs when cells shrink due to water loss in high-salt environments. High to low concentration causes bursts, low to high causes the cells to shrink

Reactive Oxygen Species (ROS)

• ROS, such as superoxide (O2-) and hydrogen peroxide (H2O2), are harmful derivatives of oxygen.
• Aerobic organisms must have protective mechanisms against ROS damage to cell components.

Required Elements for Microbial Growth

• Essential elements include Carbon, Oxygen, Hydrogen, and Nitrogen. Additional elements Sulfur, Phosphorus, Potassium, Magnesium, Calcium, and Iron.

Limiting Nutrients

• A limiting nutrient is present in the lowest concentration relative to need.
• It dictates the maximum amount of microbial growth.

Fastidious Microbes

• These microbes require growth factors due to their complicated nutritional needs.

Energy and Carbon Sources

• Photoautotrophs: Energy from sunlight, carbon from CO2.
• Chemolithoautotrophs: Energy from inorganic chemicals (H2, NH3, NO2-, Fe2+, H2S), carbon from CO2.
• Photoheterotrophs: Energy from sunlight, carbon from organic compounds.
• Chemoorganoheterotrophs: Energy and carbon from organic compounds (sugars, amino acids, etc.).

Media Types

• Complex medium: Contains a variety of ingredients with inexact composition.
• Chemically defined medium: Composed of exact amounts of pure chemicals.
• Selective media: Inhibits the growth of specific species.
• Differential media: Contains a substance that microbes change in a recognizable way.

Atmospheric Conditions

• Aerobic: Most obligate aerobes and facultative anaerobes incubated in air.
• Microaerophilic: Requires lower O2 concentration than achieved by candle jar.
• Anaerobic: Obligate anaerobes are sensitive to O2.

Enrichment Culture

• It isolate organisms present in small fractions of a mixed population.
• It provides conditions promoting the growth of target species and increase their relative concentration.

Cell Counting Methods

• Direct cell counts.
  • Counts total cells (living and dead).
  • Direct microscope count use a counting instrument to count cells in suspension.
• Viable cell counts.
  • Only counts cells capable of multiplying.
• Plate counts
  • plate counts measures the concentration of viable cells by counting colonies on an agar plate.
    • Spread plate method uniformly spreads cells on an agar surface.
    • Pour plate method mixes cells with melted agar before hardening.
  • Colony forming units (CFU) is the final unit used for measurement.
  • Membrane filtration concentrates microbes then the filter is incubated on agar.
• Measuring biomass.
  • Turbidity (cloudiness) is a rapid method that correlates with cell number.
  • Total weight tedious but accurate for filamentous microorganisms.
• Detecting cell products .
  • Can be done by including a pH indicator in the culture medium.

Principles of Sterilization, Disinfection, and Others

• Sterilization removes or destroys all microorganisms and viruses on or in a product. Sterile does not consider prions, but it is used for skin.
• Disinfection eliminates most or all pathogens on or in a material, and is used on inanimate objects.
• Pasteurization is a brief heat treatment that reduces spoilage organisms and destroys pathogens in food items without changing the product.
• Decontamination reduces pathogens to a safe level.
• Sanitization reduces microbial populations to meet health standards.
• Preservation delays spoilage of perishable products.

Microbial Growth Control

• Daily life: Washing with soaps and detergents, cooking, cleaning, and refrigeration.
• Healthcare settings: Minimizing microbial populations due to HAIs; requires sterile instruments, given patient susceptibility and high pathogen concentrations.
• Microbiology labs: Require rigorous control methods due to routine work with cultures.
• Food production: Reduce microbes while maintaining longer product quality, done through, removing, or inhibiting microbes.
• Water treatment ensures pathogens is removed.
• Other industries: Prevent contamination that affects product quality or safety.

Factors Influencing Antimicrobial Procedure Selection

• Microbe type: Resistance varies (e.g., bacterial endospores are highly resistant).
• Number of microbes: Population size affects required treatment time.
• Environmental conditions: Dirt and grease can interfere with antimicrobial action.
• Risk for infection: Medical instruments categorized by risk (critical, semicritical, non-critical).
• Composition of the item: Influences appropriate methods.

Methods of Sterilization and Disinfection

• Boiling:
  • Destroys most microorganisms but does not sterilize; endospores survive .
• Pasteurization:
  • Destroys heat-sensitive spoilage organisms in food and beverages.
• Sterilization using pressurized steam (autoclave):
  • Uses pressurized steam to kill endospores by raising temperature to 121°C at 15 psi for 15 minutes.
• Commercial canning process:
  • Uses industrial-sized autoclaves (retorts) to destroy Clostridium botulinum endospores.
• Dry heat:
  • Hot air ovens are less effective than moist heat, requiring longer times and higher temperatures.
  • Incineration is a method of dry heat sterilization.

Filtration Methods

• Depth filters:
  • Use thick, porous materials (e.g., cellulose) and electrical charges to trap cells, but do not sterilize.
• Membrane filters:
  • Use small pore sizes (0.2 μm) to remove bacteria, but does not sterilize. 0.02-0.05 μm to remove viruses and sterilize.
• HEPA filters:
  • Remove nearly all airborne particles >0.3 μm, but does not sterilize.

Radiation Methods for Destroying Microorganisms

• Gamma radiation:
  • Ionizing radiation that damages DNA and cell membranes.
• Ultraviolet radiation:
  • Damages DNA by destroying microbes directly and only kills living things.
• Microwaves:
  • Do not affect microorganisms directly; lethal due to heat.
  • Effective only if the substance contains water.
• High pressure:
  • Used as an alternative to pasteurization, around 120,000 psi.

Disinfectant Levels

• Sterilants: Destroy all microbes/sporicidal; for heat-sensitive critical instruments.
• High-level disinfectants: Destroy viruses/vegetative cells; for semi-critical instruments.
• Intermediate-level disinfectants: Destroy vegetative bacteria, mycobacteria, fungi, and most viruses; for non-critical instruments.
• Low-level disinfectants: Destroy fungi and vegetative bacteria except mycobacteria and enveloped viruses; disinfect furniture, floors, and walls.

Factors for Selecting Germicidal Chemicals

• Toxicity: Weigh benefits against risks.
• Activity: Consider presence of organic material.
• Compatibility: With the material being treated.
• Residues: Can be toxic or corrosive.
• Cost & Availability:
• Storage/Stability:
• Environmental risk:

Germicidal Chemicals

• Alcohols (ethanol & isopropanol):
  • Easy/inexpensive but rapid evaporation limits contact time, used as antiseptics and disinfectants.
• Aldehydes (glutaraldehyde, ortho-phthalaldehyde, formaldehyde):
  • Destroys all microbes but is irritating; glutaraldehyde and ortho-phthalaldehyde sterilize medical instruments, while formalin preserves biological specimens.
• Biguanides:
• Ethylene oxide gas:
  • Penetrates hard to reach places and fabrics and does not damage moisture sensitive material.
  • Toxic, explosive; used to sterilize medical devices.
• Halogens: Tincture of iodine and iodophors can be used as disinfectants or antiseptics.
  • Metals:
• Ozone: Uses unstable oxygen form to disinfect water.
• Peroxygens (hydrogen peroxide and peracetic acid):
  • biodegradable and less toxic; used to sterilize containers and disinfect medical devices.
• Phenolic compounds: Used in household disinfectants; hexachlorophene and triclosan used to be common, now limited due to safety concerns.
• Quaternary ammonium compounds:

Preserving Perishable Products

• Chemical preservatives:
  • Prevent or slow growth; must be non-toxic.
• Low temperature storage:
  • Refrigeration inhibits pathogens and spoilage organisms (some psychotrophs/psychrophiles still grow).
  • Freezing stops microbial growth.
• Reducing available water:
  • Salting and drying limits growth.

Catabolism vs Anabolism

• Catabolism:
  • Degrading compounds to release energy, captured as ATP.
• Anabolism:
  • Biosynthesis processes to assemble macromolecules, using ATP.

Energy Sources

• Photosynthetic organisms:
  • Harvest energy from sunlight to synthesize organic compounds from CO2.
• Chemoorganoheterotrophs:
  • Obtain energy from degrading organic compounds to make other organic compounds.

Components of Metabolic Pathways

• Metabolic pathways transform starting compounds (substrates) into end products.
• Enzymes: Act as catalysts to speed up reactions, highly specific ("lock & key" model), substrates fit into active site with help of coenzymes and cofactors, distorting enzyme shape or preventing binding of substrate, and are not changed.
• ATP is a central energy currency in cells.
• Chemical energy sources and terminal electrion acceptors are involved in electron transfer.
• Precursor metabolites are intermediate molecules used either as subunits of macromolecules or to generate ATP.

Central Metabolic Pathways

• Glycolysis:
  • Oxidizes glucose to pyruvate, generating ATP and reducing power.
• Transition Step:
• The TCA cycle: -cyclic pathway incorporating acetyl-CoA and generates ATP, CO2, and reducing power.

Cellular Respiration vs. Fermentation

• Cellular respiration: Utilizes an electron transport chain and a terminal electron acceptor.
• Fermentation: Does not use an electron transport chain or terminal electron acceptor.

Enzyme Active Site

• It is where substrates bind and undergo a conformational change, leading to their breakdown.

Cofactors vs. Coenzymes

• Cofactors: Non-protein components required for enzyme activity.
• Coenzymes: Non-protein organic compounds that assist enzymes, acting as carriers of small molecules or electrons.

Factors Influencing Enzyme Activity

• Temperature: A 10°C increase approximately doubles enzymatic reaction speed up to a point.
• pH: Enzymes function best at a specific pH (slightly above 7).

Allosteric Regulation

• Distorts enzyme shape, preventing or enhancing substrate binding, regulatory molecules are end products of metabolism.

Enzyme Inhibitors

• Competitive inhibition binds to the active site.
• Noncompetitive inhibition changes enzyme shape.

ATP Yields of Central Metabolic Pathways

• Glycolysis: 2 usable ATP.
• TCA cycle: 2 usable ATP.
• Electron transport chain: 1 NADH yields 3 ATP, 1 FADH2 yields 2 ATP.

Electron Transport Chain (ETC)

• Complex 1 receives electrons from NADH and pumps protons.
• Complex 2 receives electrons from FADH2.
• Complex 3 transfers electrons from Complex 1/2 and pumps protons.
• Complex 4 receives electrons, pumps protons, and forms water.
• ATP synthase: Protons flow down the concentration gradient through ATP synthase, generating ATP.

ETC Location

• Prokaryotes: Cytoplasmic membrane.
• Eukaryotes: Inner mitochondrial membrane.

Proton Motive Force

• The buildup of hydrogen ions drives the ATP synthase and allows for energy generation.

Fermentation:

• Used when respiration is not an option, or to recycle NAD+.
  • Lactic acid: Pickles, kimchi.
  • Ethanol: Bread, wine, beer.
  • Butyric acid.
  • Propionic acid: Swiss cheese.
  • Mixed acids.
  • 2,3-butanediol.

DNA vs. RNA

• DNA:
  • Complete set of genetic information (genome),uses ATCG, double stranded helix, codons (3 specifies an amino acid) and longer.
• RNA:
  • Complete genetic information in viruses, uses AUCG, mRNA, rRNA, tRNA, usually single strand, and shorter.

Importance of Gene Regulation

• Regulates the production of mRNA.

DNA Replication

• DNA must be replicated before cell division in DNA replication, which is then transcribed into RNA, then translated into proteins.

Transcription

• Requires RNA polymerase, which synthesizes single-stranded RNA using DNA as a template.
• Sigma factors: help RNA polymerase attach to right spot.
• Promoters: are the 3' start.
• Terminators: Are the 5' end.

Translation

• Translation is the process of decoding information in mRNA via ribosomes.
• Includes start (AUG) and stop codons.

Prokaryotic vs. Eukaryotic Gene Expression

• Prokaryotes: mRNA lacks a cap or poly-A tail, transcripts lack introns.
• Eukaryotes: mRNA has caps and tails, contains introns, and is monocistronic.

Quorum Sensing

• It senses cell density to coordinate gene expression.

Two-Component Regulatory Systems

• It is an important mechanism cells use to detect and react to changes in the external environment.

Antigenic vs. Phase Variation

• Antigenic variation: Changes in surface proteins.
• Phase variation: Switching genes on or off.

Enzyme Types

• Constitutive enzymes: Synthesized constantly.
• Inducible enzymes: Produced only when needed.
• Repressible enzymes: Routinely produced but turned off when not required.

Lac Operon

• Inducers helps turns on transcription, because it binds to activator which changes its shape to allow it to bind to the site, and RNA polymerase.
• Repressors blocks transcription, repressor is altered so it no longer binds to the operator, so the operon will be transcribed.