Biol-106 Exam #2: Microbial Growth and Control

Microbial Growth and Binary Fission

  • Binary Fission Process: This is the primary method of bacterial reproduction, where a single cell divides into two genetically identical daughter cells through several distinct steps:

    • 1. DNA Replication: Chromosomal DNA replicates at the origin of replication.
    • 2. Copy Movement: The DNA copies move toward opposite ends (poles) of the cell.
    • 3. Cell Elongation: The cell lengthens to accommodate the two sets of genetic material.
    • 4. FtsZ Ring Formation: The FtsZ protein forms a ring at the middle of the cell, marking the site of division.
    • 5. Septum Formation: A physical partition called the septum begins to form.
    • 6. Inward Growth: The cell wall and plasma membrane grow inward along the septum.
    • 7. Division: The cell finally splits into two separate, genetically identical daughter cells.

  • Phases of the Exponential Growth Curve:

    • Lag Phase: The initial period where cells are adapting to their new environment; there is intense metabolic activity but little to no cell division.
    • Log (Exponential) Phase: A period of rapid cell division and growth; the population increases at its maximum rate.
    • Stationary Phase: Nutrient levels decline and waste products accumulate; the rate of cell growth equals the rate of cell death.
    • Death Phase: The period where cells die faster than they can divide due to the exhaustion of nutrients and toxic waste accumulation.

Methods of Determining Cell Number

  • Direct Cell Count: Uses a microscope and specialized counting chamber to count cells directly in a liquid sample.
    • Limitation: It cannot distinguish between living and dead cells.
  • Fluorescent Staining: Employs fluorescent dyes that bind to cells.
    • Capability: It can differentiate live cells from dead cells based on membrane integrity.
  • Coulter Counter: An electronic device that counts cells by measuring changes in electrical resistance as they pass through a small opening.
  • Viable Plate Count: Measures only living cells capable of reproduction by counting the number of colonies that grow on an agar plate.
    • Reporting Unit: Results are reported as CFU (Colony Forming Units).
  • Turbidity: Measures the cloudiness of a culture; as cell number increases, turbidity increases.
  • Spectrophotometer: A device used to measure the amount of light absorbed or scattered by a culture.
    • Optical Density: Higher absorbance (optical density) indicates a larger cell population.

Alternative Methods of Cell Division

  • Fragmentation: Occurs when a parent cell or organism breaks into several pieces, with each fragment growing into a new individual.
    • Common in: Filamentous bacteria, fungi, and algae.
  • Budding: Involves the formation of a small outgrowth (bud) on the parent cell. The bud enlarges, receives a copy of the genetic material, and eventually detaches.
    • Common in: Yeast and certain bacteria.
  • Asexual Nature: Both methods are forms of asexual reproduction allowing population increase without mating or genetic exchange.

Biofilms and Microbial Communities

  • Definition: A biofilm is a structured community of microorganisms attached to a surface and embedded within a self-produced matrix.
  • Extracellular Polymeric Substance (EPS): This matrix holds the community together. It is composed of:
    • Polysaccharides
    • Proteins
    • Nucleic Acids (DNA)
    • Water
  • Locations: Biofilms form on living tissues, medical devices (catheters, implants), teeth (dental plaque), and pipes.
  • Benefits for Bacteria:
    • Protection from environmental stresses, antibiotics, and disinfectants.
    • Protection from the host immune system.
    • Enhanced communication through quorum sensing.
    • Efficient exchange of genetic material.
    • Better access to nutrients.
  • Resistance: Biofilms are significantly more resistant and difficult to eliminate than free-living (planktonic) bacteria.

Oxygen Requirements for Microbial Growth

Microbes are classified based on their use or tolerance of oxygen, which depends on their possession of detoxifying enzymes like Superoxide Dismutase (SOD) and Catalase.

  • Obligate Aerobes: Require oxygen to survive for aerobic respiration.
    • Example: Mycobacterium tuberculosis, Pseudomonas aeruginosa.
  • Obligate Anaerobes: Are harmed or killed by oxygen because they lack neutralizing enzymes.
    • Example: Clostridium difficile, Clostridium botulinum, Clostridium tetani.
  • Facultative Anaerobes: Can grow with or without oxygen. They grow better in oxygen due to higher ATP production via aerobic respiration but can switch to fermentation or anaerobic respiration.
    • Example: Escherichia coli, Staphylococcus aureus, Yeast.
  • Aerotolerant Anaerobes: Do not use oxygen for metabolism but tolerate it because they possess some detoxifying enzymes. They rely on fermentation.
    • Example: Lactobacillus, Streptococcus species.
  • Microaerophiles: Require oxygen levels lower than those found in the atmosphere; high concentrations are toxic.
    • Example: Campylobacter jejuni, Helicobacter pylori.

Reactive Oxygen Species (ROS) and Detoxification

During aerobic metabolism, oxygen is converted into highly reactive and toxic molecules that damage DNA, proteins, and membranes.

  • Major ROS:
    • 1. Superoxide Radical (O2−O_2^-): Produced during normal respiration; highly reactive.
    • Neutralization: Superoxide Dismutase (SOD) converts it into hydrogen peroxide.
    • 2. Hydrogen Peroxide (H2O2H_2O_2): Less reactive than superoxide but can form more dangerous compounds.
    • Neutralization: Catalase or Peroxidase breaks it down into water and oxygen.
    • 3. Hydroxyl Radical (OHOH): The most reactive and damaging species; destroys proteins, DNA, and membranes.
    • Protection: No specific enzyme directly detoxifies it; cells prevent its formation by removing O2−O_2^- and H2O2H_2O_2 first.

Bacterial Identification and Environmental Requirements

  • Identification Methods:

    • Gram Stain:
    • Gram-Positive: Purple; thick peptidoglycan layer.
    • Gram-Negative: Pink; thin peptidoglycan and an outer membrane (e.g., E. coli).
    • Hemolysis Patterns:
    • Alpha Hemolysis: Partial RBC destruction; green color.
    • Beta Hemolysis: Complete RBC destruction; clear zone.
    • Gamma Hemolysis: No hemolysis.
    • Motility Assay: Determines if bacteria move using flagella.

  • Environmental Factors Affecting Growth:

    • pH:
    • Neutrophiles: Grow near pH 77 (most human pathogens).
    • Acidophiles: Grow in acidic environments (e.g., Sulfolobus, Lactobacillus).
    • Alkaliphiles: Grow in basic environments (e.g., Vibrio cholerae).
    • Temperature:
    • Psychrophiles: Cold-loving.
    • Mesophiles: Moderate temperatures (20∘C20^{\circ}C to 45∘C45^{\circ}C); includes human pathogens.
    • Thermophiles: Hot temperatures (above 45∘C45^{\circ}C).
    • Hyperthermophiles: Extremely hot (often above 80∘C80^{\circ}C; e.g., hot spring bacteria).
    • Salt:
    • Halophiles: Require high salt concentrations (e.g., Staphylococcus aureus).
    • Pressure:
    • Barophiles (Piezophiles): Adapted to extreme high pressure (deep ocean).

Control of Microbial Growth

  • Biological Safety Levels (BSL):

    • BSL-1: Minimal risk; e.g., non-pathogenic E. coli.
    • BSL-2: Moderate risk; e.g., Staphylococcus aureus.
    • BSL-3: Airborne pathogens; highly dangerous; e.g., Mycobacterium tuberculosis.
    • BSL-4: Lethal, exotic pathogens; e.g., Ebola virus.

  • Methods of Control:

    • Sterilization: Destroys all microorganisms, including endospores.
    • Disinfection: Kills most pathogens on nonliving surfaces; does not sterilize.
    • Sanitization: Reduces microbes to safe public health levels.
    • Antisepsis: Use of chemicals on living tissue.
    • Degerming: Mechanical removal of microbes (e.g., handwashing).
    • Autoclaving: Steam under pressure at 121∘C121^{\circ}C for 15 minutes15\,\text{minutes}; achieves sterilization.
    • Pasteurization: Uses heat to reduce pathogens in food.
    • Refrigeration/Freezing: Refrigeration slows growth; freezing stops growth (but may not kill).
    • Desiccation: Drying to remove water.
    • Lyophilization: Freeze-drying for preservation.
    • Radiation:
    • Ionizing: (X-rays, Gamma rays) damages DNA and proteins.
    • Non-ionizing: (UV light) causes thymine dimers in DNA.
    • Filtration: Physical removal of microbes from air or liquids.

  • Chemical Control Agents:

    • Phenolics: Disrupt membranes and denature proteins.
    • Heavy Metals: (Silver, Mercury) inactivate proteins and enzymes.
    • Halogens: (Chlorine, Iodine) strong oxidizing agents.
    • Alcohols: (Ethanol, Isopropanol) denature proteins and dissolve lipids.
    • Soaps: Mechanically remove microbes by emulsifying oils.
    • Quaternary Ammonium Compounds (Quats): Disrupt cell membranes.
    • Peroxides: Produce reactive oxygen species (free radicals) that damage DNA and proteins.

History and Action of Antimicrobial Drugs

  • Pre-Antibiotic Drugs:

    • Compound 606 (Salvarsan): Developed by Paul Ehrlich (1909); first successful chemotherapeutic agent to treat syphilis.
    • Prontosil: First commercially successful sulfa drug.
    • Sulfanilamide: The active component of Prontosil; blocks folic acid synthesis.
    • Quinolines: (e.g., Quinine) used to treat malaria.

  • The Development of Penicillin:

    • Alexander Fleming: Discovered Penicillium mold killing bacteria in 1928.
    • Howard Florey & Ernst Chain: Successfully purified and mass-produced penicillin in the late 1930s/early 1940s.
    • Dorothy Hodgkin: Used X-ray crystallography to determine the molecular structure of penicillin.

  • Drug Classifications:

    • Bacteriostatic: Inhibits growth; relies on the immune system to clear infection (e.g., tetracyclines).
    • Bactericidal: Directly kills bacteria; preferred for severe infections.
    • Narrow-Spectrum: Targets a limited group (e.g., only Gram-positive); less damage to normal microbiota.
    • Broad-Spectrum: Targets many species; used when pathogen is unknown; risk of superinfection.

Antibiotic Mechanisms of Action

  • Cell Wall Inhibitors: Prevent peptidoglycan synthesis/assembly.

    • Penicillins & Cephalosporins: Block peptidoglycan cross-linking.
    • Bacitracin: Blocks transport of cell wall components.
    • Glycopeptides (Vancomycin): Bind to cell wall precursors.
    • Monobactams: Beta-lactams primarily for Gram-negatives.
    • Carbapenems: Broad-spectrum beta-lactams.
    • Isoniazid: Blocks mycolic acid synthesis in mycobacteria.

  • Protein Synthesis Inhibitors: Target 70S ribosomes.

    • 30S Subunit: Aminoglycosides (cause misreading of mRNA), Tetracyclines (block tRNA attachment).
    • 50S Subunit: Macrolides, Chloramphenicol (block peptide bond synthesis), Lincosamides, Oxazolidinones (block initiation).

  • Nucleic Acid Inhibitors:

    • Fluoroquinolones: (Ciprofloxacin) inhibit DNA gyrase/topoisomerase.
    • Rifamycins: (Rifampin) block RNA polymerase.
    • Metronidazole: Damages DNA.

  • Metabolic Inhibitors:

    • Sulfonamides & Trimethoprim: Inhibit folic acid synthesis pathway.

  • Beta-Lactam Structure:

    • Consists of a four-membered cyclic amide ring.
    • Target: Penicillin-binding proteins (PBPs/transpeptidases).
    • Resistance: Beta-lactamase enzymes hydrolyze the ring.

Specialized Antimicrobial Agents

  • Antifungals: Target Ergosterol (the fungal cholesterol equivalent).

    • Imidazoles/Triazoles: Block ergosterol synthesis.
    • Polyenes (Amphotericin B, Nystatin): Bind ergosterol and form pores in the membrane. Note: Amphotericin B side effect is nephrotoxicity.

  • Antiprotozoals:

    • Artemisinin: Contains an endoperoxide bridge; reacts with heme iron to produce free radicals; first-line for malaria.

  • Antihelminthics:

    • Benzimidazoles: (Albendazole, Mebendazole) inhibit microtubule formation.
    • Avermectins: (Ivermectin) open glutamate-gated chloride channels, causing paralysis.

  • Antivirals:

    • Acyclovir: Nucleoside analog; inhibits viral DNA polymerase (for HSV/VZV).
    • RT Inhibitors: Block RNA to DNA conversion (for HIV).
    • Protease Inhibitors: Prevent viral maturation.
    • Integrase Inhibitors: Block DNA insertion into host genome.
    • Fusion Inhibitors: Prevent entry into host cells.

Measuring Antimicrobial Effectiveness

  • Kirby-Bauer Disk Diffusion: Measures the zone of inhibition (larger zone = greater susceptibility).
  • Minimum Inhibitory Concentration (MIC): Lowest concentration preventing visible growth.
  • Minimum Bactericidal Concentration (MBC): Lowest concentration that kills the organism.

Epidemiology

  • Core Definitions:

    • Morbidity: State of illness in a population.
    • Mortality: Measurement of death rates.
    • Incidence: Number of new cases in a specific time period.
    • Prevalence: Total number of existing cases (old and new).

  • Disease Patterns:

    • Sporadic: Occurs occasionally and irregularly (e.g., Tetanus).
    • Endemic: Constantly present at baseline levels (e.g., Malaria in tropics).
    • Epidemic: Significant increase above expected levels in a specific area (e.g., Measles outbreak).
    • Pandemic: Worldwide epidemic (e.g., COVID-19).

  • Pioneers:

    • John Snow: Father of epidemiology; linked 1854 cholera to Broad Street water pump.
    • Florence Nightingale: Used statistics to prove poor sanitation caused war deaths.
    • Joseph Lister: Father of antiseptic surgery; used carbolic acid to reduce mortality.