Microbial Growth, Biofilms, and Genetic Processes: Study Notes for Microbiology

Clinical Specimens and Nosocomial Infections

Prevention and Collection

Proper collection and handling of clinical specimens are essential to prevent nosocomial infections (hospital-acquired infections). Using aseptic techniques and correct sampling methods reduces contamination and ensures accurate diagnosis.

• Proper Specimen Collection: Use sanitary wipes, wash hands, and collect midstream urine to avoid contamination. Store samples appropriately to prevent metabolic changes.

• Aseptic Technique: Prevents introduction of external microbes during collection.

• Time Delivery: Rapid transport to the laboratory is crucial, as delays can affect microbial viability.

Example: Urine sample collection should avoid contamination from the urethra and be refrigerated if not processed immediately.

Culture Media

Types and Purposes

Culture media provide nutrients for microbial growth and can be tailored for specific diagnostic or research purposes.

• Defined Media: Exact chemical composition is known; used for controlled experiments.

• Complex Media: Contains nutrient-rich extracts (e.g., tryptic soy agar, TSA) but composition varies; supports a wide range of microbes.

• Anaerobic Media: Designed for growth of anaerobes (organisms that cannot tolerate oxygen).

• Transport Media: Maintains viability of organisms during transport to the laboratory.

• Selective Media: Inhibits growth of certain microbes while allowing others to grow, based on specific characteristics (e.g., pH, nutrients).

Example: TSA at pH 7 inhibits some fungi, which require a lower pH for growth.

Phases of Microbial Growth

Growth Curve and Characteristics

Microbial populations in batch culture exhibit distinct growth phases, each with unique physiological characteristics.

• Lag Phase: Cells adapt to new environment; metabolic activity but no division.

• Log (Exponential) Phase: Rapid cell division, high metabolic and enzymatic activity, binary fission, and synthesis of cellular components (e.g., peptidoglycan, phospholipids).

• Stationary Phase: Nutrient depletion and waste accumulation halt population growth; cell division equals cell death.

• Death Phase: Cell death exceeds new cell formation; population declines.

Example: Bacteria are most susceptible to penicillin during the log phase, as they are actively synthesizing peptidoglycan.

Microbial Nutrition and Growth Requirements

Energy and Carbon Sources

Microbes are classified by their energy and carbon sources:

• Phototrophs: Use sunlight to convert CO2 to sugars.

• Heterotrophs: Obtain carbon from organic molecules.

• Chemotrophs: Derive energy from chemical compounds (e.g., humans are chemoheterotrophs).

Oxygen Requirements

Microbes vary in their oxygen needs and tolerance:

• Obligate Aerobes: Require oxygen for growth.

• Obligate Anaerobes: Cannot tolerate oxygen.

• Facultative Anaerobes: Can use oxygen but also grow without it.

• Microaerophiles: Require low levels of oxygen.

Toxic Forms of Oxygen

Oxygen metabolism can produce toxic byproducts:

• Singlet Oxygen: Highly reactive form of oxygen.

• Superoxide Radical (O2-): Unstable, requires detoxification.

• Peroxide (H2O2): Can damage cellular components.

Microbes possess enzymes (e.g., superoxide dismutase, catalase) to neutralize these toxic forms.

Nitrogen, Phosphorus, and Sulfur Requirements

Essential for synthesis of amino acids, nucleic acids, and other cellular components.

• Nitrogen Fixation: Conversion of atmospheric N2 to ammonia by certain bacteria (e.g., Rhizobium in legumes).

• Phosphorus: Required for nucleic acids and ATP.

• Sulfur: Needed for some amino acids (methionine, cysteine).

Physical Factors Affecting Growth

• Temperature: Affects enzyme activity and membrane fluidity.

  • Psychrophiles: 5–20°C

  • Mesophiles: 15–45°C

  • Thermophiles: 40–80°C

• pH: Most bacteria prefer neutral pH; extremes can denature proteins.

• Water Availability: Cells must be hydrated; capsules and endospores help retain water and resist osmotic stress.

Biofilms

Definition and Importance

Biofilms are complex microbial communities attached to surfaces and embedded in a self-produced extracellular matrix. They are highly relevant in clinical and environmental settings.

• Include bacteria, protozoa, algae, and fungi.

• Represent up to 80% of infections, especially on medical devices (e.g., catheters, pacemakers).

• Highly resistant to antibiotics and disinfectants.

Characteristics of Biofilms

• Can form on biotic (living) or abiotic (non-living) surfaces.

• Heterogeneous: Different regions have varying metabolic activity and oxygen concentrations.

• Complex social structure: Cells communicate via quorum sensing and share resources.

• Biofilms can reduce the effectiveness of antibiotics by limiting penetration and altering metabolic states.

Common Examples

• Dental plaque

• Slippery slime on river stones

• Biofilms on medical devices

Biofilm Development: Model Steps

1. Initial Attachment: Microbes adhere to a surface.

2. Microcolony Formation: Cells divide and form clusters.

3. Matrix Production: Secretion of extracellular polymeric substances (EPS) stabilizes the biofilm.

4. Maturation: Biofilm develops complex architecture (e.g., mushroom-like structures).

5. Dispersion: Cells or clusters detach to colonize new sites.

Biofilm Prevention and Disruption

• Red algae can produce compounds that disrupt quorum sensing.

• Some agents can increase biofilm sensitivity to antibiotics.

Table: Comparison of Planktonic vs. Biofilm Bacteria

Property	Planktonic Bacteria	Biofilm Bacteria
Antibiotic Sensitivity	High	Low
Metabolic Rate	High	Variable (often lower)
Social Structure	Individual	Community, communication via quorum sensing
Clinical Relevance	Acute infections	Chronic, device-associated infections

Microbial Genetics

Genomes and Genetic Information

All cells have chromosomes made of DNA. Viruses may have DNA or RNA genomes. The genome is the complete set of genes in an organism.

• Genotype: The genetic makeup (all genes present).

• Phenotype: Observable traits resulting from gene expression.

DNA Replication

DNA replication is the process of copying the genetic material before cell division. It is semi-conservative, meaning each new DNA molecule contains one old and one new strand.

• Occurs in the 5' to 3' direction.

• Requires enzymes: helicase (unwinds DNA), primase (synthesizes RNA primer), DNA polymerase (extends new strand), ligase (joins fragments).

• Leading strand: synthesized continuously.

• Lagging strand: synthesized discontinuously in Okazaki fragments.

Equation:

$\text{DNA}_{\text{parent}} \xrightarrow{\text{Replication}} \text{DNA}_{\text{daughter}}$

Transcription and Translation

• Transcription: DNA is transcribed into messenger RNA (mRNA) by RNA polymerase.

• Translation: Ribosomes read mRNA codons and assemble amino acids into proteins.

Central Dogma of Molecular Biology:

$\text{DNA} \xrightarrow{\text{Transcription}} \text{RNA} \xrightarrow{\text{Translation}} \text{Protein}$

Genetic Variation and Horizontal Gene Transfer

• Mutation: Changes in DNA sequence caused by chemicals, radiation, or errors in replication.

• Horizontal Gene Transfer: Movement of genetic material between organisms other than by descent.

  • Transformation: Uptake of free DNA from the environment.

  • Transduction: Transfer of DNA by bacteriophages (viruses).

  • Conjugation: Direct transfer of DNA via cell-to-cell contact (e.g., plasmids).

Example: Griffith's experiment demonstrated transformation in Streptococcus pneumoniae.

Microbial Control Terminology

Definitions

• Sterilization: Complete removal or destruction of all microbes, including endospores.

• Aseptic: Free of contamination by pathogens.

• Disinfection: Reduction of microbial load on inanimate objects.

• Antisepsis: Reduction of microbes on living tissue (e.g., skin).

• Degerming: Physical removal of microbes (e.g., scrubbing).

• Sanitization: Reduction of microbes to safe levels on public surfaces.

• Pasteurization: Use of heat to reduce microbial load in food and beverages.

Physical and Chemical Methods of Control

• Heat: Denatures proteins and disrupts membranes (e.g., autoclaving, pasteurization).

• Radiation: Damages DNA (e.g., UV, gamma rays).

• Filtration: Removes microbes from liquids and air.

• Chemicals: Alcohols, phenolics, and other agents disrupt membranes and proteins.

Example: Autoclaving uses pressurized steam at 121°C to sterilize media and equipment.

Evaluating Effectiveness

• Use of disk diffusion assays to test chemical agents.

• Consideration of environmental conditions (e.g., temperature, organic load).

Additional info: Some explanations and examples were expanded for clarity and completeness based on standard microbiology curricula.