Grading Exams

  • The instructor has not finished grading the exams yet.
  • Expects to have them graded by Thursday and will review them in class.

Learning Objectives

  • Introduction to microbial genetics learning objectives.
  • Microbial genetics PowerPoint will be used in the lesson.
  • Focus on culturing bacteria and related laboratory techniques.

Review of Bacterial Growth

  • Previous topics discussed include quorum sensing and biofilm formation.
  • Transition into culturing bacteria in the lab.
Characteristics Influencing Bacterial Growth
  • Important characteristics to consider when growing bacteria include:
    • Temperature
    • Salt concentration
    • pH of the medium

Media Terminology

  • Definition of medium: The culture environment that can be either liquid or solid, which provides necessary nutrients for bacterial growth.
  • Media is the plural form of medium.

Types of Media

  1. Defined Media (or Synthetic Medium)

    • All components are specific and completely purified.
    • No natural source derivatives included.
    • Example: A defined medium for E. coli can contain glucose, buffering agents, ammonia nitrogen source, and salts.

  2. Complex Media

    • Derived from natural sources and contains not fully purified components.
    • Example: MacConkey medium (derived from bile salts) and Blood agar (unsanitized blood)
    • Indicators of complex media include terms like infusion, digest, serum, or references to specific organs.

Relevant Examples

  • Tryptocase Soy Agar and Broth

    • Classified as complex media due to the inclusion of soy (tryptocase).
    • Useful for growing various bacteria, not limited to a single species.

  • LB medium (Luria-Bertani)

    • Typically used for specific strains of E. coli, likely defined but requires confirmation.

Media Classification by Functionality

  1. Selective Media

    • Promotes the growth of a specific organism while inhibiting others.
    • Example: Phenylethyl alcohol agar (PEA) selects for gram-positive bacteria by inhibiting gram-negative bacteria like E. coli.

  2. Differential Media

    • Allows differentiation of organisms based on their growth behaviors, without selective inhibition.
    • Example: MacConkey agar shows E. coli (lactose fermenter) as pink whereas Salmonella (non-fermenter) remains colorless.
    • Blood agar is also labeled as differential, allowing identification of hemolytic bacteria.

Bacterial Growth Assessment

Growth Phases in Bacterial Cultures

  • Lag Phase: Slow initial growth.
  • Exponential Phase: Rapid bacterial division; plot typically shows a straight line on a log scale.
  • Stationary Phase: Rate of division equals cell death; no net change.
  • Death Phase: Greater cell death than division occurs, leading to a decline in viable cells.
Implications of Growth Phases
  • The growth phase affects bacterial activity, often optimizing enzyme studies or antimicrobial development potential.

Bacterial Division Concepts

  • Bacteria divide primarily through binary fission, which can lead to:
    • Arithmetic Growth: Slow, stepwise addition (one cell to two, etc.).
    • Exponential Growth: Rapid multiplication (1 → 2 → 4 → 8 → 16 and so on).

Growth Curve Presentation

  • Growth curves are plotted using the logarithm of viable cell counts vs. time to provide a clearer data representation.
  • Production of antibiotics by bacteria typically occurs during the stationary phase — a response to limiting nutrients.

Culture Counting Methods

  1. Serial Dilution and Plate Count

    • Useful method for estimating live bacteria in a culture.
    • Countable plates should ideally present between 30-300 colonies.
    • Common notation: TMTC (Too Numerous To Count) if over 300 colonies are present.

  2. Microscopy for Direct Count

    • Utilizing a cytometer for counting bacteria, providing direct cell numbers; requires precision in methodology.

  3. Filtration Method

    • Best used for small sample sizes, such as in drinking water testing.
    • Not applicable for larger volumes due to clogging filters.

  4. Turbidity Measurement

    • A rough estimate of bacteria in cultures measured by cloudiness, used as a comparative measure only.

Genetic Framework in Bacteria

Bacterial Genetic Structure

  • Haploid Nature: Bacteria usually contain one circular chromosome, containing all genetic information organized in the nucleoid.
  • Presence of plasmids, additional small DNA molecules that may confer additional traits like antibiotic resistance.

Central Dogma Revisited: Transcription and Translation

  • In bacteria, transcription of DNA to mRNA directly leads to simultaneous translation into proteins without compartmentalization:
    • No five-prime cap or polyadenylation necessary, unlike eukaryotic cells.

Operon Structures and Functions

Key Terms:
  • Constitutive Genes: Always active under normal conditions.
  • Operon: A grouping of related coding sequences under a single regulatory promoter and operator control.
Operon Types:

  1. Repressible Operon: Active by default, can be repressed when end product (e.g., tryptophan) concentration is sufficiently high.

    • Tryptophan as a co-repressor binds to the repressor protein, which then attaches to the operator to halt transcription.

  2. Inducible Operon: Inactive by default but activated by the presence of an inducer (e.g., lactose).

    • Active repression occurs until lactose binds to the repressor, disabling it and allowing transcription when glucose is absent.

Comparison of Operons

  • Both operons feature promoters and operators but differ in regulation mechanisms, with one responding to nutrient availability and the other to end product concentration.

Mutations and Genetic Testing

Definitions and Concepts:

  • Mutagens: Factors (chemical or physical) that induce mutations (e.g., radiation).
  • Auxotrophs: Mutated bacteria with altered nutritional requirements compared to their prototrophic (wild-type) parent.
Ames Test Overview
  • Developed by Bruce Ames to screen for potential mutagenic chemicals using auxotrophs of Salmonella:
    1. controls for spontaneous mutations.
    1. Tests if substances can revert auxotrophs back to histidine-producing prototrophs, thus indicating mutagenicity.
Importance of Chemical Testing
  • Identifying mutagens is crucial for public safety; fostering understanding about their carcinogenic potential hinges on additional testing in animal models post-chemical evaluation.

Upcoming Topics

  • Continued exploration of operons and enhanced dynamics of gene regulation.
  • What will be needed for deeper understanding leading into the next class session.