Quantitative and Single-Cell Microbiology

Quantitative and Single-Cell Microbiology

  • Lecture by: Nela Nikolic, University of Southampton

  • Course: BIOL2038 BIOL2044

  • Date: 4 March 2025

Learning Objectives

  • Quantitative Techniques in Microbiology: Understand various methods used in assessing microbial populations.

  • Single-Cell Analysis Importance: Describe why studying individual microbial cells is critical.

  • Mechanisms of Individuality in Bacteria: Provide examples where individuality manifests among single cells.

Measuring Microbial Traits

  • Handprint Microbiota: Describes methods to measure the microbial community composition.

Standard Microbiology Techniques

  • Bulk Measurements: Evaluate bacterial growth and traits.

    • Optical Density (A600): Light absorbance at this wavelength measures bacterial density in cultures.

    • Culturing Methods: Utilized in tubes, flasks, and microplates.

Population Growth of Bacteria

  • Binary Fission: Primary method of bacterial reproduction where one cell divides into two.

    • Estimating Generations (n):

      • Formula: N2 = N1 * 2^n

      • Logarithmic Transformation: ln(N2) - ln(N1) for calculating the number of generations.

    • Growth Rate (μ):

      • Derived from cell increase during a specific time interval (t).

      • Formula: μ = (N2/N1) / (t2-t1) for exponential growth phase.

Data Distribution

  • Mean Value Implications: Discuss how averages can obscure diverse data distributions and important variations.

Phenotypic Heterogeneity

  • Definition: Variations in traits despite genetic similarity due to environmental or stochastic influences.

    • Examines the individuality in bacteria: genetically identical, yet phenotypically diverse.

    • Can be assessed through single-cell techniques.

Examples of Phenotypic Heterogeneity

  • Dormant Cells:

    • State of metabolic inactivity or growth arrest.

  • Persister Cells:

    • Tolerate antibiotics and can endure harsh conditions.

    • Mechanisms of dormancy and persistence identified (Balaban et al. 2019).

Single-Cell Microbiology Techniques

  • Light Microscopy: Techniques such as brightfield and phase contrast used for cell size and morphology.

    • Fluorescence Microscopy: Employs fluorescent dyes and probes for enhanced imaging.

  • Flow Cytometry:

    • Analyzing single cells rapidly (10,000-100,000 cells/min). Measures specific light scattering and fluorescence.

    • Enables fluorescence-activated cell sorting for categorizing cells based on traits.

  • NanoSIMS:

    • A precision technique using stable isotopes to study metabolic activities of individual cells. Analysis of carbon and nitrogen isotopes.

Measurements with Single-Cell Techniques

  • Allows assessment of variations in:

    • Gene expression

    • Cell morphology

    • Metabolic activity

    • Growth rates

    • Motility of cells

Metabolic Heterogeneity Example

  • Methylorubrum extorquens PA1: Growth observed under a single-carbon source environment using fluorescence microscopy.

Research in Virulence Heterogeneity

  • Salmonella Typhimurium: Evaluation of virulence through PfliC-gfp utilizing time-lapse microscopy.

Time-Lapse Microscopy in Microfluidics

  • Microfluidic Devices: Enable observation of bacterial behavior under controlled conditions such as:

    • Confined growth environments.

    • Continuous nutrient influx.

Droplet-Based Microfluidic Technology**

  • Enables analysis of bacterial populations in droplets, supporting various growth states:

    • Analysis can be performed on single cells and aggregates without nutrient flow.

    • Supports motility studies in 3D environments.

Examples of Current Research

  • Investigation into bacterial responses to RNA phage, with results found in a recent preprint by Nikolic et al. 2023.

Follow-Up Assignments

  • Required readings related to microbial ecology, stress response, and antiphage mechanisms for further understanding.

  • Suggested further reading: Ackermann M (2015) on phenotypic heterogeneity.