Quantitative techniques and single-cell microbiology

What does handprint microbiota contain?

Skin bacteria: possibly

Micrococcus luteus (small yellow colonies),

Staphylococcus epidermidis (small, thick white colonies),

Staphylococcus aureus (golden colonies)

Fungus:

Fluffy structure

(colonies differ in shape, size, colour)

How to determine bacterial growth or any other traits?

Bulk measurements

• Clear media = more turbid media = higher density of bacteria

• Compare e,g, if a strain grows better than the other strain in diff. conditions

Absorbance at (?) to assess the density of bacterial cells in batch culture using bulk measurements?

Absorbance at 600 nm A₆₀₀ (optical density)

Where are bacteria cultivated?

Culture tubes, flasks, microplates

Population growth graph

Measuring binary fission by calculating (estimate) the number of generations n

Calculate (estimate) the number of generations n from the increase in the number of cells N (or optical density)

Measuring binary fission by calculating the bacteria growth rate µ

Calculate the growth rate µ from the increase in the number of cells N during time t (t₂-t₁), in the exponential phase

Why natural logarithm (ln) used when calculating the bacteria growth rate µ?

Describe the exponential nature of bacterial growth (population doubles at regular intervals)

→ (ln) linearizes exponential growth by converting the multiplicative way of exp. growth to an additive one

Data distribution: Problems of averages in data

Averages can hide important variations in data, and they don't take into account the distribution of data.

Data distribution: Averages in data example

Mean, median, mode, or other statistical measures that summarize data.

Phenotypic heterogeneity: Cell-to-cell phenotypic variation and temporal variation snapshot - What happen in both variation?

• Fate of individual cells would be different in time

• Diff temporal variation when measuring

Exhibiting individuality in bacteria means

Phenotypic differences between genetically identical cells, even in homogeneous environments

→ Phenotypic heterogeneity/variation

Causes of phenotypic heterogeneity (diff gene expression): 2 Non-genetic sources

• Diff. Environmental stimuli - diff. response

• Stochastic gene expression – cellular & biochemical processes vary between cells and over time

(chemical reaction define w few mol. → noisy biochem processes)

Examples of phenotypic heterogeneity: Dormant cells v.s. persister cells at promoting individuality

Dormant cells: Metabolically inactive state/growth arrest

Persister cells: Dormancy and other molecular mechanisms underlie persistence

Examples of phenotypic heterogeneity: 2 types of persistence in persister cells

Triggered persistence: by stress conditions (env.)

e.g. limitation of different nutrients, high cell number, acid stress, immune factors and exposure to immune cells

Spontaneous persistence (by chance & rare): Few persister cells are made when the culture is in steady-state expo. growth

Examples of phenotypic heterogeneity: Speciality of persister cells

Cells tolerant to antibiotics (antibiotic persistence) and other stressors

(no active ribosome to be targeted when persisting(resting))

Why do persistent bacteria need to be looked out for in antimicrobial treatment?

• Need to make sure all disease-causing cells are eradicated

• OR ELSE, surviving persistence might cause reinfection (they can become active again)

Single-cell microbiology techniques: Light microscopy types

• Brightfield (low-contrast)

• Phase contrast

• Fluorescence

→ use 40x (100x) magnification

→ need Environmental control

Single-cell microbiology techniques: Time-lapse measurement

Understand temporal variability by allowing cells grow in a temp. across time

Single-cell microbiology techniques: Brightfield/phase contrast microscopy limitation?

Rely on transmitted light

Single-cell microbiology techniques: Phase contrast usage?

Enhances contrast for transparent and colorless specimens

Single-cell microbiology techniques: Fluorescence microscopy usage?

Uses high-intensity light (such as UV or blue light) to excite fluorescent molecules (fluorophores) within the sample.

Single-cell microbiology techniques: What does fluorescent dyes show?

Specific metabolic state of the cell/ membrane potential/ viability of the population

Single-cell microbiology techniques: What does fluorescent probes show?

Oligonucleotides hybridise bacterial cells

→ detect 16sRNA

-» diversity in population to quantify the abundance of diff species in a population

Fluorescent gene reporter systems usage (GFP)

Report and estimate on gene expression (regulated or constitutive gene expression)

What does regulated gene expression refers to?

Genes that are turned on or off in response to specific signals or conditions (respond to stimuli)

Usage of fluorescent reporters for regulated gene expression

To monitor these changes by linking the reporter gene to regulatory sequences of the gene of interest

When is the fluorescent reporter expressed? and what does it show?

When the gene is activated

→ which allows gene expression visualization and quantification in real time

What does Constitutive gene expression refer to?

Genes that are continuously expressed at a constant level

Usage of fluorescent reporters for constitutive gene expression

By linking them to constitutive promoters, which drive continuous expression of the reporter gene

→ providing a constant fluorescent signal.

Single-cell microbiology techniques: Flow cytometry - how it works

• Cells pass through a narrow channel one at a time

• Light is used to illuminate the cells in the channel

• A series of sensors detect the specific light scattering and fluorescent characteristics of each cell

• Fluorescence-activated cell sorting

Single-cell microbiology techniques: Flow cytometry - Analyse rate

Analyze 10,000-100,000 bacterial cells in 1 minute

(faster than microscopy)

What is happening in Flow cytometry results

3 infos:

1. Forward scatter cell size - both prop.

2. Side scatter cell complexity - tells how known, fluid or granular a cell is

→ dot graph: dot = measurements for a cell

3. Fluorescence signal - histogram distribution

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Last graph:

Grey = non stressful condition

Black = stressful

→ increase fluorescence = increase gene expression => also population arising (w diff. fluorescent intensities subpop.)

Single-cell microbiology techniques: NanoSIMS usage

Investigating metabolic complexity of a microbial population or entire community

• Cells utilize substrates labeled with stable isotopes, e.g., ²H,¹³C,¹⁵N

• Analysis of metabolic activity of single cells

Single-cell microbiology techniques: NanoSIMS (full name)

Nanoscale secondary ion mass spectrometry

Single-cell microbiology techniques: NanoSIMS - what samples used

Microbial sample or culture that is incubated with substrates labeled with stable isotopes

e.g. carbohydrates (source of carbon) or ammonium salts or nitrogen gas (source of nitrogen)

Single-cell microbiology techniques: NanoSIMS - how it works

NanoSIMS

• uses an ion source to produce a primary beam of ions

• where primary ion beam scans and erodes through entire sample on filter.

→ Scattered secondary ions are transmitted through a mass spectrometer, where the masses are measured.

Single-cell microbiology techniques: NanoSIMS - Analysing substrate assimilation

By measuring incorporation of those stable isotope into biomass of microbial cells.

Single-cell microbiology techniques: What can be measured?

Variation in:

• gene expression

• cell size (morphology)

• metabolism

• growth (elongation rate)

• motility

Example of phenotypic heterogeneity in metabolic functions: Methylorubrum extorquens (a member of a phyllosphere community) - what it shows

• Growth on methanol (Single-carbon source environment)

• Increase in fluorescence = increase in expression of methanol dehydrogenase (fused with reporter gene for MxaF-GFP)

→ NOT ALL CELLS hv same fluorescence intensities = not all use methanol - reflecting variation in growth rates of single cells

Heterogeneity in virulence: Salmonella Typhimurium - gene fliC

• Flagellin gene fliC encodes the majority of flagellum

• fliC promoter controls GFP expression during microcolony growth

• FliC is a virulence factor of Salmonella that is essential for bacterial adhesion to epithelial cells and for bacterial motility.

(motile? = need Flagellin gene fliC)

Time-lapse microscopy in a microfluidic device

(Kasugamycin inhibits protein synthesis at the step of translation initiation)

Droplet-based microfluidic technology

Time-lapse microscopy in a microfluidic device v.s.

Droplet-based microfluidic technology

• Bacterial cells growing in 1D or 2D

• Confined space, no motility

• Continuous inflow of fresh nutrients

• Easy switch from one medium to another

• 3D analysis

• Motile bacteria

• No inflow of fresh nutrients

• Supports bacteria growing as planktonic cells or aggregates

• Analysis of single cells and clumps

• Droplets can be sorted and analyzed

Examples of current research with Time- lapse microfluidic devices

Why designing and choosing phage experiments important in medical studies?

All bacteria are susceptible to phages

Phage is virulent in the experiment until lysis

→ heterogeneous response show

->=> cannot extrapolate impact phages hv on the community just w phage sequences

=> phage could impose diff. response of bacteria (avoid results of antibiotic resistance)

===> avoid phage resistance and tolerance