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 Single-Cell Microbiology: Tools,
Technologies, and Applications
Dr. Rajesh Kumar
Ph.D (Dairy Microbiology)
Molecular Biology Unit
Dairy Microbiology Division
N.D.R.I

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 Introduction
Traditionally the field of microbiology has been focused on
studies at population level.

 Information obtained mostly from population level data.

 Development of new tools & techniques for studying

individual microbial cell.

 Single-Cell Techniques reveal:-

i) Environmental distribution

ii) Invisible processes

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 Cont…

Single-Cell Methods:-

 Direct micro or nanoscale measurement.

 Microphysiological studies of metabolite.

 Protein elemental localization.

 Intracellular water dynamics.

 Offers discrete microbial observation.

 Microbial viability phenomenon.

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 Microbial Heterogenity
 Variability is hallmark of biological systems.

 Important practical consequences:-

 Antibiotic & biocide resistance.
 Productivity & Stability of fermentation.
 Potential of pathogen.

Phenotypic plasticity forms the basis of successful

‘life style strategy’.

 Types of Heterogenity:-
Genetic.
Biochemical/Metabolic
Physiological
Behavioral
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 Genetic Heterogenity
 Phage-related phenomena.

 Copy number of mobile genetic elements.

 Intracellular genetic heterogeneity.

 Asymmetric distribution of genetic material.

 Accumulation of DNA damage due to cell aging.

 Loss of gene silencing.

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 Biochemical / Metabolic Heterogenity
‘‘Individual cellular differences in macromolecular
composition’’

 Phenotypic expression of genetic phenomena of

mutants.

 Random transcription events and "noise“.

 Asymmetric distribution of proteins between mother

and daughter cells.

 Retention of oxidatively damaged proteins within

mother cell.

 Quantity of certain macromolecular components.

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 Physiological Heterogenity

 Stems primarily through cell cycle.

 Driven by microenvironmental factors.

 Describe morphological differences b/w individual

cells:-
a) Difference in size & shape.
b) Internal characteristics.
e.g. -
Yeast Bacteria
•Size differences •Differences in cell volume
•Bud scarring •Cell shape
•Surface wrinkling •Buoyant density
•Variation in vacuole size •Nucleoid morphology
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 Behavioral Heterogenity

 Observable consequence of cell-to-cell variation.

 Stem from:-
*Genetic mutation.
*Stochastic processes.

 Responses to chemotactic or phototactic stimuli.

 Measurement of swimming speed or direction.

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 ADVANTAGES OF SINGLE-CELL APPROACHES

 Plate counting and light microscopy.

 Recent technological and methodological innovations:-

 Advances in computing or imaging technologies
 Development of culture-independent methods

 Use of Single-Cell Approaches:-

Revealing Cryptic Processes.

Observing Discrete and Dynamic Events within Living Cells.

Relating Microscopic, Mesoscopic, and Macroscopic Observations.

Caveat: the "Uncertainty Principle“. pdfMachine 10

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 Revealing Cryptic Processes

Microorganisms  carryout a no. of processes


have substantial impact on human life

 Urgent need of proper set of tools to acess:-

Gene transfer & environmental distribution.

Biochemical interactions b/w microbial cells

or
b/w pathogens & their hosts.

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 Observing Discrete & Dynamic evnts within living cells

Previously bacterial cell "...amorphous vessel…’’


Now much more complex than previously imagined.

 Discrete subcellular domains


regulate
distinct biochemical or genetic processes

 Certain proteins change their "subcellular address“.

 Actin polymerization in Listeria monocytogenes.

 Protease secretion in Vibrio cholerae.

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 Relating Microscopic, Mesoscopic, and
Macroscopic Observations

Coordinated multicellular activities:-

Aggregation.
Development of specialized structures.
Colony pattern formation.
:-
• fruiting-body development in myxobacteria.
• mound and slug formation in Dictyostelium discoideum.
• chiral colony morphology in Bacillus subtilis.

Single-cell resolution enable connections b/w these

mesoscopic or macroscopic phenomena and their
microscopic, cellular origins.
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 A Caveat: the "Uncertainty Principle"

Single-Cell methods enable the observation of living cells.

 However, this expose cells to:-

*Potentially toxic fluorescent dyes * intense light

*electric or magnetic energies *physical manipulation.
*experience of increased metabolic load.

 This, in effect, is the biological equivalent of

Heisenberg's "uncertainty principle".

 Bridson and Gould … …"quantal microbiology"

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 Tools and Technologies
Fluorescence
 Rapid & more sensitive than colorimetric techniques.

 Facilitate staining of microbial cells according to their

individual properties.

 Compatible with living cells.

Principle:- e-

Photon fluorescence

e-

Fluorophore
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 Fluorescence Staining techniques

Allow observation of:-

 Protein expression & behavior

 Substrate uptake

 Binding and release of individual

chemoattractant molecules to cell surface
receptors

 Selective degradation of uniparental DNA

within newly formed algal zygotes

 Bacterivory and drug efflux

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 Fluorescence ratio Imaging Microscopy
Provide insights into dynamic cellular events:-

 Physiological responses of spoilage organisms to chemical

stress.
or
 Cellular inactivation b/c of antimicrobial treatment.

CFSE  Probe.

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 Fluorescent nanocrystals or Quantum dots

Incompatible spectral or chemical properties can place

practical constraints on the fluorescent dye.

Advantages:-
Including large extinction coefficients
Reduced susceptibility to photobleaching.

Most intriguing properties:-

Narrow, size-dependent emission spectra

May be excited with a single UV light source.

Can be directed to specific tissues or cell types.

Long-term labeling of live cells

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 Targeting of Quantum dots into specific tissues of mouse

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 Fluorescence insitu hybridization & Immuno fluorescence

Fluorescently labeled nucleic acid probes are hybridized to

complementary rRNA targets.

The aggregate signal leads to the sequence-specific

fluorescence of target cells.

Recent FISH-based methods:-

Detect low-copy-number targets on plasmid or
chromosomal DNA.

Different from rRNA-targeted FISH b/c it utilizes

polynucleotide probes.

The resulting signal characterized by the formation of a fluorescent

"halo" around the periphery of target cells.Thus named RING-FISH.
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 Cont…
Fluorescently labeled antibodies:-

 Detection of diagnostic molecular binding events.

 Can be directed against surface antigens or against

internal targets.

 Used for discrete localization of specific proteins

within individual cells.

 Unlike FISH, it does not require cell permeabilization

so can be used on living cells

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 Green fluorescent protein and related reporters

Versatile tool for in vivo visualization of protein expression,

localization, and functionality.

The true power of GFP is as a visual reporter of dynamic

events occurring in living cells.

In Escherichia coli regular pole-to-pole oscillation for GFP-MinD was

observed Raskin and de Boer theroized that cell may use this protein as a
"measuring device" to continuously probe the location of the center of the
cell.

 Construction of whole-cell sensors for in situ monitoring

of:-
•Cytoplasmic viscosity & internal pH of bacterial cells
•Protein diffusion rates in living cells.
•Investigation of quorum-based interspecies communication.
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 Cont…

Introduction of fluorescence-shifted spectral variants:-

Chimeric "nanosensor“ proteins based on the fusion of

ECFP a bacterial maltose binding periplasmic protein, and
EYFP. (Fehr et al)

Nanosensors  bind to maltose  confermational change


More efficient FRET from ECFP to EYFP

In S. cerevisiae, changes in ECFP/EYFP FRET ratios enabled

maltose uptake and compartmentation.

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 Flow cytometry
 A powerful fluorescence-based diagnostic tool.

 Enables rapid analysis of entire cell populations.

 Specific Flow cytometers add to the versatility of this

method.

Cells in a liquid sample  Passed individually


Data collected & saved  Intense light source

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  Facilitates valuable  Elucidating that
insights into connections apoptosis is not limited
between single-cell and to eukaryotes but may
population-level. also be active in
prokaryotic systems.

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 Laser Scanning Cytometry

 Solid-phase cytometric technology for collecting laser-

induced fluorescence from cell samples on membrane filter.

 Well suited for the observation of cellular properties as

a function of time.

e.g. *monitoring the kinetics of fluorescence staining in

living cells.
*observing interactions between neighboring cells.

 Ability to concentrate cells prior to analysis gives

definite practical advantages over flow cytometry.

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 Cont…

LSC instruments provide:-

 Rapid means of counting, quantifying, and

recording the distribution of fluorescent events on
a filter.

 Visual information on both cell morphology and

the spatial distribution of fluorescence within each
cell.

Exposure to excitation source for long periods can

create photobleaching of fluorescent labels

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 Single-cell determination of yeast glycogen content by
image cytometry.

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 Single-molecule analysis of cyclic AMP receptor
occupancy on the surface of a Dictyostelium discoideum
cell during chemotaxis.

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 Scanning Probe Microscopies
Can yield information on both the topography & physcio-
chemical properties of a sample surface.
The SPM family of tools:-
1) STM 2) AFM 3) SECM 4) MFM

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 Atomic force microscopy

Modes of imaging:- contact, noncontact, and tapping .

Contact imaging  "dragging" the tip across the sample.

Noncontact imaging 

Based on electrostatic deflection of the probe tip. Used

to investigate charge development on biological surfaces.

Tapping-mode imaging 

Alternative method for measurements of "soft" biological

surfaces.
Chemically functionalized tips oscillates over surface thus
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Applications:-
Measure discrete interaction forces in the piconewton range.

Ultrastructural studies of surfaces of living microbial cells.

AFM cantilevers with functionalized tips can be used as "nanobiosensors"

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 Scanning Electrochemical Microscopy
 SPM based tool for mapping redox activity in living
cells.

 Well established technique for electrochemical

charecterization of single living microbial cell.

 Scanning tip is an ultramicroelectrode designed for

measuring charge transfer reactions.

 Redox maps are generated as tip is scanned in x-y

plane above an electrochemically active cell.
e.g.
Electrochemical visualization of O2 production in single
algal protoplasts on exposure to light.
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 Microspectroscopic Methods

 Single cell resolution methods, enable observation of

target properties within specific cells.

 Provide biochemical information on overall macromolecular

composition of cells.

 Minimal sample preparation requirement enable analysis

for living cells.

Raman microspectroscopy
Microbeam analysis
Electrorotation

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 Raman microspectroscopy

Raman effect:-

Induced emission of light resulting from the inelastic

scattering of a small number of photons.

Raman spectra provide information on molecular vibrational

states.

Energy range:-
UV (e.g., 257 nm), visible, and infrared excitation
frequencies.

Common visible sources

Argon-ion lasers ( 514 nm) and helium-neon lasers ( 632 nm).
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 Cont…
Applications:-
To investigate biochemical differences b/w cells in morphologically
heterogenous cultures of Clostridia.

Reagentless identification of individual bacterial spores.

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 Microbeam analysis

Include sensitive methods for characterizing single cell elemental

composition.

Allow measurement of concentration, chemical state & cellular

location of biologically relevant inorganic nutrients.

Elemental map of an individual cell can generate in a single pass.

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 Micromanipulation
Used to address a cell to a specific position in a liquid medium.

Allow stable positioning of cells for observations during Single cell

assay.

Used to measure forces exerted by microorganisms on its

environment.

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 Microcapillary Electrophoresis
 Isoelectric focusing of whole cells.

 Electrophoretic separation of intracellular analytes

from a single cell after lysis.

Single cell  loading into capillary  lysed  analyzed

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 Conclusion AND Future Perspectives

 “high-tech”methods form recurrent themes in Single-Cell

Microbiology.

 Availability of high-throughput sequencing methods and

increased computing power has fueled a rapid pace of
discovery in genomics, proteomics and related fields.

 Use of Mathematical modeling in Single-Cell Microbiology:-

How to control & improve microbial fermentations.

Represent an additional source to test hypothesis with

economy, speed & flexibility.

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 Cont…

“Still this is only the scratching the surface regarding

the complexity of microbial cells, there is much more to
explore in future of Single-Cell Microbiology.”

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