Methods in Microbial Ecology
? Microbial ecology focus on two major issues,(1)
biodiversity,including the isolation,identification,
and quantification of microorganisms in various
habitats,and (2) microbial activity,that is,what are
microorganisms doing in their habitats.
? We begin here with a consideration of assessing
biodiversity through enrichment and isolation,and
then consider nonculture methods of identification
and enumeration based on fluorescent antibodies
and nucleic acid probes.
Enrichment and Isolation Methods
? In this method,a medium and a set of incubation conditions are used that are
selective for the desired organism and are counterselective for the undesired
organisms.
? 1,The Winogradsky column, for isolation of purple and green phototrophic bacteria
and other anaerobes,the Winogradsky column has traditionally been used,
Winogradsky column have been used to enrich for a variety of prokaryotes,both
aerobes and anaerobes,The great advantage of a column,besides the cultures,is that
it can be spiked with a particular compound whose degradation one wishes to study
and then allowed to select from the inoculum for an organism or organisms that can
degrade it.
? 2,From enrichments to pure cultures, the objective of an enrichment culture study
is usually to obtain a pure culture,Pure cultures can be obtained in many ways,but
the most frequently employed means are the streak plate,the agar shake,and liquid
dilution methods.
Identification and Quantification,Nucleic Acid Probes,
Fluorescent Antibodies,and Viable counts
? Staining and fluorescent antibodies,Microorganisms
can be identified and enumerated by direct
microscopic examination of the habitat,However,
special procedures are needed to make
microorganisms visible in opaque habitats.
? Nucleic acid probes,A very powerful approach to
the identification and quantification of
microorganisms in nature is the use of nucleic acid
probes.
Visualization of bacterial microcolonies (bacterial cells appear as
greenish-yellow dots) on the surface of soil particles by use of the
fluorescent antibody technique,Cells are about 1 μm in diameter.
Microbial Activity Measurements,
Radioisotopes and Microelectrodes
? Radioisotopes are very useful in measuring specific
microbial processes at high sensitivity and also in
obtaining information on the turnover rates of
chemical species in nature.
? Microbial ecologists have used small glass
electrodes,referred to as microelectrodes,to study
the activity of microorganisms in their
microenvironments,
Use of radioisotopes to
measure microbial
activity in nature,
(a) Photosynthesis
measured in natural
seawater with 14CO2
(b) Sulfate reduction in
mud measured with 35SO42-
(c) Methanogenesis
measured in mud with
acetate labeled in either
the methyl (14CH3COO-)
or the carboxyl
(CH314COO-) carbon.
Microelectrodes,
(a) Schematic
drawing of an
oxygen
microelectrode,
(b) Photo of
microelectrodes
being used in a
microbial mat
Microbial Activity Measurements,Stable Isotopes
Recovery of Single Cells or cell Organelles from Complex Natural Mixtures Using
Micromanipulation,By use of an inverted phase-contrast microscope and a
micromanipulator,a microorganism can be recovered for direct molecular analysis,
(a) The bacterium to be isolated is placed below the micromanipulator tip (diameter 5
to 10 μm) and a slight vacuum is drawn,(b) The desired single bacterium is drawn up
into the micropipette tube and is ready for molecular analysis.
Combining Micromanipulation for Isolation of Single Cells or Organelles with the Polymerase
Chain Reaction (PCR),(a) Recovery of an endosymbiotic mycoplasma from single cell of the
flagellate Koruga bonitaby micromanipulation (bar=10μm) and (b) phylogenetic analysis of the
recovered mycoplasma following PCR amplification and sequencing of the PCR products,with
the bar indicating 10% estimated sequence divergence,Lactobacillus acidophilus is the outgroup
reference,This approach makes it possible to link a specific microorganism or organelle,isolated
from a natural environment,to its molecular sequence and phylogenetic information,Flagellate
(F),capillary tube (Ct).
Preparing a phylogenetic distance-matrix
tree fromm 16S ribosomal RNA
sequences,For illustrative purposes,only
short sequences are shown,but it should
be considered that the sequences in (a)
are representative of the entire 16SrRNA,
The evolutionary distance (ED) in (b) is
calculated as the percentage of
nonhomologous sequence between the
RNAs of any total two organisms,The
corrected ED is a statistical correction
necessary to account for either back
mutations to the original genotype or
additional forward mutations at the same
site that could have occurred,The tree (c)
is ultimately generated by computer
analysis of the data to give the best fit,
The length of the branches separating any
two organisms is proportional to the
evolutionary distance between them.
Rooted universal phylogenetic tree as determined from comparative ribosomal RNA
sequencing,The data support the separation of three domains,two of which (Bacteria and
Archaea) contain only prokaryotic representatives,The location highlighted in red is the root of
the tree,which represents the position of the universal ancestor of all cells.
Example of methods
that would be used for
identification of a
newly isolated enteric
bacterium,using
conventional
microbiological
methods (the example
given shows the
procedures that would
be used for
identifying E.coli),
Note that most of the
analyses here require
that the organisms be
grown in pure culture
and that solely
phenotypic criteria be
used in the
identification of
biochemical tests.
Genomic hybridization as a taxonomic tool,(a) DNA is prepared from test organisms,One of the DNAs is labeled
(shown here as radioactive phosphate in the DNA of Organism1),(b) Actual hybridization experiment,All
combinations are tried and excess unlabeled DNA is added in each experiment,All combinations are tried and excess
unlabeled DNA is added in each experiment to prevent labeled DNA from reannealing with itself,Following
hybridization,hybridized DNA is separated from unhybridized DNA before measuring radioactivity in the hybridized
DNA only,(c) Results,Radioactivity in the control is taken as the 100% hybridization value.
In this Teld of water
cress,the world’s
biggest selling
biopesticide,Bacillus
thuringiensis
(Bt) toxin,was
overcome by the
evolution of
resistance in
diamondback moths,
This pesticide is
engineered into
millions of acres of
crop plants,and so the
ability of insects to
evolve resistance has
created anxiety in the
biotechnology
industry.
Developmental
cascade of antibiotics
used to treat dangerous
Staphylococcus
infections,Dates
reflect evolution of
resistance to each drug
requiring search for
more powerful
alternatives.
A cocktail of
antibiotics is used to
treat a patient
with septicaemia
(above,top),a
bloodstream
infection,
Staphylococcus aureus
(above) has
become resistant to
almost all antibiotics
Flow chart of the recombinant approach for drug discovery,The
stages involved from taking an environmental sample (for example,soi)
to obtaining an active biomolecule.
The Ability of This Planet to Sustain Life Is
Completely Dependent on Microbial Activity
?Microbes make up more than 50% of the Earth’s biomass.
?Microbes are found in diverse environments.
?Microbes use various substrates as energy sources.
?As the foundation of the biosphere,microbes control Earth’s
biogeochemical cycles and affect soil productivity,water
quality,and global climate.
Opportunities
?Clean energy.
?Reducing climate change.
?Cleaning up the environment.
?The revolution in our understanding
already
has begun.
Recent Progresses in Metagenomics
?Reconstruction of microbial genomes from the environment.
?Large scale genome sequencing of environmental samples,
?Screening for antibacterial activity.
?Screening for industrial enzymes.
?Expression of functional genes across genus.
?Exploring microbial diversity.
?A strategy for cloning bacterial operons.
?Discovery of new genes.
? Microbial ecology focus on two major issues,(1)
biodiversity,including the isolation,identification,
and quantification of microorganisms in various
habitats,and (2) microbial activity,that is,what are
microorganisms doing in their habitats.
? We begin here with a consideration of assessing
biodiversity through enrichment and isolation,and
then consider nonculture methods of identification
and enumeration based on fluorescent antibodies
and nucleic acid probes.
Enrichment and Isolation Methods
? In this method,a medium and a set of incubation conditions are used that are
selective for the desired organism and are counterselective for the undesired
organisms.
? 1,The Winogradsky column, for isolation of purple and green phototrophic bacteria
and other anaerobes,the Winogradsky column has traditionally been used,
Winogradsky column have been used to enrich for a variety of prokaryotes,both
aerobes and anaerobes,The great advantage of a column,besides the cultures,is that
it can be spiked with a particular compound whose degradation one wishes to study
and then allowed to select from the inoculum for an organism or organisms that can
degrade it.
? 2,From enrichments to pure cultures, the objective of an enrichment culture study
is usually to obtain a pure culture,Pure cultures can be obtained in many ways,but
the most frequently employed means are the streak plate,the agar shake,and liquid
dilution methods.
Identification and Quantification,Nucleic Acid Probes,
Fluorescent Antibodies,and Viable counts
? Staining and fluorescent antibodies,Microorganisms
can be identified and enumerated by direct
microscopic examination of the habitat,However,
special procedures are needed to make
microorganisms visible in opaque habitats.
? Nucleic acid probes,A very powerful approach to
the identification and quantification of
microorganisms in nature is the use of nucleic acid
probes.
Visualization of bacterial microcolonies (bacterial cells appear as
greenish-yellow dots) on the surface of soil particles by use of the
fluorescent antibody technique,Cells are about 1 μm in diameter.
Microbial Activity Measurements,
Radioisotopes and Microelectrodes
? Radioisotopes are very useful in measuring specific
microbial processes at high sensitivity and also in
obtaining information on the turnover rates of
chemical species in nature.
? Microbial ecologists have used small glass
electrodes,referred to as microelectrodes,to study
the activity of microorganisms in their
microenvironments,
Use of radioisotopes to
measure microbial
activity in nature,
(a) Photosynthesis
measured in natural
seawater with 14CO2
(b) Sulfate reduction in
mud measured with 35SO42-
(c) Methanogenesis
measured in mud with
acetate labeled in either
the methyl (14CH3COO-)
or the carboxyl
(CH314COO-) carbon.
Microelectrodes,
(a) Schematic
drawing of an
oxygen
microelectrode,
(b) Photo of
microelectrodes
being used in a
microbial mat
Microbial Activity Measurements,Stable Isotopes
Recovery of Single Cells or cell Organelles from Complex Natural Mixtures Using
Micromanipulation,By use of an inverted phase-contrast microscope and a
micromanipulator,a microorganism can be recovered for direct molecular analysis,
(a) The bacterium to be isolated is placed below the micromanipulator tip (diameter 5
to 10 μm) and a slight vacuum is drawn,(b) The desired single bacterium is drawn up
into the micropipette tube and is ready for molecular analysis.
Combining Micromanipulation for Isolation of Single Cells or Organelles with the Polymerase
Chain Reaction (PCR),(a) Recovery of an endosymbiotic mycoplasma from single cell of the
flagellate Koruga bonitaby micromanipulation (bar=10μm) and (b) phylogenetic analysis of the
recovered mycoplasma following PCR amplification and sequencing of the PCR products,with
the bar indicating 10% estimated sequence divergence,Lactobacillus acidophilus is the outgroup
reference,This approach makes it possible to link a specific microorganism or organelle,isolated
from a natural environment,to its molecular sequence and phylogenetic information,Flagellate
(F),capillary tube (Ct).
Preparing a phylogenetic distance-matrix
tree fromm 16S ribosomal RNA
sequences,For illustrative purposes,only
short sequences are shown,but it should
be considered that the sequences in (a)
are representative of the entire 16SrRNA,
The evolutionary distance (ED) in (b) is
calculated as the percentage of
nonhomologous sequence between the
RNAs of any total two organisms,The
corrected ED is a statistical correction
necessary to account for either back
mutations to the original genotype or
additional forward mutations at the same
site that could have occurred,The tree (c)
is ultimately generated by computer
analysis of the data to give the best fit,
The length of the branches separating any
two organisms is proportional to the
evolutionary distance between them.
Rooted universal phylogenetic tree as determined from comparative ribosomal RNA
sequencing,The data support the separation of three domains,two of which (Bacteria and
Archaea) contain only prokaryotic representatives,The location highlighted in red is the root of
the tree,which represents the position of the universal ancestor of all cells.
Example of methods
that would be used for
identification of a
newly isolated enteric
bacterium,using
conventional
microbiological
methods (the example
given shows the
procedures that would
be used for
identifying E.coli),
Note that most of the
analyses here require
that the organisms be
grown in pure culture
and that solely
phenotypic criteria be
used in the
identification of
biochemical tests.
Genomic hybridization as a taxonomic tool,(a) DNA is prepared from test organisms,One of the DNAs is labeled
(shown here as radioactive phosphate in the DNA of Organism1),(b) Actual hybridization experiment,All
combinations are tried and excess unlabeled DNA is added in each experiment,All combinations are tried and excess
unlabeled DNA is added in each experiment to prevent labeled DNA from reannealing with itself,Following
hybridization,hybridized DNA is separated from unhybridized DNA before measuring radioactivity in the hybridized
DNA only,(c) Results,Radioactivity in the control is taken as the 100% hybridization value.
In this Teld of water
cress,the world’s
biggest selling
biopesticide,Bacillus
thuringiensis
(Bt) toxin,was
overcome by the
evolution of
resistance in
diamondback moths,
This pesticide is
engineered into
millions of acres of
crop plants,and so the
ability of insects to
evolve resistance has
created anxiety in the
biotechnology
industry.
Developmental
cascade of antibiotics
used to treat dangerous
Staphylococcus
infections,Dates
reflect evolution of
resistance to each drug
requiring search for
more powerful
alternatives.
A cocktail of
antibiotics is used to
treat a patient
with septicaemia
(above,top),a
bloodstream
infection,
Staphylococcus aureus
(above) has
become resistant to
almost all antibiotics
Flow chart of the recombinant approach for drug discovery,The
stages involved from taking an environmental sample (for example,soi)
to obtaining an active biomolecule.
The Ability of This Planet to Sustain Life Is
Completely Dependent on Microbial Activity
?Microbes make up more than 50% of the Earth’s biomass.
?Microbes are found in diverse environments.
?Microbes use various substrates as energy sources.
?As the foundation of the biosphere,microbes control Earth’s
biogeochemical cycles and affect soil productivity,water
quality,and global climate.
Opportunities
?Clean energy.
?Reducing climate change.
?Cleaning up the environment.
?The revolution in our understanding
already
has begun.
Recent Progresses in Metagenomics
?Reconstruction of microbial genomes from the environment.
?Large scale genome sequencing of environmental samples,
?Screening for antibacterial activity.
?Screening for industrial enzymes.
?Expression of functional genes across genus.
?Exploring microbial diversity.
?A strategy for cloning bacterial operons.
?Discovery of new genes.
