CHAPTER 18
Methods in Microbial Ecology
Microbial diversity – isolation, identification, and quantification
Microbial activity – in the habitat
Methods
Enrichment and isolation
Cell staining
Gene isolation and characterization
Enrichment culture technique- medium and culture conditions favors
growth of desired organism and countersellect for undesired organisms
Azotobacter (N2-fixing bacterium) was first bacterium isolated by
enrichment techniques
Culture-Dependent Analyses of
Microbial Communities
Enrichment and Isolation
• Microbial ecology deals with how
microorganisms interact with one another and
their environment.
• The enrichment culture technique is a
means of obtaining microorganisms from
natural samples. Hundreds of different
enrichment strategies have been devised
(Table 18.1).
• A classic enrichment strategy is shown in Figure 18.1.
•Azotobacter selection
• The Sergei Winogradsky column is a miniature anoxic ecosystem that
can be used as a long-term source of bacteria for enrichment culture
purposes (Figure 18.2).
Gradient of H2S
Thiosperillum
Jenense
Winogradysky columns used
for isolation of sulfate
reducers
Chromatium
okenii
Chlorobium
limicola
• Although the enrichment culture is a powerful tool, in
most enrichments there exists a bias, and sometimes a
very severe bias, in the outcome. Enrichment bias can be
demonstrated by comparing the results obtained in
dilution cultures with classical liquid enrichment.
•Dilution eliminate rapidly growing but quantitatively
insignificant organisms
Isolation in Pure Culture
• Once a successful enrichment culture has been
established, a pure culture can be obtained by
conventional microbiological procedures, including
streak plates, agar shakes, and dilution methods.
• In the most probable number (MPN) technique (Figure 18.4), pure
cultures can be obtained from repeated serial dilutions.
Laser tweezers allow one to "pick" a cell from a microscope field and
literally move it away from contaminants (Figure 18.5).
Molecular (Culture-Independent)
Analyses of Microbial Communities
Viability and Quantification Using
Staining Techniques
• DAPI is a general stain for identifying
microorganisms in natural samples. It
stains DNA.
• Some stains can differentiate live versus
dead cells, and fluorescent antibodies that
are specific for one or a small group of
related cells can be prepared.
DAPI stained cells
Live = green
Dead = red
P. fluorscens tagged with GFP
• The green fluorescent protein makes cells
autofluorescent and is a means for tracking
cells introduced into the environment. Unlike
in pure cultures, morphologically similar cells
may actually be quite different genetically in
natural samples.
Genetic Stains
• A variety of fluorescent-staining methods employ the power
of nucleic acid probes and thus are highly specific in their
staining properties. These include phylogenetic staining,
chromosome painting, and reverse transcription fluorescent in
situ hybridization (FISH).
Ammonia oxiding
bacteria = red
Nitrite oxiding
bacteria green
Sewage sludge stained with three
probes, red, green
and purple
Confocal
micrograph
Linking Specific Genes to
Specific Organisms Using PCR
• The polymerase chain reaction (PCR) can be used to amplify
specific target genes such as small subunit ribosomal RNA
genes or key metabolic genes (Figure 18.13).
• The polymerase chain reaction
(PCR) can be used to amplify
specific target genes such as small
subunit ribosomal RNA genes or
key metabolic genes.
• Denaturing gradient gel electrophoresis (DGGE)
can be used to resolve slightly to greatly different
versions of these genes present in the various species
inhabiting a natural sample.
First PCR – six distinct
rRNA sequences
Separated by second PCR
And DGGE
Environmental Genomics (Metagenomics)
• Environmental genomics
involves shotgun sequencing
and analysis of the collective
genomes of the organisms
present in a microbial
community.
• In environmental genomics, all
genes in the microbial
community—the metagenome—
are sampled.
Measuring Microbial Activities
in Nature
Radioisotopes and
Microelectrodes
• The activity of microorganisms
in natural samples can be
assessed very sensitively using
radioisotopes and/or
microelectrodes (Figure
18.18a).
• In most cases, measurements are of the net activity
of a microbial community rather than of a population
of a single species (Figure 18.16).
• Radioisotopes can be used as measures of microbial
activity in a microscopic technique called
microautoradiography (MAR).
Stable Isotopes
• Isotope fractionation can reveal the biological
origin of various substances.
• Fractionation is a result of the activity of enzymes
that discriminate against the heavier form of an
element when binding their substrates.
12C
is preferentially fixed compared to 13C by the enzyme systems