Methods in Microbial Ecology Lecture 16 Feb 27, 2006

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Methods in Microbial EcologyLecture 16Feb 27,
2006
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Microbial ecology

• The study of how microorganisms interact with
  each other and with their environment.
• Biodiversity isolation, identification and
  quantification of microorganisms in their native
  habitats
• Microbial activity what are the organisms
  actually doing in their native habitats

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Culture-dependent analysis

• In nature, microorganisms exist in mixed
  cultures called microbial communities
• To isolate and study a particular organism,
  researchers must isolate microorganisms from
  nature and establish them in pure cultures
• Enrichment culture is a selective medium and
  incubation procedure that selects for desired
  organisms and against undesired ones. Success
  requires a proper inoculum, culture medium and
  incubation conditions

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Enrichment and isolation of Azotobacter

• Azotobacter is a fast-growing nitrogen-fixing
  bacterium
• It is isolated in culture conditions including
  oxygen, and no fixed nitrogen supply

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Winogradsky column

• A miniature anoxic ecosystem that is used as a
  long-term inoculant source for enrichment
  cultures
• Particularly good for enrichment of purple and
  green phototrophic bacteria, sulfate reducing
  bacteria
• Preparation 1) fill a large glass cylinder
  half full with organic-rich mud supplemented with
  calcium carbonate and calcium sulfate. 2) Top up
  with lake, pond or sea water. 3) Cap loosely and
  grow in muted sunlight

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Test the hypothesis that a particular organism
exists in the column by supplementing with a
compound that it can degrade
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• A mixture of organisms generally develops
• Algae and cyanobacteria appear in the upper part
  of water column and produce oxygen
• Anoxic decomposition in the mud creates a
  gradient of hydrogen sulfide which favors growth
  of green and purple sulfur bacteria
• Purple nonsulfur bacteria may grow at the
  interface between oxic and anoxic zones

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Enrichment bias

• Dominant organism in an enrichment culture does
  not necessarily represent the dominant organism
  in a natural habitat.
• Laboratory cultures often misrepresent natural
  communities. Lab cultures favour fastest-growing
  species
• Dilution of the inoculum prior to enrichment
  often yields a different culture. Why?

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Pure culture isolation agar shakes

• Dilution of a mixed culture in tubes containing
  molten agar. Individual colonies become
  separated and embedded in the agar
• Useful for purifying anaerobes such as
  phototrophic sulfur bactera and sulfate-reducing
  bacteria from Winogradsky columns.
• Pure cultures are generally obtained after
  several serial dilutions

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Pure culture isolation Most Probable Number
technique

• Serial dilution of inoculum in liquid medium
  until the final tube shows no growth.
• Used to test numbers of microorganisms in foods,
  wastewater and other samples where cell numbers
  are measured routinely
• Medium can be highly selective for individual
  species or complex enough to get an idea of total
  cell numbers

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MPN in this example is 105 106 recoverable
cells per gram of sample
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High-tech pure cultures-Laser tweezers

• Apparatus consists of an inverted light
  microscope, powerful infrared laser and
  micromanipulation device
• Laser creates a force pushing down on small
  objects (such as a single cell) that can be moved
  away from contaminants
• Useful for isolating slow-growing or rare
  species

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• Cells in capillary tubes can be optically
  trapped, isolated. Tube is broken and the cell
  is flushed out into sterile medium to initiate a
  pure culture

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Molecular analysis viability and
quantification staining

• DAPI 4,6-diamido-2-phenylindole is a
  fluorescent stain useful for quantitating cells
  in opaque habitats
• DAPI binds to DNA and fluoresces bright blue,
  making cells easy to count
• Aquatic samples can be counted after the sample
  is passed through a filter
• DAPI staining does not distinguish living from
  dead cells or between different microbial groups

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Viability staining

• Methods of fluorescent staining that distinguish
  between living and dead cells. Example
  LIVE/DEAD Bac Light
• Based on the integrity of the cytoplasmic
  membrane
• Green fluorescent dye penetrates all cells. Red
  dye containing propidium iodide penetrates only
  those cells with severely damaged membrane. Red
  cells dead cells
• Procedure does not work in natural habitats
  because of nonspecific fluorescence of background
  materials

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Fluorescent antibodies-immunofluorescence

• Exploits the specificity of antibodies to
  recognize a particular cell-surface protein of an
  organism
• Used for identifying an organism in a complex
  community including many species, such as soil or
  a clinical sample
• Preparation of antibodies against a particular
  organism is expensive and time-consuming
• Many clinically relevant antibodies are
  available commercially

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Detection of Sulfobolus acidocaldarius on the
surface of soil particles by immunofluorescence
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Limitations of cell staining/microscopy

• Very small cells may be overlooked
• Morphologically similar but genetically distinct
  species may be confused
• Genetic stains solve this problem by detecting
  specific gene sequences that can be quite
  specific, or quite promiscuous

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Genetic Stains - FISH

• Fluoresent in situ hybridization uses
  fluorescently-labelled single-stranded DNA or RNA
  probes to bind directly to its complementary
  sequence in a nucleic acid.
• Phylogenetic staining using FISH uses probes
  that bind directly to signature sequences
  within 16S (prokaryotes) or 18S (eukaryotes)
  rRNA. Degree of specificity of the probes can be
  altered to detect individual species or entire
  domains. Can therefore be used to track related
  organisms

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Chromosome painting

• FISH technology used to detect the presence of
  specific genes in a sample. Example is a
  nitrogen-fixing organism present? Look for
  nitrogenase
• Must have a fluorescent probe specific for the
  class of genes of interest can distinguish
  photosynthetic organisms, nitrogen fixers,
  hydrogen bacteria etc.
• Used to estimate the numbers of different types
  of cells in a natural sample

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In situ reverse transcription

• Used to determine whether a given gene is being
  expressed in a sample at a particular time
• Involves the use of a probe that binds to the
  mRNA of the gene of interest. Therefore only the
  expressed gene is detected
• Allows researchers to study factors affecting
  gene expression in natural populations and
  habitats

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PCR linking genes to specific organisms

• Biodiversity of a habitat can be monitored
  without culturing or observing cells. Isolate
  characteristic gene sequences instead!
• Isolate total DNA from habitat and clean up
  contaminants. Use PCR to amplify gene sequences
  of interest (often 16S RNA)
• Sequence individual PCR products or separate
  them by denaturing gradient gel electrophoresis

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• The most abundant members of a microbial
  community may never have been seen in laboratory
  culture

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Measuring microbial activity in
nature-radioisotopes

• Direct chemical measurements are often
  sufficient to detect microbial activities
  transformation of lactate and sulfate to hydrogen
  sulfide by sulfate-reducing bacteria
• Radioisotopes are used to determine the fate of
  portions of molecules, turnover rate, or for
  extreme sensitivity

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Controls such as the killed cell control
guarantee that transformation of a radiolabeled
compound is due to a microbial rather than a
strictly chemical process
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Microelectrodes

• Tiny glass electrodes (2-100 ?m diameter) to
  measure pH, oxygen, carbon dioxide, hydrogen,
  hydrogen sulfide. Often used to measure chemical
  reactions in microbial mats
• Layered microbial communities with cyanobacteria
  in the uppermost layer, anoxygenic phototrophs in
  middle layers and chemoorganotrophs (especially
  sulfur reducing) as light becomes limiting at the
  bottom
• Microbial mats are found in hot springs and
  intertidal zones

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Stable isotopes

• Isotopes that are not radioactive. Carbon and
  sulfur are the most commonly used in microbiology
• 12C is 95, 13C is 5
• 32S is common, 34S is rare
• Biochemical reactions tend to favour the lighter
  isotope. Therefore cells become enriched in 12C
  and depleted in 13C relative to a standard.

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Isotopic fractionation discrimination against
the heavier isotope
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Use of isotopic fractionation in microbial ecology

• Isotopic composition of a sample indicates its
  past biological activity
• Life can be inferred from organic carbon in
  rocks 3.5 billion years old
• Sulfides in lunar rocks provide evidence against
  past life on the moon

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