Methods in Microbial Ecology Lecture 16 Feb 27, 2006 - PowerPoint PPT Presentation

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Methods in Microbial Ecology Lecture 16 Feb 27, 2006

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The study of how microorganisms interact with each other and with their ... 2) Top up with lake, pond or sea water. 3) Cap loosely and grow in muted sunlight ... – PowerPoint PPT presentation

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Title: Methods in Microbial Ecology Lecture 16 Feb 27, 2006


1
Methods in Microbial EcologyLecture 16Feb 27,
2006
2
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

3
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

4
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

5
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

6
Test the hypothesis that a particular organism
exists in the column by supplementing with a
compound that it can degrade
7
  • 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

8
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?

9
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

10
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

11
MPN in this example is 105 106 recoverable
cells per gram of sample
12
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

13
  • 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

14
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

15
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

16
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

17
Detection of Sulfobolus acidocaldarius on the
surface of soil particles by immunofluorescence
18
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

19
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

20
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21
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

22
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23
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

24
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

25
  • The most abundant members of a microbial
    community may never have been seen in laboratory
    culture

26
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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28
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
29
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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32
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.

33
Isotopic fractionation discrimination against
the heavier isotope
34
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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