Experimental Ecology

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Vincent OFlaherty.
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Experimental Ecology

• What is present, where is it and what is it
  doing?
• Numbers, Biomass and Metabolic Activity are the
  fundamental basic biotic parameters of microbial
  ecosystems
• Much needs to be done to improve our accuracy and
  sensitivity in measuring key parameters -
  especially re scale.

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• All approaches that are currently used have
  advantages and disadvantages - if these are
  appreciated the best use can be made of data

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What do we want to know??

• What microbes are present? Detection/identificatio
  n
• Where are they? Detection/localisation
• How many of each population are present (No.s of
  cells or mass of cells) Numbers/biomass
• What are they doing and how is activity
  influenced by changes in the environment?
  Activity/metabolism

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Methods - Summary

• Detection and Numbers
• Culture-based methods for detection and
  enumeration
• Non-culture and non-DNA based methods for
  detection, enumeration - immunology and lipids
• DNA(molecular)-based methods for detection,
  localisation and enumeration
• 2. Methods for determination of Biomass
• 3. Activity and metabolism determinations

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Precautions

• Need to know limitations of each measurement
  procedure e.g. knowing that a total viable
  count typically enumerates around 1 of a
  microbial community
• A combination of methods usually gives the best
  results
• In some cases numbers, biomass and activity show
  proportional correlations - mostly they do not -
  how to interpret this??

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Sampling

1. Destructive sampling - removing a sample from the
   environment for analysis in the lab. -very
   important to be as non-invasive as possible e.g.
   soil cores, tissue biopsies, sea water, sediment,
   rumen fluid etc etc.
2. Micro and mesocosms -model systems
3. Field studies - at present very difficult and
   expensive to undertake

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1. Detection and Enumeration of Microbes in the
Environment

• Culture-based
• Immunology-based
• Membrane lipid
• Genotypic Methods

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Culture-based detection methods

• Organisms must be recovered from environmental
  samples - recognisable and specific phenotypes
  must be expressed during in vitro culture
• Classical approach - selective plating and
  enrichment procedures - useful because they may
  provide physiological information useful in
  analysing microbes ecological function

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Culture vs Counts
Habitat Typical microscopic counts Typical cultivability ()
Soil 109-1010 cells/g 0.01-0.1
Lakes/rivers 106-107 cells/ml 0.01-0.1
Ground water 104-105 cells/ml lt1
Marine (surface) 104-106 cells/ml 0.001-0.1
Marine (depth) 104-105 cells/ml ND
Sediments 106-109 cells/g lt1
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Why cant we grow environmental bacteria?

• Little is known about the specific growth
  requirements of most microbes - e.g. O2 levels,
  nutrients, co-factors, cross-feeding with other
  populations
• Many microorganisms in the environment will have
  a very low metabolic activity or are quiescent
• Most aggregates contain a zone of proliferation
  and a zone of quiescence e.g. biofilms -these
  microbes are not growing but are not dead -
  waiting for favourable conditions

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BioLOG System

• Method of rapid testing of environmental samples
  - can simultaneously assay for a range of
  metabolic characteristics
• Results are based on a colour-change and thus can
  be read automatically - rapid

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• Based on the pattern of substrate utilisations -
  a statistical analysis can be carried out - gives
  a physiological profile of the sample
• Available for G(-) , G() specific or general use
• Most frequently used culture-based method in
  ecological studies - labour, time and money

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• Advantages Cultivation media are formulated to
  take advantage of specific traits of organism
  i.e. nutritional capabilities and/or resistance
  to specific antibiotics - target microorganisms
  are favoured over others
• Can detect growth automatically in broth - more
  sensitive than plates
• Only viable bacteria will grow
• Can work further with isolated bacteria

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• Problems - unculturability, totally artificial
  environment
• Cant examine interactions of mixed group of
  microbes
• Lab. and in vivo phenotype may well be different

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Immunological detection methods

• Based on the fact that bacterial cell wall
  polymers such as proteins and lipopolysaccharides
  have strong antigenic properties
• Can be used to raise antibodies, usually in
  rabbits

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• After repeated exposure to antigen counts of
  antibody become very high
• Can be harvested from serum for use to detect
  antigens in samples

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• Labelled with either fluorochrome, biotin or gold
  and analysed using fluorescence or electron
  microscopy - can be very specific if monoclonal
  antibodies are used - specific for one bindng
  site, polyclonal antibodoes are more common
• Usually cells immobilised on slides and
  antibodies added - observe using a microscope or
  detect electronically. Also Direct
  immunofluorescence used to detect organisms in a
  variety of environments - water, soils, root
  surfaces etc.

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• Advantages Can be used to detect viable but
  non-culturable microorganisms can be used to
  count microbes can be automated can be used in
  situ in samples
• Problems cross-reactivity, cant raise
  antibodies if you dont have a pure culture and
  so cant predict if any other microbe will also
  react change of antigenic properties is response
  to environment sometimes not very sensitive can
  be very time-consuming

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Membrane lipid analysis

• Bacteria can be characterised on the basis of
  different lipids that are found in their
  membranes - Number of carbons, saturation,
  branching all characteristic of different
  organisms
• The fatty acids that are important for bacterial
  identification are the branched chain fatty acids
  containing from 9 to 20 carbons
• Lipids are extracted from the sample and treated
  by attaching an ester group- so they can be
  dissolved

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• Methylated phospholipid ester-linked fatty acids
  - (PL)FAME or PLFA profiles
• Consists of esterification of the lipids and
  injection, separation, identification and
  quantitation (using known standards) of the fatty
  acid methyl esters by gas chromatography (GC)
• Can read the outputs as peaks - profile of
  community structure - individual microbes will
  have individual profiles ( again can do stats)

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• Using this approach a signature profile can be
  obtained for samples
• Community members are identified also some info
  on their physiological state e.g. - a ratio of gt
  1 of trans to cis- isomers of monosaturated PLFAs
  is indicative of starvation or other
  environmental stress

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• Advantages Important chemotaxonomic approach,
  culture independent Statistically valid
  straightforward and rapid, many samples can be
  processed, and change can be observed over time
• Problems Organisms which lack signature profiles
  will not be distinguished, not very sensitive and
  environmental conditions (substrate, temperature
  etc.) can cause major changes in the patterns

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Genotypic Detection Methods

• Based on the ability to detect specific signature
  gene sequences of organisms in the environment -
  detect sequence unique to a microbe gt detect
  microbe
• Extremely valuable in detection of the microbial
  communities present in the environment
  increasingly being used to infer function - main
  method of community analysis currently employed
• Also used in phylogeny - determination of the
  evolutionary relationship between microbes

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Principals of genotypic detection methods

• Methods are based on the fact that nucleic acids
  are made up of 4 bases arranged in a specific
  order
• Base sequences are conserved from one generation
  to the next
• DNA molecules are double-stranded

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• A nucleic acid sequence will only stick or
  hybridise to a complimentary sequence
• DNA and RNA can be made single stranded or
  denatured by raising the temperature
• Two detection approaches used Nucleic acid
  Probes and DNA Amplification

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• Probing and Amplification are linked as you need
  to know the target sequence before you design a
  probe
• Must recover sequence information, analyse it and
  use it to produce probes
• Sequences got from the environment through 1.
  Extraction of nucleic acids and 2. Amplification
  via PCR

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Extraction of Nucleic Acids

• Two approaches to isolating DNA from the
  environmental samples
• 1. Isolation of microbial cells followed by cell
  lysis and purification of nucleic acid (Cell
  extraction)
• 2. Direct lysis of microbial cells in the
  environmental matrix followed by nucleic acid
  purification (Direct extraction)

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• For water samples cells can be collected by
  filtration and then lysed to obtain nucleic acids
  - cells subjected to enzymatic lysis and/or
  phenol-chloroform extraction
• Cell extraction methods also developed for soils
  - normally combine vortexing, centrifugation
  steps
• Direct DNA extraction increasingly favoured for
  environmental studies - more representative of
  populations present - crude extracts purified to
  remove interfering substances

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PCR

• Mimics the natural DNA replication in microbes
• Uses polymerase to synthesise a complimentary
  strand of DNA/RNA from a single strand
• Small sequences (primers) added to create double-
  stranded template
• A series of amplification cycles used to increase
  initial target

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• Target sequence amplified can detect very low
  initial numbers
• Amplified DNA can be used for probing or can be
  cloned and/or sequenced
• Sequencing and comparison with known sequences
  provides information on diversity and types of
  microbe present and also can be used to design
  probes

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• Advantages of PCR no culture, allows detection
  of very low starting numbers, applicable to a
  wide range of samples, allows the sequencing of
  amplified target
• Problems sampling is destructive, need to know
  some sequence information on target, does not
  distinguish between viable and non-viable, can be
  inhibited easily, absolutely dependent on success
  of nucleic acid extraction

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Nucleic acid Probes

• Probes and nucleic hybridisation techniques used
  to detect target sequences diagnostic of specific
  groups of organisms in environmental samples
• Probe is a relatively short nucleotide sequence
  that can hybridise with a homologous sequence in
  the target micro-organism
• Can be designed to target either DNA (chromosome)
  or RNA (usually the rRNA)

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How probes work

• Sequence of events is that nucleic acids are
  extracted from the sample, denatured and
  immobilised - e.g. on a nitrocellulose filter
• Labeled probe is then added and allowed to
  hybridise
• Unbound probe is then washed off and finally
  hybrids are detected

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• Normally carry out hybridisation on an
  immobilised target or probe on a solid phase e.g.
  - nitrocellulose or nylon filter surface
• Normally probe is labelled (32P) and after
  hybridisation and washing can detect target
  binding by autoradiography
• Relative amounts of nucleic acid can be
  quantified by comparison with signal obtained
  with universal probe - variations include use of
  Dot blot manifold etc.

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• Advantages Do not require culture, applicable to
  a wide range of samples, can be quantitative
• Problems destructive, requires some sequence
  information, may not detect low-numbers very well
  (combination with PCR overcomes this), no
  distinction between viable and non-viable

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In-situ hybridisation

• Alternative approach is to carry out specific
  hybridisation between labelled probe and specific
  target sequence inside intact cell with minimum
  sample disturbance
• Most direct method - morphology of the cell
  fixed, membrane made permeable to allow
  penetration of probe (usually with
  paraformaldehyde)

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• Fixed cells bound to glass slide and hybridised
  with oligonucleotide probe in a moist chamber -
  probes can be labelled with radioactivity, biotin
  combined with antibodies etc
• Most commonly labelled with a flourescent dye
  like fluorescein (green) or rhodamine (red)

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FISH

• Fluorescent signals detected by epifluorescence
  or confocal laser scanning microscope (much more
  detail)
• Excellent technique for detection of
  unculturables e.g. symbionts of protozoa etc.
• Very useful for identifying bacteria in complex
  environments - soil, biofilms, activated sludge
  etc.

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• Advantages no culture, can detect both
  culturable and unculturable organisms, localise
  specific cells within a community, estimate
  numbers
• Problems difficulties in getting clean
  hybridisation with some samples, cells have to be
  fixed to get probe in, need sequence information
  on target microbes

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Reporter Genes

• Genetic markers used to track specific
  genetically modified microbial populations in the
  environment - genetic element that permits
  detection of an unrelated biological function
  e.g. lacZ gene useful and commonly employed - can
  cleave X-gal to create a blue pigment readily
  visable on plates - versatile biomarker
• Also green fluorescent protein and
  bioluminescence genes used for this purpose

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1 (b) Determination of numbers

• Direct counts - either stains or nucleic acid
  probes
• Viable Counts

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• Numbers obtained by direct counts typically 2
  orders of magnitude higher than counts obtained
  by cultural techniques and applicable to a
  variety of habitats without culture-based biases
• Numbers of specific microbes can be estimated
  using fluorescent antibody or gene probes
• Multiple populations in the same sample can be
  counted by using several probes with different
  colours

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Stains used for direct counts

• Acridine Orange (water) - nucleic acid
• DAPI (water/solids) - DNA stain
• Fluorescein isothiocyanate (FITC) - protein stain

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Dead or Alive?

• Very important to determine if cells that you are
  counting are viable - are they alive or dead -
  number of procedures attempt to do this
• i.e. use of 2-p-indophenyl-3p-nitrophenyl-5-ph
  enyl tetrazolium chloride (INT) which deposits
  red dye in cells that have active dehydrogenases
• Similar respiration assay involves the use of
  5-cyano-2,3-ditolyl tetrazolium chloride (CTC)

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• Also membrane potential-sensitive fluorochromes
  can distinguish between active, injured (dying)
  and dead cells
• rRNA targeted probes - bind to ribosomes - these
  are present in live cells only

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• Using such methods it appears most of the cells
  observed by direct microscopy are alive - viable
  but non-culturable, concept first introduced by
  Rita Colwell in 1987
• Demonstrated organisms carry out active
  metabolism and retain virulence
• Use of gene probes/PCR etc. can classify
  unculturables - can infer properties based on
  cultured homologues - need to be careful!

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• Otherwise, Plate count and MPN the two basic
  approaches used to cultivate viable organisms-
  both rely on separation of microorganisms into
  individual reproductive units
• All viable count procedures are selective - the
  degree of selectivity varies with the particular
  viable count procedure - impossible to get a
  Total Viable Count