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  1. 1. Use the left mouse button to move forward through the show Use the right mouse button to view the slides in normal view , edit or print the slides The following slides are provided by Dr. Vincent O’Flaherty.
  2. 2. Experimental Ecology <ul><li>What is present, where is it and what is it doing? </li></ul><ul><li>Numbers, Biomass and Metabolic Activity are the fundamental basic biotic parameters of microbial ecosystems </li></ul><ul><li>Much needs to be done to improve our accuracy and sensitivity in measuring key parameters - especially re: scale. </li></ul>
  3. 3. <ul><li>All approaches that are currently used have advantages and disadvantages - if these are appreciated the best use can be made of data </li></ul>
  4. 4. What do we want to know?? <ul><li>What microbes are present? Detection/identification </li></ul><ul><li>Where are they? Detection/localisation </li></ul><ul><li>How many of each population are present (No.’s of cells or mass of cells) Numbers/biomass </li></ul><ul><li>What are they doing and how is activity influenced by changes in the environment? Activity/metabolism </li></ul>
  5. 5. Methods - Summary <ul><li>Detection and Numbers: </li></ul><ul><ul><li>Culture-based methods for detection and enumeration </li></ul></ul><ul><ul><li>Non-culture and non-DNA based methods for detection, enumeration - immunology and lipids </li></ul></ul><ul><ul><li>DNA(molecular)-based methods for detection, localisation and enumeration </li></ul></ul><ul><li>2. Methods for determination of Biomass </li></ul><ul><li>3. Activity and metabolism determinations </li></ul>
  6. 6. Precautions <ul><li>Need to know limitations of each measurement procedure e.g. knowing that a “total viable count” typically enumerates around 1% of a microbial community </li></ul><ul><li>A combination of methods usually gives the best results </li></ul><ul><li>In some cases numbers, biomass and activity show proportional correlations - mostly they do not - how to interpret this?? </li></ul>
  7. 7. Sampling <ul><li>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. </li></ul><ul><li>Micro and mesocosms -model systems </li></ul><ul><li>Field studies - at present very difficult and expensive to undertake </li></ul>
  8. 8. 1. Detection and Enumeration of Microbes in the Environment <ul><li>Culture-based </li></ul><ul><li>Immunology-based </li></ul><ul><li>Membrane lipid </li></ul><ul><li>Genotypic Methods </li></ul>
  9. 9. Culture-based detection methods <ul><li>Organisms must be recovered from environmental samples - recognisable and specific phenotypes must be expressed during in vitro culture </li></ul><ul><li>Classical approach - selective plating and enrichment procedures - useful because they may provide physiological information useful in analysing microbes ecological function </li></ul>
  10. 10. Culture vs Counts <1 10 6 -10 9 cells/g Sediments ND 10 4 -10 5 cells/ml Marine (depth) 0.001-0.1 10 4 -10 6 cells/ml Marine (surface) <1 10 4 -10 5 cells/ml Ground water 0.01-0.1 10 6 -10 7 cells/ml Lakes/rivers 0.01-0.1 10 9 -10 10 cells/g Soil Typical cultivability (%) Typical microscopic counts Habitat
  11. 11. Why can’t we grow environmental bacteria? <ul><li>Little is known about the specific growth requirements of most microbes - e.g. O 2 levels, nutrients, co-factors, cross-feeding with other populations </li></ul><ul><li>Many microorganisms in the environment will have a very low metabolic activity or are quiescent </li></ul><ul><li>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 </li></ul>
  12. 12. BioLOG System <ul><li>Method of rapid testing of environmental samples - can simultaneously assay for a range of metabolic characteristics </li></ul><ul><li>Results are based on a colour-change and thus can be read automatically - rapid </li></ul>
  13. 13. <ul><li>Based on the pattern of substrate utilisations - a statistical analysis can be carried out - gives a “physiological profile” of the sample </li></ul><ul><li>Available for G(-) , G(+) specific or general use </li></ul><ul><li>Most frequently used culture-based method in ecological studies - labour, time and money </li></ul>
  14. 14. <ul><li>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 </li></ul><ul><li>Can detect growth automatically in broth - more sensitive than plates </li></ul><ul><li>Only viable bacteria will grow </li></ul><ul><li>Can work further with isolated bacteria </li></ul>
  15. 15. <ul><li>Problems - “unculturability”, totally artificial environment </li></ul><ul><li>Can’t examine interactions of mixed group of microbes </li></ul><ul><li>Lab. and in vivo phenotype may well be different </li></ul>
  16. 16. Immunological detection methods <ul><li>Based on the fact that bacterial cell wall polymers such as proteins and lipopolysaccharides have strong antigenic properties </li></ul><ul><li>Can be used to raise antibodies, usually in rabbits </li></ul>
  17. 17. <ul><li>After repeated exposure to antigen counts of antibody become very high </li></ul><ul><li>Can be harvested from serum for use to detect antigens in samples </li></ul>
  18. 18. <ul><li>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 </li></ul><ul><li>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. </li></ul>
  19. 19. <ul><li>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 </li></ul><ul><li>Problems: cross-reactivity, can’t raise antibodies if you don’t have a pure culture and so can’t 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 </li></ul>
  20. 21. Membrane lipid analysis <ul><li>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 </li></ul><ul><li>The fatty acids that are important for bacterial identification are the branched chain fatty acids containing from 9 to 20 carbons </li></ul><ul><li>Lipids are extracted from the sample and treated by attaching an ester group- so they can be dissolved </li></ul>
  21. 22. <ul><li>Methylated phospholipid ester-linked fatty acids - (PL)FAME or PLFA profiles </li></ul><ul><li>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) </li></ul><ul><li>Can read the outputs as peaks - profile of community structure - individual microbes will have individual profiles ( again can do stats) </li></ul>
  22. 23. <ul><li>Using this approach a signature profile can be obtained for samples </li></ul><ul><li>Community members are identified also some info on their physiological state e.g. - a ratio of > 1 of trans to cis- isomers of monosaturated PLFAs is indicative of starvation or other environmental stress </li></ul>
  23. 24. <ul><li>Advantages: Important chemotaxonomic approach, culture independent; Statistically valid; straightforward and rapid, many samples can be processed, and change can be observed over time </li></ul><ul><li>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 </li></ul>
  24. 25. Genotypic Detection Methods <ul><li>Based on the ability to detect specific signature gene sequences of organisms in the environment - detect sequence unique to a microbe => detect microbe </li></ul><ul><li>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 </li></ul><ul><li>Also used in phylogeny - determination of the evolutionary relationship between microbes </li></ul>
  25. 27. Principals of genotypic detection methods <ul><li>Methods are based on the fact that nucleic acids are made up of 4 bases arranged in a specific order </li></ul><ul><li>Base sequences are conserved from one generation to the next </li></ul><ul><li>DNA molecules are double-stranded </li></ul>
  26. 28. <ul><li>A nucleic acid sequence will only stick or hybridise to a complimentary sequence </li></ul><ul><li>DNA and RNA can be made single stranded or denatured by raising the temperature </li></ul><ul><li>Two detection approaches used: Nucleic acid Probes and DNA Amplification </li></ul>
  27. 29. <ul><li>Probing and Amplification are linked as you need to know the target sequence before you design a probe </li></ul><ul><li>Must recover sequence information, analyse it and use it to produce probes </li></ul><ul><li>Sequences got from the environment through: 1. Extraction of nucleic acids and 2. Amplification via PCR </li></ul>
  28. 30. Extraction of Nucleic Acids <ul><li>Two approaches to isolating DNA from the environmental samples: </li></ul><ul><li>1. Isolation of microbial cells followed by cell lysis and purification of nucleic acid (Cell extraction) </li></ul><ul><li>2. Direct lysis of microbial cells in the environmental matrix followed by nucleic acid purification (Direct extraction ) </li></ul>
  29. 31. <ul><li>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 </li></ul><ul><li>Cell extraction methods also developed for soils - normally combine vortexing, centrifugation steps </li></ul><ul><li>Direct DNA extraction increasingly favoured for environmental studies - more representative of populations present - crude extracts purified to remove interfering substances </li></ul>
  30. 32. PCR <ul><li>Mimics the natural DNA replication in microbes </li></ul><ul><li>Uses polymerase to synthesise a complimentary strand of DNA/RNA from a single strand </li></ul><ul><li>Small sequences (primers) added to create double- stranded template </li></ul><ul><li>A series of amplification cycles used to increase initial target </li></ul>
  31. 34. <ul><li>Target sequence amplified – can detect very low initial numbers </li></ul><ul><li>Amplified DNA can be used for probing or can be cloned and/or sequenced </li></ul><ul><li>Sequencing and comparison with known sequences provides information on diversity and types of microbe present and also can be used to design probes </li></ul>
  32. 35. <ul><li>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 </li></ul><ul><li>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 </li></ul>
  33. 36. Nucleic acid Probes <ul><li>Probes and nucleic hybridisation techniques used to detect target sequences diagnostic of specific groups of organisms in environmental samples </li></ul><ul><li>Probe is a relatively short nucleotide sequence that can hybridise with a homologous sequence in the target micro-organism </li></ul><ul><li>Can be designed to target either DNA (chromosome) or RNA (usually the rRNA) </li></ul>
  34. 37. How probes work <ul><li>Sequence of events is that nucleic acids are extracted from the sample, denatured and immobilised - e.g. on a nitrocellulose filter </li></ul><ul><li>Labeled probe is then added and allowed to hybridise </li></ul><ul><li>Unbound probe is then washed off and finally hybrids are detected </li></ul>
  35. 38. <ul><li>Normally carry out hybridisation on an immobilised target or probe on a solid phase e.g. - nitrocellulose or nylon filter surface </li></ul><ul><li>Normally probe is labelled ( 32 P) and after hybridisation and washing can detect target binding by autoradiography </li></ul><ul><li>Relative amounts of nucleic acid can be quantified by comparison with signal obtained with universal probe - variations include use of Dot blot manifold etc. </li></ul>
  36. 40. <ul><li>Advantages: Do not require culture, applicable to a wide range of samples, can be quantitative </li></ul><ul><li>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 </li></ul>
  37. 41. In-situ hybridisation <ul><li>Alternative approach is to carry out specific hybridisation between labelled probe and specific target sequence inside intact cell with minimum sample disturbance </li></ul><ul><li>Most direct method - morphology of the cell fixed, membrane made permeable to allow penetration of probe (usually with paraformaldehyde) </li></ul>
  38. 42. <ul><li>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 </li></ul><ul><li>Most commonly labelled with a flourescent dye like fluorescein (green) or rhodamine (red) </li></ul>
  39. 43. FISH <ul><li>Fluorescent signals detected by epifluorescence or confocal laser scanning microscope (much more detail) </li></ul><ul><li>Excellent technique for detection of unculturables e.g. symbionts of protozoa etc. </li></ul><ul><li>Very useful for identifying bacteria in complex environments - soil, biofilms, activated sludge etc. </li></ul>
  40. 46. <ul><li>Advantages: no culture, can detect both culturable and unculturable organisms, localise specific cells within a community, estimate numbers </li></ul><ul><li>Problems: difficulties in getting “clean” hybridisation with some samples, cells have to be fixed to get probe in, need sequence information on target microbes </li></ul>
  41. 50. Reporter Genes <ul><li>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 </li></ul><ul><li>Also green fluorescent protein and bioluminescence genes used for this purpose </li></ul>
  42. 52. 1 (b): Determination of numbers <ul><li>Direct counts - either stains or nucleic acid probes </li></ul><ul><li>Viable Counts </li></ul>
  43. 53. <ul><li>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 </li></ul><ul><li>Numbers of specific microbes can be estimated using fluorescent antibody or gene probes </li></ul><ul><li>Multiple populations in the same sample can be counted by using several probes with different colours </li></ul>
  44. 54. Stains used for direct counts <ul><li>Acridine Orange (water) - nucleic acid </li></ul><ul><li>DAPI (water/solids) - DNA stain </li></ul><ul><li>Fluorescein isothiocyanate (FITC) - protein stain </li></ul>
  45. 56. Dead or Alive? <ul><li>Very important to determine if cells that you are counting are viable - are they alive or dead - number of procedures attempt to do this </li></ul><ul><li>i.e. use of 2-[ p -indophenyl]-3[ p -nitrophenyl]-5-phenyl tetrazolium chloride (INT) which deposits red dye in cells that have active dehydrogenases </li></ul><ul><li>Similar respiration assay involves the use of 5-cyano-2,3-ditolyl tetrazolium chloride (CTC) </li></ul>
  46. 57. <ul><li>Also membrane potential-sensitive fluorochromes can distinguish between active, injured (dying) and dead cells </li></ul><ul><li>rRNA targeted probes - bind to ribosomes - these are present in live cells only </li></ul>
  47. 58. <ul><li>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 </li></ul><ul><li>Demonstrated organisms carry out active metabolism and retain virulence </li></ul><ul><li>Use of gene probes/PCR etc. can classify unculturables - can infer properties based on cultured homologues - need to be careful! </li></ul>
  48. 59. <ul><li>Otherwise, Plate count and MPN the two basic approaches used to cultivate viable organisms- both rely on separation of microorganisms into individual reproductive units </li></ul><ul><li>All viable count procedures are selective - the degree of selectivity varies with the particular viable count procedure - impossible to get a “Total Viable Count” </li></ul>