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The following slides are provided by Dr. Vincent O’Flaherty.

Experimental EcologyExperimental 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.

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

What do we want to know?? What do we want to know??

• What microbes are present? Detection/identification

• 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

Methods - SummaryMethods - Summary

1. 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

1. 2. Methods for determination of Biomass

2. 3. Activity and metabolism determinations

PrecautionsPrecautions

• 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??

SamplingSampling

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

1. Detection and Enumeration of Microbes in the Environment

1. Detection and Enumeration of Microbes in the Environment

• Culture-based

• Immunology-based

• Membrane lipid

• Genotypic Methods

Culture-based detection methods

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

Culture vs CountsCulture 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 <1

Marine (surface)

104-106 cells/ml 0.001-0.1

Marine (depth) 104-105 cells/ml ND

Sediments 106-109 cells/g <1

Why can’t we grow environmental bacteria?

Why can’t 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

BioLOG SystemBioLOG 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

• 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

• 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

• Problems - “unculturability”, totally artificial environment

• Can’t examine interactions of mixed group of microbes

• Lab. and in vivo phenotype may well be different

Immunological detection methods

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

• After repeated exposure to antigen counts of antibody become very high

• Can be harvested from serum for use to detect antigens in samples

• 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.

• 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, 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

Membrane lipid analysisMembrane 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

• 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)

• 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 > 1 of trans to cis- isomers of monosaturated PLFAs is indicative of starvation or other environmental stress

• 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

Genotypic Detection MethodsGenotypic Detection Methods

• Based on the ability to detect specific signature gene sequences of organisms in the environment - detect sequence unique to a microbe => 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

Principals of genotypic detection methods

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

• 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

• 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

Extraction of Nucleic AcidsExtraction 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)

• 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

PCRPCR

• 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

• 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

• 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

Nucleic acid ProbesNucleic 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)

How probes workHow 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

• 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.

• 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

In-situ hybridisationIn-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)

• 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)

FISHFISH

• 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.

• 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

Reporter GenesReporter 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

1 (b): Determination of numbers1 (b): Determination of numbers

• Direct counts - either stains or nucleic acid probes

• Viable Counts

• 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

Stains used for direct countsStains used for direct counts

• Acridine Orange (water) - nucleic acid

• DAPI (water/solids) - DNA stain

• Fluorescein isothiocyanate (FITC) - protein stain

Dead or Alive?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]-3[p-nitrophenyl]-5-phenyl 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)

• 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

• 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!

• 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”