Methods in microbial ecology L18 Flashcards
how are isolation chips used
collect environmental sample
put iChip plate in diluted environmental sample - capture one bacterium per well
put iChip plate in a breathable membrane (bacterial communication)
iChip back in environment
what can be used for fluorescent staining
DAPI acridine orange (AO)
what are DAPI and AO like
nonspecific
what do DAPI and AO do
stain nucleic acids
used for enumeration of microorganism samples
what is a downside of fluorescent staining
Cannot differentiate between live and dead cells
Cannot give any information on species or function
what does DAPI preferentially stain
dsDNA
what colour does AO emit when bound to dsDNA
green fluorescence
what colour does AO emit when bound to ssDNA or RNA
red fluoresence
what do viability stains provide information on
differentiate between live and dead cells
what are the dyes used in viability stains
green and red
what does green dye in viability stain indicate
penetrates all cells
green cells live
what does red dye in viability stain indicate
penetrates only dead cells
red is dead
what cant viability stains give information on
species or function
what is a fluorescent gram stain
Gram negative and Gram-positive bacterial are simultaneously stained with the membrane-permeant SYTO 9 dye and hexidium iodide (which cant penetrate Gram negatives)
what colour is gram -ve in fluorescent gram stain
fluoresce green
what colour is gram +ve in fluorescent gram stain
fluoresce red
what can STYO dyes be used for
stain RNA and DNA in live and dead eukaryotic cells
as well as in Gram-positive and Gram-negative bacteria
what is an advantage of fluorescent gram stain
don’t need to heat fix all –could potentially get bacteria back that has been prepared for staining
how do bacterial gram stains and viability stains stain and differentiate
Differentially stains many Gram-positive and Gram-negative bacterial species and, at the same time, discriminates live from dead cells on the basis of plasma membrane integrity
what colour stain is produced from bacterial gram stain and viability if bacteria with intact cells
blue with DAPI
what colour stain is produced from bacterial gram stain and viability if bacteria with damaged cells
green with SYTOX green
background remains virtually non-fluorescent
what does the texas red-X dye labelled WGA component bind to
component selectively binds to the surface of MANY Gram-positive bacteria
what are specific staining emthods
Fluorescent antibodies can be used to specifically tag cells
Green fluorescent protein (GFP) can be genetically engineered into cells to make them autofluorescent
what is disadvantage of Fluorescent antibodies
time consuming and expensive
what is Green fluorescent protein (GFP) used for
- track bacteria
- act as a reporter gene
- used on live samples
what is an advantage of green fluorescent protein
special information
what is a downside of green fluorescent protein
have to be able to culture the bacteria to make the probes
what is FISH
fluorescent in situ hybridization)
what is the FISH probe like
Nucleic acid probe is DNA or RNA complimentary to a sequence in a target gene or RNA
what is FISH like when target isa high copy number
Robust when target is high copy number, e.g. ribosomes, high copy number plasmid genes
how does FISH typically work
Isolate total community DNA
PCR rRNA genes and analyse amplicons
Design ‘specific’ probes based on rRNA gene sequences
what causes each probe to have a different colour fluoresce
Phylogenetics and structure of microbial populations
what does FISH do to cells
kills bacterial cells
what does FISH analysis show
where specific microbes are in relation to each other
but no direct information on function
what is the red dye represent in FISH
ammonia-oxidising bacteria
what is the green dye represent in FISH
nitrite-oxidising bacteria
what methods are used to look at community diversity
PCR
DNA sequencing
what can PCR be used for
detect copies of specific genes or to amplify groups of related genes which can then be identified on the basis of their sequence differences
how can PCR detect copies of specific genes / amplify groups
denaturing profiles (e.g. DGGE) which separates genes based on gross DNA composition (e.g. A:C:G:T) or on specific sequence data
what is traditional microbial genomics
Sequence the genome of one (cultured) organism at a time – annotate functional genes
what occurs in metagenomics
extract sequence data from microbial communities as they exist in nature
bypass the need for culture techniques
sequence all DNA in sample
what does metagenomics not provide
Does not give any spatial information
what can be detected in metagenomics
- all genes in a sample can be detected
- picture of gene pool in environment
- detect genes that would not be amplified by current PCR primers
- phylogenetic and potential metabolic diversity of organisms in an environment
how can the substrate range of single cultured species or communities be assessed
by phenotypic profiling e.g. biolog plates
how can individual metabolites produced by organisms be measured
(e.g. CO2, lactate) using chemical or physical assays
what are biolog plates for
originally identification
now as a research tool
what are radioisotopes provide
higher sensitivity chemical analysis can be achieved
what control must be used in radioisotopes
Proper killed cell controls must be used
what can be used with radioisotopes to indicate which organisms utilising radioactive substrate
Radioisotopes can also be used in combination with FISH
FISH micro-autoradiography (FISH-MAR)
what can microelectrodes measure
wide range of activity
pH, oxygen, CO2, and others can be measured
what are the microelectrodes like
Small glass electrodes, quite fragile
where are microelectrodes placed
Electrodes are carefully inserted into the habitat (e.g., microbial mats)
what are stable isotopes
non-radioactive isotopes of an element
what are stable isotopes used for
study microbial transformations in nature
what happens in isotope fractionation
Carbon and sulfur are commonly used
Lighter isotope is incorporated preferentially over heavy isotope
Indicative of biotic processes
Isotopic composition of a material reveals its past biology (e.g., carbon in plants and petroleum)
how can we see if nitrogen is fixed
N14 in air
used N15 see if taken up
what is SIP
stable isotopes probing
what does SIP do
links specific metabolic activity to diversity using a stable isotope
how do we know is microorganisms are metabolizing stable isotopes
ncorporate it into their cellular constituents e.g. DNA
DNA with 13C can then be used to identify the organisms that metabolized the 13C-labelled substrates
what happens in stable isotope probing
environmental sample fed with C13
cells may not metabolise C13, some will extract DNA
those that dont metabolise - 12C-DNA
those that do metabolise 13C-DNA
separate light (12C) from heavy (13C) DNA
ultracentrifuge DNA
remove and analyse
what is metabolomics
large-scale study of small molecules, commonly known as metabolites, within organisms
what is a metabolome
small molecules and their interactions within a biological system
what does mass spectrometry allow us to analyse
analyse more than one compound at a time by measuring their molecular weight
what are the metabolite concentrations like in metabolomics
Reactions take place continuously, so concentrations of metabolites are considered to be very dynamic
how are the characteristics of individual molecules measured
mass spectrometer converts them to ions so that they can be moved about and manipulated by external electric and magnetic fields
what are the three essential functions for mass spectrometer
small sample is ionized, usually to cations by loss of an electron by using the Ion Source
ions sorted and separated according to their mass and charge using the Mass Analyzer
separated ions measured by Detector
results on a chart
what is a NanoSIMS function
high-resolution imaging and mass spectrometry
determining the isotopic and elemental composition in microscopic target samples
what does NanoSIMS and in situ hybridisation techniques combined give
offers a powerful method of linking metabolic capacity to phylogenetic identity in cell samples
what is the NanoSIMS and FISH function and phylogeny
Tracking uptake of substrate in microbial cells by combining FISH and NanoSIMS analyses
Cells incubated with 13 C-labelled substrate before recovery and preservation
Halogen labelled (127 I) phylogenetic probes hybridized to the cells of interest
Simultaneous imaging of 13 C and 127 I using NanoSIMS allows mapping of cell metabolic function to identity
what cant GFP be used for
in ‘real world’ communities
