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Physiology of Microbes > Methods in microbial ecology L18 > Flashcards

Methods in microbial ecology L18 Flashcards

1
Q

how are isolation chips used

A

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

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2
Q

what can be used for fluorescent staining

A 
DAPI 
acridine orange (AO)
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3
Q

what are DAPI and AO like

A

nonspecific

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4
Q

what do DAPI and AO do

A

stain nucleic acids

used for enumeration of microorganism samples

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5
Q

what is a downside of fluorescent staining

A

Cannot differentiate between live and dead cells

Cannot give any information on species or function

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6
Q

what does DAPI preferentially stain

A

dsDNA

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7
Q

what colour does AO emit when bound to dsDNA

A

green fluorescence

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8
Q

what colour does AO emit when bound to ssDNA or RNA

A

red fluoresence

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9
Q

what do viability stains provide information on

A

differentiate between live and dead cells

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10
Q

what are the dyes used in viability stains

A

green and red

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11
Q

what does green dye in viability stain indicate

A

penetrates all cells

green cells live

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12
Q

what does red dye in viability stain indicate

A

penetrates only dead cells

red is dead

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13
Q

what cant viability stains give information on

A

species or function

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14
Q

what is a fluorescent gram stain

A

Gram negative and Gram-positive bacterial are simultaneously stained with the membrane-permeant SYTO 9 dye and hexidium iodide (which cant penetrate Gram negatives)

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15
Q

what colour is gram -ve in fluorescent gram stain

A

fluoresce green

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16
Q

what colour is gram +ve in fluorescent gram stain

A

fluoresce red

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17
Q

what can STYO dyes be used for

A

stain RNA and DNA in live and dead eukaryotic cells

as well as in Gram-positive and Gram-negative bacteria

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18
Q

what is an advantage of fluorescent gram stain

A

don’t need to heat fix all –could potentially get bacteria back that has been prepared for staining

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19
Q

how do bacterial gram stains and viability stains stain and differentiate

A

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

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20
Q

what colour stain is produced from bacterial gram stain and viability if bacteria with intact cells

A

blue with DAPI

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21
Q

what colour stain is produced from bacterial gram stain and viability if bacteria with damaged cells

A

green with SYTOX green

background remains virtually non-fluorescent

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22
Q

what does the texas red-X dye labelled WGA component bind to

A

component selectively binds to the surface of MANY Gram-positive bacteria

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23
Q

what are specific staining emthods

A

Fluorescent antibodies can be used to specifically tag cells

Green fluorescent protein (GFP) can be genetically engineered into cells to make them autofluorescent

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24
Q

what is disadvantage of Fluorescent antibodies

A

time consuming and expensive

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25
what is Green fluorescent protein (GFP) used for
- track bacteria - act as a reporter gene - used on live samples
26
what is an advantage of green fluorescent protein
special information
27
what is a downside of green fluorescent protein
have to be able to culture the bacteria to make the probes
28
what is FISH
fluorescent in situ hybridization)
29
what is the FISH probe like
Nucleic acid probe is DNA or RNA complimentary to a sequence in a target gene or RNA
30
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
31
how does FISH typically work
Isolate total community DNA PCR rRNA genes and analyse amplicons Design ‘specific’ probes based on rRNA gene sequences
32
what causes each probe to have a different colour fluoresce
Phylogenetics and structure of microbial populations
33
what does FISH do to cells
kills bacterial cells
34
what does FISH analysis show
where specific microbes are in relation to each other | but no direct information on function
35
what is the red dye represent in FISH
ammonia-oxidising bacteria
36
what is the green dye represent in FISH
nitrite-oxidising bacteria
37
what methods are used to look at community diversity
PCR | DNA sequencing
38
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
39
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
40
what is traditional microbial genomics
Sequence the genome of one (cultured) organism at a time – annotate functional genes
41
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
42
what does metagenomics not provide
Does not give any spatial information
43
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
44
how can the substrate range of single cultured species or communities be assessed
by phenotypic profiling e.g. biolog plates
45
how can individual metabolites produced by organisms be measured
(e.g. CO2, lactate) using chemical or physical assays
46
what are biolog plates for
originally identification | now as a research tool
47
what are radioisotopes provide
higher sensitivity chemical analysis can be achieved
48
what control must be used in radioisotopes
Proper killed cell controls must be used
49
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)
50
what can microelectrodes measure
wide range of activity | pH, oxygen, CO2, and others can be measured
51
what are the microelectrodes like
Small glass electrodes, quite fragile
52
where are microelectrodes placed
Electrodes are carefully inserted into the habitat (e.g., microbial mats)
53
what are stable isotopes
non-radioactive isotopes of an element
54
what are stable isotopes used for
study microbial transformations in nature
55
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)
56
how can we see if nitrogen is fixed
N14 in air | used N15 see if taken up
57
what is SIP
stable isotopes probing
58
what does SIP do
links specific metabolic activity to diversity using a stable isotope
59
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
60
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
61
what is metabolomics
large-scale study of small molecules, commonly known as metabolites, within organisms
62
what is a metabolome
small molecules and their interactions within a biological system
63
what does mass spectrometry allow us to analyse
analyse more than one compound at a time by measuring their molecular weight
64
what are the metabolite concentrations like in metabolomics
Reactions take place continuously, so concentrations of metabolites are considered to be very dynamic
65
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
66
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
67
what is a NanoSIMS function
high-resolution imaging and mass spectrometry | determining the isotopic and elemental composition in microscopic target samples
68
what does NanoSIMS and in situ hybridisation techniques combined give
offers a powerful method of linking metabolic capacity to phylogenetic identity in cell samples
69
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
70
what cant GFP be used for
in ‘real world’ communities

Physiology of Microbes (21 decks)

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