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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
Q

what is Green fluorescent protein (GFP) used for

A
  • track bacteria
  • act as a reporter gene
  • used on live samples
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26
Q

what is an advantage of green fluorescent protein

A

special information

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27
Q

what is a downside of green fluorescent protein

A

have to be able to culture the bacteria to make the probes

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28
Q

what is FISH

A

fluorescent in situ hybridization)

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29
Q

what is the FISH probe like

A

Nucleic acid probe is DNA or RNA complimentary to a sequence in a target gene or RNA

30
Q

what is FISH like when target isa high copy number

A

Robust when target is high copy number, e.g. ribosomes, high copy number plasmid genes

31
Q

how does FISH typically work

A

Isolate total community DNA
PCR rRNA genes and analyse amplicons
Design ‘specific’ probes based on rRNA gene sequences

32
Q

what causes each probe to have a different colour fluoresce

A

Phylogenetics and structure of microbial populations

33
Q

what does FISH do to cells

A

kills bacterial cells

34
Q

what does FISH analysis show

A

where specific microbes are in relation to each other

but no direct information on function

35
Q

what is the red dye represent in FISH

A

ammonia-oxidising bacteria

36
Q

what is the green dye represent in FISH

A

nitrite-oxidising bacteria

37
Q

what methods are used to look at community diversity

A

PCR

DNA sequencing

38
Q

what can PCR be used for

A

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
Q

how can PCR detect copies of specific genes / amplify groups

A

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
Q

what is traditional microbial genomics

A

Sequence the genome of one (cultured) organism at a time – annotate functional genes

41
Q

what occurs in metagenomics

A

extract sequence data from microbial communities as they exist in nature
bypass the need for culture techniques
sequence all DNA in sample

42
Q

what does metagenomics not provide

A

Does not give any spatial information

43
Q

what can be detected in metagenomics

A
  • 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
Q

how can the substrate range of single cultured species or communities be assessed

A

by phenotypic profiling e.g. biolog plates

45
Q

how can individual metabolites produced by organisms be measured

A

(e.g. CO2, lactate) using chemical or physical assays

46
Q

what are biolog plates for

A

originally identification

now as a research tool

47
Q

what are radioisotopes provide

A

higher sensitivity chemical analysis can be achieved

48
Q

what control must be used in radioisotopes

A

Proper killed cell controls must be used

49
Q

what can be used with radioisotopes to indicate which organisms utilising radioactive substrate

A

Radioisotopes can also be used in combination with FISH

FISH micro-autoradiography (FISH-MAR)

50
Q

what can microelectrodes measure

A

wide range of activity

pH, oxygen, CO2, and others can be measured

51
Q

what are the microelectrodes like

A

Small glass electrodes, quite fragile

52
Q

where are microelectrodes placed

A

Electrodes are carefully inserted into the habitat (e.g., microbial mats)

53
Q

what are stable isotopes

A

non-radioactive isotopes of an element

54
Q

what are stable isotopes used for

A

study microbial transformations in nature

55
Q

what happens in isotope fractionation

A

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
Q

how can we see if nitrogen is fixed

A

N14 in air

used N15 see if taken up

57
Q

what is SIP

A

stable isotopes probing

58
Q

what does SIP do

A

links specific metabolic activity to diversity using a stable isotope

59
Q

how do we know is microorganisms are metabolizing stable isotopes

A

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
Q

what happens in stable isotope probing

A

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
Q

what is metabolomics

A

large-scale study of small molecules, commonly known as metabolites, within organisms

62
Q

what is a metabolome

A

small molecules and their interactions within a biological system

63
Q

what does mass spectrometry allow us to analyse

A

analyse more than one compound at a time by measuring their molecular weight

64
Q

what are the metabolite concentrations like in metabolomics

A

Reactions take place continuously, so concentrations of metabolites are considered to be very dynamic

65
Q

how are the characteristics of individual molecules measured

A

mass spectrometer converts them to ions so that they can be moved about and manipulated by external electric and magnetic fields

66
Q

what are the three essential functions for mass spectrometer

A

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
Q

what is a NanoSIMS function

A

high-resolution imaging and mass spectrometry

determining the isotopic and elemental composition in microscopic target samples

68
Q

what does NanoSIMS and in situ hybridisation techniques combined give

A

offers a powerful method of linking metabolic capacity to phylogenetic identity in cell samples

69
Q

what is the NanoSIMS and FISH function and phylogeny

A

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
Q

what cant GFP be used for

A

in ‘real world’ communities

Physiology of Microbes (21 decks)

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