22.1 enrichment isolation –the separation of individual organisms from the mixed community...
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22.1 Enrichment
• Isolation – The separation of individual organisms from
the mixed community
• Enrichment Cultures– Select for desired organisms through
manipulation of medium and incubation conditions
• Inocula– The sample from which microorganisms will be
isolated
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Figure 22.1
Mineral salts mediumcontaining mannitol butlacking NH4
, NO3, or
organic nitrogen.
Soil
NH4
+NH4 plate
NH4 plate
NH4 plate
+NH4 plate
Incubateaerobically
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22.1 Enrichment
• Enrichment Cultures– Can prove the presence of an organism in a
habitat
– Cannot prove an organism does not inhabit an environment
• The ability to isolate an organism from an environment says nothing about its ecological significance
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Animation: Enrichment CulturesAnimation: Enrichment Cultures
• The Winogradsky Column – An artificial microbial ecosystem (Figure 22.2)
– Serves as a long-term source of bacteria for enrichment cultures
– Named for Sergei Winogradsky
– First used in late 19th century to study soil microorganisms
22.1 Enrichment
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Figure 22.2
Gradients Column
Lake orpond water
Mudsupplementedwith organicnutrientsand CaSO4
O2
H2S
Foil cap
Algae andcyanobacteria
Purple nonsulfurbacteriaSulfurchemolithotrophs
Patches of purplesulfur or greensulfur bacteria
Anoxicdecompositionand sulfatereduction
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• Enrichment bias– Microorganisms cultured in the lab are
frequently only minor components of the microbial ecosystem
• Reason: the nutrients available in the lab culture are typically much higher than in nature
• Dilution of inoculum is performed to eliminate rapidly growing, but quantitatively insignificant, weed species
22.1 Enrichment
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22.2 Isolation
• Pure cultures contain a single kind of microorganism– Can be obtained by streak plate, agar shake, or
liquid dilution (Figure 22.3)
• Agar dilution tubes are mixed cultures diluted in molten agar– Useful for purifying anaerobic organisms
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Figure 22.3
Colonies Paraffin–mineraloil seal
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22.2 Isolation
• Most-probable-number technique – Serial 10 dilutions of inocula in a liquid media
– Used to estimate number of microorganisms in food, wastewater, and other samples (Figure 22.4)
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Animation: Serial Dilutions and a Most Probable Number AnalysisAnimation: Serial Dilutions and a Most Probable Number Analysis
Figure 22.4
1 ml(liquid)or 1 g(solid)
Enrichment cultureor natural sample
Dilution
1 ml 1 ml 1 ml 1 ml 1 ml
9 ml ofbroth
Growth GrowthNo
growth
1/10(101) 102 103 104 105 106
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22.2 Isolation
• Flow cytometry – Uses lasers
– Suspended cultures passed through specialized detector
– Cells separated based on fluorescence
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22.5 PCR Methods of Microbial Community Analysis
• Specific genes can be used as a measure of diversity
– Techniques used in molecular biodiversity studies (Figure 22.12)
• DNA isolation and sequencing• PCR• Restriction enzyme digest • Electrophoresis• Molecular cloning
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Figure 22.12
Microbialcommunity
PCR
Extract totalcommunity DNA
Amplify by PCRusing fluorescentlytagged primers
Restrictionenzymedigest andrun on gel
Amplify 16S RNAgenes using generalprimers (for example,Bacteria-specific) ormore restrictiveprimers (to targetendospore-formingBacteria)
Excise bandsand clone 16SrRNA genes Excise
bands
Generatetree fromresultsusingendospore-specificprimers
Generatetree fromresultsusingendospore-specificprimers
Sample1 2 3 4
Gel
All 16SrRNAgenes
Sample1 2 3 4
T-RFLPgel
Different16S rRNAgenes
DGGE gel
Sample1 2 3 4
Sequence Sequence
Env 1
Env 2
Env 3
Bacillus subtilis
Bacillus megaterium
Clostridium histolyticum
Bacillus cereus
DNA
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22.5 PCR Methods of Microbial Community Analysis
• DGGE: denaturing gradient gel electrophoresis separates genes of the same size based on differences in base sequence (Figure 22.13)– Denaturant is a mixture of urea and
formamide
– Strands melt at different denaturant concentrations
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Figure 22.13
PCR amplification
DGGE
1 2 3 4 5 6 7 8
1 2 3 4 5 6 7 8
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22.5 PCR Methods of Microbial Community Analysis
• T-RFLP: terminal restriction fragment length polymorphism– Target gene is amplified by PCR
– Restriction enzymes are used to cut the PCR products
• ARISA: automated ribosomal intergenic spacer analysis (Figure 22.14)– Related to T-RFLP
– Uses DNA sequencing
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Figure 22.14
Position 1
16S rRNA gene
1540 1 2900
23S rRNA gene
50–1500bp
Position 1513 Position 23
Reverse PCR primer
ITS region
Forward PCR primercontaining fluorescenttag ( )
PCR
Gel analysis
Community DNA
1000
750
500
250
0400 450 500 550 600 650 700 750 800 850 900 950 1000 1050 1100 1150 1200 1250
Flu
ore
scen
ce
Fragment size (base pairs)
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22.5 PCR Methods of Microbial Community Analysis
• Results of PCR phylogenetic analyses– Several phylogenetically distinct prokaryotes
are present• rRNA sequences differ from those of all
known laboratory cultures
– Molecular methods conclude that less than 0.1% of bacteria have been cultured
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22.6 Microarrays and Microbial Diversity: Phylochips
• Phylochip: microarray that focuses on phylogenetic members of microbial community (Figure 22.15)– Circumvents time-consuming steps of DGGE and
T-RFLP
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Figure 22.15
Positive Weak positive
Negative
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22.7 Environmental Genomics and Related Methods
• Environmental genomics (metagenomics) – DNA is cloned from microbial community and
sequenced
– Detects as many genes as possible
– Yields picture of gene pool in environment
– Can detect genes that are not amplified by current PCR primers
– Powerful tool for assessing the phylogenetic and metabolic diversity of an environment (Figure 22.16)
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Figure 22.16
Communitysampling approach
Environmentalgenomics approach
Outcomes
Single-gene phylogenetic tree Total gene pool of the community
1. Identification of all gene categories2. Discovery of new genes3. Linking of genes to phylotypes
Phylogenetic snapshotof most members of the community
1.
Identification of novelphylotypes
2.
Amplify single gene,for example, geneencoding 16S rRNA
Restriction digest total DNA andthen shotgun sequence, ORsequence directly (withoutcloning) using a high throughputDNA sequencer
Extract totalcommunity DNA
Microbialcommunity
Sequence andgenerate tree Assembly and
annotation
DNA
Partialgenomes
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III. Measuring Microbial Activities in Nature
• 22.8 Chemical Assays, Radioisotopic Methods, and Microelectrodes
• 22.9 Stable Isotopes• 22.10 Linking Specific Genes and Functions to
Specific Organisms
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22.8 Chemical Assays, Radioisotopes, & Microelectrodes
• In many studies, direct chemical measurements are sufficient (Figure 22.18)– Higher sensitivity can be achieved with
radioisotopes• Proper killed cell controls must be used
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Figure 22.18Sulfate reduction Photosynthesis
Sulfate reduction 14C-Glucose respiration
Formalin-killed controlLight
Lactate
H2S
La
cta
te o
r H
2S
14C
O2
inc
orp
ora
tio
n14
CO
2 e
vo
luti
on
H2
35S
H2 present
H2 absentKilled
KilledDark
Killed
Time Time
Time Time
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22.8 Chemical Assays, Radioisotopes, & Microelectrodes
• Microelectrodes– Can measure a wide range of activity
– pH, oxygen, CO2, and others can be measured
– Small glass electrodes, quite fragile (Figure 22.19)
– Electrodes are carefully inserted into the habitat (e.g., microbial mats)
• Measurements taken every 50–100m (Figure 22.20)
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Figure 22.19 Gold Glass Platinum
5 m
Membranes Glass
Bacteria Cathode Nutrient solution
50–100 m NO3 N2O 2e 1 N2O H2O N2 2 OH
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Figure 22.20Oxygen (O2) concentration (M)
Seawater
Oxic sediment
Anoxic sediment
Nitrate (NO3) concentration (M)
Dep
th i
n s
edim
ent
(mm
)
0 100 200 300
O2 NO3
0
5
10
0 4 8 12
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