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 © 2012 Pearson Education, Inc.

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Page 1: 22.1 Enrichment Isolation –The separation of individual organisms from the mixed community Enrichment Cultures –Select for desired organisms through manipulation

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

© 2012 Pearson Education, Inc.

Page 2: 22.1 Enrichment Isolation –The separation of individual organisms from the mixed community Enrichment Cultures –Select for desired organisms through manipulation

Figure 22.1

Mineral salts mediumcontaining mannitol butlacking NH4

, NO3, or

organic nitrogen.

Soil

NH4

+NH4 plate

NH4 plate

NH4 plate

+NH4 plate

Incubateaerobically

© 2012 Pearson Education, Inc.

Page 3: 22.1 Enrichment Isolation –The separation of individual organisms from the mixed community Enrichment Cultures –Select for desired organisms through manipulation

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

© 2012 Pearson Education, Inc.

Animation: Enrichment CulturesAnimation: Enrichment Cultures

Page 4: 22.1 Enrichment Isolation –The separation of individual organisms from the mixed community Enrichment Cultures –Select for desired organisms through manipulation

• 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

© 2012 Pearson Education, Inc.

Page 5: 22.1 Enrichment Isolation –The separation of individual organisms from the mixed community Enrichment Cultures –Select for desired organisms through manipulation

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

© 2012 Pearson Education, Inc.

Page 6: 22.1 Enrichment Isolation –The separation of individual organisms from the mixed community Enrichment Cultures –Select for desired organisms through manipulation

• 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

© 2012 Pearson Education, Inc.

Page 7: 22.1 Enrichment Isolation –The separation of individual organisms from the mixed community Enrichment Cultures –Select for desired organisms through manipulation

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

© 2012 Pearson Education, Inc.

Page 8: 22.1 Enrichment Isolation –The separation of individual organisms from the mixed community Enrichment Cultures –Select for desired organisms through manipulation

Figure 22.3

Colonies Paraffin–mineraloil seal

© 2012 Pearson Education, Inc.

Page 9: 22.1 Enrichment Isolation –The separation of individual organisms from the mixed community Enrichment Cultures –Select for desired organisms through manipulation

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)

© 2012 Pearson Education, Inc.

Animation: Serial Dilutions and a Most Probable Number AnalysisAnimation: Serial Dilutions and a Most Probable Number Analysis

Page 10: 22.1 Enrichment Isolation –The separation of individual organisms from the mixed community Enrichment Cultures –Select for desired organisms through manipulation

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

© 2012 Pearson Education, Inc.

Page 11: 22.1 Enrichment Isolation –The separation of individual organisms from the mixed community Enrichment Cultures –Select for desired organisms through manipulation

22.2 Isolation

• Flow cytometry – Uses lasers

– Suspended cultures passed through specialized detector

– Cells separated based on fluorescence

© 2012 Pearson Education, Inc.

Page 12: 22.1 Enrichment Isolation –The separation of individual organisms from the mixed community Enrichment Cultures –Select for desired organisms through manipulation

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

© 2012 Pearson Education, Inc.

Page 13: 22.1 Enrichment Isolation –The separation of individual organisms from the mixed community Enrichment Cultures –Select for desired organisms through manipulation

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

© 2012 Pearson Education, Inc.

Page 14: 22.1 Enrichment Isolation –The separation of individual organisms from the mixed community Enrichment Cultures –Select for desired organisms through manipulation

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

© 2012 Pearson Education, Inc.

Page 15: 22.1 Enrichment Isolation –The separation of individual organisms from the mixed community Enrichment Cultures –Select for desired organisms through manipulation

Figure 22.13

PCR amplification

DGGE

1 2 3 4 5 6 7 8

1 2 3 4 5 6 7 8

© 2012 Pearson Education, Inc.

Page 16: 22.1 Enrichment Isolation –The separation of individual organisms from the mixed community Enrichment Cultures –Select for desired organisms through manipulation

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

© 2012 Pearson Education, Inc.

Page 17: 22.1 Enrichment Isolation –The separation of individual organisms from the mixed community Enrichment Cultures –Select for desired organisms through manipulation

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)

© 2012 Pearson Education, Inc.

Page 18: 22.1 Enrichment Isolation –The separation of individual organisms from the mixed community Enrichment Cultures –Select for desired organisms through manipulation

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

© 2012 Pearson Education, Inc.

Page 19: 22.1 Enrichment Isolation –The separation of individual organisms from the mixed community Enrichment Cultures –Select for desired organisms through manipulation

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

© 2012 Pearson Education, Inc.

Page 20: 22.1 Enrichment Isolation –The separation of individual organisms from the mixed community Enrichment Cultures –Select for desired organisms through manipulation

Figure 22.15

Positive Weak positive

Negative

© 2012 Pearson Education, Inc.

Page 21: 22.1 Enrichment Isolation –The separation of individual organisms from the mixed community Enrichment Cultures –Select for desired organisms through manipulation

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)

© 2012 Pearson Education, Inc.

Page 22: 22.1 Enrichment Isolation –The separation of individual organisms from the mixed community Enrichment Cultures –Select for desired organisms through manipulation

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

© 2012 Pearson Education, Inc.

Page 23: 22.1 Enrichment Isolation –The separation of individual organisms from the mixed community Enrichment Cultures –Select for desired organisms through manipulation

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

© 2012 Pearson Education, Inc.

Page 24: 22.1 Enrichment Isolation –The separation of individual organisms from the mixed community Enrichment Cultures –Select for desired organisms through manipulation

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

© 2012 Pearson Education, Inc.

Page 25: 22.1 Enrichment Isolation –The separation of individual organisms from the mixed community Enrichment Cultures –Select for desired organisms through manipulation

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

© 2012 Pearson Education, Inc.

Page 26: 22.1 Enrichment Isolation –The separation of individual organisms from the mixed community Enrichment Cultures –Select for desired organisms through manipulation

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)

© 2012 Pearson Education, Inc.

Page 27: 22.1 Enrichment Isolation –The separation of individual organisms from the mixed community Enrichment Cultures –Select for desired organisms through manipulation

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

© 2012 Pearson Education, Inc.

Page 28: 22.1 Enrichment Isolation –The separation of individual organisms from the mixed community Enrichment Cultures –Select for desired organisms through manipulation

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

© 2012 Pearson Education, Inc.