LECTURE PRESENTATIONS
For BROCK BIOLOGY OF MICROORGANISMS, THIRTEENTH EDITION
Michael T. Madigan, John M. Martinko, David A. Stahl, David P. Clark
Chapter 22
Lectures by
John Zamora
Middle Tennessee State University
© 2012 Pearson Education, Inc.
Methods in
Microbial Ecology
I. Culture-Dependent Analyses of
Microbial Communities
• 22.1 Enrichment
• 22.2 Isolation
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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 medium
containing mannitol but
lacking NH4, NO3, or
organic nitrogen.
Soil
+NH4 plate
Incubate
aerobically
NH4 plate
NH4
+NH4 plate
NH4 plate
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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
Animation: Enrichment Cultures
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22.1 Enrichment
• 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
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Figure 22.2
Gradients Column
O2
Lake or
pond water
Mud
supplemented
with organic
nutrients
and CaSO4
Foil cap
Algae and
cyanobacteria
Purple nonsulfur
bacteria
Sulfur
chemolithotrophs
Patches of purple
sulfur or green
sulfur bacteria
H2S
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Anoxic
decomposition
and sulfate
reduction
22.1 Enrichment
• 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
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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
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Paraffin–mineral
oil seal
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)
Animation: Serial Dilutions and a Most Probable Number Analysis
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Figure 22.4
1 ml
(liquid)
or 1 g
(solid)
Enrichment culture
or natural sample
Dilution
1 ml
1 ml
1 ml
1 ml
No
growth
Growth
Growth
1 ml
9 ml of
broth
1/10
(101)
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102
103
104
105
106
22.2 Isolation
• Axenic culture can be verified by
– Microscopy
– Observation of colony characteristics
– Tests of the culture for growth in other media
• Laser tweezers are useful for
– Isolating slow-growing bacteria from mixed
cultures (Figure 22.5)
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Figure 22.5
b
a
Fb
Fa
Cell
Beam focus
a
b
Objective lens
of microscope
Laser beam
Optically trapped cell
Capillary
tube
Mixture of cells
Laser
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Severing point
22.2 Isolation
• Flow cytometry
– Uses lasers
– Suspended cultures passed through
specialized detector
– Cells separated based on fluorescence
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II. Culture-Independent Analyses of
Microbial Communities
• 22.3 General Staining Methods
• 22.4 Fluorescent In Situ Hybridization (FISH)
• 22.5 PCR Methods of Microbial Community
Analysis
• 22.6 Microarrays and Microbial Diversity:
Phylochips
• 22.7 Environmental Genomics and Related
Methods
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22.3 General Staining Methods
• Fluorescent staining using DAPI or acridine
orange (AO)
– DAPI-stained cells fluoresce bright blue
(Figure 22.6a)
– AO-stained cells fluoresce orange or greenishorange (Figure 22.6b)
– DAPI and AO fluoresce under UV light
– DAPI and AO are used for the enumeration of
microorganisms in samples
– DAPI and AO are nonspecific and stain nucleic
acids
– Cannot differentiate between live and dead cells
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Figure 22.6
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22.3 General Staining Methods
• Viability stains: differentiate between live and
dead cells (Figure 22.7)
–
–
–
–
–
Two dyes are used
Based on integrity of cell membrane
Green cells are live
Red cells are dead
Can have issues with nonspecific staining in
environmental samples
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Figure 22.7
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22.3 General Staining Methods
• Fluorescent antibodies can be used as a cell tag
– Highly specific
– Making antibodies is time consuming and
expensive
• Green fluorescent protein can be genetically
engineered into cells to make them
autofluorescent (Figure 22.8)
– Can be used to track bacteria
– Can act as a reporter gene
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Figure 22.8
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22.4 Fluorescent In Situ Hybridization
(FISH)
• Nucleic acid probe is DNA or RNA complementary
to a sequence in a target gene or RNA
• FISH: fluorescent in situ hybridization
(Figure 22.9)
– Phylogenetics of microbial populations
(Figure 22.10)
– Used in microbial ecology, food industry, and
clinical diagnostics
– CARD-FISH
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Figure 22.9
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Figure 22.10
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22.4 Fluorescent In Situ Hybridization
(FISH)
• CARD-FISH
– FISH can be used to measure gene
expression in organisms in a natural sample
(Figure 22.11)
– A FISH method that enhances the signal is
called catalyzed reporter deposition FISH
(CARD-FISH)
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Figure 22.11
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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
Microbial
community
Extract total
community DNA
Amplify by PCR
using fluorescently
tagged primers
DNA
PCR
Restriction
enzyme
digest and
run on gel
Sample
1 2 3 4
Sample
1 2 3 4
Gel
Amplify 16S RNA
genes using general
primers (for example,
Bacteria-specific) or
more restrictive
primers (to target
endospore-forming
Bacteria)
All 16S
rRNA
genes
T-RFLP
gel
Sample
1 2 3 4
Excise bands
and clone 16S
rRNA genes
Different
16S rRNA
genes
DGGE gel
Excise
bands
Sequence
Sequence
Bacillus subtilis
Generate
tree from
results
using
endosporespecific
primers
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Generate
tree from
Bacillus cereus
results
Bacillus megaterium
using
Env 2
endosporeClostridium histolyticum specific
primers
Env 1
Env 3
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
2
1
3
4
5
6
7
8
6
7
8
PCR amplification
1
2
DGGE
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3
4
5
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
1
1540
23S rRNA gene
ITS region
16S rRNA gene
Forward PCR primer
containing fluorescent
tag ( )
2900
Reverse PCR primer
50–1500bp
Position 1513
Position 23
PCR
Community DNA
Fluorescence
Gel analysis
1000
750
500
250
0
400
450
500
550
600
650
700
750
800
850
900
950
Fragment size (base pairs)
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1000
1050
1100
1150
1200
1250
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
Negative
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Weak positive
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
Microbial
community
Extract total
community DNA
DNA
Community
sampling approach
Environmental
genomics approach
Restriction digest total DNA and
then shotgun sequence, OR
sequence directly (without
cloning) using a high throughput
DNA sequencer
Amplify single gene,
for example, gene
encoding 16S rRNA
Sequence and
generate tree
Assembly and
annotation
Outcomes
Single-gene phylogenetic tree
1. Phylogenetic snapshot
of most members of
the community
2. Identification of novel
phylotypes
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Partial
genomes
Total gene pool of the community
1. Identification of all gene categories
2. Discovery of new genes
3. Linking of genes to phylotypes
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 22.18
Sulfate reduction
Lactate
incorporation
H2S
14CO
2
Lactate or H2S
Formalin-killed control
Photosynthesis
14C-Glucose
Dark
H2 absent
14CO
2
H2 present
respiration
evolution
Sulfate reduction
H2 35S
Killed
Time
Time
Killed
Light
Killed
Time
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Time
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 50–100mm
(Figure 22.20)
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Figure 22.19
Gold
Glass
Platinum
5 mm
Membranes
50–100 mm
NO3
2e 1 N2O  H2O
N2O
Bacteria
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Glass
Cathode
N2  2 OH
Nutrient solution
Figure 22.20
Oxygen (O2) concentration (mM)
100
200
0
300
Depth in sediment (mm)
Seawater
0
NO3
O2
Oxic sediment
5
Anoxic sediment
10
0
8
4
Nitrate (NO3) concentration (mM)
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12
22.9 Stable Isotopes
• Stable isotopes: nonradioactive isotopes of an
element
– Used to study microbial transformations in nature
– Isotope fractionation
• Carbon and sulfur are commonly used
• Lighter isotope is incorporated preferentially over
heavy isotope (Figure 22.22)
• Indicative of biotic processes
• Isotopic composition reveals its past biology
(e.g., carbon in plants and petroleum; Figure 22.23)
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Figure 22.22
Enzyme substrates
12CO
2
13CO
2
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Enzyme that
fixes CO2
Fixed carbon
12C
organic
13C
organic
Figure 22.23
Marine carbonate
Atmospheric CO2
Calvin cycle plants
Methane
Petroleum
Cyanobacteria
Purple sulfur bacteria
Green sulfur bacteria
Recent marine sediments
3.5 billion-year-old rocks
80 70 60 50 40 30 20 10
 13C (0/00)
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0
10
22.9 Stable Isotopes
– Isotope fractionation (cont’d)
• The activity of sulfate-reducing bacteria is
easy to recognize from their fractionation of
sulfur in sulfides (Figure 22.24)
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Figure 22.24
Igneous rocks
Marine
sulfate
Sedimentary sulfide
Meteoritic sulfide
Lunar sulfides
Sulfide from marine mud
Elemental
sulfur
30
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20
10
10
0
 34S (0/00)
20
30
22.10 Linking Specific Genes and
Functions to Specific Organisms
• Analysis of single cells by secondary ion mass
spectrophotometry (SIMS)
– High-energy ion beam impacts a sample
– Secondary ions released (sputtering)
– Mass spectrometry of secondary ions
(Figure 22.25)
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Figure 22.25
Ion
source
Primary
ions
Sample
10
Mass
spectrometer
Magnet
ABCDE Multiple
detectors
Secondary
ions
Outer shell of sulfate-reducing bacteria cells
(green) burned away by primary ion beam
20
13C (0/00)
30
40
Inner core of methanotrophic
Archaea cells (red) exposed
50
60
70
0
2
4
8
6
Cycles of sputtering and data collection
(increasing depth into aggregate)
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22.10 Linking Specific Genes and
Functions to Specific Organisms
• Flow cytometry and multiparametric analysis
– Natural communities contain large
populations
– Flow cytometer examines specific cell
parameters very fast (Figure 22.26)
• Cell size
• Cell shape
• Fluorescence
– Parameters can be combined and analyzed
(multiparametric analysis)
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Figure 22.26
Cells
labeled
by FISH
Sample
stream
Nozzle
Light scatter and
fluorescence detector
Laser
Deflection
plates
Sorted
samples
Waste
(non labeled cells)
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Induces charge on
selected droplets
22.10 Linking Specific Genes and
Functions to Specific Organisms
• Radioisotopes used as measures of microbial
activity in a microscopic technique called
microautoradiography (MAR)
• Radioisotopes can also be used with FISH
– FISH microautoradiography (FISH-MAR)
• Combines phylogeny with activity of cells
(Figure 22.27)
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Figure 22.27
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22.10 Linking Specific Genes and
Functions to Specific Organisms
• Stable isotope probing (SIP): links specific
metabolic activity to diversity using a stable
isotope (Figure 22.28)
– Microorganisms metabolizing stable isotope
(e.g., 13C) incorporate it into their DNA
• DNA with 13C can then be used to identify the
organisms that metabolized the 13C
– SIP of RNA also possible
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Figure 22.28
13C-DNA
This cell
metabolizes
13C substrate
12C-DNA
Environmental
sample
These cells do not
metabolize 13C
substrate
Separate
light (12C)
from heavy
(13C) DNA
12C-DNA
13C-DNA
Colin Murrell
Extract
DNA
Feed 13C
substrate
Ultracentrifuge
tube with DNA
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Remove and
analyze (PCR
16S rRNA or
metabolic genes,
or do genomics)
22.10 Linking Specific Genes and
Functions to Specific Organisms
• Single-cell genomics
• Multiple displacement amplification (MDA)
– Links metabolic function to individual cells
(Figure 22.29)
– Amplifies DNA from a single organism
– Uses cell sorting
– Uses bacteriophage DNA polymerase
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Figure 22.29
Label cells by FISH
Isolate fluorescent
cells by flow cytometry
Extract DNA
DNA
PCR
Assay for specific
genes (16S rRNA
genes, metabolic
genes, etc.)
Multiple
displacement
Phage
amplification (MDA)
DNA
and sequencing
Primers polymerase
A
B
C
Sequence genome
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