methods in microbial ecology chapter 22
TRANSCRIPT
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LECTURE PRESENTATIONS
For BROCK BIOLOGY OF MICROORGANISMS, THIRTEENTH EDITION
Michael T. Madigan, John M. Martinko, David A. Stahl, David P. Clark
Lectures by
John Zamora
Middle Tennessee State University
© 2012 Pearson Education, Inc.
Methods in
Microbial Ecology
Chapter 22
I. Culture-Dependent Analyses of
Microbial Communities
• 22.1 Enrichment
• 22.2 Isolation
© 2012 Pearson Education, Inc. Marmara University – Enve303 Env. Eng. Microbiology – Prof. BARIŞ ÇALLI
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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 (singular Inoculum)
– The sample from which microorganisms will be
isolated
© 2012 Pearson Education, Inc. Marmara University – Enve303 Env. Eng. Microbiology – Prof. BARIŞ ÇALLI
Figure 22.1 The isolation of Azotobacter.
© 2012 Pearson Education, Inc. Marmara University – Enve303 Env. Eng. Microbiology – Prof. BARIŞ ÇALLI
Mineral salts mediumcontaining mannitol butlacking NH4
, NO3, or
organic nitrogen.
Soil
NH4
+NH4 plate
NH4 plate
NH4 plate
+NH4 plate
Incubateaerobically
Selection for
aerobic N2-fixing
bacteria usually
results in the
isolation of
Azotobacter or its
relatives.
By contrast,
enrichment with
fixed forms of
nitrogen such as
NH4+ rarely results
in isolating
nitrogen-fixing
bacteria because
there is no
selective pressure
for nitrogen
fixation.
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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
© 2012 Pearson Education, Inc.
Animation: Enrichment Cultures
Marmara University – Enve303 Env. Eng. Microbiology – Prof. BARIŞ ÇALLI
• 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. Marmara University – Enve303 Env. Eng. Microbiology – Prof. BARIŞ ÇALLI
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Figure 22.2 The Winogradsky column.
© 2012 Pearson Education, Inc. Marmara University – Enve303 Env. Eng. Microbiology – Prof. BARIŞ ÇALLI
Gradients Column
Lake orpond water
Mudsupplementedwith organicnutrientsand CaSO4
O2
H2S
Foil cap
Algae andcyanobacteria
Purple nonsulfurbacteriaSulfurchemolithotrophs
Patches of purplesulfur or greensulfur bacteria
Anoxicdecompositionand sulfatereduction
Photo of Winogradsky columns
that have remained anoxic up to
the top; each column had a
bloom of a different phototrophic
bacterium.
Thiospirillum
jenense (purple
sulfur bacteria)
Chromatium
okenii (purple
sulfur bacteria)
Chlorobium
limicola (green
sulfur bacteria)
The column is incubated in a location that
receives subdued sunlight. Anoxic
decomposition leading to sulfate reduction
creates the gradient of H2S.
• 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. Marmara University – Enve303 Env. Eng. Microbiology – Prof. BARIŞ ÇALLI
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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
© 2012 Pearson Education, Inc. Marmara University – Enve303 Env. Eng. Microbiology – Prof. BARIŞ ÇALLI
Figure 22.3 Pure culture methods
© 2012 Pearson Education, Inc. Marmara University – Enve303 Env. Eng. Microbiology – Prof. BARIŞ ÇALLI
Colonies Paraffin–mineraloil seal
• Organisms that form distinct
colonies on plates are usually easy
to purify.
• Colonies of phototrophic purple
bacteria in agar dilution tubes; the
molten agar was cooled to app. 45oC
before inoculation.
• A dilution series was established from
left to right, eventually yielding well-
isolated colonies.
• The tubes were sealed with a 1:1
mixture of sterile paraffin and mineral
oil to maintain anaerobiosis
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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)
© 2012 Pearson Education, Inc.
Animation: Serial Dilutions and a Most Probable Number Analysis
Marmara University – Enve303 Env. Eng. Microbiology – Prof. BARIŞ ÇALLI
Figure 22.4 Procedure for a most-probable-number (MPN) analysis
© 2012 Pearson Education, Inc. Marmara University – Enve303 Env. Eng. Microbiology – Prof. BARIŞ ÇALLI
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
• Axenic culture (grown under sterile conditions) 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)
© 2012 Pearson Education, Inc. Marmara University – Enve303 Env. Eng. Microbiology – Prof. BARIŞ ÇALLI
Figure 22.5 The laser tweezers for the isolation of single cells
© 2012 Pearson Education, Inc. Marmara University – Enve303 Env. Eng. Microbiology – Prof. BARIŞ ÇALLI
Laser beam
b
b
a
a
Fb Fa Cell
Beam focus
Objective lensof microscope
Optically trapped cell
Capillarytube
Laser
Severing point
Mixture of cells
• A laser beam strongly focused
on a very small object such as
a cell creates downward
radiation forces that allow the
cell to be dragged in any
direction.
• The laser beam can lock onto
a single cell present in a
mixture in a capillary tube and
drag the optically trapped cell
away from the other cells.
• Once the desired cell is far
enough away from the other
cells, the capillary is severed
and the cell is flushed into a
tube of sterile medium.
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22.2 Isolation
• Flow cytometry
– Uses lasers
– Suspended cultures passed through
specialized detector
– Cells separated based on fluorescence
© 2012 Pearson Education, Inc. Marmara University – Enve303 Env. Eng. Microbiology – Prof. BARIŞ ÇALLI
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
© 2012 Pearson Education, Inc. Marmara University – Enve303 Env. Eng. Microbiology – Prof. BARIŞ ÇALLI
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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 greenish-orange (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
© 2012 Pearson Education, Inc. Marmara University – Enve303 Env. Eng. Microbiology – Prof. BARIŞ ÇALLI
Figure 22.6 Nonspecific fluorescent stains
© 2012 Pearson Education, Inc. Marmara University – Enve303 Env. Eng. Microbiology – Prof. BARIŞ ÇALLI
(a) DAPI and (b) acridine orange (AO) staining showing microbial communities inhabiting
activated sludge in a municipal wastewater treatment plant. With acridine orange, cells
containing low RNA levels stain green.
(c) SYBR Green–stained sample of Puget
Sound (Washington, USA) surface water
showing green-fluorescing bacterial cells. The
large cells near the center of the field are
0.8–1.0 m in diameter
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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
© 2012 Pearson Education, Inc. Marmara University – Enve303 Env. Eng. Microbiology – Prof. BARIŞ ÇALLI
Figure 22.7 Viability staining
© 2012 Pearson Education, Inc. Marmara University – Enve303 Env. Eng. Microbiology – Prof. BARIŞ ÇALLI
Live (green) and dead (red) cells of Micrococcus luteus (cocci) and Bacillus
cereus (rods) stained by the LIVE/DEAD BacLight Bacterial Viability Stain
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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
– Can be used to track bacteria
– Can act as a reporter gene
© 2012 Pearson Education, Inc. Marmara University – Enve303 Env. Eng. Microbiology – Prof. BARIŞ ÇALLI
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
© 2012 Pearson Education, Inc. Marmara University – Enve303 Env. Eng. Microbiology – Prof. BARIŞ ÇALLI
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Figure 22.9 Morphology and genetic diversity
© 2012 Pearson Education, Inc. Marmara University – Enve303 Env. Eng. Microbiology – Prof. BARIŞ ÇALLI
The photomicrographs shown here, produced by (a) phase-contrast and (b) a technique
called phylogenetic FISH, are of the same field of cells. Although the large oval cells are
of a rather unusual morphology and size for prokaryotic cells and all look similar in
phase-contrast microscopy, the phylogenetic stains reveal that there are two genetically
distinct types (one stains yellow and one stains blue).
Figure 22.10 FISH analysis of sewage sludge
© 2012 Pearson Education, Inc. Marmara University – Enve303 Env. Eng. Microbiology – Prof. BARIŞ ÇALLI
(a) Nitrifying bacteria. Red, ammonia-oxidizing bacteria; green, nitrite-oxidizing bacteria.
(b) Confocal laser scanning micrograph of a sewage sludge sample. The sample was
treated with three phylogenetic FISH probes, each containing a fluorescent dye (green,
red, or purple) that identifies a particular group of Proteobacteria. Green-, red- or purple-
stained cells reacted with only a single probe; other cells reacted with multiple probes to
give blue or yellow.
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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)
© 2012 Pearson Education, Inc. Marmara University – Enve303 Env. Eng. Microbiology – Prof. BARIŞ ÇALLI
Figure 22.11 Catalyzed reporter deposition FISH (CARD-FISH) labeling of Archaea
© 2012 Pearson Education, Inc. Marmara University – Enve303 Env. Eng. Microbiology – Prof. BARIŞ ÇALLI
Archaeal cells in this preparation fluoresce intensely (green) relative to DAPI-
stained cells (blue)
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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
© 2012 Pearson Education, Inc. Marmara University – Enve303 Env. Eng. Microbiology – Prof. BARIŞ ÇALLI
Figure 22.12 Steps in single-gene biodiversity analysis of a microbial community
© 2012 Pearson Education, Inc. Marmara University – Enve303 Env. Eng. Microbiology – Prof. BARIŞ ÇALLI
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
© 2012 Pearson Education, Inc. Marmara University – Enve303 Env. Eng. Microbiology – Prof. BARIŞ ÇALLI
Figure 22.13 PCR and DGGE gels
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
– Related to T-RFLP
– Uses DNA sequencing
© 2012 Pearson Education, Inc. Marmara University – Enve303 Env. Eng. Microbiology – Prof. BARIŞ ÇALLI
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
© 2012 Pearson Education, Inc. Marmara University – Enve303 Env. Eng. Microbiology – Prof. BARIŞ ÇALLI
Figure 22.15 Phylochip analysis of sulfate-reducing bacteria diversity
Positive Weak positive
Negative
© 2012 Pearson Education, Inc. Marmara University – Enve303 Env. Eng. Microbiology – Prof. BARIŞ ÇALLI
Each spot on the microarray shown has an oligonucleotide complementary to a sequence
in the 16S rRNA of a different species of sulfate-reducing bacteria. After the microarray is
hybridized with 16S rRNA genes PCR amplified from a microbial community and then
fluorescently labeled, the presence or absence of each species is signaled by fluorescence
(positive or weak positive) or nonfluorescence (negative), respectively.
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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
© 2012 Pearson Education, Inc. Marmara University – Enve303 Env. Eng. Microbiology – Prof. BARIŞ ÇALLI
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
© 2012 Pearson Education, Inc. Marmara University – Enve303 Env. Eng. Microbiology – Prof. BARIŞ ÇALLI
Figure 22.18 Microbial activity measurements
© 2012 Pearson Education, Inc. Marmara University – Enve303 Env. Eng. Microbiology – Prof. BARIŞ ÇALLI
Sulfate reduction Photosynthesis
Sulfate reduction 14C-Glucose respiration
Formalin-killed controlLight
Lactate
H2S
Lacta
te o
r H
2S
14C
O2
inco
rpo
rati
on
14C
O2
evo
luti
on
H2
35S
H2 present
H2 absentKilled
KilledDark
Killed
Time Time
Time Time
Chemical
measurement:
Lactate and H2S
transformations
during sulfate
reduction.
Radioisotopic
measurement:
sulfate reduction
measured with 35SO4
2-
Radioisotopic
measurement:
photosynthesis
measured with 14CO2
Radioisotopic
measurement
production of 14CO2 from 14C-
glucose.
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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 50–100mm
(Figure 22.20)
© 2012 Pearson Education, Inc. Marmara University – Enve303 Env. Eng. Microbiology – Prof. BARIŞ ÇALLI
Gold Glass Platinum
5 mm
Membranes Glass
Bacteria Cathode Nutrient solution
50–100 mm NO3 N2O 2e + N2O H2O N2 2 OH
Figure 22.19 Microelectrodes
© 2012 Pearson Education, Inc. Marmara University – Enve303 Env. Eng. Microbiology – Prof. BARIŞ ÇALLI
Biological microsensor for the detection of nitrate (NO3-). Bacteria
immobilized at the sensor tip denitrify NO3- (or NO2
-) to N2O, which is
detected by reduction to N2 at the cathode
The platinum rod functions as a
cathode and when voltage is
applied, O2 is reduced to H2O,
generating a current. The current
resulting from the reduction of O2
at the gold surface of the
cathode is proportional to the O2
concentration in the sample.
Note the scale of the electrode. Schematic drawing of an
O2 microelectrode.
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Figure 22.20 Depth profiles of oxygen and nitrate. Data obtained using the lander equipped with
microelectrode sensors for remote chemical characterization of deep-sea sediments.
© 2012 Pearson Education, Inc. Marmara University – Enve303 Env. Eng. Microbiology – Prof. BARIŞ ÇALLI
Oxygen (O2) concentration (mM)
Seawater
Oxic sediment
Anoxic sediment
Nitrate (NO3) concentration (mM)
De
pth
in
se
dim
en
t (m
m)
0 100 200 300
O2 NO3
0
5
10
0 4 8 12
Figure 22.21 Deployment of deep-
sea lander. The lander is equipped
with a bank of microelectrodes to
measure distribution of chemicals in
marine sediments.
22.9 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)
• The activity of sulfate-reducing bacteria is easy to
recognize from their fractionation of sulfur in
sulfides
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Figure 22.22 Mechanism of isotopic fractionation with carbon as an example
Enzyme substrates Fixed carbon Enzyme that fixes CO2
12CO2
13CO2
12Corganic
13Corganic
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Enzymes that fix CO2 preferentially fix the lighter isotope (12C). This results in fixed
carbon being enriched in 12C and depleted in 13C relative to the starting substrate. The
size of the arrows indicates the relative abundance of each isotope of carbon.
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 Flow cytometric cell sorting
© 2012 Pearson Education, Inc. Marmara University – Enve303 Env. Eng. Microbiology – Prof. BARIŞ ÇALLI
Cellslabeledby FISH
Sample stream
Nozzle
Laser
Deflectionplates
Sortedsamples
Waste(non labeled cells)
Light scatter andfluorescence detector
Induces charge onselected droplets
As the fluid stream exits the nozzle, it is
broken into droplets containing no more than
a single cell. Droplets containing desired cell
types (detected by fluorescence or light
scatter) are charged and collected by
redirection into collection tubes by positively
or negatively charged deflection plates.
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)
© 2012 Pearson Education, Inc. Marmara University – Enve303 Env. Eng. Microbiology – Prof. BARIŞ ÇALLI
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Figure 22.27 Fluorescent in situ hybridization (FISH) combined with microautoradiography (MAR).
© 2012 Pearson Education, Inc. Marmara University – Enve303 Env. Eng. Microbiology – Prof. BARIŞ ÇALLI
An uncultured filamentous cell
belonging to the Gammaproteobacteria
is shown to be an autotroph (as
revealed by MAR-measured uptake of 14CO2).
Uptake of 14C-glucose by a mixed
culture of Escherichia coli (yellow cells)
and Herpetosiphon aurantiacus
(filamentous, green cells).
MAR of the same field of cells shown
in (b). Incorporated radioactivity
exposes the film and shows that
glucose was assimilated mainly by
cells of E. coli