Methods in Microbial Ecology Chapter 22

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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


Figure 22.1 The isolation of Azotobacter.


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


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


cultures.html

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Figure 22.2 The Winogradsky column.


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


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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


Figure 22.3 Pure culture methods


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)


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


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

dilutions.html

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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)


Figure 22.5 The laser tweezers for the isolation of single cells


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


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 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


Figure 22.6 Nonspecific fluorescent stains


(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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• 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


Figure 22.7 Viability staining


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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• 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


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 Morphology and genetic diversity


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


(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)


Figure 22.11 Catalyzed reporter deposition FISH (CARD-FISH) labeling of Archaea


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


Figure 22.12 Steps in single-gene biodiversity analysis of a microbial community


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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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


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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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



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


Figure 22.15 Phylochip analysis of sulfate-reducing bacteria diversity

Positive Weak positive

Negative


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


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


Figure 22.18 Microbial activity measurements


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)


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


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.


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


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


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)


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Figure 22.27 Fluorescent in situ hybridization (FISH) combined with microautoradiography (MAR).


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

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Methods in Microbial Ecology Chapter 22