Methods in Microbial Ecology

Brock Biology of Microorganisms, Twelfth Edition

Madigan / Martinko / Dunlap / Clark

Copyright 2009 Pearson Education Inc., publishing as Pearson Benjamin Cummings

I. Culture-Dependent Analyses of Microbial Communities

22.1 Enrichment and Isolation

22.2 Isolation in Pure Culture
22.1 Culture Dependant Microbial Community Analysis

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
The Isolation of Azotobacter

Figure 22.1
Some Enrichment Culture Methods
22.1 Enrichment and Isolation

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

22.1 Enrichment and Isolation

The Winogradsky Column

An artificial microbial ecosystem
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
Schematic View of a Typical Winogradsky Column

Figure 22.2a
Photo of Winogradsky Column: Remained Anoxic Up to Top

A bloom of different phototrophic bacterium

1: Thiospirillum jenense
2: Chromatium okenii
3: Chlorobium limicola

1 2 3 Figure 22.2b
Some Enrichment Culture Methods
Some Enrichment Culture Methods
22.1 Enrichment and Isolation

Enrichment bias
Microorganisms cultured in the lab are frequently only
minor components of the microbial ecosystem
Because 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
22.2 Isolation in Pure Culture

Pure cultures contain a single kind of microorganism

Can be obtained by streak plate, agar shake, or liquid dilution
Agar dilution tubes are mixed cultures diluted in molten
agar
Useful for purifying anaerobic organisms
Most-probable number technique
Serial 10X dilutions of inocula in a liquid media
Used to estimate number of microorganisms in food,
wastewater, and other samples
22.2 Isolation in Pure Culture

Animation: Serial Dilutions and a Most Probable Number Analysis

Procedure for a Most-Probable Number Analysis

Figure 22.4
22.3 Pure Culture Methods

Figure 22.3
22.2 Isolation in Pure Culture

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
Principle of the Laser Tweezers

Figure 22.5a
The Laser Tweezers for the Isolation of Single Cells

Figure 22.5b
II. Culture-Independent Microbial Community Analysis

22.3 General Staining Methods

22.4 FISH
22.5 Linking Specific Genes to Specific Organisms
Using PCR
22.6 Environmental Genomics
22.3 General Staining Methods

Fluorescent staining using DAPI or acridine orange (AO)

DAPI (4-6-diamidino-2-phenylindole) stained cells
fluoresce bright blue
AO stained cells fluoresce orange or greenish-orange
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
Nonspecific Fluorescent Stains: Photomicrograph of DAPI

Figure 22.6a
Nonspecific Fluorescent Stains: Acridine Orange

Figure 22.6b
22.3 General Staining Methods

Viability stains: differentiate between live and dead

cells
Two dyes are used

- Green dye: penetrates all cells

- Red dye: penetrates only dead cells

Based on integrity of cell membrane
Green cells are live
Red cells are dead
Can have issues with nonspecific staining in
environmental samples
Viability Staining

Figure 22.7
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 (GFP) can be genetically
engineered into cells to make them autofluorescent
Can be used to track bacteria
Can act as a reporter gene
Fluorescent Antibodies as a Cell Tag

Sulfolobus acidocaldarius attached to

the surface of solfatra soil particles.
Figure 22.8
The Green Fluorescent Protein

Pseudomonas fluorescence attached to barley roots.

Figure 22.9
22.4 FISH

Nucleic acid probe is DNA or RNA complimentary to a

sequence in a target gene or RNA
FISH (fluorescent in situ hybridization)
Phylogenetics of microbial populations
Used in microbial ecology, food industry, and clinical diagnostics
ISRT-FISH (in situ reverse transcription-FISH)

- Use cDNAs as probes

CARD-FISH (catalyzed reported deposition FISH)

- Peroxidase is attached to the probe

- Treat with tyramide after hybridization

: converted into a very reactive intermediate that binds to adjacent
proteins and fluoresces
Morphology and Genetic Diversity

Phase contrast Phylogenetic FISH

Figure 22.10a
FISH Analysis of Sewage Sludge: Nitrifying Bacteria

Red: ammonia-oxidizing bacteria

Green: nitrite-oxidizing bacteria

Figure 22.11a
FISH Analysis of Sewage Sludge

Figure 22.11b
In-situ Reverse Transcription

Stained with DAPI

Figure 22.12b
Stained with ISRT probe
22.5 Linking Genes to Specific Organisms Using PCR

Specific genes can be used as a measure of diversity

PCR, DGGE, molecular cloning, and DNA sequencing and
analysis are tools used to look at community diversity
DGGE (denaturing gradient gel electrophoresis)
separates genes of the same size based on differences
in base sequence
Denaturant is a mixture of urea and formamide
Strands melt at different denaturant concentrations

- Use gels with different gradient of the denaturant

Steps in Single Gene Biodiversity Analysis

Figure 22.13
PCR and DGGE Gels

Figure 22.14a
PCR and DGGE Gels

Figure 22.14b
22.5 Linking Genes to Specific Organisms Using PCR

T-RFLP (Terminal Restriction Fragment Length Polymorphism)

Target gene is amplified by PCR using a primer set in which
one of the primers is end-labeled with a fluorescent dye
Restriction enzymes are used to cut the PCR products
Molecular methods demonstrate that less than 0.5% of
bacteria have been cultured
Phylochip: microarrays that focus on phylogenetic members of
microbial community
Circumvents time-consuming steps of DGGE and T-RFLP
Phylochip Analysis of Sulfate-Reducing Bacteria Diversity

Figure 22.15
22.6 Environmental Genomics

Environmental Genomics (metagenomics)

DNA is cloned from microbial community and sequenced
Idea is to detect as many genes as possible
All genes in a sample can be detected
Yields picture of gene pool in environment
Can detect genes that would not be amplified by current
PCR primers
Powerful tool for assessing the phylogenetic and metabolic
diversity of an environment
Single Gene Versus Environmental Genomics

Figure 22.16
III. Measuring Microbial Activities in Nature

22.7 Chemical Assays, Radioisotopic Methods, and

Microelectrodes
22.8 Stable Isotopes
22.7 Chemical Assays, Radioisotopes, & Microelectrodes

In many studies direct chemical measurements are

sufficient
Higher sensitivity can be achieved with radioisotopes
Proper killed cell controls must be used
Radioisotopes can also be used with FISH
FISH microautoradiography (FISH-MAR)
Combines phylogeny with activity of cells
Microbial Activity Measurements

Figure 22.17
FISH-MAR

An autotroph using 14CO2 as a carbon source.

Figure 22.18a
FISH-MAR

FISH MAR with 14C-glucose

Figure 22.18b
22.7 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
Electrodes are carefully inserted into the habitat (e.g.,
microbial mats)
Schematic Drawing of an Oxygen Microelectrode

Figure 22.19a
Microelectrodes Being Used in a Hot Spring Microbial Mat

Figure 22.19b
Microbial Mats and the Use of Microelectrodes

Figure 22.20a
Oxygen, Sulfide, and pH Profiles in Hot Spring Microbial Mat

Figure 22.20b
22.8 Stable Isotopes

Stable isotopes: non-radioactive isotopes of an element

Can be used to study microbial transformations in
nature
Isotope fractionation
Carbon and sulfur are commonly used
Lighter isotope is incorporated preferentially over heavy
isotope
Indicative of biotic processes
Isotopic composition of a material reveals its past biology (e.g.,
carbon in plants and petroleum)
Mechanism of Isotopic Fractionation Using Carbon

Figure 22.21
Isotopic Geochemistry of 13C and 12C
Isotopic Geochemistry of 34S and 32S
22.8 Stable Isotopes

Stable isotopes probing (SIP): links specific

metabolic activity to diversity using a stable isotope
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-labelled substrates
SIP of RNA can be done instead of DNA
Stable Isotope Probing