Transcript 22.2 Isolation in Pure Culture

Chapter 22
Methods in Microbial Ecology
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
Stained with ISRT probe
Figure 22.12b
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