Download 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 Enrichment and Isolation
 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
 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
 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
 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
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
 Also applied to isolation in pure culture
Procedure for a Most-Probable Number Analysis
Figure 22.4
Pure Culture Methods
Figure 22.3
 Axenic culture (=pure 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)
 Stain nucleic acids
 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
 Viability stains: differentiate between live and dead
cells
 Two fluorescent 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
 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 (green) attached to barley
roots. Blue: cells stained with DAPI.
Figure 22.9
22.4 FISH
 Use nucleic acid probe (DNA or RNA) that is 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
Confocal laser scanning
micrograph of sewage sludge
sample. The sample was
treated with three
phylogenetic FISH probes,
each containing a different
fluorescent dye.
Figure 22.11b
In-situ Reverse Transcription (ISRT)
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
Figure 22.14b
 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
 Number of bands on the gel indicates the number of phylotypes
 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
Each spot on the microassay has an oligonucleotide complementary to
a sequence in the 16S rRNA of a different species of sulfate-reducing
bacteria.
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 with 14C-glucose
Figure 22.18b
 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
(13C/12C sample) – (13C/12C sample standard) / (13C/12C standard) x 100
Standard: A belemnite sample from the PeeDee rock formation
Isotopic Geochemistry of 34S and 32S
(34S/32S sample) – (34S/32S standard) / (34S/32S standard) x 100
Standard: An iron sulfide mineral from the Canyon Diablo meteorite
 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