chapter 19 - environmental microbiology Flashcards
Inoculum
samples where microorganisms will be isolated
Enrichment cultures
used to isolate bacteria from mixed cultures (feces/soil)
What do enrichment cultures do?
favor growth of target organisms while inhibiting growth of non-target organisms
Enrichment bias
microorganisms are often minor components of microbial ecosystem, quantity in lab is bigger than nature.
What are the techniques to isolate microorganisms ?
- Most probable-number technique
- serial dilution x10
- estimate # of microorganisms - Pure (axenic) cultures need to be verified
- microscopy cell shape/size
- test living conditions - Laser Tweezers
- laser beam that moves through a lens
- focus on cell and seperates it
- isolates slow growing bacteria - flow cytometry: counting + examining method
- suspended stream pass through electronic detector
- separate based on shape, size, fluorescent properties.
How can you cultivate uncultured microorganisms?
High-throughput Screening (HTS)
- separate one cell per well in microtitre plate.
- each cell can grow without any competition (used for slow growing species that thrive in nutrient poor environments)
- microfluidic platforms are used for cultivation
How can microscopic analyses of microbial communities be done in culture-independent bacteria?
using non specific fluorescent dyes under UV light
What dyes are used and what color do they have?
DAPI - blue
Acridine orange - orange
SYBR green - green
How are viability stains done?
green dye used to enter all cells
red dye used to enter dead cells
- provides info on abundance and viability of cells
What are fluorescent protein receptors used for?
- gene encodes fluorescent proteins controlled by the promoter.
- introduced into bacteria to then track live bacteria + bacterial processes (infections)
How can molecular analysis of microbial communities be done in culture-independent bacteria?
through nucleic acid hybridization
- probe will have reporter molecules that will bind with complementary nucleic acids. This will form a duplex formation, which will reveal the unknown.
Situ
in place; used to see microorgansims in communities.
How does FISH study phylogenetic of microbial populations?
they label oligonucleotides complementary to rRNA, as it is highly specific to species. It can be modified to measure gene expression or translational activity.
What is the PCR method of microbial community analysis?
1 - isolate DNA from environment samples using Polymerase Chains Reaction, amplification of specific genes.
How can you analyze amplified genes?
- molecular cloning
- electrophoresis
- restriction enzyme digestion
- sequencing
Phylogenetic analysis
massively parallel DNA sequencing
What are the results of phylogenetic analysis?
- rRNA sequences differ from lab ones
- new phylogenetic distinct prokaryotes
- fewer than 0.1% bacteria have been cultured (enrichment bias)
Geochip
funcitonal gene microarray
- FGA (functional gene array) target genes in different functional processes
- valuable for assessing functional composition and structure of microbial communities
Environmental multi-omics
provides integrated perspective to power discovery across multiple levels of biology
How does environmental genomics occur? (metagenomics)
1- DNA is extracted
2- it is sequenced directly, without cloning
3- assembled and then annotated (partial genomes are assembled)
What are the outcomes of environmental genomics?
1 - identification of gene categories
2- discovery of new genes
3- linking of genes to phylotypes
How does metatranscriptomics happen?
1- DNA is extracted
2- Single gene is amplified
3- remove rRNA before sequencing (>90% of RNA is rRNA)
What are the outcomes of metatranscriptomics?
1- reveals genes in community
2- reveals level of gene expression
Metaproteomics
measures diversity and abundance of different proteins
Metabolomics
comprehensive analysis of cellular and extracellular metabolites
Microsensors
measure wide range of activity (pH, oxygen)
Stable isotopes
element can have multiple nonradioactive stable isotopes
Isotopic fractionation
biological reactions prefer lighter isotopes
Examples of isotopic geochemistry
Petroleum: displays similar isotopic fractionation as plants
Stable isotope probing
- feed microorganisms with substrate labelled with heavy isotope.
- heavier DNA separates by ultracentrifugation
