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26 Cards in this Set
- Front
- Back
Culture-dependent VS culture-independent
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-Enrichment is based on culturing in a selective growth medium and the tools and methods used in this approach are referred to as culture dependent
-Culture Independent: Techniques that can tell us much about the structure and function of microbial communities in the absence of actual lab cultures |
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Enrichment cultures (e.g. isolation of
Azotobacter) |
Enrichment cultures: a medium and a set of incubation are established that are selective for the desired organism and counter-selective for undesired organisms. For best enrichment culture outcomes, resources (nutritents) and conditions (temp, pH, osmotic pressure) must mimic those habits to give the best chance.
Azotobacter is a rapidly growing bacteria capable of N2 fixation in air. |
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Winogradsky column
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Artificial ecosystem and long-term source of various bacteria for enrichment cultures
- Used to isolate phototrophic purple and green bacteria, sulfate reducing bacteria and other anaerobes - It is prepared by filling a glass cylinder about half-full with organically rich mud mixed with carbon substrates. Substrates determine which organism have been enriched. |
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Enrichment Bias
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Since nutrients available in a lab culture are typically much higher than in nature, microorganisms cultured in labs are frequently only minor components of the microbial ecosystem
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Pure cultures
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One single kind of organism. Usually derived from a mixed culture. Its inoculation can assure that only one type of organism is present
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Methods of Isolation/purification
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1) Agar Dilution: A mixed culture is diluted in tubes of often agar medium, resulting in colonies embedded in the agar. Purifies anaerobic organisms such as phototrophic sulfur bacteria
2) Streak plate (easiest): Tubes containing a thin layer of agar on the inner surface, the agar is then streaked for isolated colonies. The tubes can be flushed with an oxygen-free gas, used for isolation of anaerobic prokaryotes 3) Liquid dilutions: Used to estimate viable cell #'s |
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Most probable number (MPN)
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Estimates # of microorganisms in food, waste-water, etc. Serial 10x dilutions
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Methods for verification of axenic strains (microscopic vs genetic techniques)
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Axenic: A culture that is free from any organisms other than the ones it requires. - Microscopy - Observation of colony characteristics - Tests of the culture for growth in other media - Genetic sequencing |
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Advanced isolation methods (e.g. later tweezers, flow cytometry)
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Later tweezers and flow cytometry are useful for isolating:
- Slow growing bacteria from mixed cultures - Strains that are difficult to obtain by enrichment |
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Limitations with obtaining strains in culture
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- Small cells can be unnoticed and are overload
- Differentiation of live cell from dead cell - In the microscopic methods none reveal the phylogenetic diverstiy |
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Fluorescent dyes
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Are used to stain microorganisms from virtually any microbial habitat (DAPI)
- Dyes that stain DNA are used for enumeration of microorganisms - SYBR dye - bright fluorescence when exposed to UV light - Acridine Orange |
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Viability Stains
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Differentiate between live and dead
- Two dyes used - based on integrity of cell membrane - green cells (live) / red cells (dead) |
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Reporter genes
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-Reporter gene: GFP (Gene encoding the green fluorescence protein)
-12 different fluorescence proteins - When genes containing the fused fluorescence protein genes are transcribed and translated, both the protein of interest and the fluorescence protein are made and all cells fluoresce the characteristic colors |
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Nucleic acid probes
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DNA/RNA complementary to a sequence in a target gene or RNA. Used to identify organisms that contain a nucleic acid similar to the probe
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FISH
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Fluorescing nucleotides complimentary to rRNA sequence
- Phylogenetic of microbial populations - Can employ multiple phylogenetic probes - Used in microbial ecology, food industry and clinical diagnostics |
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Enhancement of FISH (CARD-FISH)
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- FISH can be used to measure gene expression in organisms in a natural sample
- Useful to analyze slow growing microbes |
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PCR based methods vs next-gen DNA
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- Next gen DNA sequences of not require a cloning step
- PCR products can be used directly for sequencing |
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RFLP vs DGGE
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DGGE: denaturing gradient gel. electrophoresis, separates the same size based on differences in base sequence
- DNA denatures: urea and formamide - Strands melts at different denaturant concentrations T- RFLT: terminal restriction fragment length polymorphism - Target gene is amplified by PCR - Restriction enzymes are used to cut the PCR products |
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Phylochip
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Microarray that focuses on phylogenetic members of microbial community. Constructed by affixing rRNA probes or rRNA gene-targeted oligonucleotide probes to the chip surface in a known pattern
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Metagenomics
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- DNA is cloned from microbial community and sequenced
- Detects as many genes as possible - Yields picture of gene pool in environment - Can detect gene pools in environments |
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Single cell genomics
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Amplifies DNA from a single organisms. Uses cell sorting and bacteriophage DNA polymerase
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Metatranscriptomics
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- Analyzes community RNA
- Reveals genes in a community that are actually expressed - Reveals genes in a community that are actually expressed - Reveals level of gene expression |
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Metaproteomics
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- Measures the diversity and abundance of different proteins in a community
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Multiple Displacement Amplification (MDA)
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Links metabolic functions to individual cells
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PCR
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Amplifies by using fluorescently tagged primers
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Applications of MDA
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- provides a powerful tool for linking specific metabolic functions to individual cells that have never been grown in the lab culture
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