Use LEFT and RIGHT arrow keys to navigate between flashcards;
Use UP and DOWN arrow keys to flip the card;
H to show hint;
A reads text to speech;
26 Cards in this Set
- Front
- Back
|
Culture-dependent VS culture-independent
|
-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 |
|
|
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. |
|
|
Winogradsky column
|
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. |
|
|
Enrichment Bias
|
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
|
|
|
Pure cultures
|
One single kind of organism. Usually derived from a mixed culture. Its inoculation can assure that only one type of organism is present
|
|
|
Methods of Isolation/purification
|
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 |
|
|
Most probable number (MPN)
|
Estimates # of microorganisms in food, waste-water, etc. Serial 10x dilutions
|
|
|
Methods for verification of axenic strains (microscopic vs genetic techniques)
|
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 |
|
|
Advanced isolation methods (e.g. later tweezers, flow cytometry)
|
Later tweezers and flow cytometry are useful for isolating:
- Slow growing bacteria from mixed cultures - Strains that are difficult to obtain by enrichment |
|
|
Limitations with obtaining strains in culture
|
- Small cells can be unnoticed and are overload
- Differentiation of live cell from dead cell - In the microscopic methods none reveal the phylogenetic diverstiy |
|
|
Fluorescent dyes
|
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 |
|
|
Viability Stains
|
Differentiate between live and dead
- Two dyes used - based on integrity of cell membrane - green cells (live) / red cells (dead) |
|
|
Reporter genes
|
-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 |
|
|
Nucleic acid probes
|
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
|
|
|
FISH
|
Fluorescing nucleotides complimentary to rRNA sequence
- Phylogenetic of microbial populations - Can employ multiple phylogenetic probes - Used in microbial ecology, food industry and clinical diagnostics |
|
|
Enhancement of FISH (CARD-FISH)
|
- FISH can be used to measure gene expression in organisms in a natural sample
- Useful to analyze slow growing microbes |
|
|
PCR based methods vs next-gen DNA
|
- Next gen DNA sequences of not require a cloning step
- PCR products can be used directly for sequencing |
|
|
RFLP vs DGGE
|
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 |
|
|
Phylochip
|
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
|
|
|
Metagenomics
|
- 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 |
|
|
Single cell genomics
|
Amplifies DNA from a single organisms. Uses cell sorting and bacteriophage DNA polymerase
|
|
|
Metatranscriptomics
|
- 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 |
|
|
Metaproteomics
|
- Measures the diversity and abundance of different proteins in a community
|
|
|
Multiple Displacement Amplification (MDA)
|
Links metabolic functions to individual cells
|
|
|
PCR
|
Amplifies by using fluorescently tagged primers
|
|
|
Applications of MDA
|
- provides a powerful tool for linking specific metabolic functions to individual cells that have never been grown in the lab culture
|
