L11/12-Ch22 Methods in Microbial Ecology Flashcards
How could you use the enrichment technique to enrich for a specific group of microorganisms?
Enrichment technique aims to emulate the particular ecological niche of a microbe. By changing the resources (nutrients) and conditions (temp, pH, osmotic considerations), you can choose for microorganisms living in that niche.
What are some of the limitations of the enrichment technique?
1) Some enrichment techniques yield nothing bc microbe wasn’t present in sample or resources/conditions insufficient for growth. Firm positive conclusion (microbe is present) and not firm negative conclusion (microbe is not present). 2) Isolation doesn’t demonstrate anything about importance or abundance. 3) enrichment bias
Explain how non-specific strains like DAPI and acridine orange work to stain cells.
DAPI and acridine orange bind to DNA and are strongly fluorescent when exposed to ultraviolet. DAPI-blue, acridine orange-green/orange. Give reasonable estimate for cell number in sample
What are some limitations of microscopic techniques?
1) Very small cells can go unnoticed/are near limits of resolution
2) difficulty differentiating live cells from dead cells
3) does not illuminate phylogenetic diversity
Compare and contrast DGGE, ARISA, and t-RFLP.
DGGE-separates same sized genes by denaturing profile bc differences in base sequence-overview of biodiversity, but no ID
ARISA-analyze spacer between 16s rRNA and 23s rRNA, similar to t-RFLP but no restriction enzyme
t-RFLP-single gene diversity and crude ID/phylogenetics, underestimates diversity
Describe the phylochip and how it can be used to study microbial communities. Advantages? Disadvantages?
Phylochips affix rRNA probes/oligonucleoride probes to a chip surface in a known pattern. Advantages- circumvent time consuming PCR, DGGE, cloning and sequencing, ID lots, can use 16srRNA or genes for proteins in spec. metabolic pathways Disadvantages-????
Explain how new metagenomic technologies have advanced our understanding of microbial communities.
1) Discovery of novel organisms and genes. Ex. Sargasso sea
2) find known genes in novel organisms-nitrifying Archaea
3) new strategies for enrichment/isolation-light-med proton pump
4) variation in genes associated with one phylotypea
Explain the utility of metatranscriptomics and metaproteomics.
Metascriptomics-analogous to metagenomics, analyzes sequences of total RNA. Can analyze which genes are being expressed at a specific time and place
Metaproteomics-diversity/abundance of proteins, more direct measure of cell function.
Why are radioisotopes useful in measuring microbial activities?
More sensitive, can help determine turnover rates and the fate of the molecule can be followed
How can we use isotopes to identify biologically produced sulfur versus geologically produced sulfer?
There is a preference/isotopic fractionation towards isotopically lighter sulfur in microbes. Thus if a substance is filled with mostly filled with 33S or 34S than 36S, it is probably biologically (rather than geographically) produced.
How can NanoSIMS be combined with FISH to study metabolism of cell populations?
NanoSIMS is used to track the different metabolites/substrates in individual cells of different populations. FISH is used to identify the cell…?
What are some of the advantages and disadvantages to single cell genomics?
Advantages-amplification of entire genome without the bias of predesigned “universal” primers. Provides info for linking specific metabolic functions to individual cells when combined with high-throughput DNA sequencing.
Disadvantages- stringent control over purity
