LP 10 - Methods in Microbial Ecology

Discuss how the probability of providing a complete list of organisms in a given sample increases with the number of clones that are screened

A large number of clones must be selected and sequenced to detect species that are present in small numbers in original sample.

DAPI

A nonspecific fluorescent dye used to stain microbial cells in a natural sample to obtain total cell numbers

Microelectrode

A small glass electrode for measuring pH or specific compounds such as O2 or H2S that can be immersed into a microbial habitat at microscale intervals

Appreciate that a successful enrichment culture must optimize both the resources (media) and conditions (incubation temp, etc...) to be successful

Self-explanatory. Remember: resources and conditions

Describe how serial streak plating ensures that a pure culture is isolated and maintained

Serial streak plating allows for an isolated colony (which originated from one cell and thus forms a pure culture) to be easily accessed. It can be used to inoculate a new agar plate with fresh media in order to maintain living cells that can be used in experiments.

Competent cell

A cell that has the ability to take up DNA and become genetically transformed

Nucleic acid probe

A strand of nucleic acid that can be labeled and used to hybridize to a complementary molecule from a mixture of other nucleic acids. In clinical microbiology or microbial ecology, a short oligonucleotide of unique sequence used as a hybridization probe for identifying specific genes.

Two foci of Microbial Ecology:

Biodiversity: isolate, identify, quantify microorganisms Microbial activity: measure what microorganisms are doing

State what material DAPI stains and whether it distinguishes between active and inactive cells

DAPI stains both living and dead cells (bright blue when exposed to UV radiation). DAPI stains the DNA in cells as it binds to nucleic acids

DGGE

Denaturing gradient gel electrophoresis; an electrophoretic technique that allows resolving nucleic acid fragments of the same size but that differ in sequence

Understand the role of PCR products, plasmids, competent cells and plating in cloning

Genes amplified by PCR can be used as the source DNA fragment. Cloning vectors are derived from plasmids; the plasmids are cut with a restriction enzyme that yields sticky ends. The plasmids and the PCR product strands are joined by DNA ligase. This new DNA form is the recombinant vector. A competent cell can be opened up and closed again to allow for insertion of the recombinant plasmid. This step is transformation. The culture of cells are spread plated; some plasmids contain a marker so that they do not change color when gene insertion has been accomplished. The cells with the gene can be picked from visible colonies for sequencing

FISH

Fluorescent in-situ hybridization A process in which a cell is made fluorescent by labeling it with a specific nucleic acid probe that contains an attached fluorescent dye

State an example of how genetic engineering of GFP has been used in an environmental experiment

GFP-tagged cells can be introduced into an environment, such as plant roots, and then tracked over time by microscopy to assess the effects of perturbations of an environment on the survivability of the introduced strain or to study microbial competition between the native microflora and the GFP-tagged strain GFP gene has been used as a reporter gene in lab cultures; when fused with an operon under the control of a specific repressor protein, transcriptional control of the genes of interest can be studied using fluorescence as the assay of transcription. When the genes containing the fused GFP gene are transcribed, the GFP gene is also transcribed, and cells fluoresce green Also, the gene has been inserted into plasmid DNA in order to detect the genetic transfer of plasmids

GFP

Green fluorescent protein; a protein that fluoresces green and is widely used in genetic analysis

Discuss how successful cloning is dependent on successful transformation

If transformation does not proceed, the plasmid is not incorporated into the cell and the gene will not be propagated or expressed

Streak plate

Method in which a well-isolated colony is picked and re-streaked several successive times to obtain a pure culture

MPN

Most-probable number; serial dilution of a natural sample to determine the highest dilution yielding growth.

Explain why PCR using universal primers results in products with equal base pair lengths but variable sequences.

PCR uses a forward and a reverse primer that hybridize to short sequences and allow for all the base pairs in between the two to be amplified. The sequence itself, denoted by the same nucleotide sequence bit at the beginning and the end, can be different

Define the chemical and functional meaning of a denaturing gradient in DGGE

The denaturant is a mixture of urea and formamide or temperature. When a double-stranded DNA fragment moving through the gel reaches a region containing sufficient denaturant, the strands begin to "melt" and their migration stops

Explain the purpose of a GC-clamp involved in DGGE

The GC clamp prevents the PCR product from running off the gel in DGGE once the product is denatured

Describe generally the lab procedure for enriching and isolating a pure culture from an environmental sample, including the culturing methods.

The inoculums should be diluted first to eliminate quantitatively insignificant but rapidly growing "weed" species before being enriched. A favorable environment is provided for the specific organism in a growth medium, so it will reproduce and dominate the sample. The conditions and resources should also be counterselective for undesired organisms. The media can be in liquid, or agar plates. After enrichment, a single cell or colony can be isolated by streak plate method, the agar shake, or liquid dilution. Most probable number technique: serially dilute an inoculum in a liquid medium until the final tube in the series shows no growth to obtain pure cultures

State an example of how radioisotopes may be used in an environmental experiment

The light-dependent uptake of 14CO2 into microbial cells can be measured to detect photoautotrophy. Heterotrophic activities can be measured by tracking the 14CO2 released from 14C-labeled organic compounds.

Microbial ecology

The study of microorganisms in their natural environments

Transformation

Transfer of genetic information via free DNA

Enrichment culture

Use of selective culture media and incubation conditions to isolate specific microorganisms from natural samples

Provide an experimental purpose for isolating an organism from the environment

You want to study a specific organism only, without interference from others. You want to see if a specific organism was actually present in the environmental sample (be able to measure). You want to cultivate that organism so that a culture may perform a function in an experimental design.