Chapter 22-Methods in Microbial Ecology

Microbial ecology
Microbial ecology: how microorganisms interact with one another and their environment.
Enrichment culture: technique is a means of obtaining microorganisms from natural samples.
Enrichment Culture Technique
Enrichment Culture Technique:
A medium and set of incubation conditions are established (highly selective)
Conditions are selective for desired organism and counter selective for undesired organisms
Inoculum: Sample
Resources and Conditions


The Winogradsky column is a miniature anoxic ecosystem that can be used as a long-term source of bacteria for enrichment culture purposes
Enrichment Culture
Enrichment bias is demonstrated by comparing the results obtained in dilution cultures with classical liquid enrichment.
Some organisms will grow more rapidly and “take over” culture
Isolation in Pure Culture
Once an enrichment culture is established, a pure culture can be obtained using streak plates, agar shakes, or dilution methods.
MPN: Most probable number: method of measuring the numbers of microbes in different media and conditions.



Fluorescent stains
DAPI (4’, 6-diamido2-phenylindole) is a general stain for identifying microorganisms in natural samples.
Some stains can differentiate live versus dead cells- Viability stains.
Fluorescent antibodies that are specific for one or a small group of related cells can be prepared


Fluorescent stains
The green fluorescent protein makes cells autofluorescent and is a means for tracking cells introduced into the environment.
Unlike in pure cultures, morphologically similar cells may actually be quite different genetically in natural samples.

Genetic Stains
A variety of fluorescent-staining methods employ the power of nucleic acid probes and thus are highly specific in their staining properties.

These include phylogenetic staining, chromosome painting, and reverse transcription fluorescent in situ hybridization (FISH).
FISH
The sample DNA is separated into complimentary strands within the DNA double helix structure.

The fluorescently labeled probe of interest is then added to one sample mixture and binds with the sample DNA at the target site.

The probe signal can then be seen through a fluorescent microscope as a colored spot located in the target area/cell/chromosome. 
The assay uses fluorescence-labeled peptide nucleic acid (PNA) probes that target the species-specific ribosomal RNA (rRNA) in an organism. 

Results are visualized using fluorescence microscopy to see green fluorescing cells. 
Other Methods
FISH
Chromosome painting: DNA probe used to ID cells with specific highly conserved genes within groups of bacteria.
ISRT (in situ reverse transcription):
Used to measure gene expression.
Use of a probe that hybridizes with a specific mRNA.
Electrophoresis
PCR
Environmental Genomics


Electrophoresis
Denaturing gradient gel electrophoresis (DGGE) can be used to resolve difference in genes present in the various species inhabiting a natural sample.


Radioisotopes
Isotopes: Different forms of the same element containing the same number of protons and electrons but different numbers of neutrons

Microautoradiography (MAR): Radioisotopes used as measures of microbial activity in a microscopic technique

Isotope fractionation: can reveal the biological origin of various substances

FISH MAR: Technique that allows the metabolic activity to be measured and the organism to be identified