methods in microbal ecology

What is microbial ecology?

The study of microorganisms in their natural environments and how they interact with each other and their surroundings.

What are the six major elements required for microbial life (CHONPS)?

Carbon, Hydrogen, Oxygen, Nitrogen, Phosphorus, and Sulfur.

What is a culture-dependent method?

A method that requires microorganisms to be grown in the laboratory before they are studied.

What is a culture-independent method?

A method that studies microorganisms directly from environmental samples without culturing them.

What is isolation?

The separation of an individual microorganism from a mixed microbial community.

What is a pure (axenic) culture?

A culture containing only one species of microorganism.

What is an inoculum?

The original environmental sample from which microorganisms are isolated.

What is an enrichment culture?

A culture technique that favors the growth of a desired microorganism by manipulating nutrients or incubation conditions.

Why are enrichment cultures used?

To selectively grow microorganisms with specific metabolic or physiological characteristics.

What can enrichment cultures prove?

They can prove an organism is present but cannot prove it is absent.

What is enrichment bias?

The tendency for laboratory conditions to favor microorganisms that grow well in culture but may not be abundant in nature.

Why does enrichment bias occur?

Laboratory media contain much higher nutrient concentrations than natural environments, favoring fast-growing microbes.

What are "weed species" in enrichment cultures?

Fast-growing microorganisms that dominate laboratory cultures but may not be ecologically important.

What is a Winogradsky column?

A self-contained microbial ecosystem that creates oxygen and nutrient gradients to enrich diverse microorganisms.

Why is a Winogradsky column useful?

It allows different microorganisms to grow according to their metabolic requirements.

What types of microorganisms grow in a Winogradsky column?

Phototrophs, sulfur bacteria, sulfate reducers, cyanobacteria, and other environmental microbes.

What is direct enrichment?

An enrichment method that favors fast-growing microorganisms.

What is diluted enrichment?

Repeated dilution of samples to reduce competition from fast growers and isolate less competitive microorganisms.

Why is diluted enrichment performed?

To improve isolation of abundant but slower-growing microorganisms.

How can an axenic culture be verified?

By microscopy, colony morphology, and testing growth on additional media.

What are laser tweezers used for?

Isolating individual slow-growing bacterial cells from mixed cultures.

What is fluorescent staining?

A staining method that uses fluorescent dyes to visualize microorganisms.

What does DAPI stain?

DNA.

What does Acridine Orange stain?

Nucleic acids.

What does SYBR Green I stain?

DNA.

What is a limitation of fluorescent stains?

They generally cannot distinguish between living and dead cells.

What is a viability stain?

A stain that differentiates live and dead cells based on membrane integrity.

What color indicates live cells in a viability stain?

Green.

What color indicates dead cells in a viability stain?

Red.

What does FISH stand for?

Fluorescence In Situ Hybridization.

What is the purpose of FISH?

To identify specific microorganisms using fluorescent DNA or RNA probes.

What is a nucleic acid probe?

A labeled DNA or RNA sequence complementary to a target DNA or RNA sequence.

Why is rRNA commonly targeted in FISH?

It is abundant in microbial cells, making detection easier.

What are common applications of FISH?

Microbial ecology, food microbiology, and clinical diagnostics.

What is one major advantage of FISH?

It identifies microorganisms directly in environmental samples without culturing.

What does CARD-FISH stand for?

Catalyzed Reporter Deposition Fluorescence In Situ Hybridization.

How does CARD-FISH differ from regular FISH?

It amplifies the fluorescent signal, allowing detection of rare microorganisms.

What does PCR stand for?

Polymerase Chain Reaction.

What is the purpose of PCR in microbial ecology?

To amplify DNA for identification and diversity studies.

Why is DNA isolated from environmental samples?

To analyze microbial diversity and genetic information.

What is the purpose of restriction enzyme digestion?

To cut DNA into fragments for analysis.

What is electrophoresis used for?

To separate DNA fragments based on size.

What is molecular cloning?

The insertion of DNA fragments into vectors for replication and study.

What does DGGE stand for?

Denaturing Gradient Gel Electrophoresis.

What is the purpose of DGGE?

To separate DNA fragments with different sequences and compare microbial communities.

What information does DGGE provide?

A DNA fingerprint showing microbial community composition.

What is a PhyloChip?

A DNA microarray that identifies phylogenetic members of microbial communities.

What is a GeoChip?

A functional gene microarray that detects genes involved in metabolic pathways.

What is the difference between a PhyloChip and a GeoChip?

PhyloChip identifies who is present; GeoChip identifies what functions they can perform.

What is metagenomics?

The study of all DNA extracted from a microbial community.

What question does metagenomics answer?

"What genes are present?"

What is one advantage of metagenomics?

It can detect genes that PCR may miss because it does not rely on specific primers.

What is metatranscriptomics?

The study of RNA expressed by a microbial community.

What question does metatranscriptomics answer?

"Which genes are actively being expressed?"

What is metaproteomics?

The study of proteins produced by a microbial community.

What question does metaproteomics answer?

"Which proteins are being produced?"

What is metabolomics?

The study of metabolites produced by microbial communities.

What question does metabolomics answer?

"What metabolic activities are occurring?"

What is stable isotope probing used for?

To identify microorganisms actively using a specific nutrient.

What is single-cell genomics?

Sequencing the genome of an individual microbial cell.

What is the major limitation of culture-dependent methods?

Many environmental microorganisms cannot be grown in laboratory culture.

What is the major advantage of culture-independent methods?

They detect microorganisms directly from environmental samples, including unculturable species.

What is the biggest difference between culture-dependent and culture-independent methods?

Culture-dependent methods require laboratory growth, while culture-independent methods analyze microbes directly from environmental samples.