Microbiology_Exam_3_Ch. 18

why is it incorrect to say that the GFP is a "staining" method?

its a protein

Select the option that lists the steps necessary for PCR microbial community analysis in the correct order.

microbial sample collection; DNA extraction; PCR of target genes; sorting by electrophoresis; analysis

Microarrays that have been designed to screen samples for specific groups of bacteria are called __________.

phylochips

Which of the following technical breakthroughs has enabled the development of metagenomics?

high-throughput DNA sequencing

streak plate method

isolation method of choice if organisms form colonies on agar plate

agar dilution tube method

- a mixed culture is diluted in tubes of molten agar medium, resulting in colonies embedded in agar - useful for purifying anaerobic organisms such as phototrophic sulfur bacteria and sulfate reducing bacteria from Winogradsky column - purified by successive dilutions of cell suspensions in tubes of molten agar medium - tubes were sealed by sterile paraffin and mineral oil to maintain anaerobiosis

nucleic acid probes

- a strand of nucleic acid that can be labeled and used to hybridize to a complementary molecule from a mixture of other nucleic acids - a short oligonucleotide of unique sequence used as a hybridization probe for identifying specific genes

viability staining

- differentiates live cells from dead cells - determined by whether a cell's cytoplasmic membrane is intact - green fluorescing dye penetrates all cells, viable or not - red dye, which contains the chemical propidium iodide, penetrates only those cells whose cytoplasmic membrane is no longer intact and therefore dead - green alive and red dead - BACLight Bacterial viability stain - can have issues with nonspecific staining in environmental samples

How does dilution reduce enrichment bias?

- dilution of an inoculum, then liquid enrichment or plating -eliminates quantitatively insignificant but rapidly growing "weed" species, allowing development of organisms that are more abundant but slowly growing

green fluorescent protein

- fluorescent proteins as cell tags and reporter genes - a gene encoding GFP can be inserted into the genome of the microbe - when the gene is expressed, cells fluorescence green when observed with UV microscopy - requires O2

Fluorescent in situ hybridization (FISH) can be used to determine

- how many Salmonella typhimurium cells are present in a sample of unpasteurized apple juice - the phylogenetic diversity of an environmental sample - whether a specific piece of mRNA is being produced

metatranscriptomics

- it analyzes sequences of community RNA - it reveals which genes in the community are being expressed

criteria for purity

- microscopy (axenic if gram stain and morphology of colonies are similar) - observation of colony characteristics on plates or in a dilution tubes - tests of the culture for growth in other media

phylotype

- one or more organisms with the same or related sequences of a phylogenetic marker gene - organisms that share or very closely related orthologous genes

Sorting can be accomplished by?

- physical separation by gel electrophoresis - clone library construction - next-generation sequencing technology

Metagenomics

- sampling and sequencing all of the genes in an environment - genes do not have to be amplified by PCR before being sequenced

most probable number technique

- the serial dilution of a natural sample to determine the highest dilution yielding growth - used for estimating the numbers of microorganisms in foods, wastewater, and other samples - estimating the viable cell number of a water sample

ARISA

- utilizes a fluorescently labeled oligonucleotide primer for DNA amplification

CARD-FISH

-does not aid in the measurement of microbial activities in nature - uses a fluorescently labeled tyramide molecule as a substrate for a single oligonucleotide conjugated to peroxidase - measure gene expression in organisms in a natural sample - target mRNA - enhances the signal (fluorescence) - catalyzed reporter deposition FISH - used when growth rate is limited and low rRNA/ribosomes, i.e. open oceans where cold temperatures and low nutrient concentration limit growth rates

Why is sulfate (SO42-) added to a Winogradsky column?

-for sulfate reducers as a result produces sulfide as an electron donor for photosynthetic purple and green sulfur bacteria

steps in the flow cytometry

1. cells labeled by FISH 2. sample stream 3. laser, then light scatter and fluorescence detector 4. induces charges on selected droplets 5. sorted samples 6. waste (nonlabeled cells)

cultural independent analysis

1. extraction of total community DNA

Which of the following methods allows for the genetic studies of very slow-growing prokaryotic organisms?

CARD-FISH

nonspecific fluorescent dyes/stains

DAPI, Acridine orange (AO), SYBR Green I (SYBR) - cannot differentiate between live and dead cells - stain nucleic acids - fluoresce under UV light - for microbes in environmental, food, and clinical samples

What are techniques used in molecular biodiversity studies?

DNA isolation and sequencing PCR Restriction enzyme digest Electrophoresis Molecular cloning

In single-cell genomics, what type of DNA polymerase is used in multiple displacement amplification?

DNA polymerase from a bacteriophage

FISH AND CARD FISH can be used to reveal different things about cells in nature. Explain.

FISH - phylogeny CARD-FISH - gene expression

winogradsky column

a glass column packed with organically rich sulfide-containing mud (supplemented: calcium carbonate- buffer and gypsum - sulfate) and overlaid with water to mimic an aquatic environment in which various bacteria develop over a period of months - used to isolate: phototrophic purple and green bacteria, sulfate reducing bacteria, and many other anaerobes - Algae and cyanobacteria on top of column (oxygen producing so this zone is oxic) - purple non sulfur bacteria, sulfur chemolithotrophs - patches of purple and green sulfur bacteria (sulfide as a photosynthetic electron donor from sulfate reducing bacteria as a result of decomposition) - anoxic decomposition and sulfate reduction - substrate determines which organisms are enriched from the environment

When measuring microbial community metabolic activity, __________ is always necessary.

a killed control

Fluorescent in situ hybridization (FISH)

a method employing a fluorescent dye covalently bonded to a specific nucleic acid probe for identifying or tracking organisms in the environment

Acridine orange (AO)

a nonspecific fluorescent dye used to stain microbial cells in a natural sample (orange) - cells containing low RNA levels stain green

phylogenetic FISH stains

fluoresces oligonucleotides complementary in base sequence to sequences in rRNA an investigator can identify or track an organism of interest or a domain of interest in a natural sample, or determine the percentage

orthologs

genes such as those encoding rRNAs that have changed in sequence over time as species have diverged

What is the process that will amplify DNA from a single cell isolated from a natural microbial community?

multiple displacement amplification

pure culture

one containing a single kind of microorganism

What structure in the cell is the target for fluorescent probes in phylogenetic FISH?

rRNA

How might you isolate a morphologically unique bacterium present in an enrichment culture in relatively low numbers?

selective single cell isolation via laser tweezer or flow cytometry

isolation procedures

streak plate, the agar dilution (shake), liquid dilution

selective single cell isolation

the laser tweezers and flow cytometry (used for slow growing microbes that are masked by weeds or microbes present in low amounts that will be missed

inoculum

the sample from which microorganisms will be isolated

isolation

the separation of individual organisms from the mixed community

microbial ecology

the study of microorganisms in their natural environments

species richness

the total number of different species present in a community

enrichment culture

the use of selective culture media and incubation conditions to isolate specific microorganism from natural samples - can prove the presence of an organism in a habitat - cannot prove that an organism does not inhabit an environment - says nothing about its ecological significance - select for and against selected bacteria

how does viability staining differ from stains like DAPI?

viability staining can differentiate between live and dead cells while DAPI. Acradine orange, and SYBR I green stains/dyes cannot

serial dilution of an inoculum

final tube in the series show no growth

phylotype

Genes that change over evolutionary time as organisms diverge are called orthologs. Organisms with identical or very similar orthologous genes belong to the same

Which of the following statements about phylochips is correct?

They can be made to carry probes targeting genes that encode a key metabolic function

laser tweezers

a device used to obtain pure cultures by optically trapping a single cell with a laser beam and moving it away from surrounding cells into sterile growth medium - downward forces Fa and Fb allow the laser beam to drag the cell - move away from contaminants - isolated by breaking the tube at a point between the cell and the contaminants - flushing the cell into a small tube of sterile medium

SYBR Green I

a dye that confers very bright fluorescence to all microorganisms, including viruses (green) such as aquatic virus populations

There is a small lake on campus that students like to use for recreational activities. A study has begun on the microbial community in the lake at different times of the year. In order to quickly determine the approximate number of reproducing bacteria in a sample, it would best to use __________.

a fluorescent viability stain

what is a reporter gene?

a gene incorporated into a vector because the product it encodes is easy to detect

denaturing gradient gel electrophoresis

- an electrophoretic technique capable of separating nucleic acid fragments of the same size that differ in sequence; their melting (denaturing) profile - employs a gradient of a DNA denaturant (urea and formamide) - double strand moves along, once it reaches a region containing sufficient denaturant it melts and migration stops - the bands are phylotypes

the isolation of azotobacter

medium: salts, mannitol but no nitrogen incubation: aerobically soil added: nitrogen fixation on both plates with or without ammonium ammonium added: selects against nitrogen fixing microbes

When Beijerinck enriched for nitrogen fixers, he inoculated soil into two types of liquid media: one containing mineral salts and mannitol but no nitrogen source (flask A), and one containing mineral salts, mannitol, and an ammonium salt (flask B). After incubation in the presence of air, what types of organisms did he find in each flask?

Flask A contained nitrogen fixers that could grow both with and without ammonium; flask B did not contain nitrogen fixers but did contain organisms that could use ammonium.

DAPI

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

enrichment bias

a problem with enrichment cultures in which "weed" species tend to dominate in the enrichment, often to the exclusion of the most abundant or ecologically significant organisms in the inoculum

flow cytometry

a technique for counting and examining microscopic particles by suspending them in a stream of fluid and passing them by an electronic device - asses and sort single cells by selected criteria (size, shape, or fluorescent properties) of single cells