Question 1

Taking The Measure of a Microbial Ecosystem:

Accepted Answer

has to take into account the structures and functions. Diversities of microorganisms in nature and how different guilds interact. Activities and their effects on ecosystems.

Question 2

Culture-Dependent Analyses of Microbial Communities:

Accepted Answer

include Enrichment Culture Microbiology, Classical Procedures for Isolating Microbes, and Selective Single-Cell Isolation.

Question 3

Enrichment Culture Microbiology:

Accepted Answer

Uses selective growth conditions to favor the proliferation of specific microbes from a mixed community.

Question 4

Classical Procedures for Isolating Microbes:

Accepted Answer

Obtains pure microbial cultures using techniques like streak plating, dilution, and colony selection.

Question 5

Selective Single-Cell Isolation:

Accepted Answer

Separates individual microbial cells using micromanipulation, flow cytometry, or microfluidics for targeted study.

Question 6

Inoculum:

Accepted Answer

the sample from which microorganisms will be isolated.

Question 7

Enrichment:

Accepted Answer

select for desired organisms through manipulation of medium and incubation conditions. This means favoring the growth of target organisms while inhibiting the growth of non-target organisms.

Question 8

Isolation:

Accepted Answer

the separation of individual populations from the mixed community.

Question 9

Enrichment Culture Outcomes:

Accepted Answer

Successful enrichment cultures provide the necessary nutrients and conditions for target organisms to grow and indicate their presence, while failure does not prove the organism is absent from the environment.

Question 10

Winogradsky Column:

Accepted Answer

A layered microbial ecosystem used to study diversity, nutrient cycling, and growth of phototrophic and chemotrophic microbes under light, oxygen, and nutrient gradients. Provides a long-term source of Bacteria and Archaea.

Question 11

Pure Cultures:

Accepted Answer

contain a single kind of microorganism, can then be used for molecular and physiological experiments.

Question 12

Streak Plate (Classical Procedures for Isolating Microbes):

Accepted Answer

a well-isolated colony is selected and restreaked several successive times in order to obtain a pure culture.

Question 13

Serial Dilution (Classical Procedures for Isolating Microbes):

Accepted Answer

most probable number technique, serial 10x dilutions of inoculum in a liquid medium. Used to estimate the number of microorganisms in food, wastewater, and other samples.

Question 14

Enrichment Bias Problem:

Accepted Answer

Traditional cultivation methods create enrichment bias that limits understanding of microbes in nature, as lab conditions favor fast-growing organisms over those adapted to natural environments.

Question 15

Realized vs. Fundamental Niche:

Accepted Answer

Fundamental niche indicates where an organism could live based on its physiological capabilities; realized niche is where an organism actually lives, constrained by resource limitations, competition, and environmental factors.

Question 16

Culturomics:

Accepted Answer

High-throughput cultivation method that systematically tests diverse growth conditions to culture previously unculturable microbes, reducing enrichment bias and better capturing microbial diversity from natural environments.

Question 17

Selective Single-Cell Isolation:

Accepted Answer

New techniques developed to isolate microbes from nature based on their realized niches, addressing enrichment bias by targeting specific organisms in their natural context rather than selecting for fast growth in standard lab media.

Question 18

Laser Tweezers (Selective Single-Cell Isolation):

Accepted Answer

isolating slow-growing bacteria from mixed cultures. Physically separating individual cells for culture.

Question 19

Flow Cytometry (Selective Single-Cell Isolation):

Accepted Answer

technique for counting and examining a mixture of cells by suspending them in a stream of fluid and passing through an electronic detector. Sorts by cell size, shape, or fluorescent properties.

Question 20

High-Throughput Culture (Selective Single-Cell Isolation):

Accepted Answer

physically separates individual cells for culture in a microfilter plate.

Question 21

Microfilter Wells (Selective Single-Cell Isolation):

Accepted Answer

Enable researchers to adjust culture conditions (pH, nutrients, osmotic balance) and isolate single cells per well, preventing competition. Useful for slow-growing species that thrive in nutrient-poor environments.

Question 22

Microfluidic Devices for Cultivation (Selective Single-Cell Isolation):

Accepted Answer

microfluidic devices used microfabrication techniques to construct 3200 tiny individual wells.

Question 23

Measuring Microbial Activities in Nature:

Accepted Answer

can be accomplished by 1) chemical assays, radioisotopic methods, and microsensors, 2) stable isotope fractionation, 3) stable isotope probing.

Question 24

Chemical Assays for Microbial Activity:

Accepted Answer

Directly measure metabolites like lactate, sulfate, or H₂S to quantify microbial activity, using killed-cell controls to distinguish biological from abiotic processes.

Question 25

Radioisotopic Methods:

Accepted Answer

Track microbial metabolic rates with radioactive tracers (e.g., ³⁵S, ¹⁴C) for sensitive detection of processes like sulfate reduction, photosynthesis, or respiration, with killed controls to rule out abiotic reactions.

Question 26

Microsensors:

Accepted Answer

Tiny glass electrodes measure chemical gradients (pH, O₂, CO₂) at fine spatial scales in situ, revealing microbial activity and transitions between oxic and anoxic zones in environments like sediments or mats.

Question 27

Stable Isotopes:

Accepted Answer

nonradioactive isotopes of an element. Used to study microbial transformations in nature.

Question 28

Isotope Fractionation:

Accepted Answer

carbon and sulfur are commonly used. Lighter isotope is incorporated preferentially over heavy isotope, indicative of biotic processes, isotopic composition reveals its past biology (ex: carbon in plants and petroleum).

Question 29

Carbon Isotope Fractionation:

Accepted Answer

Carbon isotope fractionation is the process by which chemical or biological reactions preferentially use the lighter ^12C isotope over ^13C, causing measurable differences in their relative abundances.

Question 30

Stable Isotope Fractionation:

Accepted Answer

Preferential partitioning of stable isotopes (e.g., ¹²C/¹³C, ¹⁶O/¹⁸O, ¹⁴N/¹⁵N) due to mass differences, creating measurable isotope ratio variations. Used to detect sulfate-reducing bacteria via sulfur fractionation in sulfides.

Question 31

Stable Isotope Probing (SIP):

Accepted Answer

Tracks microbial activity by following the incorporation of a stable isotope (e.g., ¹³C) into biomolecules.

Question 32

General Staining Methods:

Accepted Answer

Techniques that apply dyes or stains to cells or tissues to enhance contrast and visualize structures under a microscope. Examples include Gram staining, acid-fast staining, and simple staining.

Question 33

Fluorescence In Situ Hybridization (FISH):

Accepted Answer

A technique using fluorescently labeled DNA or RNA probes to bind specific sequences in cells or tissues, allowing detection and localization of particular genes or microorganisms under a fluorescence microscope.

Question 34

Fluorescent Staining:

Accepted Answer

General staining, nonspecific technique using fluorescent dyes (DAPI, acridine orange, or SYBR Green I) that bind to nucleic acids and fluoresce under UV light; used for enumeration of microorganisms in samples.

Question 35

DAPI (4',6-diamidino-2-phenylindole):

Accepted Answer

Fluorescent stain that produces bright blue fluorescence when bound to DNA in cells.

Question 36

Acridine Orange (AO):

Accepted Answer

Fluorescent stain that produces orange or greenish-orange fluorescence when bound to nucleic acids in cells.

Question 37

SYBR Green I (SYBR):

Accepted Answer

Fluorescent stain that produces green fluorescence when bound to double-stranded DNA; like all these stains, it is nonspecific and cannot differentiate between live and dead cells.

Question 38

Viability Staining:

Accepted Answer

Fluorescent staining method using two dyes that differentiate live from dead cells based on cell membrane integrity; live cells fluoresce green while dead cells fluoresce red.

Question 39

Green Fluorescent Protein (GFP):

Accepted Answer

green fluorescent proteins can be genetically engineered into cells to make them autofluorescent. Can be used to track live bacteria and bacterial processes (ex: infection). Can act as a reporter gene to identify when any given promoter is active.

Question 40

Small Subunit Ribosomal RNA (SSU rRNA):

Accepted Answer

16S rRNA in prokaryotes, 18S rRNA in eukaryotes. A molecular chronometer present in all cells, with conserved regions for comparison and variable regions to distinguish species. Easily sequenced to study microbial evolution.

Question 41

Phylogenetic Probes:

Accepted Answer

Fluorescent oligonucleotides that bind SSU rRNA sequences to identify specific microbes. Target different taxonomic levels, enabling detection and distinction in complex samples via FISH.

Question 42

FISH (Fluorescence In Situ Hybridization) Meaning:

Accepted Answer

Uses fluorescent nucleic acid probes complementary to specific rRNA sequences to identify and quantify microbes in their natural communities. Allows visualization in situ without culturing.

Question 43

FISH Applications and Capabilities:

Accepted Answer

Uses multiple phylogenetic probes with different fluorescent colors to identify and distinguish microbes. Applied in microbial ecology, food quality control, and clinical diagnostics to study communities and detect pathogens.

Question 44

Culture-Independent Genetic Analyses:

Accepted Answer

Study microbial communities directly from environmental samples without cultivation, bypassing the fact that most microbes cannot be grown in the lab. Examples include single-gene biodiversity analysis, metagenomics, and environmental multi-omics.

Question 45

Single-Gene Biodiversity Analysis:

Accepted Answer

Analyzes a single conserved gene (usually 16S rRNA for prokaryotes or 18S rRNA for eukaryotes) from a community sample to identify species and assess phylogenetic diversity.

Question 46

Metagenomics (Shotgun Genomics):

Accepted Answer

Sequences all DNA from an environmental sample without targeting specific genes, revealing both phylogenetic diversity (who is there) and functional potential (what they can do) of the microbial community.

Question 47

Environmental Multi-omics:

Accepted Answer

Combines metagenomics, metatranscriptomics (gene expression), metaproteomics (proteins), and metabolomics (metabolites) to provide a comprehensive view of both the identity and activity of microorganisms in their natural environment.

Question 48

Variable Regions (V1–V9):

Accepted Answer

Nine hypervariable regions in the 16S rRNA gene that differ between bacterial species, interspersed among conserved regions. These sequence differences allow taxonomic identification.

Question 49

V4 Region Sequencing (iSeq 100):

Accepted Answer

Targets only the V4 region using 300 bp paired-end reads (~292 bp after trimming). Each read covers 150 bp from opposite ends. Provides sufficient taxonomic resolution for many microbiome studies and is cost-effective.

Question 50

V3–V4 Region Sequencing (MiSeq):

Accepted Answer

Covers two adjacent hypervariable regions (V3 and V4) using 250 bp paired-end reads (~468 bp total after assembly). Sequencing multiple regions improves taxonomic resolution and identification accuracy.

Question 51

Primer Design for Variable Regions:

Accepted Answer

Universal PCR primers bind conserved regions flanking variable regions, allowing amplification of V4 or V3–V4 from diverse bacterial communities. Forward (F) and reverse (R) primers indicate positions in the 16S gene.

Question 52

Single-Gene Biodiversity Analysis:

Accepted Answer

A method that assesses microbial diversity by examining variations in a specific gene across all organisms in a community.

Question 53

Phylotype:

Accepted Answer

A taxonomic unit defined by sequence similarity of a marker gene (often 16S rRNA), used to group microbes when full taxonomy isn’t available.

Question 54

Evolutionary Marker Gene:

Accepted Answer

A gene conserved across organisms that is used to infer evolutionary relationships (e.g., rRNA genes).

Question 55

rRNA Gene (16S/23S):

Accepted Answer

Highly conserved ribosomal RNA genes used as universal barcodes to identify and compare microbial taxa.

Question 56

Conserved Functional Gene:

Accepted Answer

A gene involved in essential metabolic functions that is preserved across many organisms (e.g., amoA, used to study ammonia-oxidizing microbes).

Question 57

DNA Isolation:

Accepted Answer

Extraction of total community DNA from an environmental sample.

Question 58

PCR Amplification:

Accepted Answer

Selective copying of a target gene (e.g., 16S rRNA) using primers so the gene can be analyzed.

Question 59

Fluorescently Tagged Primers:

Accepted Answer

PCR primers labeled with fluorescent markers that allow detection and sizing of DNA fragments after electrophoresis.

Question 60

Restriction Enzyme Digestion (T-RFLP):

Accepted Answer

Cutting PCR products with restriction enzymes to generate terminal fragment patterns used to infer diversity.

Question 61

ARISA:

Accepted Answer

A method amplifying the region between 16S and 23S rRNA genes; differences in fragment length correspond to different microbial taxa.

Question 62

Electrophoresis (e.g., DGGE):

Accepted Answer

A technique that separates DNA fragments by size or sequence differences, producing banding patterns that represent different microbial phylotypes.

Question 63

DGGE (Denaturing Gradient Gel Electrophoresis):

Accepted Answer

Fingerprints equal-length PCR fragments by sequence. Fragments migrate through a denaturant gradient until reaching their melting point, forming band patterns that represent phylotypes and enable community comparisons.

Question 64

Band Excision:

Accepted Answer

Cutting specific DGGE bands from the gel to isolate their DNA for further sequencing.

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