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Methods in Microbial Ecology
CHAPTER OVERVIEW
This chapter describes the key techniques used to define the composition and functions of microbial
communities and their populations. Cultivation techniques are presented in the context of the unique value and
their considerable limitations. Molecular techniques for assessing microbial diversity is followed by a
discussion of techniques for assessing microbial community activities.
CHAPTER OBJECTIVES
After reading this chapter you should be able to:
•
•
•
•
•
discuss the value and drawbacks of various culturing techniques in assessing microbial communities
discuss the repercussions of viable but nonculturable organisms in the environment
describe specific techniques used for assessing microbial diversity such as FISH, DGGE, and phylochips
describe biogeochemical techniques for assessing community activities such as those using stable
isotopes
describe molecular techniques for assessing community activities
CHAPTER OUTLINE
I.
II.
Culturing Techniques
A. The great plate count anomaly is the observation that most environmental microorganisms seen in
the microscope cannot currently be grown under laboratory conditions; some may actually be
nonviable; others are viable but nonculturable (VBNC)
B. Enrichment culture techniques allow abundant growth of specific microbes under defined and
limited culture conditions
C. Most probable number (MPN) analyses use enrichment cultures to determine the number of
microbes in specific groups in a community; serial dilutions are used to set up cultures in a defined
scheme that allows for statistical estimation of abundance
D. High-throughput screening methods have been developed
1. Extinction culture technique—natural samples are diluted to extinction (1 to 10 cells per ml)
and cultured in microtiter dishes; even slight degrees of growth can be detected and with
further dilution, clonal cultures established
2. Microdroplet culture—minute droplets of gel is used to capture individual cells; growth within
cultured microdroplets is detected by flow cytometry
E. Plate wash PCR—Cultures are maintained on plates or in microtiter dishes under a wide range of
different growth conditions; colonies are then pooled and PCR is used to amplify genes of interest to
determine whether these are part of the community of culturable organisms
F. Single cells can be captured from mixed cultures using optical (laser) tweezers and then examined
genetically
Assessing Microbial Diversity
A. Microbial ecologists define microbial populations within ecosystems and determine their role in
microbial communities; while axenic cultures are preferred, due to the great plate count anomaly,
other methods are often used in measuring microbial diversity
B. Staining techniques
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1.
Direct observations of microbes with electron or light microscopy is used; fluorescent stains
(e.g., DAPI) are commonly used
2. Fluorescent in situ hybridization (FISH) uses labeled DNA probes that specifically hybridize
to genomic DNA sequences in matching organisms that can be visualized using
epifluorescence microscopy; probes can be specific for 16S rRNA phylotypes; coupling the
probe to an enzyme reporter can boost the signal (CARD-FISH)
C. Molecular techniques
1. Given the great plate count anomaly, direct examination of molecular sequences is widely used
to assess microbial diversity; efficient extraction of DNA is critical to these techniques
2. Small subunit (SSU) rRNA is commonly used as the target for PCR and the generation of
clone libraries for sequencing; internal transcribed spacer regions (ITS) between rRNA genes
also has been used; PCR biases exist that confound results to some extent
3. Hierarchical oligonucleotide primer extension (HOPE) allows the detection of multiple SSU
rRNA genes in a single sample; different primers (multiplex PCR) having poly-A extensions of
different lengths allow for discrimination of phylotypes
4. rRNA sequences are compared to online databases to group them into phylotypes and identify
closely related cultured species
5. DNA fingerprinting techniques avoid laborious sequencing; denaturing gradient gel
electrophoresis (DGGE) separates PCR amplicons of similar lengths based on melting point of
the DNA fragment, and hence the primary sequence and G+C content; related fingerprinting
techniques include single-strand conformation polymorphism (SSCP) and terminal restriction
fragment length polymorphism (T-RFLP) analyses
6. DNA reassociation—counts the number of different genomes in a community by measuring
the complexity of the DNA extract by the rate at which denatured molecules reassociate
7. Phylochip—essentially a microarray of genes of interest; phylogenetic oligonucleotide arrays
(POAs) use rRNA genes; community genome arrays (CGAs) use functional genes
III. Assessing Microbial Community Activity
A. Biogeochemical approaches
1. Measurement of growth or incorporation of radiolabeled compounds under a variety of
environmental conditions
2. Microelectrodes specific for pH, oxygen tension, hydrogen, hydrogen sulfide, and other
metabolites can be used in situ to measure microbial activities
3. Stable isotope analysis follows microbial nutrient cycling using heavy and stable
(nonradioactive) isotopes measurements to determine the origin of molecules in the
environment; relies on the distinction that enzymes make between various isotopes and
interprets the isotopic fractionation of important metabolites
4. Stable isotope probing adds nutrients labeled with heavy isotopes to monitor biogeochemical
processes
B. Molecular approaches
1. Metagenomics analysis allows the detection of new genes and the measurement of mRNA
pools in microbial communities from the environment; the entire complement of genetic
sequences from a community is cloned for analysis
2. Specific mRNAs can be measured using in situ reverse transcriptase FISH (IRST-FISH); here,
FISH probes are generated from pools of mRNA reverse transcribed to cDNA
3. Determination of which cells are performing certain metabolisms can be used by coupling
FISH to microautoradiography (MAR-FISH) where radioactive substrates are added and
localized
4. Microarray analysis of functional genes (functional gene arrays; FGAs) can be prepared to
examine the metabolic capabilities of communities
5. Reporter microbes have genetic constructs that include green fluorescent protein (or another
beacon) and change their response based on environmental conditions
6. Metaproteomics examines the array of proteins produced by a microbial community using twodimensional gel electrophoresis or nano-liquid chromatography and tandem mass spectroscopy
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TERMS AND DEFINITIONS
Place the letter of each term in the space next to the definition or description that best matches it.
____ 1.
____ 2.
____ 3.
____ 4.
____ 5.
____ 6.
____ 7.
____ 8.
____ 9.
____ 10.
____ 11.
____ 12.
____ 13.
____ 14.
____ 15.
____ 16.
____ 17.
Technique for identifying certain genes or organisms
in which fluorescently labeled DNA fragments are
hybridized to genomic DNA within cells
Used to measure a variety of ions and chemical
compounds at very high spatial resolution
A group of organisms with very similar SSU rRNA
sequences
An technique for separating DNA fragments of similar
size based on their melting points
Cultures inoculated with very high dilutions of the
original sample
A simplified, model ecosystem used to simulate larger
natural systems
A region of DNA found in between rRNA genes that
are useful for phylogenetic analyses
A technique that couples fluorescent in situ
hybridization with microautoradiography of
radiolabeled metabolites for localizing cells of a
specific phylotype, exhibiting a specific metabolism
A group of techniques for studying all of the proteins
expressed by an entire community of microorganisms
to follow biogeochemical cycling
A pure culture, consisting of only one strain of
microorganism
Use of specific culture conditions to select for
microbial strains that fit particular niches
A collection of populations found in the same
ecosystem
A technique that uses an enzyme-linked reporter to
amplify the signal from fluorescent in situ
hybridization labeling
Living environmental microorganisms observed by
microscopy that cannot currently be cultured in the
laboratory
A group of techniques for studying all of the genes
from an entire community of microorganisms
A serial dilution test designed to estimate the number
of target microbes in a sample
A technique that uses compounds labeled with
nonradioactive heavy atoms
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a.
b.
c.
d.
e.
f.
g.
h.
i.
j.
k.
l.
m.
n.
o.
p.
q.
axenic culture
CARD-FISH
community
DGGE
enrichment culture
extinction culture
FISH
Internal transcribed spacer
MAR-FISH
metagenomics
metaproteomics
microcosm
microelectrodes
most probable number
phylotype
stable isotope analysis
viable but not culturable
FILL IN THE BLANK
1.
2.
3.
4.
5.
6.
7.
Living organisms from the environment that do not grow in the laboratory are
and generate a disparity between observed and culturable organisms
called
. Microbial strains from
specific niches can be selected from mixed cultures using the
technique.
The most probable number technique can be used to measure the
of microbes with
specific metabolic abilities in environmental samples. The original sample is first
and then allowed to grow. Positive tubes are scored and the number of microbes in the
original sample is determined using
analyses.
Cultures that contain only a single strain of microorganism are considered
. These can be
started from single cells captured using
. Another source of inoculum can
be cells entrapped in beads, what are known as
. Samples can also be diluted to
to
separate cells into individual cultures.
The FISH technique used DNA probes with
labels to visualize cells that contain
complementary DNA sequences. The labeled cells can be observed using
microscopy. The
signal can be enhanced with enzyme reporters in a technique called
.
Nucleotide sequences from
genes can be used to determine the evolutionary
relationships between microorganisms and group them into
. The regions between these
genes, called
regions can also be used for phylogenetics.
The complexity of microbial communities can be measured as the rate with which DNA
takes place when community genomic extracts are melted and cooled. Microarrays called
can be used to examine microbial community structure. These are called
arrays when rRNA genes are used and
arrays when functional genes are
used.
Examination of all of the proteins expressed in a microbial community is the goal of
.A
complex pattern of proteins can be generated by separation using
. A newer technique,
chromatography is used when qualitative
information is most important.
MULTIPLE CHOICE
For each of the questions below select the one best answer.
1.
2.
3.
Approximately what percentage of
microorganisms from the environment can be
cultured in the laboratory?
a. 1%
b. 10%
c. 50%
d. nearly all
After microdroplet cultures have been
incubated, ____________ is used to examine
and enumerate labeled cells.
a. PCR
b. microscopy
c. flow cytometry
d. optical tweezers
A microbial ___________ is defined as a
group of microorganisms within an
ecosystem that are similar.
a. genus
b. species
c. community
4.
5.
6.
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d. population
For the FISH technique fluorescently labeled
__________ probes are typically used.
a. proteins
b. carbohydrates
c. oligonucleotides
d. antibodies
The identification of more than one type of
PCR product in a single reaction is called:
a. hierarchical extension
b. multiplex PCR
c. MER-FISH
d. PCR bias
The separation of DNA fragments by DGGE
is based on which of the following?
a. protein binding
b. DNA sequence
c. tertiary structure
d. isotopic signature
7.
The ability of a community to carry out
certain biogeochemical activities can be
determined using:
a. functional gene arrays
b.
c.
d.
phylogenetic oligonucleotide arrays
DNA reassociation
DGGE
TRUE/FALSE
____
____
____
____
____
____
____
____
1.
2.
3.
4.
5.
6.
7.
8.
Enrichment cultures are commonly used to grow VBNC microorganisms.
MPN can be used to estimate the abundance of microbes from different functional groups.
In the extinction culture technique, samples are diluted to a density of 1 to 10 cells per ml.
The plate wash PCR technique is useful to analyzing microdroplet cultures.
Axenic cultures are pure cultures of microbes.
Fluorescent stains are valuable to counting microbes in food and clinical samples.
FISH techniques are not used for phylogenetic analyses.
Certain nucleotide templates are more readily amplified than others, even if examining the same
gene.
____ 9. Spacer regions between ribosomal RNA genes are rarely transcribed.
____ 10. Microelectrodes can be used to measure the abundance of specific functional genes in the
environment.
____ 11. Stable isotope analyses are typically performed using radioactive elements with long half-lives.
____ 12. In situ reverse transcriptase FISH measures gene expression by examining specific mRNAs.
CRITICAL THINKING
1.
The description of microbial communities using either cultivation or culture-independent techniques have
drawbacks and advantages. Discuss these with reference to specific techniques. Which techniques would
you choose if asked to define the microbial community in a previously unexamined environment?
2.
While phylogenetic analyses of microbial communities has considerable value, do you feel that this is
suitable for defining the functional abilities of the community? How would you determine which
microbial species are performing certain biogeochemical reactions?
ANSWER KEY
Terms and Definitions
1. g, 2. m, 3. o, 4. d, 5. f, 6. l, 7. h, 8. i, 9. k, 10. a, 11. e, 12. c, 13. b, 14. q, 15. j, 16. n, 17. p
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Fill in the Blank
1. viable but nonculturable; great plate count debate; enrichment culture 2. abundance; serially diluted;
statistical 3. axenic; optical tweezers; microdroplets; extinction 4. fluorescent; epifluorescence; CARD-FISH 5.
SSU rRNA; phylotypes; internal transcribed spacer 6. reassociates; phylochips; phylogenetic oligonucleotide;
community genome 7. metaproteomics; two-dimensional gel electrophoresis; nano-liquid
Multiple Choice
1. a, 2. c, 3. d, 4. c, 5. b, 6. b, 7. a
True/False
1. F, 2. T, 3. T, 4. F, 5. T, 6. T, 7. F, 8. T, 9. F, 10. F, 11. F, 12. T
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