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Prescott’s Microbiology, 9th Edition
29
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.
LEARNING OUTCOMES
After reading this chapter you should be able to:
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explain why, to the best of our knowledge, most microorganisms resist culturing in the laboratory
describe the use of enrichment cultures
state when and how to apply the most probable number (MPN) method
describe at least two innovative approaches used to coax growth in the laboratory of previously
uncultured microbes
explain the fundamentals of flow cytometry
explain why microbial communities are often investigated in situ
describe why, when, and how FISH and CARD-FISH are used
summarize how PCR is used to take a microbial census
explain why and how DGGE is used
assess the advantages and disadvantages of DNA association studies
compare and contrast the use of phylochips with other microarray-based technologies designed to assess
microbial communities
predict which techniques would be appropriate for assessing the microbial community in a gram of forest
soil and in a milliliter of seawater
explain why microbial ecologists measure community activity
describe why microelectrode measurements are important tools in the study of microbial community
activity
compare the application of traditional stable isotope analysis with stable isotope probing
assess the advantages and disadvantages of measuring in situ with mRNA abundance
list the type of data that can be generated by MAR-FISH and compare this with functional gene arrays
describe the construction and application of reporter microbes
differentiate metagenomics from metaproteomics
CHAPTER OUTLINE
I.
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
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in any manner. This document may not be copied, scanned, duplicated, forwarded, distributed, or posted on a website, in whole or part.
Prescott’s Microbiology, 9th Edition
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
II. 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
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
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© 2014 by McGraw-Hill Education. This is proprietary material solely for authorized instructor use. Not authorized for sale or distribution
in any manner. This document may not be copied, scanned, duplicated, forwarded, distributed, or posted on a website, in whole or part.
Prescott’s Microbiology, 9th Edition
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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
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
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
Microarray analysis of functional genes (functional gene arrays; FGAs) can be prepared to
examine the metabolic capabilities of communities
Reporter microbes have genetic constructs that include green fluorescent protein (or another
beacon) and change their response based on environmental conditions
Metaproteomics examines the array of proteins produced by a microbial community using twodimensional gel electrophoresis or nano-liquid chromatography and tandem mass spectroscopy
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?
3.
What genes could be used as probes in the development of a functional microarray to determine the
microbial activity in a nitrogen limited aquatic ecosystem?
4.
What are some of the pitfalls and limitations involved in enrichment techniques to estimate populations
of bacteria in the analysis of new ecosystems?
CONCEPT MAPPING CHALLENGE
Construct a concept map using the words below: provide your own linking words.
Enrichment culture
Community activity MAR-FISH
In-situ analysis
Axenic culture
Microelectrodes
Chemostat Microdroplet culture Stable isotope analysis FISH Phylochips
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© 2014 by McGraw-Hill Education. This is proprietary material solely for authorized instructor use. Not authorized for sale or distribution
in any manner. This document may not be copied, scanned, duplicated, forwarded, distributed, or posted on a website, in whole or part.