Skip to main content
  • ASM
    • Antimicrobial Agents and Chemotherapy
    • Applied and Environmental Microbiology
    • Clinical Microbiology Reviews
    • Clinical and Vaccine Immunology
    • EcoSal Plus
    • Eukaryotic Cell
    • Infection and Immunity
    • Journal of Bacteriology
    • Journal of Clinical Microbiology
    • Journal of Microbiology & Biology Education
    • Journal of Virology
    • mBio
    • Microbiology and Molecular Biology Reviews
    • Microbiology Resource Announcements
    • Microbiology Spectrum
    • Molecular and Cellular Biology
    • mSphere
    • mSystems
  • Log in
  • My alerts
  • My Cart

Main menu

  • Home
  • Articles
    • Current Issue
    • Accepted Manuscripts
    • COVID-19 Special Collection
    • Archive
    • Minireviews
  • For Authors
    • Submit a Manuscript
    • Scope
    • Editorial Policy
    • Submission, Review, & Publication Processes
    • Organization and Format
    • Errata, Author Corrections, Retractions
    • Illustrations and Tables
    • Nomenclature
    • Abbreviations and Conventions
    • Publication Fees
    • Ethics Resources and Policies
  • About the Journal
    • About AEM
    • Editor in Chief
    • Editorial Board
    • For Reviewers
    • For the Media
    • For Librarians
    • For Advertisers
    • Alerts
    • RSS
    • FAQ
  • Subscribe
    • Members
    • Institutions
  • ASM
    • Antimicrobial Agents and Chemotherapy
    • Applied and Environmental Microbiology
    • Clinical Microbiology Reviews
    • Clinical and Vaccine Immunology
    • EcoSal Plus
    • Eukaryotic Cell
    • Infection and Immunity
    • Journal of Bacteriology
    • Journal of Clinical Microbiology
    • Journal of Microbiology & Biology Education
    • Journal of Virology
    • mBio
    • Microbiology and Molecular Biology Reviews
    • Microbiology Resource Announcements
    • Microbiology Spectrum
    • Molecular and Cellular Biology
    • mSphere
    • mSystems

User menu

  • Log in
  • My alerts
  • My Cart

Search

  • Advanced search
Applied and Environmental Microbiology
publisher-logosite-logo

Advanced Search

  • Home
  • Articles
    • Current Issue
    • Accepted Manuscripts
    • COVID-19 Special Collection
    • Archive
    • Minireviews
  • For Authors
    • Submit a Manuscript
    • Scope
    • Editorial Policy
    • Submission, Review, & Publication Processes
    • Organization and Format
    • Errata, Author Corrections, Retractions
    • Illustrations and Tables
    • Nomenclature
    • Abbreviations and Conventions
    • Publication Fees
    • Ethics Resources and Policies
  • About the Journal
    • About AEM
    • Editor in Chief
    • Editorial Board
    • For Reviewers
    • For the Media
    • For Librarians
    • For Advertisers
    • Alerts
    • RSS
    • FAQ
  • Subscribe
    • Members
    • Institutions
Microbial Ecology

New Strategies for Cultivation and Detection of Previously Uncultured Microbes

Bradley S. Stevenson, Stephanie A. Eichorst, John T. Wertz, Thomas M. Schmidt, John A. Breznak
Bradley S. Stevenson
Department of Microbiology and Molecular Genetics, Michigan State University, East Lansing, Michigan
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
  • For correspondence: steven77@msu.edu
Stephanie A. Eichorst
Department of Microbiology and Molecular Genetics, Michigan State University, East Lansing, Michigan
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
John T. Wertz
Department of Microbiology and Molecular Genetics, Michigan State University, East Lansing, Michigan
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Thomas M. Schmidt
Department of Microbiology and Molecular Genetics, Michigan State University, East Lansing, Michigan
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
John A. Breznak
Department of Microbiology and Molecular Genetics, Michigan State University, East Lansing, Michigan
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
DOI: 10.1128/AEM.70.8.4748-4755.2004
  • Article
  • Figures & Data
  • Info & Metrics
  • PDF
Loading

Article Figures & Data

Figures

  • Tables
  • FIG. 1.
    • Open in new tab
    • Download powerpoint
    FIG. 1.

    PWPCR method to detect growth and monitor isolation of targeted bacteria. Of the three medium and incubation conditions shown in this diagram (conditions A, B, and C), growth of targeted bacteria (+) is represented only in condition C.

  • FIG. 2.
    • Open in new tab
    • Download powerpoint
    FIG. 2.

    Detection of V. spinosum in a collection of diverse bacteria isolated from soil. (A) A single V. spinosum colony is shown among 94 other colonies growing on an agar plate. (B) PWPCR with Verrucomicrobia-specific primers using template in which V. spinosum colony material represented 1 part in 95 parts (i.e., plate in panel A; lane 1), 1 part in 189 parts (lane 2), 1 part in 471 parts (lane 3), 1 part in 941 parts (lane 4), and 1 part in 9,401 parts (lane 5). PWPCR of a control plate lacking V. spinosum (lane 6), negative control (no DNA; lane 7), V. spinosum DNA (50 ng; lane 8), and molecular size markers (lane M) is also shown. The positions of molecular size markers (in kilobases) are shown to the left of the gel.

  • FIG. 3.
    • Open in new tab
    • Download powerpoint
    FIG. 3.

    Influence of medium additives and incubation conditions on CFU recovered from soil (A) and on the occurrence of Acidobacteria among the isolates (B). “Air” is actually the concentration of CO2 (0.03% [vol/vol]) in normal air. Data in panel A represent the mean CFU recovered from soil among n samples. Error bars represent sample standard deviations. Data in panel B represent the frequency with which Acidobacteria were detected among the plates in panel A using PWPCR. These data were subjected to chi-square analyses with Bonferroni's error rate adjustment. Statistical significance (α = 0.10, df = 1) is indicated by an asterisk.

  • FIG. 4.
    • Open in new tab
    • Download powerpoint
    FIG. 4.

    (Left) Maximum-likelihood tree of subdivisions 1 to 4 of the phylum Acidobacteria based on 16S rRNA gene sequences from organisms in culture and PCR-generated clones from soil. Isolate designations and accession numbers are given. Isolates obtained in this study are shown in boldface type. Bootstrap values for branch points of the major subdivisions are given. Branch points conserved in all analyses with bootstrap values of >75% (closed circles) and bootstrap values of 50 to 74% (open circles) are indicated. Subdivisions 2 to 4 are labeled, bracketed, and condensed as shaded trapezia with the number of sequences represented in parentheses. 16S rRNA gene sequences of members of subdivisions 6 to 8 were used as outgroups (not shown). The scale bar represents 0.10 change per nucleotide. (Right) The scanning electron micrograph shows KBS89 cells trapped in an extracellular matrix.

  • FIG. 5.
    • Open in new tab
    • Download powerpoint
    FIG. 5.

    (A) Maximum-likelihood tree of subdivisions 1 to 4 and 6 of the phylum Verrucomicrobia based on 16S rRNA gene sequences from organisms in culture and PCR-generated clones from environmental samples. Isolate designations and accession numbers are given. Isolates obtained in this study are shown in boldface type. Bootstrap values for branch points of the major subdivisions are given. Branch points conserved in all analyses with bootstrap values of >75% (closed circles) and bootstrap values of 50 to 74% (open circles) are indicated. Subdivisions 1 to 3 and 6 are labeled, bracketed, and condensed as shaded trapezia with the number of represented sequences in parentheses to simplify presentation of the tree. 16S rRNA gene sequences of members of the phylum Planctomycetes were used as an outgroup (not shown). The scale bar represents 0.10 change per nucleotide. (B and C) The scanning electron micrograph of TAV2 (B) shows the doublet cell morphology shared by all TAV isolates, and that of TAV1 (C) shows the encapsulation of the cells in an extracellular matrix, a morphological feature not shared by TAV2, TAV3, or TAV4.

Tables

  • Figures
  • TABLE 1.

    PCR primers used in this study

    PrimerPositionsaSequence (5′→3′)Annealing tempbReference or source
    F2 CAG TCA CGA CGT TGT AAA ACG ACG GC 62.0 28
    R4*c CAG GAA ACA GCT ATG ACC ATG 28
    Acd31F15-31 GAT CCT GGC TCA GAA TC 56.7 3
    Ver53F37-53 TGG CGG CGT GGW TAA GA 61.0D. Buckleyd
    1492R1492-1510 GGT TAC CTT GTT ACG ACT T 30
    • ↵ a The positions of the target region are given using E. coli numbering system of the 16S rRNA gene. Primers F2 and R4* complement regions of the multiple cloning site of pCR2.1 or pCR4.0.

    • ↵ b Annealing temperatures optimized for the conditions used in this study are given for the forward primer of each primer pair.

    • ↵ c Primer R4* was modified from the primer in reference 28 by omission of five deoxynucleotides from the 5′ end.

    • ↵ d Personal communication.

PreviousNext
Back to top
Download PDF
Citation Tools
New Strategies for Cultivation and Detection of Previously Uncultured Microbes
Bradley S. Stevenson, Stephanie A. Eichorst, John T. Wertz, Thomas M. Schmidt, John A. Breznak
Applied and Environmental Microbiology Aug 2004, 70 (8) 4748-4755; DOI: 10.1128/AEM.70.8.4748-4755.2004

Citation Manager Formats

  • BibTeX
  • Bookends
  • EasyBib
  • EndNote (tagged)
  • EndNote 8 (xml)
  • Medlars
  • Mendeley
  • Papers
  • RefWorks Tagged
  • Ref Manager
  • RIS
  • Zotero
Print

Alerts
Sign In to Email Alerts with your Email Address
Email

Thank you for sharing this Applied and Environmental Microbiology article.

NOTE: We request your email address only to inform the recipient that it was you who recommended this article, and that it is not junk mail. We do not retain these email addresses.

Enter multiple addresses on separate lines or separate them with commas.
New Strategies for Cultivation and Detection of Previously Uncultured Microbes
(Your Name) has forwarded a page to you from Applied and Environmental Microbiology
(Your Name) thought you would be interested in this article in Applied and Environmental Microbiology.
CAPTCHA
This question is for testing whether or not you are a human visitor and to prevent automated spam submissions.
Share
New Strategies for Cultivation and Detection of Previously Uncultured Microbes
Bradley S. Stevenson, Stephanie A. Eichorst, John T. Wertz, Thomas M. Schmidt, John A. Breznak
Applied and Environmental Microbiology Aug 2004, 70 (8) 4748-4755; DOI: 10.1128/AEM.70.8.4748-4755.2004
del.icio.us logo Digg logo Reddit logo Twitter logo CiteULike logo Facebook logo Google logo Mendeley logo
  • Top
  • Article
    • ABSTRACT
    • MATERIALS AND METHODS
    • RESULTS AND DISCUSSION
    • ACKNOWLEDGMENTS
    • FOOTNOTES
    • REFERENCES
  • Figures & Data
  • Info & Metrics
  • PDF

KEYWORDS

bacteria
Digestive System
Isoptera
soil microbiology

Related Articles

Cited By...

About

  • About AEM
  • Editor in Chief
  • Editorial Board
  • Policies
  • For Reviewers
  • For the Media
  • For Librarians
  • For Advertisers
  • Alerts
  • RSS
  • FAQ
  • Permissions
  • Journal Announcements

Authors

  • ASM Author Center
  • Submit a Manuscript
  • Article Types
  • Ethics
  • Contact Us

Follow #AppEnvMicro

@ASMicrobiology

       

ASM Journals

ASM journals are the most prominent publications in the field, delivering up-to-date and authoritative coverage of both basic and clinical microbiology.

About ASM | Contact Us | Press Room

 

ASM is a member of

Scientific Society Publisher Alliance

 

American Society for Microbiology
1752 N St. NW
Washington, DC 20036
Phone: (202) 737-3600

Copyright © 2021 American Society for Microbiology | Privacy Policy | Website feedback

 

Print ISSN: 0099-2240; Online ISSN: 1098-5336