Applied Microbiology and Biotechnology

, Volume 95, Issue 3, pp 777–788 | Cite as

A method for evaluating the host range of bacteriophages using phages fluorescently labeled with 5-ethynyl-2′-deoxyuridine (EdU)

  • Sayaka Ohno
  • Hironori Okano
  • Yasunori Tanji
  • Akiyoshi Ohashi
  • Kazuya Watanabe
  • Ken Takai
  • Hiroyuki ImachiEmail author
Methods and protocols


The evaluation of bacteriophage (phage) host range is a significant issue in understanding phage and prokaryotic community interactions. However, in conventional methods, such as plaque assay, target host strains must be isolated, although almost all environmental prokaryotes are recalcitrant to cultivation. Here, we introduce a novel phage host range evaluation method using fluorescently labeled phages (the FLP method), which consists of the following four steps: (i) Fluorescently labeled phages are added to a microbial consortium, and host cells are infected and fluorescently labeled. (ii) Fluorescent cells are sorted by fluorescence-activated cell sorting. (iii) 16S rRNA gene sequences retrieved from sorted cells are analyzed, and specific oligonucleotide probes for fluorescence in situ hybridization (FISH) are designed. (iv) Cells labeled with both fluorescently labeled phage and FISH probe are identified as host cells. To verify the feasibility of this method, we used T4 phage and Escherichia coli as a model. We first used nucleic acid stain reagents for phage labeling; however, the reagents also stained non-host cells. Next, we employed the Click-iT EdU (5-ethynyl-2′-deoxyuridine) assay kit from Invitrogen for phage labeling. Using EdU-labeled T4 phage, we could specifically detect E. coli cells in a complex microbial consortium from municipal sewage. We also confirmed that FISH could be applied to the infected E. coli cells. These results suggest that this FLP method using the EdU assay kit is a useful method for evaluating phage host range and may have a potential application for various types of phages, even if their prokaryotic hosts are currently unculturable.


Phage Host range EdU Click chemistry Fluorescence in situ hybridization (FISH) 



We thank Masaru Kawato and Yuto Yashiro for help with flow cytometry techniques; Norika Meguro, Hiromi Kazama, and Masayuki Ehara for the help with 16S rRNA gene analysis; Drs. Kengo Kubota, Myong-Ok Park, Takako Nogami, Hiroshi Tsukamoto, Kazuhiko Miyanaga, Hideki Kobayashi, Tadashi Maruyama, Sanae Sakai, Takuro Nunoura, Aidan J. Synnott, Roland Hatzenpichler, and Jennifer Glass for the helpful discussions and useful comments. We greatly appreciate Professor Hideki Harada for his continuous encouragement. We also thank Yuji Suzuki at the Yokohama city office for his assistance in sampling the Kanazawa-ku municipal sewage treatment plant. This study was partly supported by grants from the Japan Society for the Promotion of Science, and the Ministry of Education, Culture, Sports, Science and Technology, Japan.

Supplementary material

253_2012_4174_MOESM1_ESM.pdf (4.9 mb)
ESM 1 (PDF 5036 kb)


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Copyright information

© Springer-Verlag 2012

Authors and Affiliations

  • Sayaka Ohno
    • 1
    • 2
  • Hironori Okano
    • 1
    • 3
  • Yasunori Tanji
    • 2
  • Akiyoshi Ohashi
    • 4
  • Kazuya Watanabe
    • 5
  • Ken Takai
    • 1
  • Hiroyuki Imachi
    • 1
    Email author
  1. 1.Subsurface Geobiology Advanced Research (SUGAR) Project, Extremobiosphere Research Program, Institute of BiogeosciencesJapan Agency for Marine-Earth Science and Technology (JAMSTEC)YokosukaJapan
  2. 2.Department of BioengineeringTokyo Institute of TechnologyYokohamaJapan
  3. 3.Department of Environmental Systems EngineeringNagaoka University of TechnologyNagaokaJapan
  4. 4.Department of Social and Environmental EngineeringHiroshima UniversityHigashi-HiroshimaJapan
  5. 5.School of Life SciencesTokyo University of Pharmacy and Life SciencesHachiojiJapan

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