Water-in-oil droplet-mediated method for detecting and isolating infectious bacteriophage particles via fluorescent staining
Bacteriophages are the most abundant entities on Earth. In contrast with the number of phages considered to be in existence, current phage isolation and screening methods lack throughput. Droplet microfluidic technology has been established as a platform for high-throughput screening of biological a...
| Main Authors: | , , , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
Frontiers Media S.A.
2023-12-01
|
| Series: | Frontiers in Microbiology |
| Subjects: | |
| Online Access: | https://www.frontiersin.org/articles/10.3389/fmicb.2023.1282372/full |
| _version_ | 1797403219581730816 |
|---|---|
| author | Miu Hoshino Miu Hoshino Yuri Ota Yuri Ota Tetsushi Suyama Yuji Morishita Satoshi Tsuneda Naohiro Noda Naohiro Noda Naohiro Noda |
| author_facet | Miu Hoshino Miu Hoshino Yuri Ota Yuri Ota Tetsushi Suyama Yuji Morishita Satoshi Tsuneda Naohiro Noda Naohiro Noda Naohiro Noda |
| author_sort | Miu Hoshino |
| collection | DOAJ |
| description | Bacteriophages are the most abundant entities on Earth. In contrast with the number of phages considered to be in existence, current phage isolation and screening methods lack throughput. Droplet microfluidic technology has been established as a platform for high-throughput screening of biological and biochemical components. In this study, we developed a proof-of-concept method for isolating phages using water-in-oil droplets (droplets) as individual chambers for phage propagation and co-cultivating T2 phage and their host cell Escherichia coli within droplets. Liquid cultivation of microbes will facilitate the use of microbes that cannot grow on or degrade agar as host cells, ultimately resulting in the acquisition of phages that infect less known bacterial cells. The compartmentalizing characteristic of droplets and the use of a fluorescent dye to stain phages simultaneously enabled the enumeration and isolation of viable phage particles. We successfully recultivated the phages after simultaneously segregating single phage particles into droplets and inoculating them with their host cells within droplets. By recovering individual droplets into 96-well plates, we were able to isolate phage clones derived from single phage particles. The success rate for phage recovery was 35.7%. This study lays the building foundations for techniques yet to be developed that will involve the isolation and rupturing of droplets and provides a robust method for phage enumeration and isolation. |
| first_indexed | 2024-03-09T02:36:19Z |
| format | Article |
| id | doaj.art-5c1a1134bad649d9861b3583d1e1326e |
| institution | Directory Open Access Journal |
| issn | 1664-302X |
| language | English |
| last_indexed | 2024-03-09T02:36:19Z |
| publishDate | 2023-12-01 |
| publisher | Frontiers Media S.A. |
| record_format | Article |
| series | Frontiers in Microbiology |
| spelling | doaj.art-5c1a1134bad649d9861b3583d1e1326e2023-12-06T08:31:08ZengFrontiers Media S.A.Frontiers in Microbiology1664-302X2023-12-011410.3389/fmicb.2023.12823721282372Water-in-oil droplet-mediated method for detecting and isolating infectious bacteriophage particles via fluorescent stainingMiu Hoshino0Miu Hoshino1Yuri Ota2Yuri Ota3Tetsushi Suyama4Yuji Morishita5Satoshi Tsuneda6Naohiro Noda7Naohiro Noda8Naohiro Noda9Department of Computational Biology and Medical Sciences, Graduate School of Frontier Sciences, The University of Tokyo, Chiba, JapanBiomedical Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Ibaraki, JapanBiomedical Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Ibaraki, JapanOn-chip Biotechnologies Co., Ltd., Tokyo, JapanBiomedical Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Ibaraki, JapanOn-chip Biotechnologies Co., Ltd., Tokyo, JapanDepartment of Life Science and Medical Bioscience, School of Advanced Science and Engineering, Waseda University, Tokyo, JapanDepartment of Computational Biology and Medical Sciences, Graduate School of Frontier Sciences, The University of Tokyo, Chiba, JapanBiomedical Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Ibaraki, JapanDepartment of Life Science and Medical Bioscience, School of Advanced Science and Engineering, Waseda University, Tokyo, JapanBacteriophages are the most abundant entities on Earth. In contrast with the number of phages considered to be in existence, current phage isolation and screening methods lack throughput. Droplet microfluidic technology has been established as a platform for high-throughput screening of biological and biochemical components. In this study, we developed a proof-of-concept method for isolating phages using water-in-oil droplets (droplets) as individual chambers for phage propagation and co-cultivating T2 phage and their host cell Escherichia coli within droplets. Liquid cultivation of microbes will facilitate the use of microbes that cannot grow on or degrade agar as host cells, ultimately resulting in the acquisition of phages that infect less known bacterial cells. The compartmentalizing characteristic of droplets and the use of a fluorescent dye to stain phages simultaneously enabled the enumeration and isolation of viable phage particles. We successfully recultivated the phages after simultaneously segregating single phage particles into droplets and inoculating them with their host cells within droplets. By recovering individual droplets into 96-well plates, we were able to isolate phage clones derived from single phage particles. The success rate for phage recovery was 35.7%. This study lays the building foundations for techniques yet to be developed that will involve the isolation and rupturing of droplets and provides a robust method for phage enumeration and isolation.https://www.frontiersin.org/articles/10.3389/fmicb.2023.1282372/fullbacteriophagewater-in-oil dropletsdroplet isolationplaque assaybacteriophage screening |
| spellingShingle | Miu Hoshino Miu Hoshino Yuri Ota Yuri Ota Tetsushi Suyama Yuji Morishita Satoshi Tsuneda Naohiro Noda Naohiro Noda Naohiro Noda Water-in-oil droplet-mediated method for detecting and isolating infectious bacteriophage particles via fluorescent staining Frontiers in Microbiology bacteriophage water-in-oil droplets droplet isolation plaque assay bacteriophage screening |
| title | Water-in-oil droplet-mediated method for detecting and isolating infectious bacteriophage particles via fluorescent staining |
| title_full | Water-in-oil droplet-mediated method for detecting and isolating infectious bacteriophage particles via fluorescent staining |
| title_fullStr | Water-in-oil droplet-mediated method for detecting and isolating infectious bacteriophage particles via fluorescent staining |
| title_full_unstemmed | Water-in-oil droplet-mediated method for detecting and isolating infectious bacteriophage particles via fluorescent staining |
| title_short | Water-in-oil droplet-mediated method for detecting and isolating infectious bacteriophage particles via fluorescent staining |
| title_sort | water in oil droplet mediated method for detecting and isolating infectious bacteriophage particles via fluorescent staining |
| topic | bacteriophage water-in-oil droplets droplet isolation plaque assay bacteriophage screening |
| url | https://www.frontiersin.org/articles/10.3389/fmicb.2023.1282372/full |
| work_keys_str_mv | AT miuhoshino waterinoildropletmediatedmethodfordetectingandisolatinginfectiousbacteriophageparticlesviafluorescentstaining AT miuhoshino waterinoildropletmediatedmethodfordetectingandisolatinginfectiousbacteriophageparticlesviafluorescentstaining AT yuriota waterinoildropletmediatedmethodfordetectingandisolatinginfectiousbacteriophageparticlesviafluorescentstaining AT yuriota waterinoildropletmediatedmethodfordetectingandisolatinginfectiousbacteriophageparticlesviafluorescentstaining AT tetsushisuyama waterinoildropletmediatedmethodfordetectingandisolatinginfectiousbacteriophageparticlesviafluorescentstaining AT yujimorishita waterinoildropletmediatedmethodfordetectingandisolatinginfectiousbacteriophageparticlesviafluorescentstaining AT satoshitsuneda waterinoildropletmediatedmethodfordetectingandisolatinginfectiousbacteriophageparticlesviafluorescentstaining AT naohironoda waterinoildropletmediatedmethodfordetectingandisolatinginfectiousbacteriophageparticlesviafluorescentstaining AT naohironoda waterinoildropletmediatedmethodfordetectingandisolatinginfectiousbacteriophageparticlesviafluorescentstaining AT naohironoda waterinoildropletmediatedmethodfordetectingandisolatinginfectiousbacteriophageparticlesviafluorescentstaining |