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Batani, Giampiero; Bayer, Kristina; Böge, Julia; Hentschel, Ute; Thomas, Torsten (2020): Cell counts (CFUs) and survival rate of cells using the initial modified FFF protocol (compared to control) after each protocol step. PANGAEA, https://doi.org/10.1594/PANGAEA.911495

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Abstract:
Despite the development of several cultivation methods, the rate of discovery of microorganisms that are yet-to-be cultivated outpaces the rate of isolating and cultivating novel species in the laboratory. Furthermore, no current cultivation technique is capable of selectively isolating and cultivating specific bacterial taxa or phylogenetic groups independently of morphological or physiological properties. Here, we developed a new method to isolate living bacteria solely based on their 16S rRNA gene sequence. We showed that bacteria can survive a modified version of the standard fluorescence in situ hybridization (FISH) procedure, in which fixation is omitted and other factors, such as centrifugation and buffers, are optimized. We also demonstrated that labeled DNA probes can be introduced into living bacterial cells using chemical transformation and that specific hybridization occurs. This new method, which we call live-FISH, was then combined with fluorescence-activated cell sorting (FACS) to sort specific taxonomic groups of bacteria from mock and natural bacterial communities and subsequently culture them. Live-FISH represents the first attempt to systematically optimize conditions known to affect cell viability during FISH and then to sort bacterial cells surviving the procedure. No sophisticated probe design is required, making live-FISH a straightforward method to be potentially used in combination with other single-cell techniques and for the isolation and cultivation of new microorganisms.
Keyword(s):
Bacillus; FACS; Live-FISH; Pseudovibrio
Supplement to:
Batani, Giampiero; Bayer, Kristina; Böge, Julia; Hentschel, Ute; Thomas, Torsten (2019): Fluorescence in situ hybridization (FISH) and cell sorting of living bacteria. Scientific Reports, 9(1), https://doi.org/10.1038/s41598-019-55049-2
Parameter(s):
#NameShort NameUnitPrincipal InvestigatorMethod/DeviceComment
1TreatmentTreatBayer, KristinaFISH steps
2TableTabBayer, Kristina
3Bacteria, abundance in colony forming unitsBacteria CFUCFU/mlBayer, KristinaBacillus sp. AU29
4SurvivalSurvival%Bayer, KristinaBacillus sp. AU29
5Bacteria, abundance in colony forming unitsBacteria CFUCFU/mlBayer, KristinaRuegeria sp. AU82
6SurvivalSurvival%Bayer, KristinaRuegeria sp. AU82
7Bacteria, abundance in colony forming unitsBacteria CFUCFU/mlBayer, KristinaPseudovibrio sp. SB55
8SurvivalSurvival%Bayer, KristinaPseudovibrio sp. SB55
Size:
65 data points

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