Label-free isolation and deposition of single bacterial cells from heterogeneous samples for clonal culturing.
The isolation and analysis of single prokaryotic cells down to 1 μm and less in size poses a special challenge and requires micro-engineered devices to handle volumes in the picoliter to nanoliter range. Here, an advanced Single-Cell Printer (SCP) was applied for automated and label-free isolation and deposition of bacterial cells encapsulated in 35 pl droplets by inkjet-like printing. To achieve this, dispenser chips to generate micro droplets have been fabricated with nozzles 20 μm in size. Further, the magnification of the optical system used for cell detection was increased. Redesign of the optical path allows for collision-free addressing of any flat substrate since no compartment protrudes below the nozzle of the dispenser chip anymore. The improved system allows for deterministic isolation of individual bacterial cells. A single-cell printing efficiency of 93% was obtained as shown by printing fluorescent labeled E. coli. A 96-well plate filled with growth medium is inoculated with single bacteria cells on average within about 8 min. Finally, individual bacterial cells from a heterogeneous sample of E. coli and E. faecalis were isolated for clonal culturing directly on agar plates in user-defined array geometry.
Laboratory for MEMS Applications, Department of Microsystems Engineering-IMTEK, University of Freiburg, Georges-Koehler-Allee 103, 79110 Freiburg, Germany.
J
J
Riba
Laboratory for MEMS Applications, Department of Microsystems Engineering-IMTEK, University of Freiburg, Georges-Koehler-Allee 103, 79110 Freiburg, Germany.
T
T
Gleichmann
Laboratory for MEMS Applications, Department of Microsystems Engineering-IMTEK, University of Freiburg, Georges-Koehler-Allee 103, 79110 Freiburg, Germany.
S
S
Zimmermann
Laboratory for MEMS Applications, Department of Microsystems Engineering-IMTEK, University of Freiburg, Georges-Koehler-Allee 103, 79110 Freiburg, Germany.|Hahn-Schickard, Georges-Koehler-Allee 103, 79110 Freiburg, Germany.|BIOSS - Centre for Biological Signalling Studies, University of Freiburg, 79110 Freiburg, Germany.
R
R
Zengerle
Laboratory for MEMS Applications, Department of Microsystems Engineering-IMTEK, University of Freiburg, Georges-Koehler-Allee 103, 79110 Freiburg, Germany.
P
P
Koltay
D018929:Cell Culture Techniques / Q000295:instrumentation* / Q000379:methods*; D002468:Cell Physiological Phenomena; D002469:Cell Separation / Q000295:instrumentation / Q000379:methods*; D002470:Cell Survival; D004926:Escherichia coli / Q000166:cytology* / Q000502:physiology; D011327:Printing; D059010:Single-Cell Analysis / Q000295:instrumentation* / Q000379:methods*