Computational protocol: General principles for the formation and proliferation of a wall free (L form) state in bacteria

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Protocol publication

[…] For snapshot microscopy, the different bacterial walled and L-form cells were immobilized on microscope slides covered with a thin film of 1% agarose in NB/MSM. The cells were imaged on a Zeiss Axiovert 200M microscope controlled by Metamorph 6 (Molecular Devices, Sunnyvale, CA) with a Zeiss ×100 Plan-Neofluar oil immersion objective.For time lapse microscopy, C. glutamicum L-form cells were imaged in ibiTreat adherent, 35 mm sterile glass bottom microwell dishes (ibidi GmbH, Munich, Germany). Briefly, an 0.1 ml sample of exponential phase C. glutamicum L-form was added to 0.5 ml of fresh NB/MSM and incubated in the microwell dish for 15 min. The cells were washed three times with NB/MSM, and 0.5 ml of fresh NB/MSM with DCS was finally added. For E. coli, L-form cells were immobilized on microscope slides covered with a thin film of 1% agarose in NB/MSM with FOS. The cells were imaged on a DeltaVision RT microscope (Applied Precision, Issaquah, WA) controlled by softWoRx (Applied Precision) with a Zeiss ×100 apo fluor oil immersion lens. A Weather Station environmental chamber (Precision Control) regulated the temperature of the stage.Pictures and videos were prepared for publication using ImageJ (http://rsb.info.nih.gov/ij) and Adobe Photoshop. […]

Pipeline specifications

Software tools MetaMorph, ImageJ
Application Microscopic phenotype analysis