Editing DSMZ Cells (section)

=== Solid&nbsp; Chinese Method ===

To determine the best agar (Beijing Shuangxuan Co., Beijing, China) concentration for plating efficiency, 1.0%, 1.5% or 2.0% (w/v) of cell-imbedded TMB-starch top agar was tried on 2.0% TMB-starch bottom agar. The sterile non-reduced TMB-starch agar was melted by heating before being reduced and dispensed into autoclaved 100-mL tissue culture flasks in the anaerobic chamber. The flasks were then sealed with butyl rubber stoppers that were sealed with open-top closures to allow manipulation with syringes, and were laid on their flat sides. The inoculum was diluted in a syringe to the desired concentration, and a sample was injected into 4 mL melted-top TMB-starch agar in Hungate tubes. The cell-imbedded top agar was then immediately transferred by syringe into flasks, and was spread equally on the bottom layer. Direct plating was performed as described previously ([http://femsle.oxfordjournals.org/content/259/2/254.long#ref-5 Braun''et al.'',1979]). Inoculated flasks were incubated upside-down at 72°C. The gas-tight flasks with bottom layers, and the Hungate tubes with 4 mL top media were stored at 4°C. Single colonies were selected using a sterile 1 mL syringe equipped with a 15 cm stainless needle with 90° bent and flattened tips. Autoclaving was typically performed at 121°C for 40 min.


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