Preparation of the Master Stock (Strain P2 (DSM1617) Provided by DSMZ) (SOP SSO 060907A)

Preparation of the master stock (strain P2 (DSM1617) provided by DSMZ) (SOP_SSO_060907a) 1) The cell pellet is dissolved in 1 ml medium containing 0.4% glucose and 0.1% tryptone as carbon and nitrogen source. The medium has preferably a higher pH (4.5) than the standard medium (pH 3.5). The cell suspension is incubated for 30 min at 20°C. 2) The suspension is split into two equal batches (in 50 ml tubes). 3) Add 5.0 ml medium to each batch. 4) Prepare 1:10 and 1:100 dilutions (this is a safety measure – in case of growth in the 1:1, the dilutions can be discarded). 5) Incubate all of them without mixing for 3 days at 79°C. 6) Centrifuge the 1:1 dilutions (8000 g, 12 min, 4°C). 7) Discard the supernatant and immediately re-suspend the pellet in 1.0 ml of medium. In order to keep the cells easier to revive, higher pH is recommended (4.5 – 4.8). See notes. 8) Transfer the suspension to sterile tubes containing 0.5 ml of 60% glycerol and store at -80°C. 9) After a couple of hours – check the stock by inoculating a culture from it.

Notes: C and N sources are added to make the conditions less stressful as compared with the minimal medium used in a standard cultivation. It also allows adaptation to glucose which would enable a switch to glucose as the only C source.

Higher pH (4.5 – 4.8) prevents the cells from too high intercellular H + content while pump systems of the cells are inactive. Cells while frozen stop keeping the proton gradient (although the cells grow in pH of 3.0 – 4.0, intracellular pH is estimated at 5.8) To check the stock – scratch the inside of the tube with a sterile yellow tip and drop the tip into 50 ml long neck flask containing 25 ml standard, pre-warmed medium (to enhance growth it might also be the 0.3% glucose 0.1% tryptone variant).

Growth from master stock takes more time than usual – lag phase might take up to a week. Master stock should only be used in order to prepare new working stock.

Preparation of the working stocks from the master stock (strain P2, DSM1617) (SOP_SSO_060907b) 1) Inoculate 50 ml long neck flask containing 25 ml standard, pre-warmed medium with 100μl of master stock (add no tryptone; for details, see notes to master stock). 2) Grow till the OD600 of 1.5. 3) Transfer the tube to a pre-set fermenter of 1.5L. (with the size of the fermenter the starting culture should be respectively increased) 4) At OD600 of 1.0, harvest the content of the fermenter to 400 ml centrifuge flasks on iced water. 5) Centrifuge (30 min, 4000 xg, 4°C) 6) Discard the supernatant and re-suspend in a 4:1 mixture of medium (pH 4.5 – 4.8) and 80% glycerol (4.0 ml of mixture per every 100 ml of culture used). 7) Divide the final mixture into Eppendorf tubes in 50 µl aliquots. 8) Store at -80°C. Note: Aliquot can be used to inoculate both 25 and 50 ml of medium.