反应详情

温度

30 °C

Culture treated as under A is incubated for about 72 seconds at 30° C. in the shaking machine until it attains a cell titre of >109 per ml of culture solution. 0.1 ml of this cell suspension is transferred into 10 ml of the following nutrient solution (nutrient solution B): 0.05% NaH2PO4, 0.20% K2HPO4, 0.05% MgSO4.7H2O, 0.02% CaCl2.2H2O, 0.005% MnSO4.4H2O, 0.005% (Fe)2SO4.7H2O, 0.10% (NH4)SO4, 0.0001% biotine, 0.10% Tween 80 and 0.10% BNC. After shaking for 18 hours at 30° C. diluted with fresh, preheated nutrient solution of the same composition to about 106 cells per ml, 1000 IU penicillin G are added and incubation for a further 7 hours is carried out. The antibioticum is then removed by centrifuging of the cells with washing with sterile, physiologic sodium chloride solution and the cells further incubated for 3 days in the above-mentioned medium with 0.2% sitosterol instead of BNC. The penicillin treatment is repeated once more and the cell suspension finally plated out on the same nutrient medium A plus 1.6% agar. After the propagation of the cells to visible colonies, the cells are transfered with the aid of the "replica stamp technique" on nutrient medium B plus 1.6% agar and those colonies are selected and used as strain cultures which are not able to continue to grow, or scarecly able, on this medium.

1. additionCulture treated as under
2. additionare added
3. waitincubation for a further 7 hours is carried out
4. customThe antibioticum is then removed by centrifuging of the cells
5. washwith washing with sterile, physiologic sodium chloride solution
6. waitthe cells further incubated for 3 days in the above-mentioned medium with 0.2% sitosterol instead of BNC