In the steel wool method, the better results were obtained by the addition of CO₂ gas.
Concerning the volume of CO₂ gas, when 10, 50, 80, 98% of jar volume were added, the results were nearly equal, and even though the volume was over 10%, the results did not fall.
Moreover if the volume of CO₂ gas was equal, the results obtained under the air pressure of -600mmHg or more were better than those obtained under the air pressure of -130mmHg or -380mmHg.
So from the viewpoint of the growth degree, the most effective results would be obtained by the following method which is combined with the report of Azuma et al. (1962, in this journal).
To 500ml of 1.2% CuSO₄⋅7H₂O solution, pH 1.5 adjusted by H₂SO₄, to which is added 0.4g of tween 80, 10g of steel wool grade 0 are poured and shake well untill the decoloration of CuSO₄ solution may occur.
Then the steel wool which has been gilded with cupper is quickly transferred to a jar having a 3-liter volume, and the air pressure is quickly reduced to -650mmHg by a vacuum pump and then the CO₂ gas is added to a 76 to 650mmHg pressure.