Strain mA of Mycoplasma pulmonis, one of the isolates from the bronchi of normal mice, grew readily in the tissue culture medium (YLE) currently used at the authors' laboratory. Since this strain was known to utilize glucose, an attempt was made to titrate it in YLE by the aid of color change of phenol red which had been added to the medium as an indicator. The full titer determined by this method was comparable with that by the conventional colony counting method. The period required to obtain an end point was 3 or 4 days in the new method, while it was 1 week in the conventional method. A serum concentration of 2% was enough to obtain the full titer. Without serum, however, no color change occurred during an observation period of 2 weeks. A starting pH of 7.2 was proved to be the most sensitive in the assay system. When YLE (pH 7.2) was compared with Chanock's PPLO broth, the superiority of the former was evident, since the period required for the end point to appear was between 3 and 4 days in the former and between 5 and 6 days in the latter. It was possible to detect a single growth unit from a material by the method with YLE, so long as the material had been filtered through an HA type of millipore filter.
The present investigation was made to clarify the action of adrenalin on the formation of inosine, which was identified as antibody-promoting factor. The results obtained were as follows. Adrenalin displayed its action at two locations in the course of metabolic degradation of ATP and adenosine. At one location, it accelerated the degradation from ATP or adenosine to inosine and hypoxanthine, and at the other it inhibited the conversion from inosine and hypoxanthine to xanthine. Especially, when adrenalin and adenosine were used together, inosine and hypoxanthine were found to have increased about 1.7 times after incubation for 40-60 minutes, but xanthine did not behave in the same manner. The optimal rate of adrenalin was 100μg per ml of basic medium.
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.