TRANSFORMATION OF BACTERIA INTO L FORMS BY AMINO ACIDS,
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the alkaline growth of a few nonlactose fermenting cultures (probably those related to Escherichia) became green when suspended in the bromthymol blue solution. Many methods were tried for studying this phenomenon: rapid transfer of young cultures to reduce the amount of hydrogen ions carried over from the previous growth; addition of buffer to the culture medium; variations in the bromthymol solution, in an effort to control diffusion of the intracellular hydroxyl ions; microscopic examination of the cells under different conditions; applying the bromthymol blue solution in a "Chapman cup" (J. Bact., 63, 147, 1952); resuspension of centrifuged packed cells; and treatment of ultrafiltered cells. Obviously the cells can be washed only in a solution which does not cause diffusion of the intracellular hydroxyl ions. The simplest method was found to be as follows: spread the growth on the surface of a plate of tergitol-7 agar and incubate up to 6 hours at 37 C. Collect a loopful of the growth and emulsify it in 0.5 ml of 0.01 per cent bromthymol blue adjusted to its pK value. Shake gently and examine after standing a few minutes. Cultures which have an intracellular acid reaction are green; those with an alkaline reaction are blue. The results depend on the balance between the intraand extracellular H and OH ions.