The separation of an inhibitor for Neisseria gonorrhoeae from commercial agars was attempted on the basis of previous work by Mueller and Hinton (1941) and Gould, Kane, and Mueller (1944). In addition, this inhibitor was compared with certain organic compounds of a similar nature by biologic tests of inhibitory activity. Until the medium of Mueller and Hinton (1941) was developed, it was thought impossible to grow N. gonorrhoeae on a simple, well-defined medium, but these investigators showed that casein hydrolyzate, meat extract, agar, and starch supported growth of the organisms as well as chocolate blood agar or ascitic fluid agar. Gould, Kane, and Mueller (1944) further showed that a fluid medium containing casein hydrolyzate, sodium chloride, magnesium sulfate, and phosphate buffer would support growth of N. gonorrhoeae, and that a similar medium with agar added would fail to grow the organisms. The addition of starch to this last medium again permitted growth, which led the authors to the hypothesis that starch neutralized an inhibitor present in the agar. Frantz (1942) also developed a similar simple fluid medium for a closely related organism, Neisseria intracellularis, and observed inhibition of growth of the organisms upon addition of agar to the medium. The experimental work which follows was begun in order to prove the hypothesis of inhibition of N. gonorrhoeae by agar, or a substance in it, that was proposed by Gould, Kane, and Mueller. EXPERIMENTAL Separation of the inhibitor from agar. In the following preparations Merck's U.S.P. agar-agar shreds were used throughout. It was decided to attempt the separation of the inhibitor from the agar by the customary methods of dialysis and extraction. Hydrated agar was dialyzed through viscose tubing against running tap water for 3 days, and a similar sample of agar was electrodialyzed, using viscose partitions, for a similar length of time. Solid hydrated agar, cut in 1.0-cm cubes, was extracted for 7 days by direct contact with large volumes of distilled water, 0.1N HCI, 0.1N NaOH, and 95 per cent ethanol. A series of 5.0-g lots of dry, shredded agar was extracted for 3 days each in an all-glass Soxhlet apparatus, having an extraction chamber of 100-ml capacity and 40-mm diameter, with 250 ml of each of the following freshly distilled cp solvents: diethyl ether, acetone, benzene, chloroform, ethanol, and methanol. The treated agar samples were incorporated into the medium of Mueller and Hinton (1941) without starch in the following proportions: 453
