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
In earlier papers (1935, a, b and c) the writer has shown that mixtures of certain amino acids will replace adequately the peptone fraction of a meat-infusion peptone broth for the propagation of two strains of diphtheria bacillus which were investigated. While there were differences in the requirements of the two, they were alike in their use of glutamic acid, and, to produce maximal growth, quantities of this amino acid greatly in excess of those of any of the other essential amino acids had to be added to the medium. This fact suggested the possibility that perhaps some impurity, rather than glutamic acid itself, might prove to be responsible for the effect. In view of the fact that the quantity of growth increased along an almost straight line up to a concentration of 0.5 per cent glutamic acid hydrochloride, and that further additions produced still greater increases, although not with the same direct proportionality, while of such other essential amino acids as tryptophane, cystine and methionine quantities of the order of 0.01 per cent were adequate for maximal growth, it was clear that 1 to 2 per cent of an active impurity in the glutamic acid might account completely for its apparent action. Moreover, although repeated recrystallizations failed to change the activity of the compound, the difficulty of obtaining many of the amino acids from natural sources in a completely pure formjstill left the possibility of the presence of an impurity to be excluded by other means.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.