A MICROBIOLOGICAL ASSAY METHOD FOR p-AMINOBENZOIC ACID

Since the establishment of p-aminobenzoic acid as a member of the B vitamin group, a considerable interest has been shown in methods of determination in natural materials. Since known chemical methods are not sufficiently sensitive, it became evident that microbiological tests should be the most practicable. The organism Clostridium acetobutylicum has been used (1) but no general assay procedure has been presented. Several bacterial strains which respond to p-aminobenzoic acid have been investigated in this laboratory, but satisfactory assay procedures with these organisms have not yet been devised. For the discovery of the test organism used in the procedure described in this paper, we are indebted to Dr. Beadle and Dr. Tatum who kindly furnished us with a culture of their p-aminobenzoic acid requiring a mutant strain of Neurospora crassa, designated by them as Neurospora crassa p-aminobenzoicless No. 1633 (2). This mold will grow optimally on a medium consisting of inorganic salts, ammonium tartrate, sucrose, biotin, and p-aminobenzoic acid. For purposes of assay, however, it has proved advantageous to supplement this basal medium with natural extracts which are either naturally low in p-aminobenzoic acid or have been treated to remove it. With such a complex medium, the possibility of interference by toxic substances or stimulatory substances other than p-aminobenzoic acid which might be present in samples to be assayed is reduced to a minimum. Since the completion of a considerable part of the experimental work described in this paper, microbiological assay methods for p-aminobenzoic acid have been published by Landy and Dicken (3) utilizing the organism Acetobacter suboxydans and by Lewis (4) using Lactobacillus arabinosus 17-5.