A specific procedure has been developed for the detection of the first two enzymes involved in the arginine dihydrolase system and the detection of the decarboxylases of arginine, glutamic acid, histidine, lysine, ornithine, phenylalanine, tryptophan, and tyrosine. A loopful of growth of each organism from dihydrolase-decarboxylase induction agar medium (or broth) was washed and incubated separately with 0.2-ml samples of three test media supplemented with different amino acids. Each spent test medium was dansylated, and the dansyl derivatives were separated by two-dimensional thin-layer chromatography on polyamide sheets. The end products (citrulline, ornithine, y-amino-n-butyric acid, and amines) produced during incubation were estimated by comparing the fluorescent intensities of end products from the spent test media and of the corresponding parent amino acids from test medium controls after thin-layer chromatography. The method is reproducible, requiring incubation of an organism in three test media for 1 h for simultaneous detection of the first two enzymes involved in the arginine dihydrolase system and of eight amino acid decarboxylases. This method has been successfully applied to gram-positive and gramnegative microorganisms and also to Mycoplasmatales. It could simplify and improve the accuracy of the corresponding biochemical tests performed in clinical laboratories for the identification and differentiation of microorganisms, and it may prove particularly useful for the differentiation of species of Pseudomonas and Mycoplasma. Enzymes involved in the arginine dihydrolase system and the amino acid decarboxylases are widely distributed in microorganisms (1, 2, 8). The microbial arginine dihydrolase system which converts arginine to ornithine via citrulline consists of three enzyme reactions: arginine + H20-* citrulline + NH3 by arginine deiminase; citrulline + Pi ornithine + carbamyl

This report presents a new approach that by-passes booster immunizations with tetanus toxoid (TT) before in vitro studies of antibody (Ab) production. The methodology for optimal TT-induced synthesis of specific IgG anti-tetanus toxoid Ab (IgG anti-TT) by peripheral blood mononuclear cells (PBMC) from randomly selected TT immune individuals without recent booster immunizations is described. PBMC from most normal immune subjects could be repeatedly induced to produce in vitro IgG anti-TT; PBMC from subjects with high TT titers are not required for this new approach. This approach uses high cell concentrations in multiple replicate microcultures and TT washout to obtain optimal IgG anti-TT synthesis. Washed cultures produced more Ab than nonwashed cultures (p less than or equal to 0.005). The readdition of TT (2.5 to 250 ng/ml) to the culture media after washout of TT on day 4 suppressed specific Ab formation, whereas diphtheria toxoid added at comparable doses did not inhibit specific Ab formation. Suppression of antibody synthesis mediated by T cells could be induced by TT per se, and was not due to binding of synthesized Ab to TT in the latter 8 days of culture. In addition, suppression could not be induced in the first 4 days of culture by IgG anti-TT, IgG, or IgM. This approach permits the analysis of antigen-specific regulatory circuits in the steady and activated immune states, and the evaluation of in vivo and in vitro effects of biologic response modifiers on specific Ab production.