Effect of Preservatives on Undenatured Bacterial Antigens. ∗

Abstract

In the preparation of undenatured bacterial antigens mass-cultures of recently isolated bacteria are washed free of metabolites and media-constituents with Locke's solution. 1 The cells are fragmented in a special ball-mill 2 and residual intact cells are removed by ultrafiltration. 3 Filtrates containing the chemically unaltered antigenic fractions of the organisms are standardized on the basis of native-protein content and are called Undenatured Bacterial Antigens or more briefly U B A. Antigens of this type have been tested clinically during the past several years. 4 - 13

Since the major desideratum of the preparative technic has been avoidance of denaturation it became important to determine just how readily the antigens were altered when exposed to such degrees of heat or chemical action as might be encountered under practical working conditions. We have shown in the case of Staphylococcus U B A 14 that thermal denaturation begins to be significant at 40°C.; the reaction obeys the mass-law and exhibits the remarkably rapid rise in rate with increase in temperature that characterizes protein denaturation in general (critical thermal increment 44,000).

In order to test the adequacy of certain antiseptics to preserve U B A solutions without causing appreciable denaturation, phenol, tricresol and merthiolate were added to aliquots of freshly prepared staphylococcal U B A in such amounts that the final concentrations were 0.3% tricresol, 0.5% phenol, 0.01%, 0.005%, and 0.002% merthiolate. The solutions were kept at room-temperature for 5 months during which time weekly samples were withdrawn for determination of their content of total, denatured, and native protein. The analytical methods have been described by Krueger and Nichols. 14

A composite graph of the data obtained in 2 experiments is shown in Fig. 1.