1. Agglutinogens of broth cultures vs. saline suspensions. Of primary importance are the results in relation to agglutinogens prepared by cultivation of the microörganism in broth as compared with those prepared by cultivating on plain solid media and suspending the bacteria in saline solution: a general review of the results observed in this study with a single strain of typhoid bacilli, indicate that suspensions in saline solution so prepared as to break up clumps and diluted to proper density, are superior to broth cultures.

  2. Saline solutions vs. distilled water. As is well known, sodium chloride exerts an important rôle in the physicochemical phenomenon of agglutination; of agglutinogens prepared by suspending typhoid bacilli in strengths of sodium chloride in sterile distilled water varying from 0.85 to 5 per cent (nos. 17, 18, 19 and 20), best results were observed with the 0.85 and 1 per cent solutions (nos. 17 and 18); agglutinogens prepared with 2 per cent solutions of sodium chloride were less susceptible to agglutination and 5 per cent solutions were decidedly less susceptible. Agglutinogens prepared with distilled water alone (no. 21) were least susceptible to agglutination and yielded some of the lowest titers.

  3. Influence of heat. Of primary importance is the effect of heating an agglutinogen upon its susceptibility to agglutination; in the majority of laboratories cultures or suspensions are usually heated at 56° to 60°C. for one-half to two hours. In order to be able to observe the most marked influence of heat, if any, antigens were heated in a water bath at 60°C. for two hours; a general survey of the results of this study indicates that heated antigens are somewhat more susceptible to agglutination than unheated antigens.

  4. Influence of chemical germicides and preservatives. In this investigation phenol, tricresol, formalin, mercurophen and glycerin were employed for chemical sterilization and preservation (antiseptic activity); with the exception of formalin in 0.1 to 5 per cent; agglutinogens prepared without preservatives were somewhat superior to those containing phenol, tricresol, mercurophen and glycerin.

  5. Kind of chemical germicide and preservative. Of the chemicals employed in the preparation of agglutinogens of B. typhosus, best results were observed with formalin; in fact the addition of 1 to 2 per cent formalin2 to suspensions in isotonic saline solution yielded the best agglutinogens of the series included in this study (nos. 9 and 10). Agglutinogens were prepared with 0.1, 0.5, 1, 2 and 5 per cent neutral formalin; comparative tests have usually shown that those containing 1 and 2 per cent were least likely to show spontaneous agglutination, were most susceptible to specific serum agglutination and were never contaminated.

    Agglutinogens prepared with phenol in 0.5 per cent and tricresol in 0.5 per cent (nos. 4 and 5) yielded similar results, but they were somewhat inferior to preservative free agglutinogens and decidedly inferior to those containing 0.1 to 2 per cent formalin. Agglutinogens prepared with 1 per cent phenol and tricresol (nos. 12 and 14) were decidedly inferior to those containing 0.5 per cent of these substances and an antigen containing 5 per cent phenol (no. 13) was almost insusceptible to agglutination and proved most unsatisfactory of all.

    Mercurophen was employed because of its high germicidal activity and freedom of precipitating and coagulating influence upon proteins including bacterial proteins; agglutinogens prepared with 1:1000 mercurophen in physiological saline solution (no. 15) and 1:2000 (no. 6) yielded results similar to those containing 0.5 per cent phenol and tricresol. An agglutinogen containing 1:5000 mercurophen (no. 16) was generally satisfactory and from the standpoints of freedom from spontaneous agglutination, susceptibility to specific agglutination and freedom from contamination ranked next to plain and formalized agglutinogens.

    The addition of 10 cc. of the best grade neutral glycerin to each 100 cc. of heated agglutinogen containing 0.5 per cent phenol (no. 3) reduced susceptibility to specific agglutination and proved inferior to the same agglutinogen prepared without the addition of glycerin (no. 1).

  6. Spontaneous agglutination. Of agglutinogens 1 to 16 preserved over a period of four to eight weeks in a refrigerator, no. 15 containing 1:1000 mercurophen, showed most tendency to spontaneous agglutination and nos. 7, 8, 9 and 10 containing 0.1 to 2 per cent formalin, least spontaneous agglutination; of the freshly prepared antigens, nos. 19 and 20, containing 2 and 5 per cent sodium chloride frequently showed spontaneous agglutination. Macroscopical tests for spontaneous agglutination were conducted with each antigen whenever employed by diluting 1 cc. with 1 cc. of 0.85 per cent saline solution and incubating at 55°C. for twenty-four hours; at the same time microscopical tests were made and occasionally antigens showed small clump of bacilli microscopically, which appeared perfectly homogenous and satisfactory to the closest scrunity with the naked eye.

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