Observations on Some Problems Encountered in the Routine Performance of the Dye Test for Toxoplasmosis

The dye test for toxoplasmosis (Sabin and Feldman, 1948) depends upon the fact that toxoplasma organisms lose their affinity for methylene blue when they are exposed to specific antibody plus a heat-labile " accessory factor" at present available only from certain human sera. Accessory factor has been shown to differ from complement in several respects. Sera containing antibodies which produce the reaction are said to have " modifying activity" and toxoplasma cells which have been affected by the reaction are referred to as " modified organisms." In trying to set up a routine for running the dye test for toxoplasmosis, it was found that results were often erratic and unpredictable. This has been the experience of other authors in widely different areas (Cathie and Dudgeon, 1953; Lelong and Desmonts, 1952). After taking particular precautions to assure that general laboratory procedures and glassware were not responsible for this situation, it was found that a percentage of routine tests was still erratic and that some had to be discarded because improper results were obtained with control sera. An attempt was made to standardize the toxoplasma suspension which is added to the test sera by washing the organisms obtained from the peritoneal exudate of infected mice and replacing the liquid portion of the exudate with one of known composition. Results obtained in preliminary experiments showed that washed organisms suspended in serumsaline were more sensitive to modification by serum than those suspended in the original exudate. This increased sensitivity was specific in so far as it was apparent only in the presence of active accessory factor. In addition, evidence was obtained that so-called negative human sera used for accessory factor often contained measurable amounts of a modifying substance. This paper presents a description of this work and some observations concerning the dye test as a whole.