Increasing color density with decreasing toxicity of tetanus toxin in the Mueller medium.

The observation was made that tetanus toxin produced in the Mueller medium became visibly darker while undergoing detoxification in the incubator. It was then considered of interest to determine whether this increase in color intensity could be correlated with detoxification. This paper deals with such a correlation. The work of Agner (1950) on diphtheria toxin reveals the possibility of some related mechanism involved with detoxification and color increase although the color could be produced separately. He demonstrated that myeloperoxidase in the presence of purified diphtheria toxin would produce a reddish brown color and detoxification of the toxin when a co-factor and dilute hydrogen peroxide were dialyzed into the bag. The color could not be removed from the toxin by dialysis. If, however, the color were produced in the absence of the toxin by the combinations of myeloperoxidase plus the co-factor in the presence of dilute hydrogen peroxide and the enzyme system then allowed to enter the bag with the toxin, the toxin would not be detoxified and the color could be removed by dialysis. The tetanus toxin was harvested from a 6-day growth of the Harvard strain of Clostridium tetani on the most recent Mueller formula (Mueller, 1951). Detoxification of the sterile,formalinized toxin was accomplished in most cases by incubating at 35 C for 14 days. Samples were removed nearly every day for density determinations on the Junior Coleman Spectrophotometer set at 500 millimicrons. The criterion for complete detoxification was the absence of tetanus symptoms in a 300 to 400 gram guinea pig for 21 days after injection of 10 ml of the material. The minimum lethal dose of the partially detoxified material was determined in guinea pigs, using death times as measurement of toxicity according to a previous method (Ipsen, 1941). Nine lots of toxin were detoxified. During detoxification at 35 C, they showed a gradual and definite rise in color density of the toxin accompanied by a steady decline in the toxicity. Eight of these lots showed complete detoxification in the density range of 0.250 to 0.300, while the ninth lot was not fully toxoided until it reached a density of between 0.300 and 0.328. In all cases the toxins were completely detoxified in a period of time varying from a minimum of 5 to a maximum of 10 days. The single lot showing a reading greater than 0.300 at the time of complete detoxification was detoxified in 8 days. This might indicate that the length of the period of incubation was not the sole factor which determined the depth of color at the earliest sign that complete detoxification had occurred. Figure 1 illustrates graphically the comparative rise in density, decrease in toxicity, and M.L.D. values at different stages for a representative lot. In order to compare the density changes during a slower rate of detoxification, a lot of toxin was detoxified by maintenance at room temperature for 68 days. Frequent samples for density readings and for progress in detoxification revealed a reduced rate of