Antigenicity of Staphylococcal Toxin Detoxified by the Photo-dynamic Action of Methylene Blue

The fact that some organisms, viruses, bacteriophages and toxins may be rendered harmless and inactive by the photodynamic action of methylene blue has been well established. Since Perdrau and Todd 1 reported that their virus of canine distemper so inactivated possessed a high antigenic property, the antigenic value of these products has become an interesting problem. Rivers 2 cautioned workers on the general principle that when the degree of inactivation could not be accurately measured one should be careful in attributing antigenicity to these so-called inactivated agents. Since titrations of minute residual amounts of active virus are still impossible, one naturally turns to bacterial toxins for an answer to this question. Lippert 3 reported failure to demonstrate antigenicity in his inactivated tetanus-toxin. Lin 4 could not completely detoxify diphtheric toxin. In this note, we present the following findings: Staphylococcal toxin can be completely detoxified by the photo-dynamic action of methylene blue and when so detoxified still possesses antigenic value.

The method of preparing toxins was a modified combination of those recommended by Parker and Dolman. 5 The medium was 0.3% agar with 4% Witte's peptone in beef broth buffered at pH 7.0. After incubation in an atmosphere containing about 20% carbon dioxide, it was filtered through cheesecloth, paper and Berke-feld candles. The final filtrate was our toxin. Only those preparations whose potency equalled or surpassed the minimal standards outlined by Dolman, 7 were employed. Gruebler's “Methylenblau” was used for detoxification.

To measured volumes of toxin, minute quantities of the stock dye-solution were added and thoroughly mixed. These mixtures, contained in large covered petri dishes, were placed on ice 20 cm. below a 100 watt lamp. At appropriate intervals, samples of these exposed mixtures and of control mixtures kept in the dark were titrated for hemolysis of rabbit-erythrocytes, dermo-necrosis and killing effect in rabbits.