Purification of Tetanic Toxin

Abstract

The methods used in the purification of tetanic toxin were based on the general principles outlined in a previous paper on the purification of diphtherial toxin. 1 The crude toxins were prepared in meat-infusion containing 2% of Bacto-peptone, and 1% dextrose. Cultures of Cl. tetani were grown 14 days under vaseline and filtered. Lots 4 and 5 were about one year old, and lots 6 and 8 were fresh when the purification was started.

Purification. The toxic preparations should be kept in a refrigerator except when reagents are added or when being centrifuged. The optimal proportions of reagents vary somewhat with different lots of toxin. These variations have been indicated in the directions for the process given below. Amounts are for the purification of one liter of crude toxin.

1. To one liter of crude toxin 100 to 125 cc. of 10% solution of ferric ammonium sulphate are added gradually. The pH is kept between 5.5 and 6.0 by the addition of normal sodium hydroxide. The resulting precipitate will dissolve in an excess of the ferric salt below pH 5.5. After settling for a few hours the precipitate is collected and washed with distilled water. The toxin is then eluted with 400 cc. of 2.5% sodium citrate at a pH of 7.5 to 8.0 for 3 days or until most of the precipitate has dissolved. The insoluble residue is centrifuged down and the pH of the supernatant adjusted to 7.0.

2. Four hundred cc. of the eluted toxin are diluted to one liter and the calculated concentration of sodium citrate adjusted to 1.5 to 2.5%.