The concentration of diphtheria toxin by acid precipitation / by A.F. Watson and U. Wallace.

  • Watson, A. F.
Date:
[1924?]
Catalogue details

Licence: In copyright

Credit: The concentration of diphtheria toxin by acid precipitation / by A.F. Watson and U. Wallace. Source: Wellcome Collection.

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    minor part in the recoveries effected. As a general rule, however, more consistent and satisfactory recoveries are effected by keeping the temperature as low as possible. 3. The effect on the yields of toxin of standing for varying periods of time before separation of precipitate. When 3 per cent, glacial acetic acid is added to toxic filtrates of B. diphtherias (PH 7‘8-8’4) a cloudiness is produced but very rarely a precipitate at once. After a few minutes, however, a precipitate begins to flocculate, the time of appearance depending on the type of medium used, temperature of precipitation, etc. Where the medium employed has been prepared by tryptic digestion of horse muscle (or other medium which has supported good growth) the precipitate is heavy and settles out in a short time. After standing for some time the precipitate becomes granular and after several days the supernatant liquid is perfectly clear, under which conditions the precipitate may be readily recovered by siphoning off this supernatant liquid. Under these conditions, however, a proportion, but not all, of the toxin is lost as will be seen from table IV. Table IV. Period of standing before centrifugation- Toxicity. Lr/500 doses per c.c. Volume concentration. Original filtrate. Concentrated solution. A. 0 days. 1429 16,666 20 B. 2 „ 1429 7,111 20 C. 4 „ 1429 10,000 20 D. 6 ,, 1429 11,111 20 F. 9 „ 1429 6,250 20 H. 3 months. 1429 7,111 20 For the experiment, four litres of the filtrate were precipitated with 3 per cent, glacial acetic acid and left at cold room temperature for varying time periods, after which they were spun down in the machine and dissolved under similar conditions. The number of Lr/500 doses per cubic centimetre of the reconstructed antigen was then determined. The effect of leaving the precipitate in the acid medium is reflected in the toxicity of the precipitated material. Though some loss of toxicity has occurred, it is much less than the general statements current as to the extreme instability of diphtheria toxin in the presence of acid would lead one to expect. The nitrogen content of the solutions remained practically constant, showing that on prolonged standing no further precipitate is produced.