S. LANDI
Connaught Medical Research Laboratories, University of Toronto, Toronto, Canada
Received for publication 15 April 1963
ABSTRACT
LANDI, S. (University of Toronto, Toronto, Canada). Preparation, purification, and stability of tuberculin. Appl. Microbiol. 11:408-412. 1963.-The method used to pro- duce "Connaught" tuberculin purified protein derivative (PPD) is described. The tuberculin PPD for the multiple- puncture method was shown to be stable for at least 24 months at 5 C; tuberculin PPD for the intracutaneous method was shown to be stable at 5 C and 24 C for a period of 18 months in the presence of Tween 80. Evans blue or brilliant vital red was added to tuberculin PPD for improved testing by the multiple-puncture method. These tinted tuberculin preparations were found to be as stable as the Connaught tuberculin PPD preparations without dye at 5 C. Freeze-dried tuberculin PPD with Plasdone as an inert base was found to be remarkably stable for a period of at least 24 months at 5, 24, and 37 C.
The ever-increasing demand for a purified form of tuberculin has stimulated interest in its preparation and in the investigation of its properties. The importance of the tuberculin skin test in tuberculosis prevention programs underlines the need for the preparation of a purified tuberculin with adequate stability. The present work deals with the purification of tuberculin and its stability for preparations used for the multiple-puncture (Heaf, 1951) and the intracutaneous (Mantoux, 1909; National Tuberculosis Association, 1961) methods. For this purpose, the purified tuberculin was tested after storage at various temperatures.
It was thought that the addition of a dye to the tuber- culin preparation used for the multipuncture method would facilitate the tuberculin test and increase its accuracy. It was necessary, therefore, to determine the effect of the dye on the stability of the preparation as compared with the stability of an identical preparation without dye.
It was also thought of interest to determine the effect of freeze-drying on the potency and stability of purified tuberculin.
MATERIALS AND METHODS
Organism and medium. The "Johnston" strain of Mycobacterium tuberculosis var. hominis, isolated in 1920 in Toronto, Canada, from a case of human tuberculosis, was used for the production of tuberculin purified protein derivative (PPD). The "Johnston" strain showed the characteristic surface growth of M. tuberculosis var. hominis on Long's synthetic medium (Long and Seibert, 1926) as well as virulence for guinea pigs and mice and nonvirulence for rabbits. The cultures of the organism were maintained by weekly subculture on Long's synthetic medium.
For tuberculin production, the medium was dispensed in 150-ml amounts into Roux bottles. These bottles were sterilized in an autoclave by treatment for 10 min in flowing steam, followed by 30 min at a steam pressure of 15 lb/in.2
Preparation of tuberculin. The organisms for inocula- tion were grown as a surface pellicle in small Erlenmeyer flasks on Long's synthetic medium at 37 ± 0.5 C for 8 to 10 days. The pellicle was used for inoculating approx- imately 300 Roux bottles of medium. After inoculation, these bottles were incubated at 37 ± 0.5 C for 6 to 6.5 weeks. When the culture was ready to be harvested, it was tested for purity by transferring 0.50 ml from each Roux bottle into 10 ml of beef infusion broth and incubating at 37 0.5 C. If contamination appeared in any tube, the contents of the corresponding bottle were discarded.
After the Roux bottles had been tested for purity, they were steamed for 3 hr in a flowing-steam cabinet (100 C) and allowed to stand overnight at room temperature. The contents of the bottles were then passed through a sterile eight-layer gauze pad or a flannelette cloth placed over a filter-paper pulp pad to separate the medium containing tuberculoprotein from the bacterial growth. The crude filtrate, light amber in color, was then filtered through a Berkefeld candle. A sterility test was carried out on this filtrate, and phenol was added to it as a preservative (0.35%, final concentration). The filtrate was then stored at refrigerator temperature until purification time.
Purification of tuberculin. Following the method of Green (1946), 40% trichloroacetic acid was added to the filtrate to give a final concentration of 4%. The total protein was precipitated and allowed to sediment over- night in a refrigerator (5 C); the supernatant was siphoned off and discarded. The sedimented sludge was poured into 250-ml centrifuge cups and centrifuged at 840 × g for 20 min. The sludge was washed with 1% trichloroacetic acid and centrifuged again at 1880 X g for 15 min. The residue was washed again with 1% trichloroacetic acid and centri- fuged at 3350 × g for 15 min, and finally washed with cold 0.30% KH2PO4 and centrifuged at 4000 rev/min for [end of text]