R. J. WILSON,1 M.A., M.D., D.P.H.
THE usual basis of liquid media in which a substantial yield of Phase 1 TBordetella pertussis organisms can be obtained in the absence of blood is an acid or enzyme hydrolysate of casein. The first of these media was developed by Hornibrook (1) who added starch, salts and a sulphur-containing amino acid or peptide to a casein hydrolysate. Using a modification of Hornibrook's medium, vaccine production in a liquid medium was commenced in the Connaught Medical Research Laboratories in 1941 and was reported by the author with notes by Farrell and Taylor (2, 3). However, for metabolic and antigenic studies of B. pertussis, a chemically defined medium is more useful.This report describes the development, over a period of years, of a chemically defined medium which will support a reasonably good growth of Phase 1 organisms (4, 5).
PREPARATION OF THE MEDIUM (TABLE I)
The amino acids and glutathione are placed in 500 ml. of deionized water in a 1-liter Erlenmeyer flask (the amino acids may be dissolved by heating to 80° C. in a water-bath). Five ml. of a 0.1% solution of ascorbic acid, the L-glutamine and the adenine are added to the flask. A 15-ml. volume of a solution containing 0.002% each of guanine, hypoxanthine, thymine, uracil and xanthine is next added.* To the flask containing the amino-acid mixture is then added 10 ml. of a 0.005% solution of 2-deoxy-D-ribose. The inorganic salts, excepting CuSO4, are dissolved in 200 ml. of deionized water which is then combined with the amino-acid mixture; 5 ml. of a 0.1% solution of the CuSO4 is then added. The remaining additions consist of the tris (hydroxymethyl)- aminomethane (Sigma 121) for buffering, the adenosine triphosphate, the liver coenzyme concentrate, and finally, 1 ml. of a 0.1% solution of niacin. The medium is adjusted to pH 7.1 (with HCI) and the solution is made up to a volume of 1 liter with deionized water. The chemically defined medium is sterilized by ultrafiltration (ultrafine fritted disc glass filter (Pyrex)) and then dispensed aseptically, in 50-ml. amounts, into sterile L-tubes, each containing 10 mg. of activated charcoal powder (British Drug Houses (Canada) Ltd.), sterilized by autoclaving. [End of Text]
* This solution is prepared as follows: 10 mg. of each of the substances is placed in one 1Connaught Medical Research Laboratories, University of Toronto, Toronto, Ontario. flask and 450 ml. of deionized water and 2 ml. of N/10 NaOH are added. The material is dissolved by heating the contents of the flask to 80° C. in a water-bath. The solution is then transferred to a volumetric flask and the volume made up to 500 ml. with deionized water. 518