Development of a tetrazolium salt assay for rapid determination of viability of BCG vaccines

Standardisation and control of the live Mycobacterium bovis BCG (BCG) Vacci ne is performed as specified by the World Health Organisation (WHO) and the European Pharmacopoeia (EP). The conventional Viable count for control of potency of BCG vaccine is performed by culturing on solid medium. This assa y method is not only time consuming but may give variable results. A tetraz olium salt assay has been developed and evaluated as a potential additional , or replacement, test for determining number of viable organisms. The tetr azolium salts 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and 2,3-bis- (2-methoxy-4-nitro-5-sulphenyl)-(2H)-tetrazolium-5-carbo xanilide (XTT) used as alternative substrates in the ass ay both gave more rapid and reproducible results than the conventional viable count. XTT show ed greater sensitivity than MTT with a lower detection limit of about 7 x 1 0(4) colony forming units (c.f.u.) ml(-1). The XTT assay has proven effecti ve for determining viability of suspensions prepared from several BCG vacci ne substrains, covering a range of viable units, without the need for modif ication. This assay is easily performed and takes just 48 h to produce an e stimate of viable cell content compared with 3 weeks for the conventional m ethod. (C) 1999 Published by Elsevier Science Ltd. All rights reserved.