Development of a tetrazolium salt assay for rapid determination of viability of BCG vaccines.
Standardisation and control of the live Mycobacterium bovis BCG (BCG) vaccine 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 assay method is not only time consuming but may give variable results. A tetrazolium salt assay has been developed and evaluated as a potential additional, or replacement, test for determining number of viable organisms. The tetrazolium 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-carb oxanilide (XTT) used as alternative substrates in the assay both gave more rapid and reproducible results than the conventional viable count. XTT showed greater sensitivity than MTT with a lower detection limit of about 7x10(4) colony forming units (c.f.u.) ml(-1). The XTT assay has proven effective for determining viability of suspensions prepared from several BCG vaccine substrains, covering a range of viable units, without the need for modification. This assay is easily performed and takes just 48 h to produce an estimate of viable cell content compared with 3 weeks for the conventional method.
Kairo
S K
SK
Division of Bacteriology, National Institute for Biological Standards and Control, Potters Bar, Hertfordshire, UK.
Bedwell
J
J
Tyler
P C
PC
Carter
A
A
Corbel
M J
MJ
D001500:BCG Vaccine / Q000737:chemistry / Q000276:immunology*; D002452:Cell Count; D003114:Colony-Forming Units Assay; D004396:Coloring Agents / Q000737:chemistry; D005612:Freeze Drying; D007202:Indicators and Reagents; D009163:Mycobacterium bovis / Q000254:growth & development* / Q000276:immunology*; D013778:Tetrazolium Salts / Q000737:chemistry*; D013844:Thiazoles / Q000737:chemistry; D014613:Vaccines, Attenuated / Q000737:chemistry / Q000276:immunology
D001500:BCG Vaccine; D004396:Coloring Agents; D007202:Indicators and Reagents; D013778:Tetrazolium Salts; D013844:Thiazoles; D014613:Vaccines, Attenuated; C059087:2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-((phenylamino)carbonyl)-2H-tetrazolium hydroxide; C022616:thiazolyl blue