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Open AccessArticle

Optimisation of Mycobacterium bovis BCG Fermentation and Storage Survival

TB Research Group, Public Health England, National Infection Service, Porton Down, Salisbury, Wiltshire SP4 0JG, UK
Department of Epidemiological Sciences, Animal and Plant Health Agency, Woodham Lane, New Haw, Addlestone, Surrey KT15 3NB, UK
Department of Bacteriology, Animal and Plant Health Agency, Woodham Lane, New Haw, Addlestone, Surrey KT15 3NB, UK
Faculty of Health and Medical Sciences, University of Surrey, Guildford, Surrey GU2 7XH, UK
Author to whom correspondence should be addressed.
Pharmaceutics 2020, 12(9), 900;
Received: 20 July 2020 / Revised: 2 September 2020 / Accepted: 10 September 2020 / Published: 22 September 2020
(This article belongs to the Special Issue Tuberculosis Vaccine Research and Development)
Mycobacterium bovis Bacillus Calmette–Guérin (M. bovis BCG) was generated over a century ago for protection against Mycobacterium tuberculosis (Mtb) and is one the oldest vaccines still in use. The BCG vaccine is currently produced using a pellicle growth method, which is a complex and lengthy process that has been challenging to standardise. Fermentation for BCG vaccine production would reduce the complexity associated with pellicle growth and increase batch to batch reproducibility. This more standardised growth lends itself to quantification of the total number of bacilli in the BCG vaccine by alternative approaches, such as flow cytometry, which can also provide information about the metabolic status of the bacterial population. The aim of the work reported here was to determine which batch fermentation conditions and storage conditions give the most favourable outcomes in terms of the yield and stability of live M. bovis BCG Danish bacilli. We compared different media and assessed growth over time in culture, using total viable counts, total bacterial counts, and turbidity throughout culture. We applied fluorescent viability dyes and flow cytometry to measure real-time within-culture viability. Culture samples were stored in different cryoprotectants at different temperatures to assess the effect of these combined conditions on bacterial titres. Roisin’s minimal medium and Middlebrook 7H9 medium gave comparable, high titres in fermenters. Flow cytometry proved to be a useful tool for enumeration of total bacterial counts and in the assessment of within-culture cell viability and cell death. Of the cryoprotectants evaluated, 5% (v/v) DMSO showed the most significant positive effect on survival and reduced the negative effects of low temperature storage on M. bovis BCG Danish viability. In conclusion, we have shown a reproducible, more standardised approach for the production, evaluation, and storage of high titre, viable, BCG vaccine. View Full-Text
Keywords: BCG; fermentation; flow cytometry; calcein violet; sytox green; cryoprotectant BCG; fermentation; flow cytometry; calcein violet; sytox green; cryoprotectant
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MDPI and ACS Style

Pascoe, J.; Hendon-Dunn, C.L.; Birch, C.P.; Williams, G.A.; Chambers, M.A.; Bacon, J. Optimisation of Mycobacterium bovis BCG Fermentation and Storage Survival. Pharmaceutics 2020, 12, 900.

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