A. A standard curve of fluorescent absorbencies at 640nm vs. varying amounts of SRB-BCG mixture. B. The amount of BCG powder within the MNA was evaluated in the basis of the standard curve in A Each symbol represents the amount of BCG in nine microneedles cut from each patch and the horizontal lines are the means of 6 patches with 95% CI (confidence interval). Results of three independent experiments designated as 1, 2, and 3 were shown each with 6 MNAs and 9 microneedles in each MNA. C. A shelf-life of BCG-MNAs was determined on day 0, 30, 60, and 90 after MNA fabrication and storage at room temperature and compared with pre-loaded BCG vaccine. Each symbol represents the number of live bacilli or colony forming units (CFUs) per 24 microneedles and the short horizontal lines are the means of 6 patches. NS, no significant difference. Statistical significance was analyzed by ANOVA D.BCG-MNA could pierce into the skin of C57BL/6 mice efficiently after 90 days of storage in room temperature, as suggested by generation of an array of micropoles in the skin with the relevant area enlarged (lower panel). E. Histological skin sections were prepared 2 hr after insertion of a BCG-MNA stored for 90 days in room temperature to show skin penetration of the individual microneedles (arrows), one of which is enlarged.