The vaccine was tested for sterility using a modified version of the
British Pharmacopoeia standard (Anonymous, 2005). Ten milliliter of
tryptone soy broth (TSB, Oxoid) and marine Shieh's broth (MSB) and
25 ml of fluid thioglycollate medium (FTM, Oxoid) were inoculated
with 500 μl of the formalin-inactivated suspension. After 7 days incubation
at 25 °C for TSB and MSB and 35 °C for FTM, the broth
media were subcultured on to tryptone soy agar (TSA, Oxoid), marine
Shieh's agar (MSA) and FTM and incubated for a further 5 days at the
same temperatures as previously stated. Absence of growth on the
agar plates or FTMwas interpreted as absence of culturable bacteria in
the vaccine preparation; five replicates for each broth were used for
testing.