I work with the cell-free supernatant of a Bacillus subtilis bacterial culture. After centrifugation, I use 0.2 micrometer filters to collect the supernatant.

I noticed that spores pass through these filters, so I collected a fresh supernatant after centrifugation and a first filtration. I filtered a second time after 4 hours of incubation for the first filtered supernatant, thinking that most of the spores will give the vegetative form and they will not have time to produce more spores, and although I did not succeed in getting rid of the spores....Have you any suggestion that may help ? Thank you so much !