The reason this centrifugation/resuspension step is done has a simple reason: It concentrates the bacteria in a much smaller volume which is much easier to handle afterwards. For a maxiprep you typically have a culture volume between 250 and 500ml, depending on the copy number of the plasmid. After centrifugation the pellet is resuspended in 10ml of Buffer P1, for lysis another 10ml of buffer P2 are added, for neutralization 10ml of P3. Imagine doing this with 500ml culture volume - adding 500ml of each P2 and P3. This might work for a miniprep, but definitely not for larger volumes. Additionally, the centrifugation steps are carried out at high g numbers - typically something around 15.000xg. Doing this with this really high volumes is complicated and needs big centrifuges, which a lot of people are not trained anymore to operate or have available.

Metacyclic forms placed in 25 cm2 T-type (T-25) flasks rapidly attached to the bottom of the flasks and transformed to bloodstream forms during the initial 24 h and continued to proliferate. The bloodstream forms isolated from the infected mouse blood by means of diethylaminoethyl cellulose (DE52) column chromatography also continued to proliferate in the flasks. Cultures were maintained by replacing the medium every 24 h. Every 4-5 days, the attached bloodstream forms were resuspended in fresh medium by gentle pipetting and then were subcultured. The method was further simplified by initiating primary cultures directly with 10 microliters of the tail blood of infected mice in 24-well culture plates and then by subcultivating either in wells or in T-25 flasks. The shortest population doubling time, 9 h, was achieved by seeding subcultures with 10(6) bloodstream forms/ml. The bloodstream forms propagated in this system were morphologically similar to those seen in infected mouse blood, they were covered with a surface coat as examined by electron microscopy and they were infective to mice. Published in Parasitology (1991)