Crosbie’s procedure was used to strictly deposit one cell into
a single culture well, where cells were sorted into odd row
numbers of a multi-well plate, leaving the even rows of the
culture plate blank [6]. After each sorting cycle, each well was
thoroughly mixed with a pipette, and half the volume was
transferred to the adjacentwell on an even row. Ifmore than one
cell was originally sorted i.e. if growth occurred in both the
collected and the transferred wells, this represented amisplacement
of sorted cells and was rejected e a procedure that
improved the probability that subsequent cultures were generated
from single-sorted cells. Following inoculation and colony
formation, 250 mL of medium from the well was analyzed using
anEpicsAltraflowcytometer todetermine thepurity of isolated
strains. Expo32 version 1.2B for Altra (Beckmann Coulter) softwarewasused
for data acquisition and processing. 10 mLofeither
3-mmor 10-mmfluorospheres (Beckmann Coulter) were added to
samples as an internal cell size reference standard.