Every new batch of LJ media was controlled for sterility and for growth, using M. tuberculosis (ATCC H37Ra) and M. gordonae (ATCC 14470).
To this end we used the T. harzianum strains as a control sample for clonal sterility and seven representative strains of species from the Harzianum-Catoptron Clade which were genetically most distant to H. lixii and T. harzianum as a control for interspecific sterility.
Each plate included growth controls, sterility controls, DMSO controls, and controls with known antibiotics.
Wells without inoculum added were used for sterility control, and the positive controls comprised of inoculated growth medium without the substances.
Finally, each well (except for sterility controls) was inoculated with 5 μl of a cell suspension containing approximately 104 cells from an exponentially growing culture corresponding to each transformed strain under evaluation (overnight growth in SC-Ura and refreshed in YEPD).
With each batch of Columbia Agar base plates made, control plates were evaluated for sterility.
A number of wells was reserved in each plate for sterility control (no inoculate added) and inocula viability (no TQ added).
K. rhizophila has been widely used as a quality-control strain (American Type Culture Collection-9341) for sterility testing, and a recent case of K. rhizophila infection presented as persistent BSI associated with a damaged CVC in a girl with Hirschsprung's disease; advanced molecular methods were used for the identification in this case.
Quality control included TLC, HPLC, GC, pH control, Ge-breakthrough, half-life, endotoxin content, and control of sterility.
Nevertheless, for the two mutants analyzed, the segregations of female sterility in the offspring of the first generation cross and in backcrosses support a genetic control of sterility rather than epigenetic control.
