Nearly 40% of the starting suspension of yeast cells were recovered when cells were slowly frozen in an 8% DMSO-containing solution and this procedure was selected for long term storage of mutant pools. Although specialized Poziotinib clinical trial cooling apparatuses can be used to control the freezing rate, we found that simple placement of vials of cells within readily and cheaply obtained styrofoam containers (such as those used for shipments of molecular biology enzymes) was sufficient. Figure 2 Gradual freezing in DMSO maximizes recovery of cryopreserved Histoplasma yeast. R428 mouse WU15 yeast were frozen in varying concentrations of glycerol (A) or DMSO (B). Histoplasma yeast were grown

to late log/early stationary phase in rich medium and added to the appropriate glycerol- or DMSO-containing solutions before freezing. Final cryoprotectant concentrations

indicated along the x-axis of each graph. Vials were placed immediately Adriamycin at -80°C (rapid freeze) or were placed into a styrofoam container before placement at -80°C (slow freeze). Frozen cell aliquots were thawed after 1 week or 9 weeks and recovery measured as the number of viable cfu relative to the number present before freezing. Generation of mutant pools Insertion mutants were generated in the NAm 2 Histoplasma strain WU15 by co-cultivation of Agrobacterium tumefaciens and Histoplasma yeast cells. Co-cultures were plated onto filters and Histoplasma transformants selected Glycogen branching enzyme by transferring filters to medium containing hygromycin to which resistance is provided by sequences within the T-DNA element [23]. Transformant yeast cells were collected and suspensions from individual plates combined to create pools derived from 100 to 200 independent mutant colonies. Yeast cell suspensions were diluted into fresh medium and allowed to grow for 24-48 hours. Twenty-four pools were prepared representing roughly 4000 insertion mutants. A portion of each culture was reserved for nucleic acid isolation and the remainder frozen in aliquots and stored at -80°C. Nucleic acids were purified from

each pool, diluted to 50 ng/ul, and stored at -20°C until analysis by PCR. With an estimated 9000-10,000 genes encoded by the Histoplasma genome, this collection does not represent the number of insertion mutants required for saturation of the genome. We used two probability functions to estimate the size of the library required for a 95% chance of isolating an insertion in a particular locus in the 40 megabase NAm 2 genome. Both calculations assume no bias in insertion sites. Based on the number of predicted genes, the Poisson approach estimates a library of approximately 30,000 insertions would be required. The single study in which multiple alleles of a single locus were isolated in Histoplasma (five AGS1 alleles isolated in a screen of 50,000 insertions; [23]) supports the Poisson calculation; five alleles would be the most probable number of alleles based on a 9000 or 10,000 target estimate.