A serum-free cultivation broth and recovery agar for use in 0.1 micron-rated filter characterization and validation studies for biopharmaceutical applications were developed using Acholeplasma laidlawii as the test microorganism. Selection criteria were (A) a single-stage, serum-free broth medium to support high-titer cell growth and yield a cellular morphology suitable for bacterial challenge testing within 24 h and (B) a serum-free agar growth medium that can recover cells using conventional enumeration techniques. Different formulation components at various concentrations were screened for their effects on growth, cell size, and recovery on membrane filters. An optimized broth, Glucose Hydrolysate Broth, containing glucose, polypeptone, bovine serum albumin, 2-Amino-2-(hydroxymethyl)-1,3-propanediol (Tris base), oleic acid, and palmitic acid produced small 370 X 406 nanometer monodisperse cells at titers in excess of 1 x 10(9) colony-forming units per milliliter (cfu mL(-1)) within 20-24 h of incubation. Glucose Mycoplasma Agar, containing glucose, mycoplasma broth base, bovine serum albumin, Tris base, oleic acid, and palmitic acid, produced colonies approximately 1 mm in size and facilitated recovery on 0.2-microm polyvinylidene fluoride membrane filters. A comparison of recovery of A. laidlawii on Glucose Mycoplasma Agar pour-plate versus membrane filters resulted in an 89 +/- 4% recovery. Linearity across the target recovery range was 0.9992 (r2). Presence/absence tests of filtrate from each 0.2-microm assay membrane resulted in absence of growth as compared to controls, thus demonstrating the suitability of using 0.2-microm membrane filters for recovery of A. laidlawii.