A survey of the literature reveals that several broth and agar /solid media containing a variety of supplements are used in isolation and culture of Mycobacterium paratuberculosis. Commonly used solid media include Herrold's egg yolk media (HEYM), Lowenstein-Jensen (LJ), Middlebrook 7H10. Broth media include, Dubos broth (DB) and Middlebrook 7H9. All media are supplemented with mycobactin J and variably contain glycerol, bovine serum and detergents such as Tween-80. In this study we attempted to identify the combination of media and supplements supporting the fastest and/or most abundant growth of recent fecal isolated M. paratuberculosis strains within a 6-week period. The test supplements included, egg yolk, lecithin, pyruvate, vitamins, casamino-acids, glycerol, Tween-80, Tyloxapol, bovine serum, mycobactin-J or bovine lactoferrin, hemoglobin, transferin, and hemin. The results suggest that the type and concentration of detergent, egg yolk and glycerol had most significant effect on growth of the bacterium, and that Dubos media does not support growth of M. paratuberculosis.