Background: Although BCG is the most widely administered vaccine in the world, there have never been as many cases of TB as there are now. Globally, more than 8.8 million people developed active TB and 1.4 million—many of them—died in 2010. It is estimated that half of pulmonary TB cases arise from latent Mtb infection, making the study of latency and reactivation of utmost importance. Methods: Widely administered BCG vaccines and a gene modified recombinant BCG (rBCG) strain, AERAS-422, were used as models to investigate the growth promoting function of resuscitation-promoting factors (Rpfs) in different bacilli culture phases. Different supernatant fractions were prepared by ultrafiltration, and the promoting function of each fraction containing secreted Rpf(s) was evaluated by growth curve monitoring and colony counting on 7H10 agar plates. Results: The promoting effect of culture supernatants was mainly associated with the high molecular weight fraction (>30 kDa), which stimulated bacterial growth, but did not extend the exponential phase of stimulated culture. Anti-RpfB antibody showed significant growth restriction of the tested cultures. When comparing rBCG cultures containing 7H9 medium, the 10 - 30 kDa fraction, or the >30 kDa fraction, only the >30 kDa fraction was displayed with down-regulation of the secretion of RpfC, D and E. In colony counting tests, the plates containing the >30 kDa fraction had total countable colony numbers 2 to 3 fold higher than the plates with the 10 - 30 kDa fraction, and colonies appeared one to two weeks earlier than on the regular plates. The potential applications of the prepared supernatant fractions containing RpfA and RpfB are discussed, which may include accelerating diagnosis of Mtb infection and future TB vaccine development.