Detectable levels from low starting inoculums of F. tularensis are difficult to achieve in vitro. Various studies have shown that the spent culture medium of pathogenic bacteria, such as Mycobacterium tuberculosis, ALK5 Inhibitor II stimulated enhanced growth of dormant and small inoculum cultures. More importantly, studies conducted by Halmann and colleagues have described the presence of a growth-initiation substance in the culture VE-821 filtrate using small inocula of virulent strains of F. tularensis. Therefore, we tested if the spent culture filtrate of F. tularensis vaccine strain would stimulate and enhance the growth of a small number of F. tularensis cell. Overnight cultures of F. tularensis were serially diluted and spotted on standard culture medium supplemented with and without spent culture filtrate. F. tularensis cultured on spent culture filtrate resulted in robust growth at all dilutions in contrast to standard culture medium, which only supported the growth of 1021�C1022 dilutions. The main challenge in diagnostics and pre-analytical processing of F. tularensis in food and environmental matrices is the presence of other bacteria, which may interfere with F. tularensis growth. Furthermore, the possibility remained that F. tularensis spent culture filtrate may stimulate inter-species growth of bacteria found in food and would consequently limit its�� utility as a diagnostic aid. To determine if F. tularensis spent culture medium promoted growth of other bacterial species, we compared the CFU/mL of bacteria found within lettuce and soil on spent filtrate supplemented medium and standard medium alone. We observed no difference in CFU/mL between standard medium supplemented with or without spent filtrate. However, the possibility remains that the spent culture filtrate may inhibit the growth of certain bacterial species, while other bacterial communities may continue to exist and fill the vacant niche. In fact, we did observe changes to colony phenotype when the spent culture filtrate was added to lettuce homogenates. Next, we investigated our ability to detect F. tularensis by real-time PCR. Specifically, does the addition of spent culture filtrate in relation to the limit of detection and does the inclusion of soil and lettuce bacteria interfere with ? F. tularensis cultured with spent filtrate showed a 2.5�C3.5 fold increase in cells per mL at a starting inoculum of 1 bacterium per mL compared to standard medium alone. Addition of soil bacteria to F. tularensis cultured with spent filtrate reduced the overnight growth of F. tularensis an average of 1.5 fold; however, a 2 fold increase was observed when compared against F. tularensis mixed with soil bacteria in standard medium. Given that the spent culture filtrate resulted in enhanced detection at low inoculums, we focused on detection of F. tularensis at 1�C102 cells/mL spiked in lettuce bacteria. Supplementation of TSB and L-cysteine with spent culture filtrate resulted in increased growth of F. tularensis mixed with lettuce bacteria at all inoculums in comparison to standard medium.