A novel 2-in-1 culture system for the isolation of Mycobacterium avium subsp. paratuberculosis

Culture remains the ‘gold standard’ detection method for viable Mycobacterium avium subsp. paratuberculosis (MAP). While liquid culture methods speed up detection time, culture on a solid medium is needed to obtain MAP colonies. The aim of this study was to develop a ‘2-in-1’ culture system with liquid andsolid media in the same flask, to potentially increase MAP colony numbers and colony formation rates compared to solid media alone. Tissue culture flasks (25 ml) were prepared with 6 ml agar (7H10, 7H11 or HEYM) and 2 ml broth (7H9 or Pozzato). Once MAP inoculum was added to the broth, flasks were incubatedin a vertical position for 1-4 weeks, then incubated in a horizontal position for 24 h to inoculate the agar surface, before being returned to a vertical position for incubation until colonies appeared. Different liquid-solid media combinations and periods of incubation in broth were investigated. The optimum medium combination and incubation period in broth varied depending on the concentration of MAP added. When using a 10¹-10² cfu MAP inoculum, colonies formed more rapidly in flasks containing 7H9 broth with HEYM,and an incubation period in broth of 3-4 weeks resulted in significantly higher growth scores (p ≤ 0.01). However, a 10⁴-10⁵ cfu MAP inoculum became visible earlier in flasks containing Pozzato broth with 7H10 medium, and longer incubation periods merely extended the time required to observe colonies. To date, although the 2-in-1 system has not improved MAP colony formation rates compared to culture on agar alone, final colony counts were increased, and solid media didn’t dry out. Experiments were completed with laboratory-grown MAP strains, so further work is required to optimise this novel 2-in-1 culture system and to determine whether colony formation rates upon primary isolation of MAP can be improved compared to culture on agar only.