Optimization of the composition of a solid culture medium for Mycobacterium avium subsp. paratuberculosis using factorial design and response surface methodology

Abstract

Aim: To develop an optimized solid culture medium for improved growth of Mycobacterium avium subsp. paratuberculosis (MAP).

Methods and results: Seven medium constituents (factors) were assessed at various concentrations for their ability to positively affect MAP growth. The factors tested were Tween 80, egg yolk, casitone, taurocholic acid, Mycobactin J, agar and either OADC or ADC supplement. After an initial screening of individual factors, a fractional factorial design and a response surface methodology (RSM) central composite design were used to assess the effects of multiple factors simultaneously and design a new solid culture medium. MAP growth became visible on streak plates of the optimized solid medium 2 weeks earlier than on Herrold's egg yolk medium (HEYM).

Conclusions: MAP grew faster on the optimized solid medium than on HEYM. It consisted of Middlebrook 7H9 broth with 1.0% Tween 80, 0.019% casitone, 1.4% bacteriological agar, 10% egg yolk, 10% ADC and 1.65 μg ml^-1 Mycobactin J.

Significance and impact of the study: This is the first study to use an RSM approach to optimize the composition of a solid medium for MAP culture. The new medium could improve MAP culture in future by reducing incubation times and increasing MAP colony numbers.

Keywords: Mycobacterium avium subsp. paratuberculosis; egg yolk; growth supplements; medium composition optimization; response surface methodology; solid culture.