Development of an improved selective agar medium for isolation of Yersinia pestis

Abstract

Existing media designed for selective isolation of clinically important members of the genus Yersinia were found to be unsatisfactory for the growth and isolation of Yersinia pestis. We report the development of a new selective agar medium (termed BIN) that supports the growth of Y. pestis. The development of the formulation of this medium was based on a fluorescence screening system designed for monitoring bacterial growth on semisolid media, using a green fluorescent protein-expressing strain. High-throughput combinatorial experiments can be conducted for the quantitative evaluation of the effect of different medium components on growth. Generation of fluorescence plots in this system, using microplates, allowed the quantitative evaluation of the growth rate of Y. pestis EV76 cultures in different agar compositions. The final BIN formulation is based on brain heart infusion agar, to which the selective agents irgasan, cholate salts, crystal violet, and nystatin were introduced. It was found that BIN agar is more efficient in supporting colony formation and recovery of Y. pestis than are the conventional semisolid media MacConkey agar and Yersinia-selective agar (cefsulodin-irgasan-novobiocin agar). The advantage of BIN over other media has been also demonstrated in recovering virulent Y. pestis from the mixed bacterial populations found in decaying carcasses of infected mice. The BIN medium is suggested as a selective medium for isolation and recovery of Y. pestis from various backgrounds.

FIG. 1.

Growth of the GFP-expressing Y. pestis EV76(pGFPuv) strain on commercially available semisolid media. (A) Fluorescence emission curves of agar cultures on the indicated media, incubated at 28°C. (B) Fluorescence of bacterial suspensions containing 10⁸ CFU/ml in PBS prepared from the indicated agar cultures. RFU, relative fluorescence units.

FIG. 2.

Fluorescence curves of the GFP-expressing Y. pestis EV76 strain on BHIA containing various levels of selective components, incubated at 28°C. The concentrations of cefsulodin, novobiocin, crystal violet, irgasan, and cholate salts (equal amounts of sodium cholate and sodium deoxycholate) are indicated in micrograms per milliliter. The concentration of nystatin is indicated in units per milliliter (USP).

FIG. 3.

Growth of Y. pestis EV76(pGFPuv) on BIN agar at 28°C. The growth of this strain on BHIA is presented for comparison. RFU, relative fluorescence units.