The method used for the isolation of spores from environmental samples was that described in OIE Terrestrial Manual 2012 [15 ], with some modifications. For culturing and isolation of B. anthracis the TSMP medium was used, consisting in the semi-selective Columbia blood agar added with trimethoprim (16 mg/lt), sulfamethoxazole (80 mg/lt), methanol (5 ml/lt) and polymyxin (300,000 units/lt). Based on our experience, TSMP has the same efficacy of PLET in isolating B. anthracis (data not shown). Briefly, to each 7.5 gram aliquot of soil sample were added 22.5 ml of deionized sterile water. After 30 minutes of washing by vortexing, the suspension was incubated at 64°C for 20 min to eliminate any vegetative forms of soil contaminants [16 (link)].From each sample, 10 ml of supernatant were collected and dilutions of 1:10 and 1:100 were made using normal saline solution.
Subsequently, 10 plates of TMSP were seeded with the undiluted suspension (100 μl/plate), 10 plates with the 1:10 dilution and 10 plates with the 1:100 dilution. After 24 and 48 hours of incubation at 37°C, each plate was examined for the presence of suspect colonies of B. anthracis and of contaminants. All colonies were counted. B. anthracis colonies were identified by Gram staining, colony morphology and anthrax-specific PCRs [17 (link)].
Subsequently, 10 plates of TMSP were seeded with the undiluted suspension (100 μl/plate), 10 plates with the 1:10 dilution and 10 plates with the 1:100 dilution. After 24 and 48 hours of incubation at 37°C, each plate was examined for the presence of suspect colonies of B. anthracis and of contaminants. All colonies were counted. B. anthracis colonies were identified by Gram staining, colony morphology and anthrax-specific PCRs [17 (link)].
Free full text: Click here
