Ground Anthrax Bacillus Refined Isolation

The Ground Anthrax Bacillus Refined Isolation (GABRI) was performed as described by [36 (link)]. After incubation at 37°C for 24 hours the suspected colonies appearing whitish and without hemolysis were picked and spread on Columbia blood agar. To reduce the chance for mutations during in vitro cultivation, bacteria were inactivated (for DNA extraction) after a single passage on solid growth medium. In rare cases of impurities another incubation step on selective agar was repeated. A few colonies were scraped off the plate and transferred into a 1.5 ml reaction tube filled with 500 μl 2% (v/v) Terralin PAA (Schülke & Mayr). Colony clumps were dissolved by rotating the inoculation loop or by pipetting. The reaction tube was filled up completely with 2% (v/v) Terralin PAA and incubated for 30 min for a proper inactivation of B. anthracis endospores which are vulnerable to peracetic acid [37 (link)], the active agent of Terralin PAA. Following centrifugation at 6000 x g for 2 minutes the supernatant was removed and the pellet resuspended in 1 ml phosphate-buffered saline (PBS). After two more washing steps with 1 ml PBS the pellet was stored at -20°C until further use. Work involving live B. anthracis was performed in a biosafety level 3 laboratory within a class III safety cabinet (glove box).
For each soil sample up to three aliquots were processed and from these ≥3 isolates (if present) were further processed in order to obtain a representative subpopulation. Names of single isolates comprise sampled position, their depths, and a running number. C1/5 cm– 1, for instance, indicates the first isolate from position 1 of burial site C in 5 cm depth. Strains which were further characterized are listed in Table 1.