BBL™ Brain Heart Infusion Broth, BD

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	Brain Heart Infusion broth (BD BBL Brain Heart Infusion Modified and 1.0 g/L cysteine-HCL pH 7.4).

Protocol tips
Brain Heart Infusion broth (BD BBL Brain Heart Infusion Modified and 1.0 g/L cysteine-HCL pH 7.4).

Publication protocol

C. botulinum ATCC
3502 Hall 174 (HA+, toxin type A1), C. botulinum ATCC 19397 NCTC 7272 (HA+, toxin type
A1), C. botulinum ATCC 17841 McClung 1347 (HA+, toxin type B1), C. botulinum ATCC
35415 Langeland (orfX+, toxin type F1), and C. sporogenes ATCC 3584 (non-toxigenic). These
four strains of C. botulinum and the C. sporogenes strain were selected because of the availability of genomic sequence data that could provide predicted protein information for the strains
(Accession numbers noted in Table 1). In addition, the strains were all within C. botulinum
Group I (which appear similar to each other using genomic sequence comparisons, described
below and Fig 1), express any one of three toxins BoNT/A, BoNT/B, or BoNT/F, and represent
the two toxin clusters (HA+ or orfX+).
Samples were generated by inoculating 100 mL TPGY broth (50.0 g/ L trypticase peptone,
5.0 g/ L Bacto peptone, 4.0 g/ L glucose, 20.0 g/L yeast extract and 1.0 g/L cysteine-HCL pH
7.4) or Brain Heart Infusion broth (BD BBL Brain Heart Infusion Modified and 1.0 g/L cysteine-HCL pH 7.4). These media were selected as they are commonly used for culturing C. botulinum. A single bacterial colony was used to inoculate an overnight culture of 100 ml TPGY.
One ml of the culture at an OD600 of 1.0 (Thermospectronic spectrophotometer model 40001/
4), was used to inoculate a 100 ml bottle of TPGY or BHI media. Cultures were incubated
anaerobically at 30˚C, and two 10ml samples were removed at three points: when the OD600 =
0.4, OD600 = 0.8, and at 5 days post-inoculation. These three time points were chosen to represent an early and late exponential, and stationary phase cultures, to determine if there were
observable differences between these growth phases. The culture aliquots were autoclaved for
60 minutes, then frozen at -70˚C prior to protein analysis. In this experimental design, each
strain/medium combination was cultured twice on two separate days to generate biological
replicates for protein analysis

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