Mycobacterium bovis BCG Cultivation and Drug Susceptibility Assay

Mycobacterium bovis (BCG, Tokyo strain) was purchased from Japan BCG Laboratory (Tokyo, Japan) and maintained in BD BACTEC™ MGIT™ medium supplemented with OADC Enrichment and PANTA antibiotic mixture (Becton Dickinson Co., 245122, Franklin Lakes, NJ) at 37 °C. Mid-log-phase BCG was prepared at concentrations of 1000 CFU/µl for time-lapse microscopy and 10,000 CFU/µl for drug susceptibility testing in MGIT media by using a bacterium counting chamber (A161, SANSYO, Tokyo, Japan). RFP, INH, PZA, SM, EB, LVFX, TH, and CS were obtained from FUJIFILM Wako (Tokyo, Japan). KM and EVM were obtained from Nacalai Tesque (Tokyo, Japan) and Asahi-Kasei Pharma (Tokyo, Japan), respectively. DLM and BDQ were obtained from Selleck (Houston, TX). These chemicals were dissolved in deionized water (INH, PZA, SM, EB, TH, KM, EVM, and CS), methanol (RFP, TH), 0.1 N hydrochloride (LVFX), or dimethyl sulfoxide (DLM, BDQ), and then sterilized using 0.22-µm MILLEX-GV syringe filters (Millipore, Burlington, MA) prior to being added to MGIT media under aseptic conditions. Perfluorocarbon (Fluorinert™, FC-3283) was obtained from 3M (Maplewood, MN). Live/Dead™ BacLight™ bacterial viability reagent was purchased from Thermo Fisher Scientific (L13152, Waltham, MA). Detailed information on the antibacterial agents used in this study is shown in Supplementary Table 1.

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Kikuchi S., Yamashige Y., Hosoki R., Harata M, & Ogawa Y. (2023). Near-field sensor array with 65-GHz CMOS oscillators can rapidly and comprehensively evaluate drug susceptibility of Mycobacterium. Scientific Reports, 13, 3825.

Publication 2023

MILLEX-GV syringe filters

Antibacterial agents Antibiotic Aseptic Bacterial viability Dimethyl sulfoxide Fluorinert Methanol Microscopy Mycobacterium bovis Perfluorocarbon Strain Susceptibility Syringe

Corresponding Organization : Hyogo University

Other organizations : Kyoto University, Tohoku University

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Variable analysis

dependent variables

Drug susceptibility of Mycobacterium bovis (BCG, Tokyo strain)
Mycobacterial growth in MGIT medium

control variables

Mycobacterium bovis (BCG, Tokyo strain) culture conditions (37 °C in MGIT medium supplemented with OADC Enrichment and PANTA antibiotic mixture)
Bacterial concentrations (1000 CFU/µl for time-lapse microscopy, 10,000 CFU/µl for drug susceptibility testing)

positive controls

None specified

negative controls

None specified

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