Sensititre auto inoculator

Phenotypic DST was performed using the broth microdilution method which provides a minimal inhibitory concentration (MIC) value (Sensititre^® MYCOTB plate). A panel of 13 anti-TB drugs was tested, including INH, RPF, and EMB. Several colonies were selected from growth on solid media using a sterile loop and inoculated into a test tube containing glass beads as described previously [12 (link)]. A 0.5 McFarland standard equivalent was made after vortexing, suspensions were diluted 100-fold with the addition of 100 μL of the 0.5 Mc suspensions to 10 mL of Mueller–Hinton broth with OADC (oleic acid-albumin-dextrose-catalase). Aliquots of 100 μL of the standard 1.5 × 10⁵ CFU/mL inoculum were distributed to each well using the semiautomated SensititreTM Auto-inoculator (Thermo Fisher, Scientific Inc., San Francisco, CA, USA). MYCOTB plates were sealed and incubated at 37 °C. MIC was defined as the lowest concentration without obvious visible bacterial growth compared with positive controls, aided by the VizionTM Digital viewing system. Mtb H37Rv (ATCC 27294) was used as a quality control in each batch. Based on CLSI M62, the breakpoints for INH, RPF, and EMB were 0.12 μg/mL, 1 μg/mL and 2 μg/mL, respectively. An inconclusive result for ethambutol was an MIC of 4 μg/mL, which does not correlate with either a susceptible or resistant result.

Scientists from all laboratories received training in plate inoculation and reading at the SRL. The standard operating procedure involved preparing a suspension at a 0.5 McFarland standard in saline Tween with glass beads (Thermo Fisher, Scientific Inc., USA) from 20- to 25-day-old colonies (or no later than 14 days after visible growth) grown on Lowenstein-Jensen or 7H10 agar medium. Duplicates were tested on different days and starting from different subcultures, thus also starting from different bacterial suspensions.
Suspensions were then diluted 100-fold by the addition of 100 μl of suspension to 10 ml of enriched 7H9 broth. Aliquots of 100 μl of standard inoculum (1.5 × 10⁵ CFU/ml; approximate range, 5 × 10⁴ to 5 × 10⁵ CFU/ml) were dispensed into wells by use of a semiautomated Sensititre Autoinoculator (Thermo Fisher, Scientific Inc., USA). Each plate was then sealed using a manufacturer-supplied rectangle of transparent plastic that remained in place throughout the 21-day incubation period.