bThe Sensititre MycoTB plate (TREK Diagnostic Systems, Cleveland, OH) uses a microtiter plate MIC format for susceptibility testing of Mycobacterium tuberculosis complex isolates against first-and second-line antituberculosis agents. Categorical agreement versus the agar proportion method for 122 M. tuberculosis complex isolates was 94% to 100%. T he gold standard method for Mycobacterium tuberculosis complex susceptibility testing is the 1% indirect agar proportion method (APM), which is laborious and requires 2 to 3 weeks for results (4, 7). This two-site study evaluated a new system, the Sensititre MycoTB plate (MycoTB), for susceptibility testing of M. tuberculosis complex. The plate uses a 96-well microtiter broth format and contains 12 lyophilized first-and second-line antimycobacterial drugs. In contrast to other M. tuberculosis complex susceptibility methods, which test one or two critical concentrations of a drug, the MycoTB plate examines a range of drug concentrations and produces an MIC result. Recently, Abuali et al. evaluated the MycoTB plate using 37 M. tuberculosis complex isolates (1). Our study confirms and extends these results by testing a larger number of isolates, including singly and multiply resistant isolates, and provides precision data for the MycoTB plate. We also compared a manual, mirror plate-reading method with a commercially available, manual plate reader with data management software (Vizion System, TREK Diagnostics).(This study was presented in part at the 50th Annual Interscience Conference on Antimicrobial Agents and Chemotherapy Boston, MA, 12 to 15 September 2010.)One hundred twenty-two M. tuberculosis complex isolates (59 from site 1 and 63 from site 2) were tested using the APM and the MycoTB plate. An additional 15 M. tuberculosis complex "challenge" strains with known susceptibility patterns were provided by the Centers for Disease Control and Prevention (CDC) and tested using the MycoTB plate. Inoculum preparation and plating were performed using a biological safety cabinet (BSC) inside a biosafety level 3 (BSL3) laboratory as previously described (5). Several colonies were selected from Middlebrook 7H10 medium using a sterile loop and inoculated into a test tube containing saline-Tween and glass beads (TREK Diagnostics). After being vortexed for 30 to 60 s, the inoculum was allowed to settle for 15 min and adjusted to a 0.5 McFarland standard equivalent using a nephelometer. The numbers of CFU were determined for each isolate tested to verify that the inoculum was within a specific targeted amount (ϳ10 5 CFU/ml). One hundred microliters of the inoculum was transferred to 11 ml of Middlebrook 7H9 broth containing oleic acid-albumin-dextrose-catalase (TREK Diagnostics) and vortexed for 20 s. One hundred microliters was transferred to the MycoTB plate wells containing the lyophilized antibiotics. MycoTB plates were covered with plastic seals provided by the manufacturer, and the entire outer surface of the plate was disinfected with a tuberculocidal agent. Plates were in...

The rapid detection of antimicrobial resistance is important in the effort to control the increase in resistant Mycobacterium tuberculosis (Mtb). Antimicrobial susceptibility testing (AST) of Mtb has traditionally been performed by the agar method of proportion or by macrobroth testing on an instrument such as the BACTEC (Becton Dickinson, Sparks, MD), VersaTREK (TREK Diagnostics, Cleveland, OH) or BacT/ALERT (bioMérieux, Hazelwood, MO). The agar proportion method, while considered the "gold" standard of AST, is labor intensive and requires calculation of resistance by performing colony counts on drug-containing agar as compared to drug-free agar. If there is ≥1% growth on the drug-containing medium as compared to drug-free medium, the organism is considered resistant to that drug. The macrobroth methods require instrumentation and test break point ("critical") drug concentrations for the first line drugs (isoniazid, ethambutol, rifampin, and pyrazinamide). The method described here is commercially available in a 96 well microtiter plate format [MYCOTB (TREK Diagnostics)] and contains increasing concentrations of 12 antimicrobials used for treatment of tuberculosis including both first (isoniazid, rifampin, ethambutol) and second line drugs (amikacin, cycloserine, ethionamide, kanamycin, moxifloxacin, ofloxacin, para-aminosalicylic acid, rifabutin, and streptomycin). Pyrazinamide, a first line drug, is not included in the microtiter plate due to its need for acidic test conditions. Advantages of the microtiter system include both ease of set up and faster turn around time (14 days) compared with traditional agar proportion (21 days). In addition, the plate can be set up from inoculum prepared using either broth or solid medium. Since the microtiter plate format is new and since Mtb presents unique safety challenges in the laboratory, this protocol will describe how to safely setup, incubate and read the microtiter plate. Video LinkThe video component of this article can be found at https://www.jove.com/video/3094/ ProtocolEach laboratory needs to perform a risk assessment in collaboration with their Institutional Biological Safety Officer to determine the appropriate biosafety level for preparation of the inoculum and for pipetting of the inoculum into the plate. In our laboratory, we utilize biosafety level 3 laboratory (BSL3) practices until the microtiter plates are inoculated, and sealed with a plastic adhesive seal. These plates are then placed inside a plastic bag, which is heat-also sealed. Incubation and interpretation reading of the microtiter plate is conducted in a biosafety level 2 laboratory (BSL2). Labeling of materials1. Label a blood agar plate, three Middlebrook 7H10 plates, Middlebrook 7H9 broth, a saline tween glass bead tube and a microtiter AST plate with the appropriate identifiers (e.g., patient name, medical record number) in the BSL2. 2. Label the blood agar and 7H10 plated as "purity check". Periodic colony counts of the inoculum are recommended to ensure that an appropriate con...

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