Maldi tof vitek ms 3.0 system

Following microorganism growth identification by the BacT/ALERT^® 3D system, blood culture media were collected from each bottle and subjected to Gram staining. Then, samples were subcultured on solid growth media, including blood agar (bioMérieux) and Sabouraud agar (Merck, Germany), and incubated at 35°C for 18–24 hours. To estimate the cell numbers in the bottles, 5 positive blood culture bottles were randomly selected. Then, 1 mL sample was aspirated from each of these bottles, serially 10‐fold diluted with sterile saline, and 50 μL of suspensions was plotted on the Sabouraud agar plate, and colonies were counted after 24 h of incubation (ranged from 7 × 10⁵ to 5 × 10⁷ CFU/mL). The rapid disc diffusion method was performed according to the RAST methodology standardized by the European Committee on Antimicrobial Susceptibility Testing. Following incubation, isolated colonies were subjected to analysis by the MALDI-TOF VITEK MS^® 3.0 system (bioMérieux, France) according to the manufacturer's instructions. Fluconazole susceptibility was assessed using a disk diffusion method according to the CLSI M44-A2 guidelines and a broth microdilution method according to the European Committee on Antimicrobial Susceptibility Testing guidelines [8 , 9 ].

The rapid identification method was performed according to the protocol proposed by Spanu et al. [10 (link)]. Each test was conducted in duplicate. An 8 ml aliquot from the blood culture bottle was centrifuged at 10,000 rpm for 2 minutes at room temperature. The supernatant was discarded, and the pellet was washed twice with 1 ml of pure water and recentrifuged. It was suspended in 1 ml of 0.1% Tween 80, incubated for 2 minutes, recentrifuged, washed twice with 1 ml of pure water, recentrifuged, suspended in 300 μl of pure water plus 900 μl of absolute ethanol, and recentrifuged. Then, 30 μl of 70% formic acid plus 30 μl of pure acetonitrile was added to the pellet, and it was thoroughly vortexed and centrifuged at 14,000 rpm for 2 minutes. A 1 μl aliquot of the supernatant was collected and applied to a steel MALDI target plate. Finally, the sample was subjected to analysis by the MALDI-TOF VITEK MS^® 3.0 system (bioMérieux, France).