Novel method for clearing red blood cell debris from BacT/ALERT® blood culture medium for improved microscopic and antimycobacterial drug susceptibility test results

Even though automation in mycobacterial culture has immensely improved the detection of organisms, identification of species and antimycobacterial susceptibility testing from blood culture bottles remain cumbersome and error-prone due to the presence of intact red blood cells (RBCs). The removal or lysis of these RBCs and excessive protein from the blood components could theoretically help improve this process. The present study reports an effective method that uses ammonium chloride (NH₄Cl) and Triton X-100 to lyse the RBCs in blood culture medium. The method was optimized by preparing various concentrations of NH₄Cl and Triton X-100, and incubation conditions, leading to eight protocols. The lysis protocol with a concentration of 150 mM of NH₄Cl, 0.5% Triton X-100, and 1% potassium bicarbonate, pH 7.0, and incubation at 37°C for 15 min was found to be optimal. This method not only made the culture medium clear, the protein concentration decreased from 753.5 ± 39.4 to 53.2 ± 4.2 mg/mL in the M. tuberculosis-spiked culture medium and in the blood culture medium inoculated with the blood from tuberculosis patients. The method had no adverse effect on mycobacteria, and no depletion of M. tuberculosis colony-forming units was found. The lysate could be used for antimycobacterial susceptibility testing with no difficulty in setting the mycobacterial concentration of inoculum to 0.5 McFarland standards. Furthermore, this method had the added advantage in the microscopy and molecular methods for the speciation of Mycobacterium sp.