Evaluation of the Inoculation Procedure Using a 0.25 McFarland Standard for the BD Phoenix Automated Microbiology System

Rapid bacterial identification (ID) and antimicrobial susceptibility tests (AST) can have a significant impact on the management of infections, especially those caused by antibiotic-resistant bacteria (1, 2, 5). However, these rapid automated methods do not overcome the need for the preparation of a suspension of the organism at a fixed density (0.5 to 0.6 McFarland standard [MF]; 1.5 × 108 CFU/ml). Subculturing the isolate to obtain the required number of colonies is the usual solution to insufficient numbers of colonies available on primary isolation agar plates, but this equates to a further 24-h delay in completing the test and, in turn, in providing results to the clinician. In these cases, the rapid automated methods offer no time advantage compared to manual methods. In order to overcome this problem, BD, for its Phoenix automated microbiology system, has recently developed new software and a database for ID using a lower inoculation density, a 0.25 MF (7.5 × 107 CFU/ml) instead of the previously used 0.5 MF, for the inoculation of panels. The new capability to determine ID involves the use of separate ID substrate databases for substrate reactions for each method. The AST inoculation density, 5 × 105 CFU/ml, is maintained following the procedure with the 0.25 MF by transfer of twice as much ID suspension (50 μl instead of 25 μl) to the AST broth. We conducted a prospective study over a 1-month period to evaluate and validate the possibility of using the 0.25 MF in our facility. This study compared inoculation with the currently used 0.5 MF, used for 6 years in our laboratory (3), to inoculation with a 0.25 MF for the exact same isolates (Table ​(Table11). TABLE 1. List of discordant results obtained by comparing methods with a 0.25 MF and a 0.5 MFa Accurate ID at the species level was achieved for 95.5% of the isolates by use of the 0.25 MF and for 95.5% of the isolates by use of the 0.5 MF. Ten of 132 clinical isolates (7.3%) had discordant ID results by the two methods (Table ​(Table1).1). No discordant identification results were observed for Staphylococcus aureus and Pseudomonas aeruginosa isolates. Differences between the two ID approaches were not statistically significant. Errors were less than 1.4% among 2,654 performed AST. No very major errors (the method with the 0.25 MF indicating sensitivity and that with the 0.5 MF indicating resistance) were found. Totals of 32 (1.2%) major errors (ME; the method with the 0.25 MF indicating resistance and that with the 0.5 MF indicating sensitivity) and 37 (1.4%) minor errors (mE; the method with the 0.25 MF indicating an intermediate result and that with the 0.5 MF indicating sensitivity or resistance) were obtained (not shown). There were 7 ME and 7 mE for Enterobacteriaceae, 5 ME and 1 mE for staphylococci, 18 ME and 18 mE for Pseudomonaceae (mainly P. aeruginosa), and 2 ME and 11 mE for enterococci. Although we used the inoculation method with a 0.5 MF as the comparative method (which is not truly a reference method), we have 6 years of experience with this system and have confidence in its accuracy for most organism-antibiotic combinations. In addition, we developed several confirmatory tests for some combinations with which the system's accuracy is weaker (3). The performance of the low-inoculum-density method based on presented results has allowed us to start to use this approach routinely in our laboratory. (Part of this work was presented at the 107th General Meeting of the American Society for Microbiology, Toronto, Ontario, Canada, 21 to 25 May 2007 [4].)