However the decrease in score between chemical extraction

However, the decrease in score between chemical extraction and direct smear from over 2.0 to 1.70–2.00 did not overall change the final correct identifications (202 vs. 194) by Biotyper 3.0 software (0.05<<0.1), (). Furthermore, other reports have previously suggested that a cut off of the score>1.70 was sufficient for accurate identification of spp. and other species. Similarly, Alatoom et al. demonstrated that a cut off of 1.70 was sufficient to identify the genus of Corynebacterium spp. extraction. Fourteen of unidentified isolates after chemical extraction were subjected to further testing by 16S rDNA sequencing (21, ). Identification using 16S rDNA sequence analysis indicated 5 of 14 unidentified isolates were not in the Bruker Biotyper version 3.0 database. These five isolates are (=1), (=1), (=1), (=2). Hence the both chemical extraction and direct smear could not identify these isolates. Another five isolates which were not identified by either chemical extraction or direct smear were (=1), spp. (=2), (=2), spp. It is noted that these were among the genus that had a relatively decreased score following direct smear (). Surprisingly, direct smear identified four isolates not identified following chemical extraction (), these four isolates had no any colony growth which could result in no identification by chemical extraction because there were not enough cell mass for extraction. Two methods have been described for the preparation of bacteria for identification by MALDI-TOF MS, one is direct colony smear on the target plates (with or without the addition of formic I-BET-762 for extraction), the other is the standard ethanol/formic acid protein extraction in which 70% formic acid is added to the ethanol-washed cell pellet, mixed, and followed by addition of acetonitrile and then supernatant is spotted to the target plate. The direct smear method is fast and simple, but the results are sometimes inconsistent compared to the stand chemical extraction. This study demonstrated anaerobic bacteria could be identified by direct smear methods. The results indicate not only that direct smear may be non-inferior to chemical extraction, but also that direct smear may allow additional identifications inaccessible to chemical extraction. The results also underline, as describe elsewhere, that the lack of certain anaerobic species such as and in the database of Biotyper 3.0 are the limit of MALDI-TOF MS. For example, the previous studies have already reported that the lack of the species in the Biotyper database led the system to identify as with high confidence score. Although the mass spectrum of is in the database, the system failed to identify this strain accurately (), this may be due to only a unique peak profile of the single strain in the database which might not be enough to represent the diversity of entire species and lead to such problems. Hence, increasing the number of representative spectra for species might lead Biotyper software to more accurately identify this species. Although company has been increasing the representatives of microorganism species of database in the systems, it may still be updated and expanded through addition of in-house spectra from encountered clinical strains. Therefore, the impact of our study was to increment the Biotyper3.0 database with the spectra of all the strains identified through 16S rDNA sequence analysis. In addition to the number of the reference strains in the database, many other factors may affect the identification by Biotyper, such as the cell wall rigidity, culture condition and bacterial culture age. Our experiments indicate that the number of cells spotted on the target plate has also been shown to influence the identification by direct smear methods (data not shown), too many bacteria cells may overwhelm the lysis of formic acid and matrix solution and hence the cells may not be properly disrupted. We found that 1×10–10 bacterial cells resulted in the good spectral resolution. Smearing cells evenly on the target plates is also critical to achieve good results.