Objective·To directly detect the bacteria in positive blood culture specimens by the separation gel tube combined with MicroflexTM matrixassisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS),perform direct antimicrobial susceptibility test based on the results of mass spectrometry,and preliminary explore the redesign of conventional positive blood culture specimen processing flow.Methods·449 positive-alarm blood culture specimens of monobacterial infections in clinical microbiology laboratory of Xirhua Hospital affiliated to Shanghai Jiao Tong University School of Medicine from September 1,2015 to August 31,2016 were collected and prepared according to the new positive blood culture specimens processing flow.The new redesigned processing flow included smear staining and microscopy,separation gel/mass spectrometry direct identification,bacteria film/mass spectrometry identification,pure colony/mass spectrometry identification,direct antimicrobial susceptibility test,and conventional antimicrobial susceptibility test,etc.According to the results of microscopic examination,identification test,and antimicrobial susceptibility test,level Ⅰ direct microscopic examination report,positive blood culture level Ⅱ (direct identification/bacteria film identification or direct antimicrobial susceptibility) report,and level Ⅲ final report were provided sequentially.Results·With the new redesigned processing flow,bacterial specieslevel identification reports for 82.2％,95.8％,and 100％ of positive blood samples could be obtain at 10 am and 17 pm on the current day and 10 am in the next morning,respectively,and be sent to clinical departments.Initial antimicrobial susceptibility reports for the bacteria that were considered as clinical significant pathogens by preliminary assessment could be provided in the next day of blood culture positive alarm with a higher coincidence rate as compared to the results of traditional antimicrobial susceptibility tests.Conclusion·The time from blood culture positive alarm to the provision of initial identification and antimicrobial susceptibility reports is shorter by 1-2 d for the redesigned processing flow than for the traditional one,which can provide fast and accurate etiologic diagnosis evidence for bloodstream infections for clinicians and is important for improving early diagnosis and treatment of clinical bloodstream infections.