AOAC 2017 First Action Methods

Note : When exchanging targets, do not leave the load port lid open for any longer than necessary. Leaving the load port lid open for extended periods does not damage the instrument but will prolong target loading by as much as 30 min. F. Operating the MALDI Biotyper System ( a ) Log on to the MALDI Biotyper System computer. ( b ) Start the MALDI Biotyper System software by first clicking on the Flex Control icon. Then click onto the RTC software icon on the desktop. ( c ) After successful startup, the main application window is displayed. ( d ) Prepare a test run according to the MALDI Biotyper System Instructions for Use. G. Analyzing Results ( a ) MALDI Biotyper System identification consists of two steps, which are performed on each test and BTS control position. ( b ) A mass spectrum is acquired from the test or BTS control position (measurement step). ( c ) The resulting mass spectrum is processed, and the resulting peak pattern is matched against reference patterns in the MALDI Biotyper reference library (identification step). ( d ) The identification step is started immediately after the associated measurement step has been completed and a mass spectrum is available. ( e ) When the identification run is started, the sample positions to be measured appear as white circles in the target display. ( f ) During the run, the appearance of the sample positions and BTS control positions in the target display reflects the success of the measurement and identification process of each test organism. ( g ) If spectrum measurement is successfully completed, the left half of the sample position is colored green. If measurement fails, the left half of the sample position is colored orange. ( h ) The coloring of the right half of the sample position indicates the score value of the identification (green, yellow, or red). The legend display explains the color coding used to indicate the status of sample positions in the target display. ( i ) Based on the Consistency Category Table of the MBT Compass User Manual, scores ≥2.0 will be presented in green and are determined to be high-confidence identification. Results presented in yellow will have values between 1.70 and 1.99 and are low-confidence identification. Results presented in red are considered not acceptable for identification. The consistency category of the identification is based on the confidence level of the best and second-best matches as described Table  2017.09K . ( j ) The two best matches should be >1.70 for the confirmation of Salmonella , Cronobacter, and Campylobacter to the genus level. Tip : Point to the information button in the top-right corner of the target display to show the color-coding legend. ( k ) When the identification procedure is completed, the organism identification result will appear in the result table. ( l ) The result table of the MALDI Biotyper System window gives a real-time overview of the identification results for the active test run. This table contains only the best-matching reference pattern for each test organism and is a summary of the complete Run Results Report. Tip : A PDF result report can be generated at any time by clicking the View Results Report button or View > Results on the menu bar.

Table 2017.09K. Identification consistency category descriptions

Identification consistency category

Description

High

Best match is high-confidence identification Second-best match is: * High-confidence identification in which the species is  identical to the best match * Low-confidence identification in which the genus is  identical to the best match * Nonidentification Requirements for high consistency are not met Best match is high- or low-confidence identification Second-best match is: * High- or low-confidence identification in which the genus  is identical to the best match * Nonidentification Requirements for high or low consistency are not met

Low

None

( m ) When measurement and identification of all sample positions have been completed, the status bar displays the status message “Finished Successfully.” ( n ) If the identification score received is below 1.7, the isolate should be reanalyzed. For 5–10% of the isolates, an extended Direct Transfer (eDT) or Extraction (EXT) procedure might be required. H. eDT ( a ) If the identification score received is below 1.7, the isolate should be reanalyzed following the eDT. Note : After inoculation of bacteria on recommended isolation media, colonies are stable for up to 12 h when held at room temperature (20–25°C). If testing is not done within 12 h, subculture the test organism before testing on the MALDI Biotyper System. ( b ) Using a sterile colony-transfer device, smear an isolated colony of bacteria as a thin film directly onto a sample position on a cleaned reusable steel target or disposable Biotarget 96. ( c ) Overlay the sample spot with 1 µL 70% aqueous formic acid and allow to dry at room temperature (20–25°C). ( d ) Select a minimum of one BTS control position on a target to inoculate with BTS solution. It is mandatory to get at least one valid BTS control per target and per run. Therefore, it is advised to select two BTS control positions. Note : After samples and BTS have dried, HCCA Matrix must be added within 30 min, or the target must be cleaned and the inoculation of sample and BTS must be repeated. ( e ) Pipet 1 µL reconstituted BTS solution onto the selected positions and dry at room temperature (20–25°C). Note : Make sure that the screw-cap tubes containing HCCA Matrix are tightly closed after use to minimize solvent evaporation. ( f ) Overlay each of the sample positions and BTS control positions with 1 µL HCAA Matrix. Use a new pipet tip to add HCCAMatrix to each inoculated sample position. ( g ) Dry the inoculated target at room temperature (20–25°C). ( h ) The inoculated target is now ready for use. Note : An inoculated target must be processed within 24 h of preparation, or the target must be cleaned and the inoculation of samples and BTS must be performed again. The sample positioning on the target can be facilitated with the MBT Pilot™ workstation. The eDT can be supported with the MBT Galaxy™.

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