ERP Micro December 2019

( e ) Pipet 1 µL of reconstituted BTS solution onto the selected positions and dry at room temperature (20–25°C). Note : Make sure that the screw-cap tube containing HCCA Matrix is tightly closed after use to minimize solvent evaporation. ( f ) Overlay each of the sample position and BTS control positions with 1 µL of HCAA Matrix. Use a new pipet tip to add matrix 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. J. Formic Acid/Ethanol EXT Sample Preparation ( a ) If the identification score received is still not conclusive after the eDT, the isolate should be reanalyzed following the EXT procedure. ( b ) Transfer 300 µL HPLC grade water to a tube. Transfer colonies from the plate agar into the water to create a cell suspension of approximately 1.5–2 McFarland. ( c ) Add 900 µL ethanol and mix suspension. ( d ) Centrifuge the suspension for 2 min at 15871–21130 g , equivalent to 13000 to 15000 rpm for Eppendorf tube centrifuged with a 5424R rotor. Decant the ethanol. ( e ) Repeat step ( d ) to remove all residual ethanol. Avoid contact with the pellet. ( f ) Air-dry the pellet for a minimum of 5 min at room temperature (20–25°C). ( g ) Add 25 µL of 70% formic acid and pipet up and down to resuspend the pellet. Vortex thoroughly and allow to dry at room temperature (20–25°C). ( h ) Add 25 µL of 100% acetonitrile and mix by pipetting up and down 2–3 times. ( i ) Centrifuge 2 min at 15 871–21130 g , equivalent to 13000 to 15000 rpm for Eppendorf tube centrifuged with a 5424R rotor. ( j ) Dispense 1 µL of sample onto the reusable steel target or the disposable biotarget. ( k ) As a valid BTS control is mandatory per target and run, it is advised to use two BTS control positions. Select the BTS control positions on a target to inoculate with BTS solution. ( l ) Pipet 1 μL of reconstituted BTS solution onto the two selected positions and dry at room temperature (20–25°C). Note : After samples and BTS have dried, HCCA Matrix portioned solution must be added within 30 min, or the target must be cleaned and the inoculation of samples BTS must be repeated. ( m ) Overlay each sample position and BTS control positions with 1 μL HCCA matrix. Use a new pipet tip to add matrix to each inoculated sample position. ( n ) Dry the inoculated target at room temperature (20–25°C). ( o ) 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. K. Generating a Run Result Report ( a ) After the acquisition of the target has been completed, click View > Results to generate a PDF report. ( b ) The PDF report header contains the Run Identifier and Run Creation Date/Time from the Run Info section. ( c ) The PDF report footer contains the report creation date/time, page count, and the appropriate area of application.

Table 2017.10D. Identification consistency category descriptions

Identification consistency category

Description

High

Best match is a 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 a high- or low-confidence identification Second-best match is: * Hhigh- 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

based on the confidence level of the best and second-best matches as described in Table 2017.10D . Tip : Point to the information button in the top-right corner of the target display to show the color-coding legend. ( j ) When the identification procedure is completed, the organism identification result will appear in the result table. ( k ) 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. ( l ) When measurement and identification of all sample positions have been completed, the status bar displays the status message Finished Successfully. ( m ) If the identification is not conclusive, the DT procedure should be repeated. For 5–10% of the isolates, an extended Direct Transfer (eDT) or Tube Extraction (EXT) may be required. I. eDT ( a ) If the identification score received is below 2.00, 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. ( 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 plate to inoculate with BTS solution. It is mandatory to get at least one valid BTS control per target plate and per run. Therefore, it is advised to select two BTS control positions. Note : After samples and BTS have dried, HCCA Matrix portioned solution must be added within 30 min, or the target must be cleaned and the inoculation of sample and BTS must be repeated.

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