pn_010317
Contents
1. Turn on the Covaris Focused ultrasonicator the associated computer and launch SonoLab 7 2 software 2 Load the Mycobacterium protocol with heat inactivation file in SonoLab if it is not already loaded or create a new file section 5 1 Covaris 010157 M220 Manual Rev E 3 Ensure the water bath has the correct amount of water and that the temperature is in the correct range 4 Centrifuge the pre filled Covaris microTUBEs with beads at 3000 RCF for 10 seconds to pellet the beads Use the Centrifuge and Heat Block adaptors to support the microTUBEs in the centrifuge 5 The microTUBEs are bar coded If you do not utilize the bar code manually label the plastic hub of the tubes for sample tracking using an indelible marker 6 Place up to 4 microTUBEs in the front row of the Prep Station Figure 1 NOTE The Prep Station was designed for workflow and has places for 4 four microTUBES to allow the user to keep track of un processed and processed samples The station enables one handed opening of the microTUBE and the back row of the Prep Station is configured to hold the microTUBE screw cap while adding sample 7 Open microTUBE Screw Cap and place Screw Cap in designated area of the Prep Station 8 Add 100 ul of high purity water to each microTUBE Part Number 010317 Rev B Page 5 Date Sept 15 2015 Patents Issued and pending 10 11 12 13 14 15 16 17 18 19 20 21 22 Add 1 uL inocul2. be processed and spotted onto the MALDI TOF target plate and analyzed within 60 minutes 23 Follow the manufacturer s directions for completing the MALDI TOF MS analysis For example Table 2 represents typical scores obtained from culture plates from Mycobacterium smegmatis prepared with both the truXTRAC protocol and with the MycoEx protocol according to manufacturer s instructions Part Number 010317 Rev B Page 6 Date Sept 15 2015 Patents Issued and pending gt e al 4 Fee dae eS o gt ee gx T T gt A S Figure 1 Prep Station with microTUBEs and Screw Caps in use Row 1 microTUBE and Screw Cap Row 2 4 microTUBEs with Screw Caps removed TYPICAL OUTPUT READINGS VARIATION Table 2 Bruker Biotyper Scores from Mycobacterium smegmatis after two day culture on Middlebrook agar plates Covaris AFA Heat inactivation Bruker MycoEx 1 2 208 2 259 2 2 187 2 227 3 2 167 2 181 4 2 243 2 210 5 1 990 1 853 6 2 155 1 795 Avg 2 158 2 088 cv 4 09 9 89 Using Bruker Mycobacteria database version 1 0 Part Number 010317 Rev B Date Sept 15 2015 Patents Issued and pending Page 7
3. PROTOCOL Covaris truXTRAC MALDI TOF Mycobacteria Kit Mycobacteria Colony Sample Preparation Protocol with a Heat Inactivation Step for Analysis on a Bruker Biotyper MALDI TOF Mass Spectrometer Revision History Part Number Revision Date Description of change 010317 B Sept 15 2015 Part number changes for starter and reorder kits 010317 A Sept 2 2015 Initial release Part Number 010317 Rev B Page 1 Date Sept 15 2015 Patents issued and pending INTENDED USE This is a Research Use Only protocol Refer to Covaris document 010157 M220 Manual Rev E for M series User Manual general instructions and maintenance of the instrument computer and software This protocol is for the sample preparation of Mycobacteria grown on agar plates or tube slants for MALDI TOF MS microorganism identification analysis with the Bruker Biotyper mass spectrometer Colonies should be clearly visible no inoculum Collect bacterial colonies using a sterile disposable 1 ul inoculation loop 4 8 mg of cells This Adaptive Focused Acoustics AFA sample preparation protocol was developed to include a heat inactivation step prior to AFA processing The heat inactivation conditions of time and temperature are identical to those of the Bruker MycoEx sample preparation protocol The Bruker database is based on samples that had this initial heat inactivation conditions and deviation from these initial conditions may adversely impact id
4. ation loop of sample 4 8 mg to the microTUBE by shaking the loop until the cells disperse into the water Place a Screw Cap onto each microTUBE and tighten Transfer the microTUBEs to the laboratory block heater Use the blue anodized Centrifuge and Heat Block Adaptors for each tube as necessary to ensure heat transfer Heat the microTUBEs for 30 minutes at 100 C Carefully remove the microTUBEs from the laboratory block heater allow to cool several minutes in the Prep Station Using the Centrifuge and Heat Block Adaptors as necessary centrifuge all sample microTUBEs at 18 000 x g for five 5 minutes Transfer the microTUBE back to the Prep Station and remove the Screw Cap Using a pipette carefully remove 75 ul of water above the beads Take care not to remove pelleted material that may be above the beads Add 100 uL of Extraction Solvent 50 acetonitrile 35 formic acid 15 H20 to each microTUBE Replace the Screw Cap onto each microTUBE and tighten Place a microTUBE with sample into the appropriate Covaris instrument and process each sample with AFA by running the Mycobacterium protocol with heat inactivation treatment Once each sample has been processed place the microTUBE in the second row of the Prep Station Centrifuge all samples at 18 000 x g for 2 minutes using the Centrifuge and Heat Block Adaptors to support the microTUBEs Spot 1 uL replicates onto the MALDI target plate or slide WARNING All samples should
5. cid 15 H20 Make up in advance using an amber glass container with screw cap Discard if not used after 30 days For formic acid 98 mass spectrometry grade use Fluka 94318 250 mL e Laboratory dry block heater capable of heating to 100 C e Sterile disposable 1 ul inoculating loops e Centrifuge fixed rotor 18 000 RCF e Variable pipette and tips 2 5 ul and 200 ul Part Number 010317 Rev B Page 3 Date Sept 15 2015 Patents Issued and pending e MALDI TOF MS Bruker Biotyper and Mycobacteria database Values mentioned in this Quick Guide are nominal values The tolerances are as follows Temperature 2 C Sample volume 5 ul RISK AND SAFETY INFORMATION The following protocol uses an organic solvent that according to the Globally Harmonized System of Classification and Labeling of Chemicals GHS is considered a hazardous chemical In the manufacturer s experience the product has no harmful effect when used and handled according to instructions truXTRAC Extraction Solvent is classified as a hazardous chemical MSDS INFORMATION IS AVAILABLE AT http covarisinc com resources msds sheets All patient samples and cultures must be considered potentially infective Only qualified laboratory personnel should perform this protocol Personnel performing this protocol are responsible for taking and following all the necessary safety precautions for handling potentially pathogenic material This would include the wearing
6. entification of microorganism SUMMARY OF AFA OPERATING CONDITIONS AFA Instrument M220 Peak Incident Power 40 Watts Duty Factor 50 Cycles per Burst 200 Duration 60 seconds Bath Temperature 18 C Processing tube volume 130 ul Note Recommended settings are subject to change without notice Contact Covaris for application to other Covaris Focused ultrasonicator systems such as 220 E220 and LE220 See http covarisinc com resources protocols for updates to this document Part Number 010317 Rev B Page 2 Date Sept 15 2015 Patents Issued and pending COVARIS SUPPLIES Item Materials Description Part Number M220 Focused M220 with computer and 500295 Ultrasonicator Software M220 Starter Kit Item Materials Description Part Number truXTRAC MALDI TOF 520194 Mycobacteria Starter Kit M220 Holder XTU Holder for 520170 M220 Holder XTU Holder Insert for microTUBE Insert 130 ul Prep Station Prep Station for microTUBE Screw Cap Centrifuge and Heat Centrifuge and Heat Block Block Adaptor Adaptor for microTUBE Screw Cap truXTRAC MALDI TOF microTUBE Acoustical Cuvette Mycobacteria Kit Reorder Kit Item Materials Description Part Number truXTRAC MALDI TOF 520170 Mycobacteria Kit microTUBE Acoustical microTUBE 130 Glass Beads Cuvette No Slit Screw Cap 25 ADDITIONAL MATERIALS SUPPLIED BY USER e High purity water e g HPLC MS grade e Extraction Solvent 50 acetonitrile 35 formic a
7. of appropriate personal protective equipment such as a laboratory coat safety glasses and gloves SAMPLE PREPARATION Organisms should be grown on agar until colonies are visually present and experiencing freshly positive growth Approximately 1 uL should be scraped from the plate using a disposable inoculation loop 4 8 mg OPERATING CONDITIONS The Covaris AFA process focuses high frequency acoustic energy through vessel walls and into a sample Such energy is influenced by objects in the acoustic path from the transducer surface to the sample For example microscopic particles in the Covaris instrument water bath may scatter the acoustic energy from the sample and reduce processing efficiency WARNING Replace water on a daily basis In addition if the daily use is high replace after 100 samples during the day Part Number 010317 Rev B Page 4 Date Sept 15 2015 Patents Issued and pending For M Series Focused ultrasonicators put the Holder XTU and the Insert XTU microTUBE 130 ul in place and fill the water bath until the water reaches the top of the holder Allow system to reach temperature Table 1 Mycobacterium protocol with heat inactivation SonoLab parameters Power Duty Cycle Cycles per Temperature burst 40W PIP 60 seconds Prepare Heating Block Dry block heaters should be preset at 100 C Verify temperature with a calibrated glass thermometer PROTOCOL WITH HEAT INACTIVATION 1
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