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Pathatrix® Salmonella spp. Kit (Individual Samples) Linked to PCR

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1. Pathatrix Salmonella spp Kit Same Day APS50SD Pathatrix 5 Pooling Salmonella spp Kit APS250P Pathatrix 10 Pooling Salmonella spp Kit APS500P Pathatrix 5 Pool DUAL E coli Salmonella spp Kit APDES250P Pathatrix DUAL Listeria Salmonella spp Kit APD50 Equipment Pathatrix Auto Instrument PATHATRIXAUTO Cartridge Rack optional for use with the Pathatrix Auto Instrument ACARTRACK holds 5 Cartridges DynaMag 2 Magnet for use with microcentrifuge tubes 123 21D Magnetic Capture Plate MAGNETICPLATE Reagents PBS 10X pH 7 4 AM9624 or AM9625 Related PCR assay MicroSEQ Salmonella spp Detection Kit 4403930 54 Pathatrix Salmonella spp Kit Individual Samples Linked to PCR or ELISA Safety WARNING GENERAL SAFETY Using this product in a manner not specified in the user documentation may result in personal injury or damage to the instrument or device Ensure that anyone using this product has received instructions in general safety practices for laboratories and the safety information provided in this document Before handling chemicals read and understand all applicable Safety Data Sheets SDSs and use appropriate personal protective equipment gloves gowns eye protection etc To obtain SDSs see the Documentation and Support section in this document Chemical safety AN WARNING GENERAL CHEMICAL HANDLING To minimize hazards ensure laborator
2. Detection direct plating optional Note We recommend streaking the sample out to generate individual colonies as opposed to spread plating 1 Using an inoculation loop streak 10 uL of the unlysed Pathatrix paramagnetic bead suspension over a well dried XLD xylose lysine desoxycholate agar plate and another 10 uL onto an appropriate second selective plate medium Note The laboratory may choose which medium to use but the second selective plate should be any other solid selective medium complimentary to XLD and especially appropriate for the isolation of lactose positive Salmonella Salmonella typhi and Salmonella paratyphi strains 2 Allow the plates to dry for approximately 10 minutes then invert and incubate at the required temperature for 18 24 hours or as recommended by the manufacturer A presumptive positive result is defined as the isolation of typical suspicious or atypical Salmonella spp colonies on the agar plates used A presumptive positive isolate should be subsequently confirmed by the use of subculture and appropriate biochemical and serological tests as detailed in USDA Microbiology Laboratory Guidebook MLG 4 04 for cooked ham or FDA Bacteriological Analytical Manual BAM Chapter 5 sections D and E for tomatoes and chocolate see References on page 58 Test result interpretation and classification The Pathatrix Salmonella spp Kit is designed as a sample preparation method for presence abs
3. Salmonella visual immunoassay or the VIDAS immunoassay testing systems or be isolated on selective agar plates CAUTION The Pathatrix kit has been evaluated on cooked ham chocolate and chopped tomato food matrices Given the wide variety of products and manufacturing procedures we recommend that you check that the composition of the matrices to be tested does not affect the reliability of the results A negative result does not guarantee the absence of target organism in the original sample and may be due to the inability of the organism to adequately reproduce to required levels in the enrichment medium with subsequent outgrowth on selective agar plates potentially due to but not limited to competitive microflora sub lethal injury or matrix inhibition Pathatrix Salmonella spp Kit Individual Samples Linked to PCR or ELISA Appendix B Background Product overview Operating The Pathatrix Auto Instrument is for indoor use only and for altitudes not exceeding conditions 2000 m 6500 ft above sea level Temperature and humidity requirements Condition Acceptable range Temperature 5 40 C Humidity Maximum relative humidity 80 for temperatures up to 31 C decreasing to 50 Pathatrix Salmonella spp Kit Individual Samples Linked to PCR or ELISA 53 C Ordering Information Related materials from Life Technologies Item Cat no Related consumable kits with associated beads
4. ecu tee A e cee elt Ande hire gon dren e Sah teen atta Kit Contelto m ie a cae bote ten ache em itachi Sued wherein ath tas dae ta Materials not included in the kit 0 00 00 ccc ee eee eee Hi CHAPTER 1 Pathatrix Salmonella spp Kit Linked to the DuPont BAX PCR System eese WOPR ON 11 252 ea a bee beer bp C Er pr tiep P estu ib Dabo As Procedural guidelines ooooocccccoccocorr ence eee e eens Sample enrichment ooo Sample preparatloh i21 verde a P lead pang emirates Samples and consumable loading ce cece ete e tent eeee Sample unloading 0c cece cece eee e teenies Detection BAX PCR protocol 0 0 c ccc cence rl Detection direct plating optional 00 o ccc ccc cence ences Test result interpretation and classification 000 0c e cece eee CHAPTER 2 Pathatrix Salmonella spp Kit Linked to the Idaho Technology R A P I D 9 LT PCR System Work OW C m Procedural guidelines cta RR UE RAM EROR SED ie Sample enrichment ooo Sample preparation a es ahi een rehi na i Aa eee ttt n Samples and consumable loading 0ce nnana neea Sample unloading 210 A a aa a b aa Sp BC eG AA Detection R A P I D 9 LT PCR protocol 00 00 c cece ccc e Detection direct plating optional 0 o ccc ccc cence ences Test result interpretation and classification 0 0 ccc cece
5. screw cap microcentrifuge tube of O Ya Bead free supernatant Transfer the bead free supernatant into the designated well of the RayAl microplate Immediately after heating capture beads for 2 min on the DynaMag 2 Magnet Vortex the tube for 10 sec and then place in a water bath at 95 C for 15 min Vortex the tube for 10 sec and then incubate the bead suspension at 37 C for 2 hr Proceed with the assay in accordance with the manufacturer s instructions If a positive ELISA result is obtained an aliquot of the Pathatrix paramagnetic beads should be plated out see the following section Detection direct plating optional 32 Pathatrix Salmonella spp Kit Individual Samples Linked to PCR or ELISA Chapter 3 Pathatrix Salmonella spp Kit Linked to the RayAl Salmonella OPTIMA System 3 Detection direct plating optional Detection direct plating optional Note We recommend streaking the sample out to generate individual colonies as opposed to spread plating 1 Streak 50 uL of the unlysed Pathatrix paramagnetic bead suspension over a well dried XLD xylose lysine desoxycholate agar plate and another 50 uL onto an appropriate second selective plate medium Note The laboratory may choose which medium to use but the second selective plate should be any other solid selective medium complimentary to XLD and especially appropriate fo
6. 10 11 12 Sample unloading At the end of the run the LED will flash red and green alternately Press the button above the appropriate Cartridge to initialize the draining step approximately 1 minute When the draining step is complete and the LED is illuminated red remove the Cartridge by pulling it out away from the instrument Remove the syringe from the Cartridge and carefully pull out the rest of the kit starting at the top and working downwards When removing the Sample and Elution Vessels from the Cartridge hold firmly by the vessels themselves to prevent spillage Place both vessels in the Tube Rack also known as the Sample Vessel Holder or rack Remove the lid from the Elution Vessel and leaving the Elution Vessel in place in the rack lift away the rest of the consumable including the Sample Vessel and discard Place the Elution Vessel in the rack and cap with a flat lid provided in the kit Leave in the rack for 1 minute to allow capture of the Pathatrix paramagnetic beads Remove all the liquid from the Elution Vessel without removing the vessel from the rack taking care not to disturb the captured Pathatrix paramagnetic beads Remove the Elution Vessel from the vessel holder use 1200 uL of pre warmed BPW to resuspend the Pathatrix paramagnetic beads and transfer 1100 uL to a microcentrifuge tube Note 100 uL of the Pathatrix paramagnetic beads can be retained for direct plating pr
7. Pathatrix paramagnetic bead suspension into the spout on the lid of the Sample Vessel Sample Vessel Spout Elution Vessel Sample Vessel Sterile 1X PBS 35 mL Sample Vessel Holder ATUBEHOLD 4 Remove the capture phase kit from the bag and orient with the valve plunger pointing left Connect the valve firmly to the lids of the Sample and Elution Vessels n Capture Phase Valve Plunger Pathatrix Salmonella spp Kit Individual Samples Linked to PCR or ELISA 45 5 Chapter 5 Pathatrix Salmonella spp Kit Linked to the bioM rieux VIDAS Immunoassay Testing System Samples and consumable loading 5 Holding both vessels lift the assembled vessels and attached capture phase kit out of the Sample Vessel Holder 6 Place the vessels into the Cartridge pushing them in firmly from the bottom upwards 7 Firmly push the remainder of the kit into the Cartridge starting with the valve followed by the capture phase and finally by the syringe 8 Push the magnet slider across into the locking position and press the magnet release button on the side of the Cartridge to ensure the kit is positioned correctly and the magnets will move away from the capture phase freely Syringe Capture Phase Sliding Magnet Holder Magnet Release Button Valve Plunger Sample Vessel Elution Vessel Cartridge Holder ACARTHOLD 9 Reset the magnets into the locking position 10 Insert the Cartridge i
8. Salmonella spp Kit Individual Samples Linked to PCR or ELISA 27 2 Chapter 3 Pathatrix Salmonella spp Kit Linked to the RayAl Salmonella OPTIMA System Sample enrichment Sample enrichment Note Certain food types and swabs sponges can benefit from an alternative enrichment strategy see Appendix A Alternative Enrichment Methods 1 Prepare a 1 10 dilution of the food sample in the appropriate prewarmed 37 1 C enrichment media in a sterile bag Note For example add 25 g of food sample to 225 mL of prewarmed media or add 325 g of food sample to 2925 mL of prewarmed media IMPORTANT It is critical that the enrichment media is prewarmed to 37 1 C prior to adding the food sample To prevent cooling all samples should then immediately be placed in a 37 C incubator 2 Homogenize the sample by massaging the sample between the fingers for 10 15 seconds to disperse any large clumps of material Narang and Cray 2006 3 Incubate at 37 1 C for a minimum of 18 24 hours Sample preparation 1 Remove the Sample Vessel and Elution Vessel from the consumable kit packaging and place into the Sample Vessel Holder 2 Partially remove the lids from both vessels making a large enough opening to allow the sample and wash buffer to be dispensed into the vessels 3 Place 50 mL of your sample in the Sample Vessel Note If the samples are highly particulate or has a high fat content a Seward plain sterile bag with inter
9. can be obtained prior to confirmation within 22 28 hours Once confirmed the results are reported as e Salmonella spp Detected in 25 325 g sample matrices e Salmonella spp Not detected in 25 325 g sample matrices Pathatrix Salmonella spp Kit Individual Samples Linked to PCR or ELISA 49 A Alternative Enrichment Methods Specific recommendations for high background microflora TT broth information 50 For certain food groups particularly where high levels of background microflora are present the Salmonella assay results can be enhanced by the use of Tetrathionate TT broth instead of Buffered Peptone Water The level of background contamination on the agar plates can be reduced by this method Examples of food groups that benefit from the use of TT broth are Raw meat Raw vegetables Salads Fruits Ready meals Materials Tetrathionate broth base is available from Oxoid Product Code CM0029 e Iodine Solution to be added following preparation and heating of TT broth base Note Prepare the iodine solution in advance to allow the iodine to dissolve but use on the same day that it is made Directions for use 1 Add 77 g of TT broth base to 1 L of distilled water and bring to a boil 2 Cool below 45 C 3 Add 20 mL of iodine solution and mix well Following boiling the prepared base can be stored for several weeks at 5 3 C but once the iodine has been added the media should be used the s
10. cece eee Pathatrix Salmonella spp Kit Individual Samples Linked to PCR or ELISA Contents Hi CHAPTER 3 Pathatrix Salmonella spp Kit Linked to the RayAl Salmonella OPTIMA System esee 27 Workflow ees eta a iR A A Ai eie ed 27 Procedural quidelin s cix tr Saath ee encloses 4 wid eels edad A 27 Sample enrichment aiu Le RM A pee 28 Sample preparation isisussusullllllllllllss ss n 28 Samples and consumable loading 000cee cece cece eect eee 29 Sample Unloading 1 22 35 5 hf ride a ke Sa sha le reses ub ald des lea ool ed dos 31 Detection Salmonella OPTIMA protocol ooooocococccccccconen rr 32 Detection direct plating optional 0 ccc ence ne ene ne enes 33 Test result interpretation and classification 00 000 cece eee eee eee eee ees 33 CHAPTER 4 Pathatrix Salmonella spp Kit Linked to the 3M TECRA M Salmonella Visual Immunoassay 35 WORKTIOW fave sete faves A EA E Lon 35 Proceduraliquidelines oc bus ia ea te 35 Sample enrichiment eni o E A RR EXER SE 36 Sample preparation 0c ee eee nent n 36 Samples and consumable loading 000c cece cece ee eee ete ees 37 Sample unloadinq cota REA EE ep ge ee Re ed 39 Detection TECRA Salmonella visual immunoassay protocol 0 00 cece eee eee 40 Detection direct plating optional 2 00 ccc nnn enn een e eens 41 Test result interpretation and classific
11. lifetech com For the latest services and support information for all locations go to www lifetechnologies com support At the website you can e Access worldwide telephone and fax numbers to contact Technical Support and Sales facilities Search through frequently asked questions FAQs Search for user documents SDSs application notes formulations handbooks certificates of analysis citations and other product support documents Obtain information about customer training Limited product warranty Life Technologies Corporation and or its affiliate s warrant their products as set forth in the Life Technologies General Terms and Conditions of Sale found on Life Technologies website at www lifetechnologies com termsandconditions If you have any questions please contact Life Technologies at www lifetechnologies com support Pathatrix Salmonella spp Kit Individual Samples Linked to PCR or ELISA 57 58 References Food Safety and Inspection Service USDA 2008 Isolation And Identification of Salmonella From Meat Poultry Pasteurized Egg and Catfish Products MLG 4 04 Microbiology Laboratory Guidebook Narang N and Cray W C 2006 Evaluation of Hand Mixing of Ground Beef and Poultry Samples as an Alternative to Stomaching for the Detection of Salmonella Food Protection Trends 26 14 19 US FDA Bacteriological Analytical Manual BAM Chapter 5 go to www fda gov Food ScienceResearch LaboratoryMethods B
12. 4 Pathatrix Salmonella spp Kit Linked to the 3M TECRA Salmonella Visual Immunoassay Test result interpretation and classification 42 Pathatrix Salmonella spp Kit Individual Samples Linked to PCR or ELISA Workflow Pathatrix Salmonella spp Kit Linked to the bioM rieux VIDAS Immunoassay Testing System Sample enrichment page 44 v Sample preparation page 44 v Samples and consumable loading page 45 v Sample unloading page 47 v Detection VIDAS9 immunoassay testing protocol page 48 v Detection direct plating optional page 49 v Test result interpretation and classification page 49 Procedural guidelines e Use aseptic technique and good laboratory practices at all times e A facemask should be worn when weighing out powders e Care must be taken when boiling agar prior to autoclaving and heat resistant gloves should be worn when handling hot flasks of liquid e Take care when handling plates or vessels that contain microorganisms e Avoid generating aerosols as pathogenic organisms may be present e Used or unused reagents used media sample enrichments as well as other contaminated disposable materials should be disposed of following procedures for infectious or potentially infectious products Sample enrichments might be contaminated with pathogenic organisms infectious to humans so all waste must be treated as biohazardous and handled and disposed using safe labora
13. I D 9 LT PCR System Test result interpretation and classification 26 Pathatrix Salmonella spp Kit Individual Samples Linked to PCR or ELISA Workflow Pathatrix Salmonella spp Kit Linked to the RayAl Salmonella OPTIMA System Sample enrichment page 28 v Sample preparation page 28 v Samples and consumable loading page 29 v Sample unloading page 31 v Detection Salmonella OPTIMA protocol page 32 v Detection direct plating optional page 33 v Test result interpretation and classification page 33 Procedural guidelines e Use aseptic technique and good laboratory practices at all times e A facemask should be worn when weighing out powders e Care must be taken when boiling agar prior to autoclaving and heat resistant gloves should be worn when handling hot flasks of liquid e Take care when handling plates or vessels that may contain microorganisms e Avoid generating aerosols as pathogenic organisms may be present e Used or unused reagents used media sample enrichments as well as other contaminated disposable materials should be disposed of following procedures for infectious or potentially infectious products Sample enrichments might be contaminated with pathogenic organisms infectious to humans so all waste must be treated as biohazardous and handled and disposed using safe laboratory practices in accordance and compliance with all appropriate regulations Pathatrix
14. USER GUIDE applied biosystems oy Life technologies Pathatrix Salmonella spp Kit Individual Samples Linked to PCR or ELISA For use with the Pathatrix Auto Instrument Catalog Number APS50 Publication Number MANO0007089 Revision 11 October 2012 o For testing of Food and Environmental samples only technologies The information in this guide is subject to change without notice DISCLAIMER LIFE TECHNOLOGIES CORPORATION AND OR ITS AFFILIATE S DISCLAIM ALL WARRANTIES WITH RESPECT TO THIS DOCUMENT EXPRESSED OR IMPLIED INCLUDING BUT NOT LIMITED TO THOSE OF MERCHANTABILITY FITNESS FOR A PARTICULAR PURPOSE OR NON INFRINGEMENT TO THE EXTENT ALLOWED BY LAW IN NO EVENT SHALL LIFE TECHNOLOGIES AND OR ITS AFFILIATE S BE LIABLE WHETHER IN CONTRACT TORT WARRANTY OR UNDER ANY STATUTE OR ON ANY OTHER BASIS FOR SPECIAL INCIDENTAL INDIRECT PUNITIVE MULTIPLE OR CONSEQUENTIAL DAMAGES IN CONNECTION WITH OR ARISING FROM THIS DOCUMENT INCLUDING BUT NOT LIMITED TO THE USE THEREOF Limited Use Label License Environmental testing quality control quality assurance testing food and agricultural testing The purchase of this product conveys to the purchaser the limited non transferable right to use the purchased amount of the product a to perform internal research for the sole benefit of the purchaser and b for environmental testing quality control quality assurance testing food and agricultural testing including reporting
15. acteriologicalAnalyticalManualBAM default htm and scroll to Salmonella Pathatrix Salmonella spp Kit Individual Samples Linked to PCR or ELISA Headquarters 5791 Van Allen Way Carlsbad CA 92008 USA Phone 1 760 603 7200 Toll Free in USA 800 955 6288 For support visit www lifetechnologies com support or email techsupport dlifetech com technologies www lifetechnologies com October 2012
16. ally appropriate for the isolation of lactose positive Salmonella Salmonella typhi and Salmonella paratyphi strains 2 Allow the plates to dry for approximately 10 minutes then invert and incubate at the required temperature for 18 24 hours or as recommended by the manufacturer A presumptive positive result is defined as the isolation of typical suspicious or atypical Salmonella spp colonies on the agar plates used A presumptive positive isolate should be subsequently confirmed by the use of subculture and appropriate biochemical and serological tests as detailed in USDA Microbiology Laboratory Guidebook MLG 4 04 for cooked ham or FDA Bacteriological Analytical Manual BAM Chapter 5 sections D and E for tomatoes and chocolate see References on page 58 Test result interpretation and classification The Pathatrix Salmonella spp Kit is designed as a sample preparation method for presence absence detection of Salmonella spp in food matrices Using the Pathatrix Salmonella spp Kit linked to the R A P 1 D LT PCR system presumptive results can be obtained prior to confirmation within 17 19 hours Once confirmed the results are reported as e Salmonella spp Detected in 25 325 g sample matrices e Salmonella spp Not detected in 25 325 g sample matrices Pathatrix Salmonella spp Kit Individual Samples Linked to PCR or ELISA 25 J Chapter 2 Pathatrix Salmonella spp Kit Linked to the Idaho Technology R A P
17. ame day and any excess should be disposed of Pathatrix Salmonella spp Kit Individual Samples Linked to PCR or ELISA Appendix A Alternative Enrichment Methods Specific recommendations for milk powder chocolate and cocoa based samples Specific recommendations for milk powder chocolate and cocoa based samples All milk powder chocolate or cocoa based samples can benefit from an alternative enrichment Use prewarmed 37 1 C sterile UHT skim milk supplemented with Brilliant Green 0 002 instead of Buffered Peptone Water as the enrichment media Milk powder samples should be incubated at 37 1 C for 22 2 hours e Chocolate and cocoa based samples should be incubated as described in the protocol at 37 1 C for 19 1 hour Specific recommendations for potentially acidic alkaline samples Samples which potentially deviate from neutral pH should be prepared as follows 1 Dilute the sample according to the sample enrichment protocol 2 Incubate for 60 5 minutes at room temperature 3 Mix by hand massaging and determine the pH 4 If necessary adjust the pH to 6 8 0 2 and mix well before determining the final pH Pathatrix Salmonella spp Kit Individual Samples Linked to PCR or ELISA 51 Background Product overview Description of target microorganisms Audience Sampling protocol Kit sensitivity 52 More than 2 400 Salmonella serotypes have been reported all of which are potent
18. ant Proceed to PCR platform e g 5 40 uL into PCR reaction RA PI D LT BAX iCycler or mixture LightCycler instrument 24 Pathatrix Salmonella spp Kit Individual Samples Linked to PCR or ELISA Chapter 2 Pathatrix Salmonella spp Kit Linked to the Idaho Technology R A P I D 9 LT PCR System Detection direct plating optional Review the amplification curves and melt peaks as described in the R A P D 9 LT PCR software Screen Shot Protocol If any Amplification Curve exhibits an increase in fluorescence and or displays a potential Melt Peak in the absence of a positive PCR software determination a repeat PCR analysis should be carried out by repeating the R A P LD 9 LT PCR steps in this section see page 24 If a positive PCR result is obtained an aliquot of the Pathatrix paramagnetic beads should be plated out see the following section Detection direct plating optional Detection direct plating optional Note We recommend streaking the sample out to generate individual colonies as opposed to spread plating 1 Using an inoculation loop streak 10 uL of the unlysed Pathatrix paramagnetic bead suspension over a well dried XLD xylose lysine desoxycholate agar plate and another 10 uL onto an appropriate second selective plate medium Note The laboratory may choose which medium to use but the second selective plate should be any other solid selective medium complimentary to XLD and especi
19. as opposed to spread plating 1 Streak 50 uL of the unlysed Pathatrix paramagnetic bead suspension over a well dried XLD xylose lysine desoxycholate agar plate and another 50 uL onto an appropriate second selective plate medium Note The laboratory may choose which medium to use but the second selective plate should be any other solid selective medium complimentary to XLD and especially appropriate for the isolation of lactose positive Salmonella Salmonella typhi and Salmonella paratyphi strains 2 Allow the plates to dry for approximately 10 minutes then invert and incubate at the required temperature for 18 24 hours or as recommended by the manufacturer A presumptive positive result is defined as the isolation of typical suspicious or atypical Salmonella spp colonies on the agar plates used A presumptive positive isolate should be subsequently confirmed by the use of subculture and appropriate biochemical and serological tests as detailed in USDA Microbiology Laboratory Guidebook MLG 4 04 for cooked ham or FDA Bacteriological Analytical Manual BAM Chapter 5 sections D and E for tomatoes and chocolate see References on page 58 Test result interpretation and classification The Pathatrix Salmonella spp Kit is designed as a sample preparation method for presence absence detection of Salmonella spp in food matrices Using the Pathatrix Salmonella spp Kit linked to the VIDAS immunoassay presumptive results
20. at may contain salmonellae Laboratory personnel must be adequately trained to handle pathogens before being permitted to analyze samples for Salmonella spp Laboratory personnel must wear appropriate protective equipment which includes but is not limited to protective eyewear face shield clothing lab coat and gloves Waste should be disposed of in compliance with local and national legislation as appropriate The Pathatrix Salmonella spp Kit provides a sample preparation method for presence absence testing based on the detection of as few as 1 10 cfu colony forming units 25 325 g of food sample An AOAC validated protocol using this kit for sample preparation followed by detection of microorganisms by selective agar plates can be downloaded from www lifetechnologies com pathatrix Pathatrix Salmonella spp Kit Individual Samples Linked to Selective Agar Plates Pub no MAN0006974 This user guide provides 5 alternative protocols for using the Pathatrix Salmonella spp Kit followed by detection of microorganisms by PCR or ELISA Presumptive results can be obtained within the following times e 20 22 hours when linked to the DuPont BAX PCR system Chapter 1 17 19 hours when linked to the Idaho Technology R A PID 9 LT PCR system Chapter 2 e 22 28 hours when linked to the RayAl Salmonella OPTIMA Chapter 3 e 22 28 hours when linked to the 3M TECRA Salmonella visual immunoassay Chapter 4 e 22 28 hours wh
21. aterial Narang and Cray 2006 3 Incubate at 37 1 C for a minimum of 16 hours Sample preparation 1 Remove the Sample Vessel and Elution Vessel from the consumable kit packaging and place into the Sample Vessel Holder 2 Partially remove the lids from both vessels making a large enough opening to allow the sample and wash buffer to be dispensed into the vessels 3 Place 50 mL of your sample in the Sample Vessel Note If the samples are highly particulate or has a high fat content a Seward plain sterile bag with internal strainer may be used Seward Product Code BA6041 STR 4 Store the individual enriched samples at 5 3 C for potential reanalysis until the test result is confirmed Note Do not store for more than 32 hours If refrigerated after pre enrichment samples should be re warmed to 37 1 C prior to removal of aliquots for analysis 20 Pathatrix Salmonella spp Kit Individual Samples Linked to PCR or ELISA Chapter 2 Pathatrix Salmonella spp Kit Linked to the Idaho Technology R A P I D 9 LT PCR System 2 Samples and consumable loading Samples and consumable loading 1 Add PBS Cat no AM9624 diluted to 1X to the fill line of the Elution Vessel approximately 35 mL of 1X PBS 2 Place the lids on the Sample and Flution Vessels making sure that the vessels are sealed all the way around 3 Ensure the Pathatrix paramagnetic beads included in the kit are fully resuspended by agitating the bead v
22. ation 00 000 ce eee eee eee eee eee ees 41 CHAPTER 5 Pathatrix Salmonella spp Kit Linked to the bioM rieux VIDAS Immunoassay Testing System 43 nA states ni PI Baan E E Pan areas Sout 43 Procedural guidelines 0 0 cece cece eee eee e cena e eee eens 43 Sarrple entIchrrienb su eee 2 eg m e 44 Sample preparation ooooooccoco eee ene nent n 44 Samples and consumable loading n s nenna annann nanne cece ees 45 Sample unloading seb b o eke eae aoe 47 Detection VIDAS immunoassay testing protocol 0 0 cece cece cece cence es 48 Detection direct plating optional 0 ccc nnn een nen eens 49 Test result interpretation and classification 000 000 cece eee eee eee ees 49 Pathatrix Salmonella spp Kit Individual Samples Linked to PCR or ELISA Contents APPENDIX A Alternative Enrichment Methods 50 Specific recommendations for high background microflora 2 2202e cece eee eeee 50 TA broth information cuo trice A o 50 Specific recommendations for milk powder chocolate and cocoa based samples 51 Specific recommendations for potentially acidic alkaline samples ssusuuue 51 APPENDIX B Background o ccocccccccccc 52 ProductovervieW aca tr Eas RM RERUM ARRA RA E 52 Description of target microorganisms issssssssslssslllslllss sss 52 Audience xs e eet e ori esee ene Dates dados 52 Sam
23. ble includes materials and equipment for using but not included in the Pathatrix consumables kits Unless otherwise indicated many of the listed items are available from major laboratory suppliers MLS Item Source Equipment Incubator 37 1 C MLS Pathatrix Auto Instrument Life Technologies Cat no PATHATRIXAUTO For use with the BAX PCR protocol Magnetic Capture Plate Life Technologies Cat no MAGNETICPLATE For use with the R A P I D 9 LT PCR protocol and VIDAS protocol DynaMag 2 Magnet Life Technologies Cat no 123 21D Forceps scissors spatula knife and or MLS scalpel Consumables Optional for environmental samples Swabs or sponges MLS Sterile bags for enrichment Whirl Pak or Nasco B01196WA Seward product code Stomacher bag or equivalent BA6041 or equivalent 8 Pathatrix Salmonella spp Kit Individual Samples Linked to PCR or ELISA Product Information Materials not included in the kit Item Source Optional for high particulate or high fat content samples Sterile filter bags for enrichment Whirl Pak or Stomacher bag or equivalent Nasco B01348WA Seward product code BA6041 STR or equivalent Microcentrifuge tubes PCR clean 1 5 mL MLS Sterile 10 uL disposable loops MLS Media See Sample Enrichment sections in the protocols page 12 20 28 36 or 44 or Appendix A on page 50 for recommendations about enrichme
24. ck and cap with a flat lid provided in the kit Leave in the rack for 1 minute to allow capture of the Pathatrix paramagnetic beads Remove all the liquid from the Elution Vessel without removing the vessel from the rack taking care not to disturb the captured Pathatrix paramagnetic beads Remove the Elution Vessel from the vessel holder add 100 uL of PBS into the Elution Vessel and resuspend the Pathatrix paramagnetic beads Appropriate aliquots of the Pathatrix paramagnetic bead suspension can then be immediately analyzed using the laboratory s chosen pathogen detection method Note The Pathatrix paramagnetic bead suspension may be retained for later testing if necessary The beads should be stored in sterile 1 5 mL microcentrifuge tubes AWAY from magnets for example away from Pathatrix vessel holders at 5 3 C for up to 24 hours Pathatrix Salmonella spp Kit Individual Samples Linked to PCR or ELISA 23 2 Chapter 2 Pathatrix Salmonella spp Kit Linked to the Idaho Technology R A P I D 9 LT PCR System Detection R A P1 D 9 LT PCR protocol Detection R A P I D 9 LT PCR protocol IMPORTANT It is solely the operator s responsibility to refer to the package insert instructions and instrument user manual for information regarding the correct set up and operation of the R A P LD 9 LT PCR system 1 Add 25 uL of the resuspended Pathatrix paramagnetic beads into a Blue Capped Bead Tube and vorte
25. d Sample preparation 1 44 Remove the Sample Vessel and Elution Vessel from the consumable kit packaging and place into the Sample Vessel Holder Loosen the lids from the vessels and partially remove leaving an opening through which to add sample and wash buffer Place 50 mL of your sample in the sample vessel Note If the samples are highly particulate and or contain a high fat content a Seward plain sterile bag with internal strainer may be used Seward Product Code BA6041 STR Store the individual enriched samples at 5 3 C for potential reanalysis until the test result is confirmed Note Do not store for more than 32 hours If refrigerated at 5 3 C prior to testing samples should be rewarmed to 37 1 C prior to removal of aliquots for analysis Pathatrix Salmonella spp Kit Individual Samples Linked to PCR or ELISA Chapter 5 Pathatrix Salmonella spp Kit Linked to the bioM rieux VIDAS Immunoassay Testing System 5 Samples and consumable loading Samples and consumable loading 1 Add PBS Cat no AM9624 diluted to 1X to the fill line of the Elution Vessel approximately 35 mL of 1X PBS 2 Place the lids on the Sample and Elution Vessels making sure that the vessels are sealed all the way around 3 Ensure the Pathatrix paramagnetic beads included in the kit are fully resuspended by agitating the bead vial for example by vortexing bead vial or inversion of sealed vial and add 50 uL of the
26. ding the food sample To prevent cooling all samples should then immediately be placed in a 37 C incubator 2 Homogenize the sample by massaging the sample between the fingers for 10 15 seconds to disperse any large clumps of material Narang and Cray 2006 3 Incubate at 37 1 C for a minimum of 16 hours Sample preparation 1 Remove the Sample Vessel and Elution Vessel from the consumable kit packaging and place into the Sample Vessel Holder 2 Partially remove the lids from both vessels making a large enough opening to allow the sample and wash buffer to be dispensed into the vessels 3 Place 50 uL of your sample in the Sample Vessel Note If the samples are highly particulate or has a high fat content a Seward plain sterile bag with internal strainer may be used Seward Product Code BA6041 STR 4 Store the individual enriched samples at 5 3 C for potential reanalysis until the test result is confirmed Note Do not store for more than 32 hours If refrigerated after pre enrichment samples should be rewarmed to 37 1 C prior to removal of aliquots for analysis 12 Pathatrix Salmonella spp Kit Individual Samples Linked to PCR or ELISA Chapter 1 Pathatrix Salmonella spp Kit Linked to the DuPont BAX PCR System 1 Samples and consumable loading Samples and consumable loading 1 Add PBS Cat no AM9624 diluted to 1X to the fill line of the Elution Vessel approximately 35 mL of 1X PBS 2 Place the
27. e TECRA microplate To proceed refer to the TECRA operating instructions ay 400 uL Transfer 400 pL of bead suspension into a standard 2 0 mL screw cap microcentrifuge tube of 00 pL N Bead free supernatant Remove 200 uL of bead free supernatant and transfer into the designated well of a TECRA plate Vortex the tube for 10 sec and then place in a water bath at 95 C for 15 min Vortex the tube for 10 sec and then incubate the bead suspension at 37 C for 2 hr Proceed with the assay in accordance with the manufacturer s instructions If a positive ELISA result is obtained an aliquot of the Pathatrix paramagnetic beads should be plated out see the following section Detection direct plating optional Pathatrix Salmonella spp Kit Individual Samples Linked to PCR or ELISA Chapter 4 Pathatrix Salmonella spp Kit Linked to the 3M TECRA Salmonella Visual Immunoassay 4 Detection direct plating optional Detection direct plating optional Note We recommend streaking the sample out to generate individual colonies as opposed to spread plating 1 Streak 50 uL of the unlysed Pathatrix paramagnetic bead suspension over a well dried XLD xylose lysine desoxycholate agar plate and another 50 uL onto an appropriate second selective plate medium Note The laboratory may choose which medium to use but the second selective plate should be any oth
28. e used Seward Product Code BA6041 STR 4 Store the individual enriched samples at 5 3 C for potential reanalysis until the test result is confirmed Note Do not store for more than 32 hours If refrigerated after pre enrichment samples should be rewarmed to 37 1 C prior to removal of aliquots for analysis 36 Pathatrix Salmonella spp Kit Individual Samples Linked to PCR or ELISA Chapter 4 Pathatrix Salmonella spp Kit Linked to the 3M TECRA Salmonella Visual Immunoassay n Samples and consumable loading Samples and consumable loading 1 Add PBS Cat no AM9624 diluted to 1X to the fill line of the Elution Vessel approximately 35 mL of 1X PBS 2 Place the lids on the Sample and Elution Vessels making sure that the vessels are sealed all the way around 3 Ensure the Pathatrix paramagnetic beads included in the kit are fully resuspended by agitating the bead vial for example by vortexing bead vial or inversion of sealed vial and add 50 uL of the Pathatrix paramagnetic bead suspension into the spout on the lid of the Sample Vessel Sample Vessel Spout Elution Vessel Sample Vessel Sterile 1X PBS 35 mL Sample Vessel Holder ATUBEHOLD 4 Remove the capture phase kit from the bag and orient with the valve plunger pointing left Connect the valve firmly to the lids of the Sample and Elution Vessels n Capture Phase Valve Plunger Pathatrix Salmonella spp Kit Indiv
29. en linked to the bioM rieux VIDAS immunoassay testing system Chapter 5 A presumptive positive isolate should be subsequently confirmed by the use of subculture as well as appropriate biochemical and serological tests as required Once confirmed the results are reported as e Salmonella spp Detected in 25 325 g sample matrices e Salmonella spp Not detected in 25 325 g sample matrices See Appendix B on page 52 for additional background information Pathatrix Salmonella spp Kit Individual Samples Linked to PCR or ELISA 7 Product Information Kit contents Kit contents The Pathatrix Salmonella spp Kit Cat no APS50 contains enough consumable components and Pathatrix paramagnetic beads to process 50 samples Item peat ar Storage volume Pre sterilized Sample and Elution 50 each Room temperature Vessel Packs Pre sterilized Capture Phase Packs 50 each Room temperature Pre sterilized Flat Cap Lids 50 each Room temperature Anti Salmonella spp Antibody 2 5 mL 50 tests 5 3 C Coated Paramagnetic Beads t The beads have a shelf life of 12 months and are labeled with an expiration date accordingly IMPORTANT Never freeze the Pathatrix paramagnetic bead suspension Beads that have been subjected to freezing temperatures may be rendered inactive Note Parts may ship separately depending on configuration and storage conditions Materials not included in the kit The following ta
30. ence detection of Salmonella spp in food matrices Using the Pathatrix Salmonella spp Kit linked to the BAX PCR system presumptive results can be obtained prior to confirmation within 20 22 hours Once confirmed the results are reported as e Salmonella spp Detected in 25 325 g sample matrices e Salmonella spp Not detected in 25 325 g sample matrices Pathatrix Salmonella spp Kit Individual Samples Linked to PCR or ELISA 17 1 Chapter 1 Pathatrix Salmonella spp Kit Linked to the DuPont BAX PCR System Test result interpretation and classification 18 Pathatrix Salmonella spp Kit Individual Samples Linked to PCR or ELISA Workflow Pathatrix Salmonella spp Kit Linked to the Idaho Technology R A P I D 9 LT PCR System Sample enrichment page 20 v Sample preparation page 20 v Samples and consumable loading page 21 v Sample unloading page 23 v Detection R A P I D 9 LT PCR protocol page 24 v Detection direct plating optional page 25 v Test result interpretation and classification page 25 Procedural guidelines e Use aseptic technique and good laboratory practices at all times e A facemask should be worn when weighing out powders e Care must be taken when boiling agar prior to autoclaving and heat resistant gloves should be worn when handling hot flasks of liquid e Take care when handling plates or vessels that may contain microorganisms e Avoid generati
31. er solid selective medium complimentary to XLD and especially appropriate for the isolation of lactose positive Salmonella Salmonella typhi and Salmonella paratyphi strains 2 Allow the plates to dry for approximately 10 minutes then invert and incubate at the required temperature for 18 24 hours or as recommended by the manufacturer A presumptive positive result is defined as the isolation of typical suspicious or atypical Salmonella spp colonies on the agar plates used A presumptive positive isolate should be subsequently confirmed by the use of subculture and appropriate biochemical and serological tests as detailed in USDA Microbiology Laboratory Guidebook MLG 4 04 for cooked ham or FDA Bacteriological Analytical Manual BAM Chapter 5 sections D and E for tomatoes and chocolate see References on page 58 Test result interpretation and classification The Pathatrix Salmonella spp Kit is designed as a sample preparation method for presence absence detection of Salmonella spp in food matrices Using the Pathatrix Salmonella spp Kit linked to the TECRA Salmonella visual immunoassay presumptive results can be obtained prior to confirmation within 22 28 hours Once confirmed the results are reported as e Salmonella spp Detected in 25 325 g sample matrices e Salmonella spp Not detected in 25 325 g sample matrices Pathatrix Salmonella spp Kit Individual Samples Linked to PCR or ELISA 41 n Chapter
32. etation and classification 34 Pathatrix Salmonella spp Kit Individual Samples Linked to PCR or ELISA 4 Workflow Pathatrix Salmonella spp Kit Linked to the 3M TECRA Salmonella Visual Immunoassay Sample enrichment page 36 v Sample preparation page 36 v Samples and consumable loading page 37 v Sample unloading page 39 v Detection TECRA Salmonella visual immunoassay protocol page 40 v Detection direct plating optional page 41 v Test result interpretation and classification page 41 Procedural guidelines e Use aseptic technique and good laboratory practices at all times e A facemask should be worn when weighing out powders e Care must be taken when boiling agar prior to autoclaving and heat resistant gloves should be worn when handling hot flasks of liquid e Take care when handling plates or vessels that contain microorganisms e Avoid generating aerosols as pathogenic organisms may be present e Used or unused reagents used media sample enrichments as well as other contaminated disposable materials should be disposed of following procedures for infectious or potentially infectious products Sample enrichments might be contaminated with pathogenic organisms infectious to humans so all waste must be treated as biohazardous and handled and disposed using safe laboratory practices in accordance and compliance with all appropriate regulations Pathatrix Sal
33. etic beads have aggregated against the magnet open the tube and remove 500 uL of the bead free supernatant and inoculate the VIDAS strip 4 To proceed please refer to the VIDAS operating instructions Standard sample 600 pL Add 700 uL of pre warmed BPW Transfer 600 uL of bead Vortex the tube for 10 sec and then Vortex the tube for 10 sec and then 37 C to resuspend the Pathatrix suspension into a standard 2 0 mL incubate the bead suspension for place in a water bath at 95 C and beads screw cap microfuge tube 2 hr at 37 C heat for 15 min to release antigens Note 100 uL of the beads can be retained for plating on 500 uL EN Bead free supernatant Immediately atter heating capture Remove 500 uL of bead free Proceed with the assay in accordance beads for 1 min on the supernatant and inoculate the with the manufacturer s instructions DynaMag 2 Magnet VIDAS strip If a positive ELISA result is obtained an aliquot of the Pathatrix paramagnetic beads should be plated out see the following section Detection direct plating optional 48 Pathatrix Salmonella spp Kit Individual Samples Linked to PCR or ELISA Chapter 5 Pathatrix Salmonella spp Kit Linked to the bioM rieux VIDAS Immunoassay Testing System 5 Detection direct plating optional Detection direct plating optional Note We recommend streaking the sample out to generate individual colonies
34. garding the correct set up and operation of the BAX PCR system 1 Pipet 25 uL of the Pathatrix paramagnetic bead suspension into BAX lysis tubes and proceed according to the standard BAX lysis procedure 2 Once the lysis step is complete immediately place the rack and tubes onto the Magnetic Capture Plate Cat no MAGNETICPLATE and leave for at least 1 minute to allow the Pathatrix paramagnetic beads to accumulate on the bottom of the tube 3 Transfer only bead free lysate into the BAX PCR reaction tubes Note Target DNA if present will be in the bead free supernatant 4 To proceed refer to the DuPont Qualicon operating instructions Standard sample 25 uL Add 100 pL PBS to resuspend the beads Transfer 25 uL of bead suspension into BAX lysis Tubes containing standard lysis reagents including protease Bead free lysate Magnetic plate Perform standard BAX lysis procedure Magnetic plate Capture beads for 1 min Transfer bead free lysate into BAX PCR Reaction Tubes If a positive PCR result is obtained an aliquot of the Pathatrix paramagnetic beads should be plated out see the following section Detection direct plating optional 16 Pathatrix Salmonella spp Kit Individual Samples Linked to PCR or ELISA Chapter 1 Pathatrix Salmonella spp Kit Linked to the DuPont BAX PCR System 1 Detection direct plating optional
35. ial for example by vortexing bead vial or inversion of sealed vial and add 50 uL of the Pathatrix paramagnetic bead suspension into the spout on the lid of the Sample Vessel uu Vessel Spout Elution Vessel Sample Vessel Sterile 1X PBS 35 mL Sample Vessel Holder ATUBEHOLD 4 Remove the capture phase kit from the bag and orient with the valve plunger pointing left Connect the valve firmly to the lids of the Sample and Elution Vessels n Capture Phase Valve Plunger Pathatrix Salmonella spp Kit Individual Samples Linked to PCR or ELISA 21 2 Chapter 2 Pathatrix Salmonella spp Kit Linked to the Idaho Technology R A P I D 9 LT PCR System Samples and consumable loading 5 Holding both vessels lift the assembled vessels and attached capture phase kit out of the Sample Vessel Holder 6 Place the vessels into the Cartridge pushing them firmly in place from the bottom upwards 7 Firmly push the remainder of the kit into the Cartridge ensuring that the valve the capture phase and the syringe are all held securely in the molded recess of the Cartridge 8 Push the magnet slider across into the locking position and press the magnet release button on the side of the Cartridge to ensure the kit is positioned correctly and the magnets can freely disengage from the capture phase Syringe Capture Phase Sliding Magnet Holder Magnet Release Button Valve Plunger Sample Vessel Elut
36. ially pathogenic Salmonella is a frequently reported cause of foodborne illness occurring in both epidemics and in isolated cases Salmonella bacteria are the causative agent for Salmonellosis Outbreaks have been associated with raw meats and poultry eggs milk and dairy products seafood coconut sauces salad dressings cocoa chocolate spices frozen products and vegetables such as hot peppers The Pathatrix Salmonella spp Kit is for professional use only and is intended for use by qualified users interested in determining the presence absence of Salmonella spp in food samples Users may include but are not limited to food producers food processors food manufacturers retailers and microbiology testing laboratories The standard food sample size used in the Pathatrix Auto system is 25 g of food diluted with 225 mL of enrichment medium We recommend that the sub samples removed for analysis are processed immediately or if storage is required that the samples are refrigerated at 5 3 C Samples should be rewarmed to 37 1 C prior to analysis with the Pathatrix Auto system The sample preparation procedure allows you to detect as few as 1 10 cfu from 25 325 g of food samples after enrichment The limitation of the Pathatrix Salmonella spp Kit is in the ability of the target to reproduce in the enrichment medium be captured by the magnet and subsequently be detected by BAX PCR R A P LD LT PCR Salmonella OPTIMA TECRA
37. ic beads Remove all the liquid from the Elution Vessel without removing the vessel from the rack taking care not to disturb the captured Pathatrix paramagnetic beads Remove the Elution Vessel from the vessel holder and use 700 uL of prewarmed BPW to resuspend the Pathatrix paramagnetic beads Transfer 600 uL to a microcentrifuge tube Note 100 uL of the Pathatrix paramagnetic beads can be retained for direct plating prior to the additional 2 hour growth step Vortex the tube for 10 seconds Incubate the tube for 2 hours at 37 1 C Remove the tube from the incubator and vortex for 10 seconds Pathatrix Salmonella spp Kit Individual Samples Linked to PCR or ELISA 47 5 Chapter 5 Pathatrix Salmonella spp Kit Linked to the bioM rieux VIDAS Immunoassay Testing System Detection VIDAS immunoassay testing protocol Detection VIDAS immunoassay testing protocol IMPORTANT It is solely the operator s responsibility to refer to the package insert instructions and instrument user manual for information regarding the correct set up and operation of the VIDAS system 1 Ensure that the tubes are securely capped and then heat in a water bath at 95 C for 15 minutes to release target antigens 2 After heating immediately place the tube into the DynaMag 2 Magnet Cat no 123 21DK for 1 minute to allow the Pathatrix paramagnetic beads to be pulled out of the lysate 3 Once the Pathatrix paramagn
38. idual Samples Linked to PCR or ELISA 37 n Chapter 4 Pathatrix Salmonella spp Kit Linked to the 3M TECRA Salmonella Visual Immunoassay Samples and consumable loading 5 Holding both vessels lift the assembled vessels and attached capture phase kit out of the Sample Vessel Holder 6 Place the vessels into the Cartridge pushing them in firmly from the bottom upwards 7 Firmly push the remainder of the kit into the Cartridge starting with the valve followed by the capture phase and finally the syringe 8 Push the magnet slider across into the locking position and press the magnet release button on the side of the Cartridge to ensure the kit is positioned correctly and the magnets will move away from the capture phase freely Syringe Capture Phase Sliding Magnet Holder Magnet Release Button Valve Plunger Sample Vessel Elution Vessel Cartridge Holder ACARTHOLD 9 Reset the magnets into the locking position 10 Insert the Cartridge into the Pathatrix Auto Instrument until in the locking position 11 Press the numbered button above the appropriate Cartridge The LED above the Cartridge will turn green indicating the run has begun approximately 14 minutes 38 Pathatrix Salmonella spp Kit Individual Samples Linked to PCR or ELISA Chapter 4 Pathatrix Salmonella spp Kit Linked to the 3M TECRA Salmonella Visual Immunoassay Sample unloading 10 11 12 13 Sample unload
39. ing At the end of the run the LED will flash red and green alternately Press the button above the appropriate Cartridge to initialize the draining step approximately 1 minute When the draining step is complete and the LED is illuminated red remove the Cartridge by pulling it out away from the instrument Remove the syringe from the Cartridge and carefully pull out the rest of the kit starting at the top and working downwards When removing the Sample and Elution Vessels from the Cartridge hold firmly by the vessels themselves to prevent spillage Place both vessels in the Tube Rack also known as the Sample Vessel Holder or rack Remove the lid from the Elution Vessel and leaving the Elution Vessel in place in the rack lift away the rest of the consumable including the Sample Vessel and discard Place the Elution Vessel in the rack and cap with a flat lid provided in the kit Leave in the rack for 1 minute to allow capture of the Pathatrix paramagnetic beads Remove all the liquid from the Elution Vessel without removing the vessel from the rack taking care not to disturb the captured Pathatrix paramagnetic beads Remove the Elution Vessel from the vessel holder and use 500 uL of prewarmed BPW to resuspend the Pathatrix paramagnetic beads Transfer 400 uL to a microcentrifuge tube Note 100 uL of the Pathatrix paramagnetic beads can be retained for direct plating prior to the additional 2 hour growth s
40. ing out powders e Care must be taken when boiling agar prior to autoclaving and heat resistant gloves should be worn when handling hot flasks of liquid e Take care when handling plates or vessels that may contain microorganisms e Avoid generating aerosols as pathogenic organisms may be present e Used or unused reagents used media sample enrichments as well as other contaminated disposable materials should be disposed of following procedures for infectious or potentially infectious products Sample enrichments might be contaminated with pathogenic organisms infectious to humans so all waste must be treated as biohazardous and handled and disposed using safe laboratory practices in accordance and compliance with all appropriate regulations Pathatrix Salmonella spp Kit Individual Samples Linked to PCR or ELISA 11 1 Chapter 1 Pathatrix Salmonella spp Kit Linked to the DuPont BAX PCR System Sample enrichment Sample enrichment Note Certain food types and swabs sponges can benefit from an alternative enrichment strategy see Appendix A Alternative Enrichment Methods 1 Prepare a 1 10 dilution of the food sample in the appropriate prewarmed 37 1 C enrichment media in a sterile bag Note For example add 25 g of food sample to 225 mL of prewarmed media or add 325 g of food sample to 2925 mL of prewarmed media IMPORTANT It is critical that the enrichment media is prewarmed to 37 1 C prior to ad
41. ion Vessel Cartridge Holder ACARTHOLD 9 Reset the magnets into the locking position 10 Insert the Cartridge into the Pathatrix Auto Instrument until it clicks into the locking position 11 Press the numbered button above the appropriate Cartridge to start the run approximately 14 minutes The associated LED will turn green to indicate the run has started 22 Pathatrix Salmonella spp Kit Individual Samples Linked to PCR or ELISA Chapter 2 Pathatrix Salmonella spp Kit Linked to the Idaho Technology R A P I D 9 LT PCR System Sample unloading 10 Sample unloading At the end of the run the LED will flash red and green alternately Press the button above the appropriate Cartridge to initialize the draining step approximately 1 minute When the draining step is complete and the LED is illuminated red remove the Cartridge by pulling it out away from the instrument Remove the syringe from the Cartridge and carefully pull out the rest of the kit starting at the top and working downwards When removing the Sample and Elution Vessels from the Cartridge hold firmly by the vessels themselves to prevent spillage Place both vessels in the Tube Rack also known as the Sample Vessel Holder or rack Remove the lid from the Elution Vessel and leaving the Elution Vessel in place in the rack lift away the rest of the consumable including the Sample Vessel and discard Place the Elution Vessel in the ra
42. ior to the additional 2 hour growth step Vortex the tube for 10 seconds Incubate the tube for 2 hours at 37 1 C Remove the tube from the incubator and vortex for 10 seconds Pathatrix Salmonella spp Kit Individual Samples Linked to PCR or ELISA 31 Detection Salmonella OPTIMA protocol Chapter 3 Pathatrix Salmonella spp Kit Linked to the RayAl Salmonella OPTIMA System Detection Salmonella OPTIMA protocol IMPORTANT It is solely the operator s responsibility to refer to the package insert instructions and instrument user manual for information regarding the correct set up and operation of the Salmonella OPTIMA system 1 Ensure that the tubes are securely capped and then heat in a water bath for 15 minutes at 95 C to release target antigens 2 After heating immediately place the tube into the DynaMag 2 Magnet Cat no 123 21D for 2 minutes to allow the Pathatrix paramagnetic beads to be pulled out of the lysate 3 Once the Pathatrix paramagnetic beads have aggregated against the magnet open the tube and transfer 1100 uL of the bead free supernatant into the designated well of the RayAl microplate 4 To proceed refer to the Salmonella OPTIMA operating instructions Standard sample ay 1 1 mL Add 1 2 mL of pre warmed BPW 37 C to resuspend the Pathatrix beads Note 100pL of the beads can be retained for plating Transfer 1 1 mL of the bead suspension into a standard 2 0 mL
43. it is positioned correctly and the magnets can freely disengage from the capture phase Syringe Capture Phase Sliding Magnet Holder Magnet Release Button Valve Plunger Sample Vessel Elution Vessel Cartridge Holder ACARTHOLD 9 Reset the magnets into the locking position 10 Insert the Cartridge into the Pathatrix Auto Instrument until it clicks into the locking position 11 Press the numbered button above the appropriate Cartridge to start the run approximately 14 minutes The associated LED will turn green to indicate the run has started 14 Pathatrix Salmonella spp Kit Individual Samples Linked to PCR or ELISA Sample unloading 10 Chapter 1 Pathatrix Salmonella spp Kit Linked to the DuPont BAX PCR System 1 Sample unloading At the end of the run the LED will flash red and green alternately Press the button above the appropriate Cartridge to initialize the draining step approximately 1 minute When the draining step is complete and the LED is illuminated red remove the Cartridge by pulling it out away from the instrument Remove the syringe from the Cartridge and carefully pull out the rest of the kit starting at the top and working downwards When removing the Sample and Elution Vessels from the Cartridge hold firmly by the vessels themselves to prevent spillage Place both vessels in the Tube Rack also known as the Sample Vessel Holder or rack Rem
44. lids on the Sample and Elution Vessels making sure that the vessels are sealed all the way around 3 Ensure the Pathatrix paramagnetic beads included in the kit are fully resuspended by agitating the bead vial for example by vortexing bead vial or inversion of sealed vial and add 50 uL of the Pathatrix paramagnetic bead suspension into the spout on the lid of the Sample Vessel m m Sample Vessel Spout Elution Vessel Sample Vessel Sterile 1X PBS 35 mL Sample Vessel Holder ATUBEHOLD 4 Remove the capture phase kit from the bag and orient with the valve plunger pointing left Connect the valve firmly to the lids of the Sample and Elution Vessels n Capture Phase Valve Plunger Pathatrix Salmonella spp Kit Individual Samples Linked to PCR or ELISA 13 1 Chapter 1 Pathatrix Salmonella spp Kit Linked to the DuPont BAX PCR System Samples and consumable loading 5 Holding both vessels lift the assembled vessels and attached capture phase kit out of the Sample Vessel Holder 6 Place the vessels into the Cartridge pushing them firmly in place from the bottom upwards 7 Firmly push the remainder of the kit into the Cartridge ensuring that the valve the capture phase and the syringe are all held securely in the molded recess of the Cartridge 8 Push the magnet slider across into the locking position and press the magnet release button on the side of the Cartridge to ensure the k
45. lysis if necessary the waste generated by the particular applications reagents and substrates used in your laboratory Ensure that the waste is stored transferred transported and disposed of according to all local state provincial and or national regulations IMPORTANT Radioactive or biohazardous materials may require special handling and disposal limitations may apply Specific chemical handling CAS Chemical Phrase 26628 22 8 Sodium Azide Sodium azide may react with lead and copper plumbing to form highly explosive metal azides Biological hazard safety 56 WARNING BIOHAZARD Biological samples such as tissues body fluids infectious agents and blood of humans and other animals have the potential to transmit infectious diseases Follow all applicable local state provincial and or national regulations Wear appropriate protective equipment which includes but is not limited to protective eyewear face shield clothing lab coat and gloves All work should be conducted in properly equipped facilities using the appropriate safety equipment for example physical containment devices Individuals should be trained according to applicable regulatory and company institution requirements before working with potentially infectious materials Read and follow the applicable guidelines and or regulatory requirements in the following In the U S U S Department of Health and Human Services guideli
46. monella spp Kit Individual Samples Linked to PCR or ELISA 35 n Chapter 4 Pathatrix Salmonella spp Kit Linked to the 3M TECRA Salmonella Visual Immunoassay Sample enrichment Sample enrichment Note Certain food types and swabs sponges can benefit from an alternative enrichment strategy see Appendix A Alternative Enrichment Methods 1 Prepare a 1 10 dilution of the food sample in the appropriate prewarmed 37 1 C enrichment media in a sterile bag Note For example add 25 g of food sample to 225 mL of prewarmed media or add 325 g of food sample to 2925 mL of prewarmed media IMPORTANT It is critical that the enrichment media is prewarmed to 37 1 C prior to adding the food sample To prevent cooling all samples should then immediately be placed in a 37 C incubator 2 Homogenize the sample by massaging the sample between the fingers for 10 15 seconds to disperse any large clumps of material Narang and Cray 2006 3 Incubate at 37 1 C for a minimum of 18 24 hours Sample preparation 1 Remove the Sample Vessel and Elution Vessel from the consumable kit packaging and place into the Sample Vessel Holder 2 Loosen the lids from the vessels and partially remove leaving an opening through which to add sample and wash buffer 3 Place 50 mL of your sample in the Sample Vessel Note If the samples are highly particulate or has a high fat content a Seward plain sterile bag with internal strainer may b
47. nal strainer may be used Seward Product Code BA6041 STR 4 Store the individual enriched samples at 5 3 C for potential reanalysis until the test result is confirmed Note Do not store for more than 32 hours If refrigerated after pre enrichment samples should be rewarmed to 37 1 C prior to removal of aliquots for analysis 28 Pathatrix Salmonella spp Kit Individual Samples Linked to PCR or ELISA Chapter 3 Pathatrix Salmonella spp Kit Linked to the RayAl Salmonella OPTIMA System 3 Samples and consumable loading Samples and consumable loading 1 Add PBS Cat no AM9624 diluted to 1X to the fill line of the Elution Vessel approximately 35 mL of 1X PBS 2 Place the lids on the Sample and Elution Vessels making sure that the vessels are sealed all the way around 3 Ensure the Pathatrix paramagnetic beads included in the kit are fully resuspended by agitating the bead vial for example by vortexing bead vial or inversion of sealed vial and add 50 uL of the Pathatrix paramagnetic bead suspension into the spout on the lid of the Sample Vessel Sample Vessel Spout Elution Vessel Sample Vessel Sterile 1X PBS 35 mL Sample Vessel Holder ATUBEHOLD 4 Remove the capture phase kit from the bag and orient with the valve plunger pointing left Connect the valve firmly to the lids of the Sample and Elution Vessels n Capture Phase Valve Plunger Pathatrix Salmonella spp Kit I
48. ndividual Samples Linked to PCR or ELISA 29 3 Chapter 3 Pathatrix Salmonella spp Kit Linked to the RayAl Salmonella OPTIMA System Samples and consumable loading 5 Holding both vessels lift the assembled vessels and attached capture phase kit out of the Sample Vessel Holder 6 Place the vessels into the Cartridge pushing them in firmly from the bottom upwards 7 Firmly push the remainder of the kit into the Cartridge ensuring that the valve the capture phase and the syringe are all held securely in the molded recess of the Cartridge 8 Push the magnet slider across into the locking position and press the magnet release button on the side of the Cartridge to ensure the kit is positioned correctly and the magnets can freely disengage from the capture phase Syringe Capture Phase Sliding Magnet Holder Magnet Release Button Valve Plunger Sample Vessel Elution Vessel Cartridge Holder ACARTHOLD 9 Reset the magnets into the locking position 10 Insert the Cartridge into the Pathatrix Auto Instrument until it clicks into the locking position 11 Press the numbered button above the appropriate Cartridge to start the run approximately 14 minutes The associated LED will turn green to indicate the run has started 30 Pathatrix Salmonella spp Kit Individual Samples Linked to PCR or ELISA Chapter 3 Pathatrix Salmonella spp Kit Linked to the RayAl Salmonella OPTIMA System Sample unloading
49. nes published in Biosafety in Microbiological and Biomedical Laboratories found at www cdc gov biosafety Occupational Safety and Health Standards Bloodborne Pathogens 29 CFR 1910 1030 found at www access gpo gov nara cfr waisidx_01 29cfr1910a_01 html Your company s institution s Biosafety Program protocols for working with handling potentially infectious materials Additional information about biohazard guidelines is available at www cdc gov In the EU Check local guidelines and legislation on biohazard and biosafety precaution and refer to the best practices published in the World Health Organization WHO Laboratory Biosafety Manual third edition found at www who int csr resources publications biosafety WHO_CDS_CSR_LYO_2004_11 en Pathatrix Salmonella spp Kit Individual Samples Linked to PCR or ELISA Documentation and Support Obtaining SDSs Safety Data Sheets SDSs are available from www lifetechnologies com support Note For the SDSs of chemicals not distributed by Life Technologies contact the chemical manufacturer Obtaining Certificates of Analysis The Certificate of Analysis provides detailed quality control and product qualification information for each product Certificates of Analysis are available on our website Go to www lifetechnologies com support and search for the Certificate of Analysis by product lot number which is printed on the box Obtaining support Support email foodsafety
50. ng aerosols as pathogenic organisms may be present e Used or unused reagents used media sample enrichments as well as other contaminated disposable materials should be disposed of following procedures for infectious or potentially infectious products Sample enrichments might be contaminated with pathogenic organisms infectious to humans so all waste must be treated as biohazardous and handled and disposed using safe laboratory practices in accordance and compliance with all appropriate regulations Pathatrix Salmonella spp Kit Individual Samples Linked to PCR or ELISA 19 2 Chapter 2 Pathatrix Salmonella spp Kit Linked to the Idaho Technology R A P I D 9 LT PCR System Sample enrichment Sample enrichment Note Certain food types and swabs sponges can benefit from an alternative enrichment strategy see Appendix A Alternative Enrichment Methods 1 Prepare a 1 10 dilution of the food sample in the appropriate prewarmed 37 1 C enrichment media in a Stomacher bag Note For example add 25 g of food sample to 225 mL of prewarmed media or add 325 g of food sample to 2925 mL of prewarmed media IMPORTANT It is critical that the enrichment media is prewarmed to 37 1 C prior to adding the food sample To prevent cooling all samples should then immediately be placed in a 37 C incubator 2 Homogenize the sample by massaging the sample between the fingers for 10 15 seconds to disperse any large clumps of m
51. nt media choice The media is supplied by several manufacturers e g Oxoid product codes shown Difco and Merck in a dehydrated form and should be prepared according to the manufacturer s instructions Buffered peptone water Oxoid product code CM0509 For samples with high background microflora Tetrathionate TT broth base Oxoid product code CM0029 For milk powder chocolate or cocoa based samples UHT skim milk Brilliant green CAS 633 03 4 Food retail store MLS Selective agar XLD agar Oxoid product code CM0469 Brilliant green agar modified Oxoid product code CM0329 Reagents PBS 10X pH 7 4 Life Technologies Cat no AM9624 or AM9625 Pathatrix Salmonella spp Kit Individual Samples Linked to PCR or ELISA Product Information Materials not included in the kit 10 Pathatrix Salmonella spp Kit Individual Samples Linked to PCR or ELISA Workflow Pathatrix Salmonella spp Kit Linked to the DuPont BAX PCR System Sample enrichment page 12 v Sample preparation page 12 v Samples and consumable loading page 13 v Sample unloading page 15 v Detection BAX9 PCR protocol page 14 v Detection direct plating optional page 17 v Test result interpretation and classification page 17 Procedural guidelines e Use aseptic technique and good laboratory practices at all times e A facemask should be worn when weigh
52. nto the Pathatrix Auto Instrument until in the locking position 11 Press the numbered button above the appropriate Cartridge The LED above the Cartridge will turn green indicating the run has begun approximately 14 minutes 46 Pathatrix Salmonella spp Kit Individual Samples Linked to PCR or ELISA Chapter 5 Pathatrix Salmonella spp Kit Linked to the bioM rieux VIDAS Immunoassay Testing System Sample unloading 10 11 12 13 Sample unloading At the end of the run the LED will flash red and green alternately Press the button above the appropriate Cartridge to initialize the draining step approximately 1 minute When the draining step is complete and the LED is illuminated red remove the Cartridge by pulling it out away from the instrument Remove the syringe from the Cartridge and carefully pull out the rest of the kit starting at the top and working downwards When removing the Sample and Elution Vessels from the Cartridge hold firmly by the vessels themselves to prevent spillage Place both vessels in the Tube Rack also known as the Sample Vessel Holder or rack Remove the lid from the Elution Vessel and leaving the Elution Vessel in place in the rack lift away the rest of the consumable including the Sample Vessel and discard Place the Elution Vessel in the rack and cap with a flat lid provided in the kit Leave in the rack for 1 minute to allow capture of the Pathatrix paramagnet
53. ove the lid from the Elution Vessel and leaving the Elution Vessel in place in the rack lift away the rest of the consumable including the Sample Vessel and discard Place the Elution Vessel in the rack and cap with a flat lid provided in the kit Leave in the rack for 1 minute to allow capture of the Pathatrix paramagnetic beads Remove all the liquid from the Elution Vessel without removing the vessel from the rack taking care not to disturb the captured Pathatrix paramagnetic beads Remove the Elution Vessel from the vessel holder add 100 uL of PBS into the Elution Vessel and resuspend the Pathatrix paramagnetic beads Appropriate aliquots of the Pathatrix paramagnetic bead suspension can then be immediately analyzed using the laboratory s chosen pathogen detection method Note The Pathatrix paramagnetic bead suspension may be retained for later testing if necessary The beads should be stored in sterile 1 5 mL microcentrifuge tubes AWAY from magnets for example away from Pathatrix vessel holders at 5 3 C for up to 24 hours Pathatrix Salmonella spp Kit Individual Samples Linked to PCR or ELISA 15 Chapter 1 Pathatrix Salmonella spp Kit Linked to the DuPont BAX PCR System Detection BAX PCR protocol Detection BAX PCR protocol IMPORTANT It is solely the operator s responsibility to refer to the package insert instructions and instrument user manual for information re
54. pling protocol sleep hex n e eK Rp har h ved rr Ele Y o n wad 52 Kit sensitiVity 254 5 2k dace acca emia he beast Eee i ete abst lee oes tig 52 Operating conditions 0 c cee e eee eee e eens 53 APPENDIX C Ordering Information oooccccocccoccccc 54 Related materials from Life Technologies 00000 cece cece or 54 APPENDIX D Safety ut irccrll errar 55 Chemicalsafety tot it tick ate nero Ie da Eu te eS di 55 Specific chemical handling 00 ccc narren rnrn nennen eanne 56 Biologicalhazard safety eere ee A MI et 56 Documentation and Support ccc ccc eee eens 57 Obtaining SDSS s 3 c0 VERRE Te PII Ew ee Fees A 57 Obtaining Certificates of Analysis 20 000 e eens 57 Obtainina support ark done RERO eek ene p yee el te ERO 57 Limited product warranty 00 60 cece e ee rm rr 57 References 52 be ccetctesraeenbadesei ane eetenenenesone Mean AREE 58 Pathatrix Salmonella spp Kit Individual Samples Linked to PCR or ELISA 5 Contents 6 Pathatrix Salmonella spp Kit Individual Samples Linked to PCR or ELISA About the kit Product Information IMPORTANT Before using this product read and understand the information in Appendix D Safety on page 55 CAUTION Salmonella spp is a Biosafety Level 2 BSL 2 organism excluding S typhi and S paratyphi which are both Biosafety Level 3 BSL 3 Care must be taken when handling samples th
55. r the isolation of lactose positive Salmonella Salmonella typhi and Salmonella paratyphi strains 2 Allow the plates to dry for approximately 10 minutes then invert and incubate at the required temperature for 18 24 hours or as recommended by the manufacturer A presumptive positive result is defined as the isolation of typical suspicious or atypical Salmonella spp colonies on the agar plates used A presumptive positive isolate should be subsequently confirmed by the use of subculture and appropriate biochemical and serological tests as detailed in USDA Microbiology Laboratory Guidebook MLG 4 04 for cooked ham or FDA Bacteriological Analytical Manual BAM Chapter 5 sections D and E for tomatoes and chocolate see References on page 58 Test result interpretation and classification The Pathatrix9 Salmonella spp Kitis designed as a sample preparation method for presence absence detection of Salmonella spp in food matrices Using the Pathatrix Salmonella spp Kit linked to RayAl Salmonella OPTIMA presumptive results can be obtained prior to confirmation within 22 28 hours Once confirmed the results are reported as e Salmonella spp Detected in 25 325 g sample matrices e Salmonella spp Not detected in 25 325 g sample matrices Pathatrix Salmonella spp Kit Individual Samples Linked to PCR or ELISA 33 3 Chapter 3 Pathatrix Salmonella spp Kit Linked to the RayAl Salmonella OPTIMA System Test result interpr
56. results of purchaser s activities in environmental testing quality control quality assurance testing food and agricultural testing for a fee or other commercial consideration No other right is hereby granted expressly by implication or by estoppel This product is for environmental testing quality control quality assurance testing food and agricultural testing and research purposes only The purchase of this product does not grant the purchaser any additional rights including without limitation the right to transfer or resell the product in any form or the right to use the product as a therapeutic agent or diagnostics test component For information on obtaining additional rights please contact outlicensing alifetech com or Out Licensing Life Technologies 5791 Van Allen Way Carlsbad California 92008 TRADEMARKS The trademarks mentioned herein are the property of Life Technologies Corporation or their respective owners in the United States and other countries BAX is a registered trademark DuPont or its affiliates R A P I D is a registered trademark of Idaho Technology Inc Stomacher is a registered trademark of Seward Limited UK TECRA and 3M are trademarks of 3M Company Corporation VIDAS is a registered Trademark of bioM rieux Whirl Pak is a registered trademark of Wheatherby Nasco Inc 2012 Life Technologies Corporation All rights reserved Contents Product Information 0cc cece cence eee eee nh AbouttheKlt
57. tep Vortex the tube for 10 seconds Incubate the tube for 2 hours at 37 1 C Remove the tube from the incubator and vortex for 10 seconds Pathatrix Salmonella spp Kit Individual Samples Linked to PCR or ELISA 39 Chapter 4 Pathatrix Salmonella spp Kit Linked to the 3M TECRA Salmonella Visual Immunoassay Detection TECRA Salmonella visual immunoassay protocol Detection TECRA Salmonella visual immunoassay protocol Standard sample Add 500 pL of pre warmed BPW 37 C to resuspend the Pathatrix beads Note 100 pL of the bead suspension can be retained for plating w 6 Immediately after heating capture beads for 2 min on the DynaMag 2 Magnet 40 IMPORTANT It is solely the operator s responsibility to refer to the package insert instructions and instrument user manual for information regarding the correct set up and operation of the TECRA Salmonella visual immunoassay system Ensure that the tubes are securely capped and then heat in a water bath at 95 C for 15 minutes to release target antigens After heating immediately place the tube into the DynaMag 2 Magnet Cat no 123 21D for 2 minutes to allow the Pathatrix paramagnetic beads to be pulled out of the lysate Once the Pathatrix paramagnetic beads have aggregated against the magnet open the tube and transfer 200 uL of the bead free supernatant into the designated well of th
58. tory practices in accordance and compliance with all appropriate regulations Pathatrix Salmonella spp Kit Individual Samples Linked to PCR or ELISA 43 Chapter5 Pathatrix Salmonella spp Kit Linked to the bioM rieux VIDAS Immunoassay Testing System Sample enrichment Sample enrichment Note Certain food types and swabs sponges can benefit from an alternative enrichment strategy see Appendix A Alternative Enrichment Methods 1 Prepare a 1 10 dilution of the food sample in the appropriate prewarmed 37 1 C enrichment media in a sterile bag Note For example add 25 g of food sample to 225 mL of prewarmed media or add 325 g of food sample to 2925 mL of prewarmed media IMPORTANT It is critical that the enrichment media is prewarmed to 37 1 C prior to adding the food sample To prevent cooling all samples should then immediately be placed in a 37 C incubator Homogenize the sample by massaging the sample between the fingers for 10 15 seconds to disperse any large clumps of material Narang and Cray 2006 Incubate at 37 1 C for a minimum of 18 24 hours Note We recommend that these sub samples for pooling are processed immediately or if storage is required that the samples are refrigerated at 5 43 C Samples should be rewarmed to 37 1 C prior to analysis on the Pathatrix Auto Instrument The remaining enriched sample should be stored until the results of the pooled sample have been determine
59. x on the Disruptor Genie vortexer equipped with the TurboMix attachment for 5 minutes on the highest setting 2 Once the bead beating lysis step is complete immediately place the Bead Tubes into the DynaMag 2 Magnet Cat no 123 21D 3 Wait for at least 1 minute to allow the Pathatrix paramagnetic bead debris to be drawn out of suspension thereby producing the bead free supernatant Note If present target DNA will be in the bead free lysate 4 Pipet 10 uL of Reconstitution Buffer into the Unknown test vial Note Once pellets have been reconstituted samples should be processed immediately 9 Pipet 10 uL of bead free supernatant into the PCR Unknown test reaction vial Mix well to fully resuspend the PCR reagent pellet Note Take care not to transfer any glass beads from the Blue Capped Bead Beating Tubes into the PCR reagent vial 6 Transfer 18 uL of the reagent sample suspension and add into the top of a glass PCR capillary tube Using the Capping Tool provided place the cap on the sample capillary tube and push down firmly 7 To proceed refer to the R A P I D 9 LT PCR operating instructions Standard sample Add 100 uL PBS to resuspend the Transfer 25 pL of bead suspension Bead beat for 5 min to release Capture beads for 2 min Pathatrix beads into Small Bead Tube with glass target DNA beads WP 540yuL Bead free supernatant Aliquot bead free supernat
60. y personnel read and practice the general safety guidelines for chemical usage storage and waste provided below and consult the relevant SDS for specific precautions and instructions Read and understand the Safety Data Sheets SDSs provided by the chemical manufacturer before you store handle or work with any chemicals or hazardous materials To obtain SDSs see the Documentation and Support section in this document Minimize contact with chemicals Wear appropriate personal protective equipment when handling chemicals for example safety glasses gloves or protective clothing Minimize the inhalation of chemicals Do not leave chemical containers open Use only with adequate ventilation for example fume hood Check regularly for chemical leaks or spills If a leak or spill occurs follow the manufacturer s cleanup procedures as recommended in the SDS Handle chemical wastes in a fume hood Ensure use of primary and secondary waste containers A primary waste container holds the immediate waste A secondary container contains spills or leaks from the primary container Both containers must be compatible with the waste material and meet federal state and local requirements for container storage After emptying a waste container seal it with the cap provided Pathatrix Salmonella spp Kit Individual Samples Linked to PCR or ELISA 55 Appendix D Safety Biological hazard safety Characterize by ana

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