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1. Error Tubes Reversed Calibration Not Complete The substrate blank and the standard were tested in reversed order or the substrate blank result was over 3000 FLU Prepare new samples and repeat the procedure The difference between the fluorescence of the duplicate readings on each calibrator must be less than 5 fluorescence units 59 Fluorophos ALP Test System User s Guide Problem Message Explanation Calibration Not 1 The calibrators may not have been Acceptable brought to temperature 38 C or the calibrators may be bad Recalibrate using new calibrators See the Calibration section in Chapter 2 2 Check Calibrator C under PMT voltage See the PMT Voltage Adjustment section in Chapter 5 3 The optical lamp and or excitation filter may require replacement Contact Advanced Instruments for Hot Line Service Value Less Than 10 Fifty seven out of 60 readings in either mU L minute 2 or minute 3 are below or equal to zero fluorescence indicating a very low result Confirm this very low result by pressing TEST and START a second time and or third time Check to make sure sam ple has been added to the substrate Repeat the entire test with new substrate and sample Error Overranged Display reads over 24 000 fluorescent units Repeat Test very high activity Repeat on dilution to con firm Error Unstable The average change in fluorescence during Reading Repeat Test
2. Cuvette Holder Cleaners re order pkg of 50 as FLA840 Heating Block optional Heating Block Thermometer optional Warranty Card Table 1 A Model FLM200 Packing List Fluorophos ALP Test System User s Guide zz _ F WORCENOS FLUOROPHOS j Figure 1 B FLMA02 ALP Starter Test Kit Quantity Part No Description 1 FLA865 25 uL Sample Pipette FLA885 75 uL Sample Pipette FLA888 2000 uL Sample Pipette FLA224 Fluorophos ALP 225 Test Reagent Set FLA250 Fluorophos ALP Calibrator Set FLA260 Fluorophos ALP Pasteurization Controls FLA280 Fluorophos ALP Daily Instrument Control 1 pack ACM205 2 ml Pipette Tips 25 1 pack FLA880 25 75 uL Pipette Tips 100 1 FLM5 Fluorophos ALP Test System User s Guide Se Se eS eS Ee Table 1 B FLMA02 ALP Starter Test Kit Packing List Installation amp Setup e Fill out the postage paid warranty card enclosed Mark the appropri ate boxes if you wish to receive additional information You may mail the card postage required outside of the United States or fax it to 781 320 8181 Step 3 Obtain additional items Items needed but not supplied by the manufacturer include e A vortex mixer e Soft lint free wiping tissues Step 4 Set up heating block a Plug the heating block cord into a grounded outlet b Switch the heating block power switch to the ON position The power light on the front panel will illuminate c Insert the heating block thermometer into the
3. This product meets the intent of Directive 2004 108 EC for Electromagnetic Compatibility Compliance was demonstrated using the following standards as listed in the Official Journal of the European Communities Con sult the Declaration of Conformance certificate shipped with the product for the latest update EN 61326 1 2006 Group 1 Class B Electrical Equip ment for Measurement Control and Laboratory Use This product meets the intent of Directive 2006 95 EC the Low Voltage Directive Compliance was demon strated using the following standards as listed in the Official Journal of the European Communities Consult the Declaration of Conformance certificate shipped with the product if required for the latest update IEC EN 61010 1 2nd edition Safety Requirements for Electrical Equipment for Measurement Control and Laboratory Use IEC EN 61010 2 010 2nd edition Safety Requirements for Electrical Equipment for Measurement Control and Laboratory Use Particular Requirements for Laboratory Equipment for the Heating of Materials Regulatory Notices Regulatory approval type Description EC Declaration This product meets the intent of Directive 2002 96 EC of Conformity as amended by 2003 108 EC for Waste Electrical and WEEE Electronic Equipment WEEE Consult the Declaration of Conformance certificate shipped with the product if required for the latest update FCC Part 15 This device complies
4. Switch the power switch to the off position and disconnect the power cord Use a small flat bladed screwdriver or similar tool to pry open the fuse holder door Remove the fuse holder inside and visually check for a blown fuse If there is any doubt test the fuses with a continuity check er or ohmmeter or simply replace them with new fuses Check error messages The software of the instrument is designed in such a way that any incomplete task will be associated with an error message many self explanatory that will help you discover the source of the problem You can find all error messages and descriptions of what they mean in Appendix B Internal diagnostics Your instrument has been equipped with a series of diagnostic tests to allow you to isolate any problem To access diagnostic checks use the following procedure a To access the SETUP menu press the SETUP switchpad at CALIB Calibration Product TEST The display will change to 1 A D Test 46 Troubleshooting amp Service b To select another test on the SETUP menu repeatedly press gt or lt until the desired item is displayed c To activate a displayed test press START d Press STOP to exit the SETUP menu The following menu choices allow you to change operating settings Please note that other options are available but are settings and are dis cussed in Chapter 2 Instrument Operation A D Test The A D test is used to check the accuracy of the
5. Applications and Limitations in Challenges to Contemporary Dairy Analytical Techniques Royal Society of Chemistry London 1984 21 Fluorophos ALP Test System User s Guide pasteurization in dairy products They demonstrated that natural milk ALP was destroyed at temperatures slightly above those necessary to destroy micro organisms pathogenic to man Reduced levels of ALP in finished dairy products therefore ensured that the product had been heat ed to the required temperature or that it had not been contaminated with raw milk during manufacture The significance of this discovery was appreciated immediately and the ALP test was soon universally accepted To this day measurement of ALP activity is the only chemistry test which can be used to confirm that dairy products have been correctly pasteurized The original Kay and Graham ALP assay used phenylphosphate as sub strate and required a 24 hour incubation for quantitative analysis Since then many modifications and improvements have been introduced Currently all quantitative ALP assays use one of three colorimetric sub strates The Scharer and Cornell methods still use phenylphosphate as in the original Kay and Graham procedure the Aschaffenburg and Mullen procedure uses p nitrophenyl phosphate and the Rutgers assay uses phenolphthalein monophosphate Quantitative methods using the original phenylphosphate substrate mea sure enzymatically liberated phenol by coupli
6. Fat 1 PROD 13 Dry Milk PROD 3 Low Fat 2 PROD 14 Sour Cream PROD 4 Skim Milk PROD 15 Product A PROD 5 Choc Milk PROD 16 Product B PROD 6 Cheese PROD 17 Product C PROD 7 Butter PROD 18 Product D PROD 8 Buttermilk PROD 19 Product E PROD 9 UHT Milk PROD 20 Product F PROD 10 Cream PROD 11 Light Cream 7 The fluorometer will display Insert ASTD START 8 Gently mix one 1 of the cuvettes containing Calibrator A and the sample 9 Open the cuvette chamber door and place Calibrator A in the cham ber Shut the door and press START 10 After thirty 30 seconds the fluorometer will make a final reading 200 FLU and display Insert ASTD START Repeat with a second sample of Calibrator A 28 ALP Milk Pasteurization Assay 11 Follow the prompts for Calibrators B and C If the calibration was successful then the fluorometer display will read CALIBRA TION COMPLETE and the channel will be calibrated for that specific product The calibration data is stored in the fluorometer until a new cali bration is performed for the selected product NOTES A If the fluorometer displays STANDARDS REVERSED instead of prompting you to proceed with the next calibra tor level check to make sure that both cuvettes contain the correct calibrator Prepare two new calibrator samples and repeat the procedure until the instrument prompts you to advance to the next calibration lev
7. Test System User s Guide Notes Xiv B Foreword Principles of Fluorescence The technology of fluorescence has been used in analytical assays for over 75 years Molecules can be excited to higher energy levels by the absorption of electromagnetic radiation such as visible light The excited molecular state is short lived and certain molecules will emit light in the process of returning to the lower energy level The emitted light is usually lower in energy than the excitation light The emission of this lower energy light is called fluorescence Light that is lower in ener gy is longer in wavelength For example light with a wave length of 560 nm has less energy than light with a wavelength of 439 nm Many fluorometric assays take advantage of the ability to mea sure the conversion of non fluorescent molecules to highly fluo rescent molecules such as enzymatic assays Inorganic moleules can be measured with fluorescence by combining them with other non fluorescent molecules that will fluoresce when attached to the inorganic ion One of the major advantages of fluorescence assays is the improved sensitivity Fluorescence assays can typically mea sure as little as 10 g mL of analyte compared to colorimetric assays which measure 107 g mL XV Fluorophos ALP Test System User s Guide Fluorometric Assays The FLM200 Fluorometer is designed to perform two fluorometric assays the Alkaline Phosphotase ALP assay a
8. a small amount of distilled water using large amounts of water can damage the optical filters Insert the cleaner all the way to the bottom of the cuvette holder and twist to clean the entire surface of the cuvette holder Repeat with two or three new cuvette holder cleaners to dry the cuvette holder Replace optical filters With the power switch in the off position connect the power cord to the instrument and the power outlet Place the power switch in the on position and allow the instrument to warm up Run controls to verify instrument performance If problems are evi dent when running control values obtain Hot Line Service as described in Chapter 5 Instrument Maintenance Reagent storage See information supplied with each reagent for storage instructions Air filter replacement becomes clogged with dust so as to affect instrument perfor mance To change air filters simply slide old filter out through back panel and replace with new filter cartridge The air filter should be replaced monthly or whenever it Optical filter replacement The fluorometer has been equipped with ALP and ACP excita tion filters and a dual filter holder assembly The assembly may be used by customers who run ALP tests ACP tests or both Because filters age and can easily be degraded by spills scratches etc the excitation and emission filters should be replaced annually To replace filter assembly 1 Shut off the fluoro
9. and press SETUP Advance the menu to 10 A D Test by pressing lt or gt or by using the number keypad Press START Change the fluorescence level to 602 2 FLU for ALP by slowly adjusting the potentiometer on the left side of the instrument enclo sure see figure 6 Wait approximately five minutes and recheck the reading Re adjust to the above limits if the reading has shifted Exit the Setup menu Figure 6 PMT Voltage Potentiometer Serial Suffix D and Later Troubleshooting amp Service NOTE Prior to calibrating or testing products check the calibration ratio of the A B and C calibrators with no milk added on an empty channel The calibration ratio should be 151 4 E7 If the fluorescence cannot be set to the required value the PMT gain will have to be changed Contact Advanced Instruments for Hot line Service For instruments with Serial Suffix prior to D A Instruments prior to Serial suffix D are not equipped with an external accessible potentiometer Adjusting the PMT voltage requires removal of the enclosure and exposure to po tentially hazardous voltages Contact Advanced Instruments for Hot line Service 49 Fluorophos ALP Test System User s Guide Notes 50 Appendix A Additional Technical Information Calibration equations Calibration ratio refers to a number used by the fluorometer for calculating unknown test values and is derived a
10. improved assay for alkaline phosphatase in fluid milk products Presented at the 102nd AOAC Annual Meeting Palm Beach FL August 29 1988 23 Fluorophos ALP Test System User s Guide The Fluorophos method has been collaboratively studied and received IMS FDA AOAC SMEDP and IDF approval and ISO approval The Fluorophos method was correlated to Salmonella and Listeria inac tivation by Eckner in 1992 The application of the Fluorophos method to the determination of both high and very low levels of ALP activity in milk cream and cheese was reported by Lechner and Regensburger in 1993 and the fluorometric method for cheese was approved by ISO in 2003 Introduction of this new approach to ALP testing will provide a rapid modern and cost effective improvement in quality assurance test ing in the dairy industry The Fluorophos ALP reaction The Advanced Instruments fluorometric assay for Alkaline Phosphatase ALP in fluid dairy products is based on a specially designed enzyme substrate called Fluorophos The high sensitivity of fluorometry per mits direct and continuous monitoring of the action of ALP on the sub strate Fluorophos is an aromatic ortho phosphoric monoester com pound which is non fluorescent in solution When acted upon by ALP Fluorophos loses its phosphate radical and becomes a highly fluorescent molecule called Fluoroyellow The reaction is kinetic and can be mea sured with a fluorom
11. in calibration ratios between different products are due to differences in fat content 2 vs 3 2 in the example above and differences in color or added flavor white vs chocolate milk In all cases use controls for verifica tion when running different products on the same channel The four general rules for determining whether separate calibration curves are required are 1 All colored flavored products will require separate calibration curves 2 The fat content of individual dairy products determines whether the products can be run on the same calibration curve the closer the fat content the more likely the products can be run on the same channel 3 When the calibration ratios of two different products are within 5 of each other the two products may be run on the same calibration curve 4 In all cases when two products are spiked with 0 1 v v mixed herd raw milk and run on their own calibration curves and give the same results within 10 then both products may be run on the same channel Reconstituting reagents Each ALP Reagent Set contains two bottles each of Fluorophos ALP Substrate and ALP Substrate Buffer These items must be used together do not intermix bottles from different reagent sets 30 ALP Milk Pasteurization Assay Allow one bottle of Fluorophos Substrate and one bottle of ALP Substrate Buffer to come to room temperature To reconstitute carefully decant the entire contents of t
12. instrument during cali bration and the calibration ratio differences between specific products are mainly due to differences in color and turbidity The fluorometer is designed to be able to run several different tests Each test has its own specific calibration procedure Please see the chapter detailing specific test s instructions for further calibration instructions Preparing reagents To run tests on the fluorometer you will need to reconstitute the appro priate reagents Please see information supplied with each reagent type for instructions on reconstitution Test procedure The fluorometer is designed to be able to run several different tests Each test has its own specific test procedure Please see the chapter detailing the performance of specific test s and product inserts for further instruc tions Changing operating settings The fluorometer has been individually set up at the factory for normal operation but the setup procedure may be required for changing the date and time customizing operating parameters or testing certain instrument functions To change settings use the following procedure a To access the SETUP menu press the SETUP switchpad at CALIB Calibration Product TEST The display will change to 1 A D Test 17 Fluorophos ALP Test System User s Guide b To select another function on the SETUP menu repeatedly press gt or lt until the desired item is displayed c To activa
13. it falls into the heating block wells immediately unplug the power cord remove the thermometer and cuvettes from the heating block and clean them separately Use a test tube brush to clean the heating block wells Take care not to scratch the anodized finish 37 Fluorophos ALP Test System User s Guide Cleanup of cuvette holder spills Failure to clean liquid spills out of the cuvette holder may result in incorrect fluorometer response due to dirty optics Ifa liquid is spilled or glass is broken such that it falls into the fluorometer cuvette chamber take the following steps before drying occurs a b A 38 Unplug the power cord immediately Remove any cuvette from the cuvette holder Use tweezers forceps etc as necessary to remove any large pieces of broken glass from the cuvette holder Remove any liquid from the cuvette holder by inserting a cuvette holder cleaner straight down firmly to the bottom of the cuvette holder Allow the cleaner to absorb liquid for about 20 seconds then remove Discard the used cleaner carefully it may have picked up sharp bits of broken glass from the cuvette holder Repeat with new cuvette holder cleaners as necessary until the cuvette chamber is empty Once all liquid has been removed from the instru ment remove filters and tip or invert fluorometer as necessary to remove any broken glass remaining in the cuvette holder Moisten a clean dry cuvette chamber cleaner with
14. only for transportation This warranty does not extend to any instrument sub jected to negligence accident abuse misuse or other im proper operation or which has been tampered with altered or repaired by anyone other than Advanced Instruments or its authorized service representatives Nor does this warranty extend to any consequential damages caused by defects or improper use or secondary damage resulting from prior problems The above provisions do not extend the original war ranty period of any instrument or part thereof which has been replaced or repaired thereunder Remedies made under the terms of this warranty in no case include any obligation or responsibility for transporta tion charges or arrangements All results calibrations effectiveness medical diagno ses and interpretations are the responsibility of the user THIS WARRANTY IS IN LIEU OF ALL OTHER By accepting and operating this instru MODEL NUMBER SERIAL NUMBER INSTALLATION DATE WARRANTIES REPRESENTATIONS AND CONDI TIONS OF ANY KIND EXPRESSED OR IMPLIED IN FACT OR BY LAW IN NO EVENT SHALL AD VANCED INSTRUMENTS BE LIABLE UNDER THIS WARRANTY OR OTHERWISE IN ANY MATTER FOR ANY REMOTE OR CONSEQUENTIAL DAMAGES RE SULTING FROM THE BREACH OF THIS WARRANTY OR CONDITION IMPLIED BY LAW OR OTHERWISE OR FOR ANY DAMAGE TO THE INSTRUMENT OR ANY PART THEREOF CAUSED BY IMPROPER INSTALLATION UNAUTHORIZED REPAIR OR AL TERATION NEGLIGENCE ABUSE OR MISUSE
15. pre warmed reconstituted substrate cuvette into the cuvette chamber and close the door Once the display has stabilized it should read lt 1200 Record this value If the value is above 1200 do not use this substrate Reconstitute a new bottle of substrate and retest Being open for more than 60 days light contamination or not being refrigerated when not in use could cause the A D result to be above 1200 This can be avoided by keeping the reconstituted substrate at 2 8 C protected from light and pouring off the amount that is going to be used into a clean vessel for pipetting Sample preparation The following sample preparation procedures have been used with the Advanced ALP Fluorophos fluorometric method 1 Whole milk skim milk low fat milk chocolate milk half and half and heavy cream Mix the sample thoroughly before testing Buttermilk sour cream and creamed cottage cheese Homogenize in a mechanical blender before testing Concentrated and dry milk products Reconstitute the product with laboratory grade water as necessary and test according to the procedure for the original product Butter To each labeled 16 x 100 mm screw cap glass test tube add a 0 5 gram butter sample taken from the center of the block Add 5 0 mL Advanced Instruments FLA005 Cheese Extraction Buffer Warm about 10 minutes in a 38 C water bath to allow the butter to dissolve in the buffer Remove the tube and gently mix by inversi
16. will be more stringent control over disposal of products and recycling of their components once they are withdrawn from use Since regulations governing the disposal of your instrument and acces sories may vary depending upon your geographic location the following guidelines are provided to assist you in identifying the options available to you once the decision has been made to replace or dispose of this product Contact the supplier who sold you the product Whether this was Advanced Instruments itself or one of its authorized dealers this supplier should be knowledgeable about the national and local regulations governing disposal and recycling of products in your area In some cases this supplier may be legally obligated to accept the product from you and arrange for proper disposal or recycling with no further involvement on your part Alternately the supplier can provide you with specific instructions for actions that you can take to dispose of the product properly 79 Fluorophos ALP Test System User s Guide Contact your local government agency responsible for waste col lection and disposal They can identify procedures and restric tions in effect to ensure proper disposal and available locations where products can be sent Contact Advanced Instruments Hot Line Service e 800 225 4034 toll free within the USA and Canada after normal business hours dial extension 2191 US 781 320 9000 elsewhere 781 3
17. with Part 15 of the FCC Rules Subpart B Class B Operation is subject to the following two conditions 1 this device may not cause harmful interference and 2 this device must accept any interference received in cluding interference that may cause undesired operation Canadian ICES 003 This Class B digital apparatus complies with Canadian ICES 003 Cet appareil num rique de la classe B est conforme la norme NMB 003 du Canada EC Declaration This product meets the intent of Directive 2002 95 EC of Conformity for Restriction of the Use of Certain Hazardous Sub RoHS stances in Electrical and Electronic Equipment as an exempt medical device per Article 2 Paragraph 1 and per Annex 1 Category 9 of Directive 2002 96 EC CB Report A CB report and certificate have been issued for this CB Certificate product The standard used was IEC 61010 1 2nd edi tion and 61010 2 010 2nd edition Fluorophos Test System Approvals as of June 2004 Covering Milk from cows sheep and goats Cheeses and Creams Regulatory Approvals 1 AOAC First Action 1991 2 AOAC International 1995 15th Ed Sec 979 13 AOAC International Final Action 1995 67 Fluorophos ALP Test System User s Guide oN A U International Dairy Federation IDF 1992 International Standards Organization ISO ISO DIS 11816 2 2001 Interstate Milk Shippers IMS 1993 FDA 8th Edition 1995 Cheese NCIMS 2001 Method ext
18. 0 to 999 9 FLU minute 1000 9999 FLU minute automatic 1 FLU automatic Less than 3 FLU per hour 12 x 75 mm round 15 minutes for cuvette chamber 30 minutes Fluorophos ALP Test System User s Guide Optics Lamp Filters Excitation Emission Storage Temperature Communications Printer Operating Temp ambient Room Humidity Test Time Dimensions Height Width Depth Dimensions Block Height Width Depth Weight Net Shipping Certification 64 90 optical bench with light source fixed filter detector quartz halogen 440 ALP nm narrow pass 405 ACP nm narrow pass 530 nm sharp cut off 32 to 158 F or 0 to 70 C RS 232C 9 pin DTE standard baud selectable 30 1200 or 9600 built in 40 column thermal 68 to 85 F or 20 to 29 C 5 80 non condensing 3 minutes inches centimeters 7 0 18 1235 32 16 5 42 inches centimeters 3 5 9 0 6 5 16 5 8 5 21 5 pounds 25 11 kilograms CE Intertek 98214 Appendix D Regulatory Notices e This product has been designed and manufactured in accordance with U S Canadian and European regulatory requirements as out lined below Modifications made to this product that are not expressly approved in writing by the manufacturer will void the user s authority to operate this product previously issued factory approvals and the user s rights under the warranty The distributor or deale
19. 16 5 x 21 5 x 9 0 cm Note that the heating block will perform optimally if placed in a draft free location e AC voltage selector Your FLM200 is equipped with a volt age selector located on the rear of the instrument within the fuse holder The setting should match the table below If not seek qualified assistance to make the required change A change may require fuse replacement see Chapter 4 Fuse replacement Measured VAC Position 100 110 100V 110 120 120V 200 235 230V 235 240 240V Fluorophos ALP Test System User s Guide AN e Electric outlet availability Your instrument and cuvette heat ing block will need to operate within five feet of a properly grounded three pronged electrical outlet capable of continu ously supplying 1 ampere at 200 240V to 1 25 amperes at 100 120V If the instrument and heating block are not ground ed properly their operation may be impaired and a safety haz ard may exist Therefore be sure to test the outlet and record the results before operating your instrument For proper instru ment operation you should also choose an outlet that is not subject to excessive electric interference or surges NOTE If your laboratory has electrical fluctuations we recom mend using a line conditioner with your instrument Step 2 Unpack the instrument To unpack the fluorometer take the following steps a Carefully unpack the fluorometer accessories and supplies and inspect them for shippi
20. 20 0811 fax Service personnel will provide you with contact information for local disposal or instructions for returning the product directly to Advanced Instruments 80 Appendix Service Log Model FLM200 Serial Number Software Revision Problem Symptom Action 81 Fluorophos ALP Test System User s Guide Date 82 E index A Diagnostic checks 6 46 Dis Enable Beeper 19 A D test 47 Dis Enable Xon Xoff 19 Air filter 39 Display panel 13 Alkaline phosphatase see ALP Display Print test 47 ALP 21 ALP pasteurization assay 21 E Calibration 27 ss os Calibration curves 30 Excitation dilter 6 High turbidity procedure 34 F Reagents reconstituting 30 Reagents storage 26 Fuse replacement 40 Sample preparation 32 Test 34 H Assistance 19 Heating block 5 13 C High turbidity test 33 Calculations l Butter and cheese 54 Fluid Dairy Products 51 Installation 1 Calibration 16 ALP Pasteurization 24 K Equations 51 Keypad 13 Frequency 16 Keypad test 47 Cuvette door 13 L D Lamp replacement 41 Daily Instrument Checks 31 Daily Instrument Control 37 M Date setting 8 Date Time 18 Maintenance 37 83 Fluorophos ALP Test System User s Guide O Operator settings Optical filters Replacement Care P Percent raw milk Photomultiplier tube voltage adjustment Pipette Maintenance Power panel Power requirements Print avg f min Printer Loading paper Paper jams Product specificatio
21. A D conversion chan nel or to monitor the A D channel for drift over time or temperature Serial Loop Test This menu item is no longer supported Display Print Test Display print test is a simple check of the display and printer Press START to run the test On entry a series of characters should scroll across the display and a single line of the same characters should print out on the printer The characters displayed have been chosen to illu minate every dot in the character matrix This makes it possible to dis tinguish any dots that no longer work on the printer as well as the dis play Press STOP to end the test Keypad Test The keypad test enables checking each switchpad on the keypad except STOP as well as the lamps of each illuminated switchpad Press START to begin the test then press each individual switchpad to be tested As each switchpad is pressed its name should be displayed and the lamp of each illuminated switchpad should light Press STOP to end the test Photomultiplier tube voltage adjustment The voltage supplied to the photomultiplier tube PMT can be adjusted to compensate for long term changes in its output 47 Fluorophos ALP Test System User s Guide For instruments with Serial Suffix D or later 1 48 Pipette 2 mL of Calibrator C into a cuvette and place the cuvette into the cuvette heater to warm at 38 C for 15 minutes Place Calibrator C into the cuvette holder close the door
22. D 344 Avg C STD 475 Calibration Ratio 142 2 NOTE The values above are examples and for instructional purposes only Do not use for the calculation of unknown values 2 Enzyme units one unit of ALP is that amount of enzyme which cat alyzes the transformation of one micromole of substrate per minute per liter of sample Because of the low levels of ALP in pasteurized fluid dairy products results are reported in milliunits per liter mU L 52 a Calculation equation Calculate the umoles FY min 0 075 mL sample using the correct ed fluorescence reading of the B calibrator Calibration Ratio used for automatic fluorometer calculations which contains 3 448 x 10 5 umoles of FY 2 mL Avg F min 0 075 mL sample 3 448 x 10 5 umoles FY min 0 075 mL Avg F of corrected B Calibrator Determine the pmoles of FY formed by one liter of sample Appendix A Additional Technical Information Avg F min 0 075 mL sample 3 448 x 10 5 13333 U L Avg F of corrected B Calibrator Convert U L into mU L Avg Avg F min 0 075 mL sample 3 448 x 10 5 13333 1000 mU L Avg F of corrected B Calibrator Simplified calculation Avg F of corrected B Calibrator Avg B STD Avg A STD 344 198 146 Avg F min 0 075 mL sample 459 7 mU L Avg F of corrected B Calibrator Using the example above 1 and the equations in 3 above the following results were calculated Fluorescen
23. Fluorophos ALP Test System Model FLM200 User s Guide Al ADVANCED INSTRUMENTS inc Two Technology Way 781 320 9000 Norwood Massachusetts 02062 USA 800 225 4034 Fax 781 320 8181 FLM5 Rev16 060110 www aicompanies com Fluorophos ALP Test System User s Guide Copyright This user s guide is copyrighted by Advanced Instruments Inc with all rights reserved Under copyright laws this guide may not be reproduced in any form in whole or part without the prior written consent of Advanced Instruments 2009 by Advanced Instruments Inc Norwood MA 02062 Advanced Instruments has reviewed this guide thoroughly All material contained within is believed reliable but the accuracy and completeness are not guaranteed or warranted and are not intended to be representa tions or warranties concerning the product described Windows is a registered trademark of Microsoft Corporation in the United States and other countries All other trademarks are the property of Advanced Instruments Hot Line Service If you have any questions regarding the proper operation of your instru ment or would like to order replacement parts please contact our Hot Line Service department by calling one of the following numbers e 800 225 4034 toll free within the USA and Canada after normal business hours dial extension 2191 e US 781 320 9000 elsewhere e 781 320 0811 fax ii Mi Table of Contents Safe Use Suppli
24. IN CLUDING USE OF IMPROPER GLASSWARE OR REAGENTS ACCIDENT OR ACT OF GOD Attention is called to the user s responsibility regarding reasonable use useful life of the instrument and immediate notification of accident outlined in the warranty duties sec tion of the user s guide and or service manual No person is authorized to assume for us any other lia bility in connection with the sale or service of this equip ment Advanced Instruments reserves the right at any time without notice to make changes in price color material specifications and models without incurring liability to modify products previously produced and to add or discon tinue models and accessories This warranty gives you specific legal rights and you may also have other rights which vary from state to state AI1YW REV O ADVANCED INSTRUMENTS ment the user and Advanced Instruments agree to the following responsibilities which constitute contractual warranties and conditions between the seller and the user for the maximum benefit and useful ness of the instrument WARRANTS THAT IT 1 Has produced equipment equal to or exceeding that of any competitive product in the same price range in standards of design material and workmanship 69 Fluorophos ALP Test System User s Guide Knows of no defects in design or materials which may cause bodily injury Will endeavor to advise the user of changes or improvements in the instrument
25. amage i Close the printer door and take up any paper slack by pulling the end of the paper If the printer paper becomes jammed under the print head a protective circuit will shut off the printer without deactivating the display or the rest of the instrument To reactivate the printer shut off the instrument power Then clear the paper jam and turn the power back on The instrument will complete the warm up cycle and the printer will report Fluorometer Ready if the paper jam has been successfully cleared Step 8 Set date and time At this time you should set the date and time functions on the instru ment To set these items use the instrument s setup mode as follows a When the fluorometer display scrolls CALIB Calibration Product TEST press the SETUP keypad to enter the setup mode The display will read 1 A D Test Installation amp Setup Use the lt or gt keypad to cycle through setup menu options until you see 2 Set Date Time At Set Date Time press START to display the current date and time Press CLEAR to clear the entire setting and display the present for mat mm dd yy or dd mm yy and use the lt keypad to toggle to the date format you require Use the gt keypad to toggle to 24hr format am or pm as required Enter the current date and time including zeros via the numeric keypad one digit at a time from left to right For example September 9 1999 would
26. an 50 4 The instrument cannot be calibrated because the fluorescence of Calibrator B is too close to the fluorescence of Calibrator C If the specified conditions exist follow the calibration procedure for high turbidity products 33 Fluorophos ALP Test System User s Guide Calibrating and testing high turbidity samples To calibrate and test high turbidity samples use the same technique described for calibration and testing of normal samples but use a 25 uL sample You can use the specially designed 25 uL pipette sup plied with your instrument to handle a sample of this size To account for the times three dilution for this procedure you will need to multiply your printed result by three NOTE If the cheese extraction buffer is also used the total dilution factor is 30 3 X 10 Test procedure Once the fluorometer is ready for use follow these instructions to run a test NOTE The test must be started within 20 seconds after addition of the product to the working substrate 1 Dispense 2 mL of the reconstituted Fluorophos ALP Substrate into each of enough fluorometer cuvettes for no more than one day s tests Preheat for at least 15 minutes in the heating block to bring the reagent to a temperature of 38 C Reconstituted Fluorophos ALP Substrate is stable for 6 hours in the heating block 2 Pipette 75 uL of the product to be tested into one of the preheated cuvettes containing reconstituted Fluorophos ALP Su
27. an unstable non level work sur face Do not operate this product with the covers removed or unsecured FCC Requirements WARNING Changes or modifications to this unit not expressly approved by Advanced Instruments could void the user s authority to operate the equipment This equipment has been tested and found to comply with the limits for a Class B digital device pursuant to Part 15 of the FCC Rules These limits are designed to provide reasonable protection against harmful interference in a residential installation This equipment generates uses and can radiate radio frequency energy and if not installed and used in accordance with the instructions may cause harmful interference to radio communications However there is no guarantee that interference will not occur in a particular installation If this equipment does cause harmful interference to radio or televi sion reception which can be determined by turning the equipment ix Fluorophos ALP Test System User s Guide off and on the user is encouraged to try to correct the interference by one or more of the following measures Reorient or relocate the receiving antenna Increase the separation between the equipment and receiver Connect the equipment into an outlet on a circuit different from that to which the receiver is connected Consult the dealer or an experienced radio TV technician for help Mi Supplies Parts amp Accessories To order
28. as the depreciation life in the Internal Revenue Service guide lines whether or not the user actual ly depreciates the instrument but not to exceed 10 years from date of delivery to the user b Only during the time the equipment has been maintained on a regular basis as prescribed by Advanced Instruments If the user is in an area which has no local service Advanced Instruments may require a local service person understood to mean the person actually per forming the hands on service of the equipment to attend and pass a reasonable maintenance and repair course Only during the time when the user has not altered the equipment in any way without written approval from Advanced Instruments Appendix E Warranty amp Warranty Duties d Only during the time when the user has not loaned leased or resold the equipment to any third party 2 Reasonable use is use a According to the instructions sup plied by Advanced Instruments assuming English reading person nel or supervision If neither the supervisor nor the operator reads English the user agrees to obtain accurate translations of the instru ment labels instructions user s guides and or manuals provided Under direct on the job supervi sion of the supervisor or other pro fessional in charge In which there are no known defects or uncorrected repairs Only for the purpose stated in the instructions provided with the instrument In wh
29. as they are developed so that the user may take steps to improve the safety and performance of his equipment throughout its useful life Will replace or repair equipment according to the guarantee on the attached warranty Will cooperate closely in common defense of any accident involving this equipment or third party suit against the user or operating personnel if advised immediately by the user of the occurrence of any accident THE USER WARRANTS THAT 1 70 The instrument will be used reason ably The instrument will be regularly maintained according to this manual including a log of all service tests and repairs performed on the equip ment and records will be kept of all requests for repair made to Advanced Instruments where such repairs were beyond the ability of local service personnel The instrument will not be altered without written approval from Advanced Instruments Advanced Instruments will be notified immediately if any injury occurs in any association with the instrument and will be allowed prompt and thorough examination of the instrument in ques tion 5 Advanced Instruments will be held harmless in cases of injury arising see definitions below a Beyond the useful life of the equipment b From unreasonable use c When Advanced Instruments is not immediately notified of said injury d From interpretation of results DEFINITIONS 1 Useful life is a The same
30. be entered 090999 Entering a two digit year will not affect instrument operation Your instrument will auto matically default to the correct four digit date and all printouts and readouts will use the correct four digit year Press ENTER to store the new setting or STOP to restore the origi nal setting If an invalid setting is attempted the instrument will reject the setting and beep when ENTER is pressed Fluorophos ALP Test System User s Guide Notes 10 FJ Instrument Operation This chapter contains information on operating the fluorometer A We have provided additional information for the Fluorophos ALP Milk Pasteurization Assay later in this user 5 guide You should read both this chapter and the test chapter before run ning any tests on the fluorometer Hazardous material cautions WARNING The specimens used in this product may be A hazardous to your health and to the health of others Follow national and local recognized safety standards and or proto cols for handling and disposing of such specimens e Ifa hazardous material is spilled on or inside the equipment the user is responsible for taking the appropriate decontami nation steps as outlined by the user s national safety council or equivalent agency for hazardous materials and those policies and procedures established within the user s place of business To avoid injury or fire hazard do not operate this product in an explosive atmospher
31. bol Definitions Record Review AN Caution Hot Surface e 4 Setup h Dangerous Voltage v Calibration Lifting Hazard Z Cancel Delete Calibrator 75 Fluorophos ALP Test System User s Guide CONTROL Control Handle With Care Negative Control Positive Control Flammable U Fragile For In Vitro Diagnostic Use Keep Dry Temperature Limit Z Date Manufactured See Instructions STERILE Sterile LOT Lot Number x Dat 7 i Date Serial Number nin a Representative SOLN Solution Part Number 76 Appendix G Symbol Definitions Tests Open Here Diluent See Instructions for kuz Low Fluid Level Temperature Guidelines Potential Puncture O Keep Hands Clear Hazard Man faetir r Electronic Equipment Dispose of Properly 77 Fluorophos ALP Test System User s Guide Notes 78 Appendix H Product Disposal and Recycling International concern about environmental pollution resulting from improper disposal of products and materials at the end of their useful life has resulted in an increase in legislation to control the methods and procedures used to handle waste electrical and electronic equipment While the regulatory status in some regions of the world has progressed to the point where formal legislation is already in effect many other regions are in the process of creating similar legislation or adopting that already in existence in other areas The result in the years ahead
32. bstrate Do not allow the sample to sit in the heating block once the product to be tested has been added to the reagent 3 Mix well with the vortex mixer 4 Place the cuvette in the fluorometer cuvette holder and close the cuvette door 5 At CALIB Calibration Product TEST press TEST 34 10 11 12 13 14 ALP Milk Pasteurization Assay If necessary use the lt or gt switchpad to select ALP Dairy and press ENTER As necessary use the lt or gt switchpad to change to an appropriate channel for the product to be tested and press ENTER again Using the fluorometer keypad type an identification number for the sample being tested and press ENTER Make sure the cuvette door is shut and press START to begin the test The display will count down 60 seconds while the substrate and sample are being stabilized at 38 C After 60 seconds the fluorometer will begin to measure and display the fluorescence of the sample in fluorescence units FLU At the end of three minutes the fluorometer will display the aver age increase in fluorescence and the ALP activity in mU L Remove the cuvette from the cuvette holder If desired test another sample in the same manner When finished with ALP testing press STOP a second time to return to the CALIB Calibration Product TEST display Interpretation of results Any test that yields a test result under 350 is considered ALP NEGA TIVE No further action
33. ce of corrected B Calibrator 146 Fluorescence Units Avg F for Test Sample 114 5 Fluorescence Units min 114 5 459 7 360 5 mU L 146 53 Fluorophos ALP Test System User s Guide Calculation for butter and cheese The extraction of 0 5 g of butter or cheese into 5 0 mL of extraction buffer results in a ten fold dilution and requires the mU L printout on the fluorometer tape to be multiplied by 10 mU L 10 mU kg Percent raw milk and ALP enzyme activity U S Federal and IDF Standards require that cows milk intended for human consumption contain less than 1 0 ug phenol mL 15 min of ALP activity This value of 1 0 ug phenol mL 15 min is equivalent to 500 mU L of ALP enzyme activity using the Fluorophos test method It is important to remember that the cut off values established by all regulatory agencies are based on residual levels of ALP activity not on weighed in val ues for the enzyme or on a percent of raw milk remaining in the finished prod uct Experience over the years has shown however that a 0 1 v v dilution of mixed herd raw milk will give approximately 1 to 2 ug phenol mL 15 min or 500 mU L of ALP activity The reason that the values obtained with a 0 1 v v raw milk dilution are approximate is that the original enzyme activity of the mixed herd raw milk sample will not always contain the same amount of enzyme sample to sample The total amount of enzyme in the mixed herd sample will vary from sea
34. check the air vents for blockage e Wipe the exterior of the product with a soft damp cloth as needed Using cleaning products other than those specified may discolor or damage the finish e Ifthe product requires service for any of the following rea sons unplug the product from the electrical outlet and refer service to a qualified service technician The power cord extension cord power strip or power input module is damaged 43 Fluorophos ALP Test System User s Guide Liquid has been spilled into the interior of the product A foreign object has fallen into the product The product has been dropped or damaged by a falling object There are noticeable signs of overheating or a burning odor The product does not operate normally when you follow the oper ating procedures The main supply fuse s or any internal fuse s continually fail A discharge of static electricity from contact with the human body or other conductor may damage system boards or static sensi r A tive devices Never perform internal maintenance without following recommended static protection procedures The product is equipped with operator accessible fuses Ifa fuse blows it may be due to a power surge or failure of a component Replace the fuse only once If the fuse blows a second time it is probably caused by failure of a component part If this occurs refer service to qualified service personnel Always replace the fuse
35. cubation followed by spectrophotometric reading of the dialysate The IDF has approved a p nitrophenyl phosphate assay in which the p nitrophenol is read after protein precipitation following a two hour incu bation These quantitative methods have served the dairy industry well and have aided in monitoring milk quality throughout the world for over fifty years In late 1988 Advanced Instruments Inc presented preliminary data on a new approach to ALP testing in dairy products The method is based on a specially designed substrate called Fluorophos which when acted upon by ALP is converted to a highly fluorescent product Fluoroyellow The quantitative Fluorophos assay is the first milk ALP test which per mits the continuous and direct measurement of the released reaction product from a fluorogenic substrate The use of this fluorometric sub strate eliminates the interferences and nonspecificity encountered in col orimetric assays and avoids the need for dialysis butanol extraction or protein precipitation which are tedious and time consuming 8 Official Methods of Analysis 16th Edition 1996 Association of Official Analytical Chemists Arlington VA 9 Detection of Phosphatase Activity IDF Standard 63 1971 International Dairy Federation Brussels Belgium 10 Detection of Phosphatase Activity IDF Standard 82A 1987 International Dairy Federation Brussels Belgium 11 Rocco R M and Bargoot F G An
36. d in item 1 a Perform ALP Test POSITIVE NEGATIVE Residual and or Reactivated ALP No further action Perform Differentiating Procedure for Residual vs Reactivated ALP above EEE Test lt Blank Test gt Blank POSITIVE RESIDUAL ALP NEGATIVE RESIDUAL ALP Figure 8 Residual ALP Test Flow Diagram REF Standard Methods for the Examination of Dairy Products 17th Ed Ed H Wehr J Frank 2004 p349 350 American Public Health Association Washington D C 58 Appendix B Troubleshooting Table Error Cuvette Door Open Test procedure is cancelled Close the cuvette door and restart the test Low Cuvette Temperature High Cuvette Temperature The temperature of the fluorometer cuvette chamber and or the contents of the cuvette in the cuvette chamber is not at 38 0 2 C Wait for the cuvette chamber temperature to stabilize and the fluorometer display to read Fluorometer Ready Repeat the test with reagents pre warmed as recommended in the instructions in the test kit Calibration Required The product channel chosen has not yet been calibrated Perform calibration Unacceptable Calib Ratio The difference in fluorescence reading between the calibrators is greater than allowed Repeat calibration with new cali brators Standards Reversed Please Repeat The calibrators were tested in reverse order Prepare new calibrators and repeat
37. e Function of major components Operation of the instrument will be quicker and easier if you become familiar with the locations and functions of the compo nents systems and controls described below before proceeding further 11 Fluorophos ALP Test System User s Guide Cuvette Door Printer Cuvette Display Panel Cuvette Testing Keypad Chamber Figure 3 Fluorometer Components and Controls Power panel Contains the power plug fuses and rocker type power switch The upper half of the switch is the power on position The fluorometer is intended to be left on with the display reading 2 A D Test during normal operating hours Serial port The Advanced Fluorometer transmits a significant amount of informa tion via the serial port Almost every item of information displayed by the instrument is also transmitted over the serial port including test results all error messages and most display data from the diagnostic menu Printer The printer is located beneath the slotted door in the top right side of the fluorometer Lift the front of the printer door and check that ade quate thermal paper is properly loaded 12 Instrument Operation Display panel The microprocessor communicates with the instrument operator by means of alphanumeric messages printed on paper tape and displayed on the illuminated display panel located at the top of the keypad Keypad The keypad contains spill proof pressure pads for operat
38. either of these switchpads presents the name of the channel on which the last test or calibration was accomplished At this point either press ENTER to select that test or use the arrow switchpads to step through the menu of available channels and press ENTER to select the required test or calibration Calibration Periodic calibration of the fluorometer is required to maintain instrument accuracy In general each product type to be tested requires its own calibration curve The calibration parameters for each calibrated product may be stored in a separate channel of the instrument memory and are used by the fluorometer for each test in that channel until changed by performing a new calibration in the same channel It is important to calibrate the instrument under the following condi tions When the fluorometer is initially installed For each different product type that is analyzed e Whenever significant service procedures are performed e g lamp or filter change When adjustments are made to bring A D Mode into specification 16 Instrument Operation The fluorometer will not allow tests to be performed using an un cali brated channel Calibration Required will be displayed when any new channel is defined However after calibration if the calibration ratios of two product types are found to be within 5 of each other they may both be run on the same channel Briefly calibration ratios are determined by the
39. el Calibration should be completed within 10 minutes from addition of product to calibrator If the calibration is not accepted by the fluorometer pre pare new calibrators with the selected product and repeat the entire calibration procedure The calibration procedure can be interrupted at any point during the calibration process by pressing STOP If no test is in progress when STOP is pressed the entire calibration will be cancelled To continue calibration from that point you must re enter the calibration mode by pressing CALIB and starting the calibration procedure from the beginning However if a test is in progress when STOP is pressed only the test in progress will be cancelled The cancelled test may be re run by inserting another standard of the same value and pressing START or the entire calibration sequence may be cancelled by pressing STOP again The fluorometer will discard the new calibration data and revert to the previous calibration status 29 Fluorophos ALP Test System User s Guide How to determine the need for different calibra tion curves calibration ratio and product type As a general rule when the calibration ratios for different products are within 5 of each other those products may be run on the same chan nel For example if a 2 Low Fat milk has a calibration ratio of 127 2 and a 3 2 Whole Milk has a calibration ratio of 133 3 both products may be run on the same channel The differences
40. ension granted for cream products Official Journal of the EU named Reference Method ISO 11816 1 6 May 2007 alternative methods must be validated against the Fluorometric method Official Journal of the French Republic 2004 Independent Laboratory Reviews 1 NS ON DH KM9 WN NIZO Holland Bundesgesundheit Adm Germany Weihenstephan Germany SMR Sweden Milk Marketing Board England and Wales Food Standards Agency UK Hannah Research Institute Scotland University of Milan Italy Instituto Nacional al Engenharia e Technologia Lisbon Portugal 10 Universidad Autonoma de Bellaterra Barcelona Spain 11 Nestle Switzerland 12 Black Gilbert Chandler Institute Australia 13 University of Tennessee U S A 14 University of Wisconsin U S A 68 Appendix E Warranty amp Warranty Duties LIMITED WARRANTY CONDITIONS Advanced Instruments warrants that for 12 months from date of shipment each new product sold or manufactured is free from defects in material and workmanship under normal installation use and service except glass plastic and parts warranted by their makers Advanced Instruments agrees that such defects will be remedied or new parts furnished in exchange for any defective part delivered by the owner transportation charges prepaid to the factory for our examination If in our judgment the part contains any such defect it will be replaced charging
41. es Parts amp Accessories Reagents Calibrators amp Controls Foreword Principles of Fluorescence Chapter 1 Installation amp Setup Step 1 Find a location for the instrument Step 2 Unpack the instrument Step 3 Obtain additional items Step 4 Set up heating block Step 5 Check excitation filter Step 6 Power up and run diagnostic test Step 7 Load printer paper Step 8 Set date and time Chapter 2 Instrument Operation Hazardous material cautions Function of major components Function and components of the heating block Using the 25 uL or 75 pL pipette Using the 25 mL pipette Instrument start up Calibration Preparing reagents Test procedure Changing operating settings vii xi xiii gt lt 9 J amp Q Q N L Se eee eee eee NNN OS Qa Qi JS QARA iii Fluorophos ALP Test System User s Guide Chapter 3 Fluorophos ALP Milk Pasteurization Assay Overview Alkaline Phosphatase ALP background information The Fluorophos ALP reaction Materials required Reagent storage stability Reagent handling Calibration How to determine the need for different calibration curves Reconstituting reagents Daily instrument checks Sample preparation Determining the need for the high turbidity test procedure Calibrating and testing high turbidity samples Test procedure Interpretation of results Chapter 4 Instrument Maintenance Daily controls Cleanup of spill
42. eter In this procedure an aliquot of dairy product is added to 2 0 mL of sub strate The rate of product formation is monitored for three minutes in a compact fluorometer thermostated at 38 C Enzyme activity is calculated 12 Rocco R M Fluorometric Determination of Alkaline Phosphatase in Fluid Dairy Products Collaborative Study J Assoc Off Anal Chem 73 Nov Dec 1990 13 Milk and Milk Based Drinks Determination of Alkaline Phosphatase Activity Fluorometric Method IDF Standard 155 1992 International Dairy Federation Brussels Belgium 14 ISO DIS 11816 1 Milk and Milk Based Drinks Determination of Alkaline Phosphatase Activity Fluorimetric Method 15 Eckner K F Fluorometric Analysis of Alkaline Phosphatase Inactivation Correlated to Salmonella and Listeria Inactivation J Food Protection 55 960 963 1992 16 Lechner E and Regensburger V Alkaline Phosphatase Activity in Milk and Milk Products Translated from Deutsche Milchwirtschaft 44 17 815 819 1993 17 ISO 11816 2 IDF 155 2 Milk and Milk Products Determination of Alkaline Phosphatase Activity Part 2 Fluorometric Method for Cheese 24 ALP Milk Pasteurization Assay O lI O O P OH ALP O ORo OH pH 10 Fluorophos Fluoroyellow NON FLUORESCENT FLUORESCENT EX 439 NM EM 560 NM Figure 5 The Fluorophos Reaction in mU L and displayed automatically at the end of the reading period Reaction rates are linear for up to thirty mi
43. etting is indicated by the symbol Press ENTER to store the new setting or STOP to restore the original setting Sub Blank Testing This option is designed for factory use and may not be used or reset in the field 18 Instrument Operation Dis Enable Beeper At lt Dis Enable press lt or gt to disable or enable the keypad beeper The current setting is indicated by the symbol Neither selection will affect the invalid selection beeper but when the keypad beeper is enabled an invalid selection will invoke two beeps when dis abled only one Press ENTER to store the new setting or STOP to restore the original setting Dis Enable Xon Xoff At lt Dis Enable press lt or gt to enable or disable serial port Xon Xoff handshaking The current setting is indicated by the symbol Press ENTER to store the new setting or STOP to restore the origi nal setting Set Serial Rate At Serial Rate xxxx the serial port baud rate may be reset by means of the lt or gt switchpad to either 300 1200 or 9600 baud Press ENTER to store the new setting or STOP to restore the original setting Zero Range Adjust The fluorometer zero ranges are individually set at the factory and should not be reset Serial Number Fluorometer serial numbers are set at the factory for instrument identifi cation and may not be reset in the field Product Test This option is designed for factory use and may not be used or re
44. f the power cord provided is replaced for any reason or if an alter nate cord is used the cord must be approved for use in the local country The power cord must be approved for the product s listed operating voltage and be rated at least 20 greater than the ampere ratings marked on the product s electrical ratings label The cord end that connects to the product must have an IEC 60320 connector Plug the product into an approved grounded electrical outlet Do not disable the power cord s grounding pin If an extension cord or power strip is used make sure that the cord or strip is rated for the product and that the total ampere ratings of all products plugged into the extension cord or strip do not exceed 80 of the cord s or strip s rating limit viii Safe Use Route power cords so that they will not be walked on tripped on or pinched by items placed upon or against them Pay particular atten tion to the plug electrical outlet and the point where the cord exits the product Do not pull on cords and cables When unplugging cords or cables grasp the corresponding connector Do not install or use this product in any area subject to extreme short term temperature variations or locations that exceed the speci fied operating environment temperatures Never use this product in a wet area To avoid injury or fire hazard do not operate this product in an explosive atmosphere Do not install or use the product on
45. g water bath for 30 minutes after the test product temperature reaches 95 C Cool rapidly b Place 5 0 mL of the unheated test product in each of two 16 x 100 mm screw cap test tubes c Add 0 1 mL of DI water to one of the test tubes to constitute a Blank sample d Add 0 1 mL of the Magnesium Acetate reagent prepared in step 2 above to the second test tube to constitute a Test sample e Cap both test tubes mix well and incubate for 1 hour at 34 C Remove the test tubes from the heated bath and cool in an ice bath for 5 minutes f Run an ALP activity test on the Blank sample g Add 1 0 mL of the Test sample to 5 0 mL of the heated and cooled test product from step 3 a above 57 Fluorophos ALP Test System User s Guide h Run an ALP activity test on the Test sample diluted product from step 3 g above 4 Interpretation of Test Results a If the ALP activity of the Test sample diluted product x 6 is equal to or greater than the ALP activity of the Blank sample the original product is considered negative for residual ALP activ ity as defined in item 1 a above indicating that the assayed activity was reactivated ALP in origin as defined in item 1 b b If the ALP activity of the Test sample diluted product x 6 is less than the ALP activity of the Blank sample the original product is considered positive for residual ALP activity as define
46. he Substrate Buffer into the Fluorophos Substrate bottle Mix by gentle inversion and allow to sit at room temperature for at least 30 minutes or until fully dissolved Daily instrument checks 1 Dispense 2 0 mL of the Daily Instrument Control into a clean labeled cuvette Place the cuvette in the 38 1 C heating block and allow to warm for fifteen 15 minutes On the fluorometer press SETUP Select menu item A D TEST by pressing lt or gt With nothing in the cuvette chamber press START The display should read 302 4 e If the value is outside of the specified range clean the excitation and emission filters and repeat the A D Test Insert the pre warmed Daily Instrument Control cuvette into the cuvette chamber and close the door Once the display has stabilized it should read 602 15 Record this value Ifthe value is outside of the specified range the value can be cor rected to 602 by slowly adjusting the potentiometer screw R15 resistor clockwise or counterclockwise as necessary Ifthe instrument is adjusted to 602 or has experienced excessive drift it should be recalibrated with the Calibrator Set P N FLA250 Dispense 2 0 mL of the reconstituted substrate into a clean labeled cuvette 31 Fluorophos ALP Test System User s Guide 10 Place the cuvette in the 38 1 C heating block and allow to warm for fifteen 15 minutes After 15 minutes insert the
47. he service technician may assist you in mak ing minor repairs over the phone providing you with recommended parts or part numbers or may have you ship the instrument for factory repair If you need to return an instrument for repair or replacement Notify our service department to obtain an RMA Be sure to contact Advanced Instruments before shipping to avoid any delays Carefully pack and send the instrument using its own specially designed carton and packaging material If you did not save the car ton and packing materials please request one from Advanced Instruments or an authorized representative Be sure to prepay for any shipment to the factory Advanced Instruments cannot accept collect shipments without prior approval Please insure the shipment or accept the damage risk 45 Fluorophos ALP Test System User s Guide Do not tip store or ship the fluorometer without first removing any liquid containing cuvette from the cuvette chamber Please use cuvette chamber cleaners to remove any liquid that may be in the cuvette chamber Troubleshooting checks Check operational requirements If you are experiencing difficulties with the instrument first carefully review the operational requirements listed in the product specifications and the recommended setup and operating procedures Check fuses You will find the power switch and fuse holder beside the power cord connector on the back panel of the instrument
48. heating block ther mometer well d Allow the heating block to warm up for at least 30 minutes to set temperature and stabilize The ready light on the front panel will illuminate e Use the heating block thermometer to check the temperature of the heating block f Ifthe temperature is not 38 1 C use the provided temperature adjustment tool and the setscrew accessible through the front panel to adjust the temperature To do this you will need to use very small increments and allow the temperature to stabilize after each adjust ment g When stable at 38 1 C the heating block is properly set up and ready to use Turn off the heating block when not in use Fluorophos ALP Test System User s Guide Step 5 Check excitation filter Your instrument is shipped with a dual excitation filter assembly which houses two filters for ALP and ACP testing respectively Determine which test s you will be running and check your filter to ensure that the correct filter is in place If testing dairy products make sure that the ALP filter is in place Facing the front of the instrument the excitation filter assembly is locat ed behind the cuvette holder and is accessible through the cuvette door see Figure 3 The two ends of the dual filter assembly are marked ACP and ALP respectively When the filter is installed the visible end of the filter will indicate which filter is currently in place If the current filter is not the o
49. hos AOAC 979 13 IDF 82 1987 mU L ug phenol mL 15 min ug PNP mL 2 hrs 0 03 0 06 0 12 0 25 0 50 lt 1 00 2 00 4 00 1000 00 0 312 0 625 1 25 2 50 5 00 10 00 20 00 40 00 10 000 00 always true in that the total ALP acti vity in mixed herd raw milk before pasteuriza tion will vary due to species and seasonal variations 4 Cutoff for acceptable milk is u1 mg phe nol mL 15 min Pasteurized Milk Ordinance PMO Grade A 1985 Revision U S Department of Health and Human Services FDA Washington D C 5 Cutoff according to Codex Alimentarius Complete mission World Health Organization July 1989 Joint FAO WHO Standards Programme Differentiating microbial from bovine milk ALP 1 Principle Alkaline Phosphatase ALP EC 3 1 3 1 synthesized by microorganisms is heat stable at 145 F or 63 C whereas bovine milk ALP is inactivated at this temperature Procedure a Pipette 1 0 mL of the milk being tested into the bottom of a labeled 16 x 150 mm glass screw cap culture tube without allow ing any of the milk to touch or adhere to the sides of the tube b Place the screw cap on the tube but do not seal completely to allow for gas expansion Place the tube in a 63 0 5 C water 55 Fluorophos ALP Test System User s Guide bath for 30 minutes or 66 C if the fat content is over 10 During this time gently mix the tube once every 10 minutes c Remove the tube from the water bath and a
50. ich the equipment has been maintained according to the instructions provided 3 Immediate notification is a Recognition that time is of the essence when any accident mal practice or product liability arises which involves Advanced Instruments equipment Notification to Advanced Instruments immediately the same day if possible in the event of injury to any person in circum stances involving Advanced Instruments equipment in which Advanced Instruments might be named as a defendant in any form of litigation Allowing Advanced Instruments or its representatives immediate full and thorough examination of Advanced Instruments equipment and all records pertaining to such equipment 71 Fluorophos ALP Test System User s Guide Notes 72 Appendix F Supplemental RS 232 Information The DB 9 RS 232 port on your instrument conforms to the DTE EIA 232 standard and can reliably communicate over shielded cable up to 10 meters in length depending on the baud rate you use Almost every item of information displayed by your instrument is also transmitted over the RS 232 port including test results all error messages and most display data from the diagnostic menu Data is transmitted asynchronously as start bit 8 data bits and 1 stop bit with no parity Each message transmitted from the communication port is terminated by the sequence Carriage Return OD Hex Line Feed 0A Hex Note that your
51. instrument is only capable of outputting information At this time there is no protocol for talking to the instru ment Sample RS 232 Setup As a typical example of a communications program setup the following instructions will describe the process necessary for using your instru ment in conjunction with Hyperterminal for Windows 95 or later oper ating systems and a null modem cable available from Advanced Instruments as shown in the diagram below You can use this procedure to be sure that your instrument and cable are operating correctly 1 Create a Hyperterminal connection using the Hyperterminal pro gram 2 While working within that connection choose the Properties selec tion from the File menu 73 Fluorophos ALP Test System User s Guide 3 Select the Configure button 4 Choose the following settings for the Configure menu and click on the OK button Bits per second See User s Guide Data bits 8 Parity None Stop bits 1 Flow control None 5 Select the Settings menu 6 Chose TTY for the Emulation option 7 Select ASCII setup 8 Set the Sending option to Echo Typed Characters Locally 9 Set the Receiving option to Wrap Lines that Exceed Terminal Width You should at this point see all instrument output in the Hyperterminal window of your computer screen DB9 FEMALE DB9 FEMALE Null Modem Cable Shield connects to metalized connector hous ing at both ends 74 Appendix G Sym
52. is required because a negative result indicates proper and complete pasteurization Any sample that yields a test result over 350 mU L is considered POSI TIVE for bovine reactivated and or microbial ALP If a sample tests positive you must perform confirmation tests to determine whether the sample is positive for bovine reactivated and or microbial ALP NOTE Refer to Appendix A for confirmatory tests Differentiating bovine residual ALP from microbial and or reactivated ALP 35 Fluorophos ALP Test System User s Guide Notes 36 EJ Instrument Maintenance Daily controls Use the Advanced Instruments Daily Instrument Control to mon itor the day to day drift of the fluorometer and to help deter mine when the instrument should be re calibrated If values fall outside of the ranges indicated then a fluorometer drift problem can be quickly identified and corrected To order Daily Instrument Control contact Advanced Instruments or an authorized representative Cleanup of spills If a liquid is spilled into the fluorometer or heating block the power cord should be unplugged immediately to avoid electrical accidents Small spills may generally be cleaned up and wiped off with the aid of a mild non abrasive detergent Do not immerse either the fluorometer or the electrical parts of the heating block in water under any circumstance Cleanup of heating block spills If a liquid is spilled or glass is broken such that
53. ituted store at 2 to 8 C 36 to 46 F for up to 60 days In the Heating Block store at 2 to 8 C 36 to 46 F for up to 6 hours Fluorophos ALP Calibrator Set Unopened store at 2 to 8 C 36 to 46 F for up to 18 months Opened store at 2 to 8 C 36 to 46 F for up to 60 days In the Heating Block store at 2 to 8 C 36 to 46 F for up to 6 hours e PhosphaCheck ALP Pasteurization Control Set Unopened store at 2 to 8 C 36 to 46 F for up to 18 months Opened store at 2 to 8 C 36 to 46 F for up to 72 hours Fluorophos ALP Daily Instrument Control Unopened store at 2 to 8 C 36 to 46 F for up to 18 months Opened store at 2 to 8 C 36 to 46 F for up to 60 days In the Heating Block store at 2 to 8 C 36 to 46 F for up to 6 hours 26 ALP Milk Pasteurization Assay Fluorophos ALP Cheese Extraction Buffer Unopened Opened store at 2 to 8 C 36 to 46 F for up to 3 years Reagent handling The reagents used in the ALP Pasteurization Assay are irritating to skin harmful if swallowed and pose danger of serious damage to health by prolonged exposure if swallowed Refer to the individual Material Safety Data Sheets for complete hazard information NOTE Do not freeze any of the above reagents Make sure that all bottles and vials are sealed tightly after use to prevent evapora A tion Store all bottles and via
54. llow the plunger to return to the release position This must be done slowly Remove the tip from the sample solution To dispense the collected reagent hold the point of the tip against the wall of the cuvette and depress the plunger slowly to the stop position After the contents of the tip have been dispensed move the tip away from the wall of the cuvette before allowing the pipette plunger to come to its release position Dispose of the used pipette tip Never reuse the same pipette tip especially between different reagents Instrument start up To start up the test system take the following steps a Power up the fluorometer and heating block Both are designed to operate at 38 C and must be turned on and allowed to warm to oper ating temperature before use Allow the heating block to warm for 30 minutes 15 Fluorophos ALP Test System User s Guide b Instrument will run start up diagnostics and count down 15 minutes while the cuvette chamber in the instrument warms to 38 C If the instrument cannot meet the required operating conditions for any reason an error message will be displayed c When the fluorometer and heating block warm up requirements have been met and the instrument is ready for operation the display will begin to scroll CALIB Calibration Product TEST at which time the operator may press CALIB to calibrate a fluorometer chan nel or TEST to begin testing a product Pressing
55. llow to cool for at least 5 minutes in a cold water bath d Re test for ALP activity The flow diagram in figure 7 will assist in interpretation of the results Perform ALP Test POSITIVE NEGATIVE milk and or microbial ALP No further action Perform Differentiating Procedure for Microbial ALP above POSITIVE NEGATIVE Microbial ALP milk properly Bovine ALP milk not pasteurized positive due to properly pasteurized microbial ALP Figure 7 Microbial ALP Test Flow Diagram REF Standard Methods for the Examination of Dairy Products 17th Ed Ed H Wehr J Frank 2004 p349 American Public Health Association Washington D C 56 Appendix A Additional Technical Information Differentiating residual from reactivated alka line phosphatase ALP in fluid milk products 1 Definitions a Residual ALP ALP enzyme remaining in the product due to raw milk contamination and or incomplete pasteurization b Reactivated ALP ALP enzyme that was inactivated due to pas teurization usually UHT treatment which recovers its activity due to storage conditions 2 Reagent Magnesium Acetate 40 1 mg of Mg mL Dissolve 35 4 g of Mg CyH307 9 4H20 in about 50 mL of DI water Warm to dissolve completely then bring to 100 mL with additional DI water This solution is stable for 6 months at 2 8 C 3 Procedure a Place 10 mL of the test product in a suitable glass container and heat in a boilin
56. ls upright to prevent spills and leakage Calibration The FLA250 Fluorophos ALP Calibrator Set consists of three calibra tors labeled A B and C The calibrators are ready to use and designed to be used directly from the bottle Calibrator set preparation 1 Mix gently by inversion prior to each use to assure optimal results 2 Label two 2 cuvettes for each of the three 3 calibrators 3 Dispense 2 0 mL of the appropriate calibrator into each clean labeled cuvette 4 Place the cuvette in the 38 1 C heating block and allow to warm for fifteen 15 minutes Calibrating with a product sample Samples should be prepared as they are for regular testing 1 Using a 75 uL pipette add a 75 uL sample of the prepared product to each of the pre warmed calibrator cuvettes 27 Fluorophos ALP Test System User s Guide 2 Cover with parafilm and mix gently by inversion or vortex mixer 3 Return each cuvette to the heating block Calibration must be com pleted within ten 10 minutes after the addition of the sample 4 Press CALIB 5 Select ALP Dairy by pressing lt or gt then press ENTER 6 Select the product type to be calibrated by pressing lt or gt or by using the numbered keys When the desired product is displayed select it by pressing ENTER e If the channel has been previously calibrated a printout of that calibration will be provided PROD 1 Whole Milk PROD 12 Heavy Cream PROD 2 Low
57. meter power switch and unplug the power cord from the power outlet 2 Carefully remove any cuvette from the cuvette holder If there is any evidence of broken glass or spills in the cuvette holder follow the cleanup procedure in this chapter 3 Facing the front of the instrument the excitation filter holder is located behind the cuvette holder Slide the excitation filter holder straight up and out of the instrument If the filter holder is too slip pery to grip use a piece of masking tape to improve your grip and provide a pull tab 4 Slide in the new filter holder Note that the ends of the dual filter assembly are marked ACP and ALP respectively When the filter is in place the label on the end of the filter will indicate which filter is currently in place If the current filter is not ALP simply remove the 39 Fluorophos ALP Test System User s Guide filter holder flip it around and replace it in such a way that the ALP filter name is showing Optical filter care Use extreme caution whenever you handle a filter or filter assembly Handle optical filters only by the edges and never on the optical sur faces The filters scratch very easily To clean the filter use a piece of lint free non abrasive tissue and a small amount of 70 isopropanol solution Be sure the filter is com pletely dry before re installing Excitation and emission filters should be replaced annually Pipette maintenance The tips supplied
58. minute 2 is not within 20 of that during minute 3 Among the reasons for this are 1 Substrate not at 38 C 2 Not a uniform well mixed sample 3 Very low result 4 Very high result Repeat the test 60 Troubleshooting Table Problem Message Explanation Readout is unstable The optical lamp and or excitation filter may require replacement Contact Advanced Instruments for Hot Line Service Calibrator C output drifts over time or instrument requires frequent readjustment The optical lamp and or excitation filter may require replacement Contact Advanced Instruments for Hot Line Service 61 Fluorophos ALP Test System User s Guide Notes 62 Appendix C Product Specifications Electrical Voltage Frequency Current 100 120 VAC 200 240 VAC Fuses Power Fluorometer Heating block Memory backup Sample Volume Sample Capacity Readout Units Results Units Auto Ranges Low High Zeroing Resolution Gain Drift Cuvette Size Warm up Time Fluorometer Heating Block 100 to 120 VAC or 200 to 240 VAC 50 or 60 Hz 1 2 Amp for fluorometer 1 4 Amp for heating block 0 7 Amp for fluorometer 1 8 Amp for heating block as listed on power label 150 Watts 30 Watts Integral lithium cell five years minimum in absence of power See test kit instructions Single sample 20 character dot matrix FLU fluorescence units See test kit instructions
59. nd the Acid Phosphotase ACP assay The Fluorophos ALP Assay is a rapid test for verifying proper pasteurization of dairy products by measuring the Alkaline Phosphotase ALP activity This User s Guide provides information specific to performing the Fluorophos ALP assay The ThermaZyme ACP assay and Fluorophos ACP Juice Pasteurization assay utilize materials reagents substrate buffers and Fluorometer setup that are different from the Fluorophos ALP assay Information specific to these tests is not included in this User s Guide To obtain more information about ThermaZyme ACP assay or Fluorophos ACP Juice Pasteurization assay please contact Advanced Instruments Inc xvi Ed Installation amp Setup AN A In order to set up your instrument properly it is important that you read and follow the steps in this chapter Please follow these steps carefully and be sure to read Chapter 2 Instrument Operation before attempting to run tests on your instrument Step 1 Find a location for the instrument When choosing a location for the fluorometer be sure to meet the following criteria Adequate space The dimensions of the instrument are 16 5 x 12 5 x 7 inches 42 x 32 x 18 cm Be sure to keep your workplace free of debris especially near the left side of the instrument where proper ventilation is needed Your instrument s cuvette heating block also requires a small amount of space 6 5 x 8 5 x 3 5 inches
60. ne you need to use simply remove the filter holder flip it around and replace it in such a way that the correct filter name is showing NOTE Use extreme caution whenever you handle a filter or filter assembly The filters scratch easily To clean your filter use a piece of lint free non abrasive tissue and a small amount of 70 isopropanol solution Be sure the filter is com pletely dry before re installing This excitation filter assembly FL2333 should be replaced annually to maintain optimal performance Step 6 Power up and run diagnostic test a Plug the fluorometer power cord into a properly grounded outlet b Switch the fluorometer power switch into the ON position The dis play will report software and hardware identification and assistance information as well as the date and time c The display will then begin a 15 minute countdown during which the cuvette chamber will stabilize at the required operating tempera ture Installation amp Setup Figure 2 Loading Printer Paper When the countdown ends the instrument will run some brief diag nostic tests to ensure the instrument is functioning properly After the diagnostic tests are successfully completed the fluorometer will display CALIB Calibration Product TEST and will be ready for use Step 7 Load printer paper A roll of printer paper and a paper roll holder are supplied with the instrument These must be installed in the pri
61. ng damage Use the enclosed packing list to verify that all items have been received Save the instrument s shipping boxes and packaging material in case future transport of the instrument becomes necessary If any item on the packing list appears to be missing from your ship ment please search carefully through and under all packing materi als If the item is not found notify your receiving department imme diately Advanced Instruments can only be responsible for items reported missing within 10 days of a shipment s arrival If you receive any damaged items save the cartons and packing material those items came in for inspection by the insurer The carri er dealer and Advanced Instruments must be notified within 24 hours in order for your warranty and insurance to apply Have the transportation company inspect items fill out a Report of Concealed Damage and file your claim Then notify Advanced Instruments immediately for repair or replacement Installation amp Setup I Figure 1 A Model FLM200 Fluorophos Test System and Supplies Quantity Part No 1 FLM200 1 1 FL0408 1 FL0425 2 rolls 1 FL2999 1 FLA020 1 pack 1 FLA802 1 FLA810 1 FLMS5 6 Description The Fluorophos Test System Power Cord as specified Paper Roll Holder Printer Maintenance Kit with Instructions Printer Paper re order package of 5 rolls as FLA835 Black Body Calibration Tool Fluorophos Cuvettes
62. ng it with a color reagent to form an indophenol The blue indophenol is removed from interfering turbidity caused by the dairy product in one of two ways First the American Public Health Association APHA and the Association of 2 Kay H D and Graham W R The phosphatase test for pasteurized milk J Dairy Res 6 191 203 1935 3 Scharer H A rapid phosphomonoesterase test for control of dairy pasteurization J Dairy Science 21 21 34 1938 4 Kosikowski F V A simple universal dairy products phosphatase test Science 110 480 481 1949 5 Aschaffenburg R and Mullen J E C A rapid and simple phosphatase test for milk J Dairy Res 16 58 67 1949 6 Kleyn D H and Lin S H C Collaborative study of new alkaline phosphatase assay system for milk J O A C 51 802 807 1968 7 Standard Methods for the Examination of Dairy Products 16th Edition 1992 American Public Health Association Washington DC 22 ALP Milk Pasteurization Assay Official Analytical Chemists AOAC have approved methods using butanol extraction followed by spectrophotometric measurement Second both AOAC and the International Dairy Federation IDF have approved methods in which the indophenol is read after protein precipi tation with barium and zinc salts Phenolphthalein monophosphate is used as substrate in the quantitative Rutgers method Liberated phenolphthalein is dialyzed into deionized water during a three hour in
63. ns Product test R Reagents Handling Preparing Reconstituting Storage Regulatory Notices RS 232 Information S Serial loop test Serial number Serial port Service 84 17 39 40 xiii 27 17 30 26 39 65 73 47 19 12 45 Service log Set serial rate Space requirements Specifications Spills Sub blank testing Symbol definitions T Time setting Troubleshooting checks Troubleshooting table U Unpacking V Ventilation requirements W Warranty duties Z Zero range adjust 81 19 63 37 18 75 46 59 69 19
64. nter as follows a b Unroll 6 8 inches 15 20 cm of paper from the paper roll Fold both corners of the end of the paper to form a triangular point approximately 2 3 inches or 5 8 cm long Crease the paper folds so that the doubled part will not be much thicker than two thicknesses of paper Insert the roller into the paper roll Open the printer door on the top right side of the instrument case Holding the roller in place with one hand use the other hand to feed the triangular pointed end of the paper from the bottom of the roll into the opening between the back of the printer frame and the print er circuit board Fluorophos ALP Test System User s Guide f Push the paper in and or use the FEED button on the keypad to feed the paper in far enough for the end to protrude from the top of the printer g Center the paper roll and roller between the V shaped roller guides behind the printer and press down firmly to seat the roller When both ends of the roller are in proper position the paper roll should be level If the paper roll is not level carefully reseat the roller between the roller guides h Press FEED or pull enough paper through the printer to take up any slack and feed the end out through the slot at the back of the win dow in the printer door NOTE The paper should only be driven or pulled forward through the printer attempting to pull the paper backward through the printer may cause printer d
65. nutes and to 2 0 raw milk The assay is complete in three minutes and utilizes a single stable sub strate reagent Detection limits down to 0 006 raw milk approximate ly equivalent to 0 06 ug phenol in the APHA phenylphosphate rapid method are possible Materials required Contents of the Fluorophos ALP Test Kit FLA225 e Fluorophos ALP Substrate 2 bottles Substrate Buffer 2 bottles e Glass Fluorometer Cuvettes Materials required but not included in the Fluorophos ALP Test Kit Fluorophos Test System with firmware version 2 0 or higher and ALP excitation filter assembly in place Fluorophos ALP Test Kit FLA124 smaller bottle size 120 mL e Fluorophos ALP Calibrator Set FLA250 e PhosphaCheck ALP Pasteurization Control Set FLA260 18 Rocco R M Fluorometric Analysis of Alkaline Phosphatase in Fluid Dairy Products J Food Protection 53 588 591 1990 25 Fluorophos ALP Test System User s Guide e Fluorophos ALP Daily Instrument Control Kit FLA280 Fluorophos ALP Cheese Extraction Buffer FLA005 only if test ing cheese e Vortex mixer e Deionized water e 2 mL pipette and disposable tips e 25 uL pipette and disposable tips e 75 uL pipette and disposable tips Reagent storage stability Protect stock working reagents and calibrators from light exposure e Fluorophos ALP Test Kit Unopened store at 2 to 8 C 36 to 46 F for up to 2 years Reconst
66. ody a Depress the plunger fully b Immerse the pipette tip about 1 4 Pi h FTIp inch or 6 7 mm into the liquid to be Wire Assembly pipetted Pipette Tip c Release the plunger smoothly and gently Figure 4 Fluorometer Pipette d Remove the pipette tip from the liq uid and wipe the outside do not wick out any fluid from the inside e Immerse the pipette tip about 1 4 inch or 6 7 mm into the liquid in the fluorometer cuvette and depress the pipette plunger fully several times f Depress the pipette plunger fully once more and while depressed remove the pipette from the cuvette To eliminate sample to sample contamination between pipetting sessions remove and replace the disposable pipette tip after each sample Occasionally the pipette plunger tip and wire assembly may require replacement Calibration and component replacement instructions are supplied with each pipette 14 Instrument Operation Using the 2 mL pipette The 2 mL pipette is a fixed volume pipette and is ready for use It is used for dispensing the ALP Test Reagent the ALP Calibrators and the ALP Daily Instrument Control Operate the pipette as follows a Place a clean tip firmly onto the pipette making sure that there is a tight seal Depress the plunger completely before placing the clean tip into the reagent to be dispensed Immerse the tip approximately 1 8 down into the reagent to be dis pensed A
67. on over parafilm or use a vortex mixer Test as for fluid 32 ALP Milk Pasteurization Assay dairy products except multiply the mU L printout by 10 to correct for the tenfold dilution as explained in Appendix B 5 Cheese To each labeled 16 x 100 mm screw cap glass test tube add a 0 5 gram cheese sample Add 5 0 mL Advanced Instruments FLA005 Cheese Extraction Buffer and macerate with a glass rod Centrifuge 10 minutes at 1000xg Sample from the upper layer and test as for fluid dairy products except multiply the mU L printout by 10 to correct for the tenfold dilution as explained in Appendix B Determining the need for the high turbidity test procedure Some dairy products such as ice cream mixes etc are so turbid i e have such a high fat content that a slight change in the calibration pro cedure is required Samples that cause this type of problem usually have greater than 10 fat and or are very highly colored products such as chocolate and blueberry ice cream mixes for example Reducing the sample volume to 25 uL will appreciably decrease turbidi ty and permit calibration and testing of these types of products The 25 uL procedure should be used when the following conditions exist 1 Results of controls and calibration are acceptable when used with other products 2 The suspect product has a very dark color and or has a fat content over 10 3 The calibration ratio when using the suspect product is less th
68. or input to the instrument Cuvette door The cuvette door located to the left of the keypad protects the cuvette testing chamber Function and components of the heating block Operation of the heating block will be quicker and easier if you become familiar with the locations and functions of the components systems and controls described below before proceeding further Power switch The rocker style power switch is located on the power entry assembly on the back of the instrument Power light The power light on the front of the heating block will illuminate when power is being received by the block Ready light The ready light on the front of the heating block will illuminate when the set temperature is reached Temperature adjustment screw The temperature adjustment screw is used in conjunction with the ther mometer to set the heating block to a desired temperature Thermometer and thermometer well The thermometer is placed in the thermometer well to monitor the heat ing block temperature setting 13 Fluorophos ALP Test System User s Guide SF ous amp Key Using the 25 uL or 75 uL pipette The Advanced positive displacement pipettes are factory set for precise fixed Calibration volume delivery and shipped ready for AA HEG use Calibration adjustment keys and detailed instructions are provided with each pipette Operate the pipette as fol lows B
69. parts and accessories contact your local Advanced Instruments Distributor or the Advanced Instruments Customer Service Department by using one of the following numbers e 800 225 4034 toll free in the USA and Canada e US 781 320 9000 elsewhere 781 320 3669 fax PART DESCRIPTION PART NO Pipette Tips 25 75 uL 100 FLA880 Fluorophos Cuvettes 250 FLA020 Printer Paper 5 rolls FLA835 25 uL Pipette FLA865 75 uL Pipette FLA885 Plunger Wire for 75 uL Pipette FLA887 Cuvette Holder Cleaners 50 cleaners FLA840 Heating Block Thermometer FLA810 20 Sample Heating Block FLA802 Service Manual FLMSSM User s Guide FLM5 Dual Excitation Filter ACP ALP Assembly FL2333 Replacement Air Filter box of 6 3D2340 xi Fluorophos ALP Test System User s Guide PART DESCRIPTION PART NO Pipette Tips 2 mL 25 ACM205 xii Mi Reagents Calibrators amp Controls To order calibrators and standards contact your local Advanced Instruments Distributor or the Advanced Instruments Customer Service Department by using one of the following numbers e 800 225 4034 U S territories and Canada e US 781 320 9000 international 781 320 3669 fax ALP Pasteurization Assay PART DESCRIPTION PART NO Cheese Extraction Buffer FLA005 ALP Test Kit FLA124 225 Test ALP Reagent Cuvettes Kit FLA225 ALP Calibrator Set FLA250 ALP PhosphaCheck Control FLA260 ALP Daily Instrument Control FLA280 xiii Fluorophos ALP
70. r may have applied additional local national or international approvals to this product Consult the distributor or dealer for more information and documentation Connections to this product must be made with shielded cables Use of non shielded cables may violate RFI EMI limits Symbol Conventions This symbol indicates conformity to relevant European direc tives This symbol indicates the product was tested to conform to Gi j relevant Canadian and U S safety standards by Intertek Testing Services NA Inc The ETL mark is approved in the neu United States as a Nationally Recognized Testing Lab NRTL by OSHA and in Canada by the Standards Council of Canada 65 Fluorophos ALP Test System User s Guide Regulatory approval type USS Safety Canadian Safety EC Declaration of Conformity EMC EC Declaration of Conformity Low Voltage 66 Description This product has been listed by ETL testing laboratories as being in compliance with the requirements of UL 61010 1 2nd edition Electrical Equipment for Laboratory Use The US in the lower right of the ETL mark demonstrates this listing This product has been listed by ETL testing laboratories as being in compliance with the requirements of CAN CSA C22 2 No 61010 1 2nd edition Safety Require ments for Electrical Equipment for Measurement Control and Laboratory Use The C in the lower left of the ETL mark demonstrates this listing
71. s Reagent storage Air filter replacement Optical filter replacement Optical filter care Pipette maintenance Fuse replacement Lamp replacement Chapter 5 Troubleshooting amp Service Service amp maintenance cautions Obtaining service Troubleshooting checks Internal diagnostics Photomultiplier tube voltage adjustment iv 21 21 21 24 25 26 27 27 30 30 31 32 33 34 34 35 37 37 37 39 39 39 40 40 40 41 43 43 45 46 46 47 Appendixes Appendix A Additional Technical Information Appendix B Troubleshooting Table Appendix C Product Specifications Appendix D Regulatory Notices Appendix E Warranty amp Warranty Duties Appendix F Supplemental RS 232 Information Appendix G Symbol Definitions Appendix H Product Disposal and Recycling Appendix I Service Log Index 51 59 63 65 69 73 75 79 81 83 Fluorophos ALP Test System User s Guide Notes vi Mi Safe Use To reduce the risk of bodily injury electric shock fire and damage to your instrument please read and observe the precau tions in this User s Guide Ifthe product is used in a manner not in accordance with the equipment design operating instructions or manufacturer s recommendations the operation of the product may be impaired to the extent that a safety hazard is created Do not attempt to perform electrical work if you are not fully qualified This manual is no
72. s follows Calibration Ratio CAL C A 2 CAL B A 4 Assume 191 is the mean of the Calibrator A fluorescence readings 317 the mean of Calibrator B and 448 the mean of Calibrator C For these averages Calibration Ratio 257 2 126 4 127 2 The fluorometer also uses the calibration ratio for linearity checks during cali bration and will only accept calibration readings which are linear and within specified ranges The linearity of the calibrators may be manually checked as follows Lin CAL A 0 umoles L FY 191 191 0 Lin CAL B 17 24 x 10 3 umoles L FY 317 191 126 Lin CAL C 34 48 x 10 3 umoles L FY 448 191 257 In every case the value of Calibrator C must be twice 10 the value of Calibrator B If not repeat calibration with fresh calibrators or a new lot of calibrators Manual calculations for fluid dairy products The Advanced FLM200 Fluorometer printout reports the ALP activity of the test sample mU L as well as the average fluorescence increase Avg F min Alternatively the ALP activity may be calculated from the print 51 Fluorophos ALP Test System User s Guide out of the calibrator values and the average increase in fluorescence of the sample as follows 1 Calibration as the calibration process is accomplished printouts simi lar to the following should result ASTD 198 ASTD 198 B STD 345 B STD 344 C STD 475 C STD 475 Avg A STD 198 Avg B ST
73. set in the field Assistance Provides the manufacturer s name address telephone fax and internet information and the instrument model software version and serial number 19 Fluorophos ALP Test System User s Guide Notes 20 Fluorophos ALP Milk Pasteurization Assay This chapter will give you information specific to the Fluorophos ALP milk assay Please read this chapter and chap ters 1 and 2 on instrument operation before running tests Overview The Fluorophos ALP Assay is a rapid test for verifying proper pasteurization of dairy products Alkaline Phosphatase ALP found naturally in raw milk is destroyed at temperatures slight ly above the temperatures necessary to destroy micro organisms pathogenic to man Reduced levels of ALP in finished dairy products ensure that the products have been heated to the prop er temperature and have not been contaminated with raw milk The Fluorophos ALP Assay allows you to measure the ALP activity in your dairy products allowing you to monitor com pleteness of pasteurization and detect raw milk contamination Alkaline Phosphatase ALP background information Numerous fluorometric analytical methods have been devel oped for analytes of interest to the dairy industry and Shipe has reviewed some of them In 1935 Kay and Graham developed an alkaline phosphatase ALP assay which could be used to detect the completeness of 1 Shipe W F Fluorometric Methods
74. son to season and possibly from breed to breed For example in order to obtain a dilution of raw milk at 0 1 v v which contains exactly 1 ug phenol mL 15 min activity the original mixed herd milk sample must have contained 1 000 ug phenol mL 15 min activity When 1 000 ug phenol mL 15 min activity is diluted to 0 1 v v x 10 000 dilution the expected value will be 1 0 ug phenol mL 15 min activity A slight variation in biological sampling of a mixed herd population could reduce the expected value for a 0 1 v v dilu tion of this original mixed herd raw milk to 0 9 ug mL 15 min The table on the next page shows the results in which a sample of mixed herd raw milk was added to heat treated milk from 0 003 to 0 4 and the samples tested by various methods including Fluorophos 54 Appendix A Additional Technical Information Raw Milk v v in Whole Milk 15 6 31 2 62 5 125 0 250 0 500 0 1000 0 2000 0 500 000 0 1 Whole milk 3 2 fat was heated to 95 C for two minutes and found to contain less than 10 mU L of ALP activity by the Fluorophos ALP method Fresh mixed herd raw milk was then added at the above v v concentrations 2 Extrapolated from dilutions made as in note 1 3 Above values are based on the assumption that the mixed herd raw milk which is used to make the above dilutions contains exact ly 5000 000 mU L or 1000 ug phe nol mL 15 min ALP activity This is not ALP ACTIVITY Fluorop
75. t a substitute for electrical training Symbol Conventions A A amp ta F The exclamation point within an equilateral triangle is intended to alert the user to the presence of important operating and maintenance servicing instructions in the literature accompanying this product The lightning flash with arrowhead symbol within an equilateral triangle is intended to alert the user to the presence of uninsulated dangerous voltage within the product s enclosure that may be of sufficient magnitude to constitute risk of electric shock to persons The static symbol within an equilateral triangle is intend ed to alert the user to the presence of internal components that could be damaged by static electricity This static symbol is intended to alert the user to the pres ence of a specific component that could be damaged by static electricity vii Fluorophos ALP Test System User s Guide 0 WO This symbol indicates the presence of alternating current AC This symbol indicates the presence of a fuse This symbol indicates the presence of protective earth ground This symbol indicates the power is ON This symbol indicates the power is OFF Additional symbol definitions are available in Appendix F General Cautions This product should be operated only with the type of power source indicated on the product s electrical ratings label Refer to the installation instructions included with the product I
76. te a displayed function press START If the function enables modifying an operating parameter the display will change to indicate the current setting either as a numeric value or the sym bol next to the current setting In most cases the setting may be changed by pressing lt or gt d Press ENTER to save the new setting or STOP to cancel the new set ting The current setting will be printed for verification and the dis play will revert to the title of the item selected indicating that the SETUP menu is again available e Press STOP to exit the SETUP menu The following menu choices allow you to change operating settings Please note that other options are available but are used in trou bleshooting and will be discussed in the next chapter Date Time At the date and time display press CLEAR to clear the entire setting and display mm dd yy hh mm am the required format or repeat edly press lt to clear the setting one digit at a time from the right Enter the desired numbers via the numeric keypad one digit at a time from the left Use gt to toggle to am or pm as required Press ENTER to store the new setting or STOP to restore the original setting The instrument will beep and reject any invalid date or time Print Avg F min At Y lt avg f min N press lt or gt to print or suppress the printing of the average increase in fluorescence which is displayed just before the test results The current s
77. tive lifetime of 10 000 hours The lamp is not a user accessible area and should be changed by AN a qualified service technician per the instructions found in the service manual for this instrument Users are cautioned that opening this instrument exposes them to Hazardous Live Voltages in excess of 600 volts AC and DC Instruments operated on an 8 hour day may need lamp replacement after approximately 1200 days of operation If the instrument is left on continuously this number falls to 416 days 41 Fluorophos ALP Test System User s Guide Notes 42 5 A Troubleshooting amp Service This chapter contains very basic information to help you solve problems that might arise with your fluorometer Please read all instructions very carefully and if a solution cannot be found in this guide contact Advanced Instruments for Hot Line Service Service amp maintenance cautions Do not perform any service or maintenance yourself except as detailed in the User s Guide e Unplug the power cord prior to opening or removing covers or else you may be exposed to electric shock excessive temperatures or mechanical hazards Voltages in excess of 600 VAC and VDC are present inside this product e Performing service or maintenance not detailed in the User s Guide with or without a Service Manual should only be done by a qualified service technician e Never restrict airflow into or out of the product Occasionally
78. with one of the same rating voltage and type Never replace the fuse with one of a higher current rating When servicing the product use only factory specified parts The product contains an integral lithium battery that is not user ser viceable WARNING When returning this product for service or shipping this 44 product to a second location remove all hazardous spec A imens and decontaminate the product before packaging for shipment If the product cannot be decontaminated consult with your shipping agent on appropriate packag ing and marking Troubleshooting amp Service Obtaining service Before contacting Advanced Instruments for Hot Line Service be sure to read through this user s guide for instructions on routine adjustments instrument care and troubleshooting If this information does not solve the problem call the appropriate number below e 800 225 4034 toll free within the USA and Canada after normal business hours dial extension 2191 e US 781 320 9000 elsewhere 781 320 0811 fax When contacting our service personnel please have the model and seri al numbers from the label on the back of the instrument and the symp toms of the problem ready You should use a telephone as close to the instrument as possible to facilitate making recommended diagnostic checks If you need to order parts or service a purchase order from your purchasing agent will be necessary After Hot Line diagnosis t
79. with the pipette are stated by the manufacturer to be chemically resistant to most reagents except chlorinated hydrocarbons If the pipette piston plunger tip becomes worn or otherwise un usable and a replacement piston is available please follow the replacement instructions supplied with the pipette Always re calibrate the pipette after replacing the piston or other internal parts referring to the calibra tion instructions supplied with the pipette To eliminate possible sample to sample contamination between pipet ting sessions remove and replace the pipette tip after each sample Fuse replacement If you determine that the instrument is not functioning because of blown fuses you will need to replace the fuses using the following procedure 1 Switch the power switch to the off position and disconnect the power cord 2 Use a small flat bladed screwdriver or similar tool to pry open the fuse holder door Remove the fuse holder inside 40 Instrument Maintenance 3 Double check the values marked on the fuses Use 5 x 20 mm 250V time delay Type T fuses For 100 120V operation use 2 Amp fuses for 200 240V operation use 1 Amp fuses 4 Re install the fuse holder into the back of the instrument and close the fuse holder door 5 Re connect the power cord and switch the power switch to the on position The instrument should start up as normal Lamp replacement The instrument contains a Halogen lamp with a rated effec

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