Milk Safety References
National Conference on Interstate Milk Shipments (NCIMS)

PHOSPHATASE TEST - FLUOROPHOS ALP TEST SYSTEM
[Unless otherwise stated all tolerances are ±5%]

Sample

1. __________ Laboratory Requirements (see CP, item 33 & 34)

   APPARATUS

2. __________ See CP, items 1-32 (as necessary)
3. __________ Cuvette Heating Block
   1. __________ Thermostatically-controlled at 38±1C
   2. __________ NIST-traceable thermometer, calibration checked as specified in CP item 3
   3. __________ Temperature checked and recorded daily
4. __________ Pipettors, fixed volume or electronic
   1. __________ 75 µL pipettor
   2. __________ 25 µL pipettor, for use with high-turbidity or high fat products (if needed)
   3. __________ Calibrated as specified in CP item 6e; records maintained
5. __________ Reagent Dispenser
   1. __________ Fixed volume 2.0 mL, calibrated and checked
   2. __________ Optionally, use 2.0 mL fixed volume or electronic pipettor to dispense reagent
   3. __________ Calibrated as specified in CP item 6e; records maintained
6. __________ Cuvettes
   1. __________ Disposable glass 12 x 75 mm, dirt and scratch free
7. __________ Fluorometer
   1. __________ Air fan in the rear unobstructed
   2. __________ Vents in the bottom base plate are unobstructed
   3. __________ Sufficient paper is on the roll in the printer
   4. __________ User's manual available
8. __________Water Bath, 34±1C and 63±1C, circulating (Confirmation procedures)

   REAGENTS

9. __________ Reagents, Handling and Storage
   1. __________ Test Reagent Set
      1. __________ Fluorophos substrate and Substrate buffer
      2. __________ Lot # _______ Rcd date _______ Exp date _______
   2. __________ Calibrator Set
      1. __________ Calibrators A, B and C
      2. __________ Lot # _______ Rcd date _______ Exp date _______
   3. __________ PhosphaCheck Pasteurization Controls Set
      1. __________ Positive and negative control
      2. __________ Lot # _______ Rcd date _______ Exp date _______
   4. __________ Daily Instrument Control
      1. __________ Lot # _______ Rcd date _______ Exp date _______
   5. __________ Reagents stored at 0-6C
   6. __________ Bottles labeled with receive and open dates

   REAGENT PREPARATION

10. __________ Working substrate
    1. __________ Prepare reagents as per manufacturer instructions, mix by inversion until fully dissolved
    2. __________ Date (mixture stable 60 days at 0-6C)
       1. __________ Bottle labeled with date prepared
       2. __________ Preparation date _______
    3. __________ Place cleaned, 2 mL reagent dispenser (item 5) on prepared reagent bottle, or cap if using 2 mL pipettor

    CALIBRATION (Required at installation and after any instrument adjustments)

11. __________ Procedure
    1. __________ Perform instrument and reagent checks (item 14) prior to proceeding
       1. __________ 1. Readings from item 14 are within specification, proceed with calibration
       2. __________ If readings not within specification, do not proceed with calibration, make appropriate adjustments or seek technical assistance and re-check
       3. __________ Record all values (initial and re-checks) on tape and in QC record
    2. __________ Check calibration ratio of Calibrators A, B and C, record Calibrator Lot # on strip and in QC record
       1. __________ 1. Label two (2) tubes each for Calibrators A, B and C
       2. __________ Add 2 mL of each calibrator to the appropriately labeled tubes
       3. __________ Heat tubes for 20 minutes to 38±1C
       4. __________ Find an empty channel
          1. __________ Press the "Calib" key _
          2. __________ Locate the channel to be used, using the "<" and ">" keys
          3. __________ Press the "Enter" key to select the channel
       5. __________ Place a tube of warmed Calibrator A (with no milk added) into the cuvette chamber, close the door and press the "Start" key
       6. __________ Continue as prompted until all six (6) tubes have been run
       7. __________ Calibration ratio should be 151±7 (when A/D mode check for Calibrator C/Daily Instrument Control is 602±6)
       8. __________ If ratio within specification continue, if not make adjustment and re-check calibration ratio
    3. __________ Invert retail containers 25 times, each inversion a full cycle down and up
    4. __________ Remove test portions (avoiding foam) within 3 min of agitation
    5. __________ To each calibrator, add 75 µL (or 25 µL) of the well-mixed product being tested
       1. __________ Prior to pipetting sample, draw up MS water once and expel to waste
       2. __________ Dry exterior of piston and tip
       3. __________ Place tip of pipettor into sample (no more than 1 cm) and draw up and expel several times
       4. __________ Draw sample into pipettor, touch off to side of container
       5. __________ Holding pipettor at 90° to lab bench and with tip at eye level, dry exterior of tip by quickly wiping from the pipettor over the tip
          1. __________ Carefully inspect the pipettor tip to insure sample volume is flush with the tip
          2. __________ If concave, re-sample
          3. __________ If convex, re-wipe as above to achieve a flush sample volume
       6. __________ Dispel 75 µL (or 25 µL) of sample 1 cm below the surface of the calibrator (do not dispense down side of cuvette)
       7. __________ With tip still below surface depress plunger three times into calibrator to completely expel sample
       8. __________ With plunger still completely depressed, remove from tube
    6. __________ Add products to calibrators one tube at a time just prior to being tested
    7. __________ Mix by vortexing
    8. __________ Place cuvette in Fluorometer, close cuvette door
    9. __________ Press the "CALIB" key on the Fluorometer keypad and follow the prompts on the display panel
    10. __________ After each reading, remove cuvette and close door immediately
    11. __________ Record lot #'s of the calibrators used on the tape printout and in the QC record book
    12. __________ Instrument calibrated for each product type to be tested, some products with similar fat content may share same channel
    13. __________ Re-calibration required if:
        1. __________ Controls out of limits
        2. __________ Adjustments made to bring A-D mode checks (item 14) into specification
        3. __________ Any significant instrument service if performed, ex. lamp or filter replaced
    14. __________ Instrument checks and calibrations within specification

    CONTROLS

12. __________ Negative Control
    1. __________ Use PhosphaCheck negative control from set in item 9c
    2. __________ Or, optionally heat a sample of product to 95±1C for 1 min with stirring (temperature control [TC] used)
       1. __________ Cool rapidly to 0-4.4C in an ice bath
       2. __________ If desired, distribute 1 mL quantities in small tubes, seal and freeze in a non-frost-free freezer, or place in a styrofoam container and place in the center of a frost-free freezer for no more than 2 months
    3. __________ Add 2.0 mL of working substrate (Reagent C) to cuvettes and heat to 38±1C for 20 min (use within 4 hours)
    4. __________ Add 75 µL of well mixed control to cuvette and immediately vortex
    5. __________ Once control is added to reagent test within 20 sec
    6. __________ Place the cuvette in the Fluorometer, close the cuvette door and press the "TEST" key on the keypad
    7. __________ Value less than (<) 20 mU/L
13. __________ Positive Control
    1. __________ Use PhosphaCheck positive control from set in item 9c
    2. __________ Or, optionally to a portion of negative control, add exactly 0.1 mL of mixed-herd raw milk and bring up to exactly 100 mL with additional negative control (as in item 12b)
       1. __________ If desired, distribute 1 mL quantities in small tubes, seal and freeze in a non-frost-free freezer, or place in a styrofoam container and place in the center of a frost-free freezer for no more than 2 months
    3. __________ Test as in items 12 c-f
    4. __________ Value between 500±150 mU/L

    INSTRUMENT AND REAGENT CHECKS

14. __________ Optionally use manufacturer's reagent kits and instructions specific for each instrument
    1. __________ Check calibration if readings are suspect (positive control value out of limits)
    2. __________ Press set-up button
    3. __________ Press left or right arrow key to get A/D option
    4. __________ Select A/D Mode
    5. __________ Zero Check
       1. __________ With no tube in the instrument, press "Start" key and take a reading
       2. __________ A/D value ____________
       3. __________ The reading must not exceed 314. If the reading exceeds 314, an instrument problem is indicated, call for technical assistance
       4. __________ Record value on printout and QC record
    6. __________ Calibrator C/Daily Instrument Control Check
       1. __________ Dispense 2.0 mL of Calibrator C (item 9b) or Daily Instrument Control (item 9d) into a 12x75 mm cuvette and allow to warm to 38±1C for 20 minutes
       2. __________ Place the cuvette with the warmed Calibrator C or Daily Instrument Control into the sample chamber and press "Start" key
          1. __________ The A/D value should be 602±15 (maximum allowable drift.
          2. __________ A/D value ____________
          3. __________ Record Lot # and value on printout and QC record
       3. __________ If the value does not fall within the acceptable range, then perform the following procedure (refer to manual, or contact manufacturer if unsure)
          1. __________ With Calibrator C or Daily Instrument Control in the sample chamber, adjust the R15 resistor until the A/D value reads 602±2 .
          2. __________ Allow the instrument to equilibrate for 15 minutes, the A/D value should still read 602±2
          3. __________ Record Lot # and value on strip and in QC record
       4. __________ If the value does not fall within and stabilize at 602±2 seek technical assistance
       5. __________ For older units requiring the instrument cover to be removed or if unsure seek technical assistance
    7. __________ Reconstituted Substrate/Buffer stability check
       1. __________ Dispense 2.0 mL of working substrate (item 10) into a 12x75 mm cuvette and allow to warm to 38±1C for 20 minutes
       2. __________ Place the cuvette with the warmed working substrate into the sample chamber and press "Start" key
          1. __________ The A/D value should be < 1,200
          2. __________ A/D value ____________
          3. __________ Record Lot # and value on printout and QC record
    8. __________ Reconstituted Substrate/Buffer contamination check
       1. __________ Dispense 2.0 mL of working substrate (item 10) into a 12x75 mm cuvette and allow to warm to 38±1C for 20 minutes
       2. __________ Place the cuvette with the warmed working substrate into the sample chamber
       3. __________ Initiate an ALP sample reading of the working substrate on an unused channel
       4. __________ The ALP value should be < 10 mU/L
          1. __________ ALP value __________
          2. __________ Record Lot # on printout and QC record
       5. __________ If the working substrate value does not fall within the acceptable range, do not use working substrate, re-check to verify, reconstitute a new set of reagents or seek technical assistance before testing samples

    TEST PROCEDURE

15. __________ Test Procedure
    1. __________ Perform all instrument and reagent checks (item 14), negative control test (item 12) and positive control test (item 13) prior to running analysis
    2. __________ Using reagent dispenser, fixed volume or electronic pipettor, dispense 2.0 mL of working substrate into labeled 12 x 75 mm glass cuvettes
       1. __________ Prime reagent dispenser (item 5) 3x prior to dispensing volumes to cuvettes to remove any bubbles from dispenser tubing
    3. __________ Warm substrate to 38±1C in the heating block for 20 min (use within 4 hours)
    4. __________ Sample agitation
       1. __________ Invert filled retail containers 25 times, each inversion a full cycle down and up
    5. __________ Remove test portions within 3 min of agitation
    6. __________ Press the "Test" key on the keypad
    7. __________ Select the product type channel and enter identifi- cation number
    8. __________ Dispense 75 µL (or 25 µL) of the well-mixed sample into the warmed substrate and immediately mix by vortexing
       1. __________ Prior to pipetting sample, draw up MS water once and expel to waste
       2. __________ Dry exterior of piston
       3. __________ Place tip of pipettor in sample (no more than 1 cm) and draw up and expel several times
       4. __________ Draw sample into pipettor, touch off to side of container
       5. __________ Holding pipettor at 90° to lab bench and with tip at eye level, dry exterior of tip by quickly wiping from the pipettor over the tip
          1. __________ Carefully inspect the pipettor tip to insure sample volume is flush with the tip
          2. __________ If concave, re-sample
          3. __________ If convex, re-wipe as above to achieve a flush sample volume
       6. __________ Dispel 75 µL (or 25 µL) of sample 1 cm below the surface of the substrate (do not dispense down side of cuvette)
       7. __________ With tip still below surface depress plunger three times into substrate to completely expel sample
       8. __________ With plunger still completely depressed, remove from tube
       9. __________ Run test within 20 sec of adding sample to reagent
    9. __________ Place the cuvette in the Fluorometer, close the cuvette door, and press the "START" key on the keypad
    10. __________ Results will display in 3 min., save tape print- out of results in record book
        1. __________ If a 25 µL sample volume was used multiply the displayed value by 3
        2. __________ Record adjusted value on printout
    11. __________ Values of ³ 350 mU/L or more of ALP activity are considered to contain approximately 0.1% (v/v) raw milk and must be confirmed

    CONFIRMATION

16. __________ Negative Control
    1. __________ Prepare separate control for each product
    2. __________ Prepare by heating sample for at least 1 min after thermometer registers 95±1C, stirring or mixing as necessary (TC used)__________
    3. Cool rapidly to 0-4.4C in an ice bath
    4. __________ This control must be less than 20 mU/L when tested
17. __________ Positive Control (See item 13)
    1. __________ Must be prepared from suspect product
18. __________ Microbial Phosphatase
    1. __________ To determine presence of microbial phosphatase, heat 1.0 mL of suspect milk at 63±1C for 30 min, stirring or mixing every 10 min (if fat content is > 10%, heat at 66±1C) [TC used]
    2. __________ Cool rapidly to 0-4.4C in an ice bath
    3. __________ Test heated portion, unheated portion, and positive and negative controls
    4. __________ Interpretation
       1. __________ If heated and unheated portions have equal activity (within ±5%), the sample is regarded negative for residual phosphatase, the activity originally measured is microbial
       2. __________ If the heated portion has significantly reduced (>5%) or no activity, the sample contains milk phosphatase activity, either residual or reactivated
19. Reactivated Phosphatase
    1. __________ Magnesium acetate solution
       1. __________ Dissolve 35.4g of Mg(C₂H₃0₂)₂·4H₂0 in 25 mL MS water warming slightly to aid solution. Pour solution into 100 mL volumetric flask, rinse original container several times and add rinses to flask. After cooling, make up to 100 mL (stable for 1 year at 0-4.4C)
    2. __________ Procedure
       1. __________ Place 10 mL of each milk or milk product sample to be tested in a boiling water bath and hold 1 min after temperature sample has reached 95±1C (TC used)
       2. __________ Cool samples rapidly to 0-4.4C in an ice bath
       3. __________ Place a 5 mL aliquot of sample (unheated) to be tested in a screw-cap test tube and add 0.1 mL MS water ("Blank" sample)
       4. __________ To a second 5 mL aliquot (unheated) in an identical tube, add 0.l mL Mg acetate solution ("Test" sample)
       5. __________ Cap tubes and incubate both aliquots for 1 hr at 34±1C
       6. __________ Remove samples from water bath and cool rapidly to 0-4.4C in an ice bath
       7. __________ Dilute 1 mL of sample containing magnesium (Test) with 5 mL (1:6 dilution) of corresponding boiled milk or milk product control (items 21b 1 & 2 above)
       8. __________ Test undiluted sample containing no magnesium (Blank) and diluted sample containing magnesium (Test) for phosphatase activity (as described in item 16)
    3. Interpretation
       1. __________ If the diluted aliquot containing magnesium (Test) has equal (±5%) or greater phosphatase activity than the undiluted aliquot containing no magnesium (Blank), the sample is regarded negative for residual phosphatase, and the phosphatase originally measured is of reactivated origin
          Dil. w/Mg (Test) ³ Undil. (Blank) = Reactivated
       2. __________ If the diluted aliquot (Test) contains less activity (< 5%) than the undiluted aliquot (Blank), the sample is considered positive for residual phosphatase
          Dil. w/Mg (Test) < Undil. (Blank) = Residual
       3. __________ A false-positive for residual phosphatase may also be obtained if a reactivatable sample has been allowed to stand at elevated temperatures (20C) for periods of 1 hr or more before testing (SPC <20,000/mL)

    REPORT

20. __________ Confirmatory Interpretation
    1. __________ Report as positive for residual phosphatase if microbial phosphatase, and reactivatable phosphatase are not present
    2. __________ Run standard/control 3x
    3. __________ Report as Not Found for residual phosphatase if:
       1. __________ If microbial phosphatase present
       2. __________ Or, if reactivatable phosphatase present
       3. __________ Or, if there is documentation that the product was treated such that reactivatable phosphatase may be present
    1. __________ Count obtained x 1000 is the cell count/mL milk
    2. __________ In reporting electronic somatic cell counts (ESCC/mL), record only first two left hand digits, raising second digit to next higher number when third digit is 6 or more
    3. __________ Report the two left hand digits (rounded)
       1. __________ If the third digit is 5 the second digit is rounded by the following rule
          1. __________ When the second digit is odd round up, raise the second digit by 1 (odd up, 235 to 240)
          2. __________ When the second digit is even round down, delete the 5 and report the second digit as is (even down, 225 to 220)
    4. __________ If count on instrument is < 100 report as < 100,000 ESCC/mL