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

PHOSPHATASE TEST - CHARM PASLITE - ALKALINE PHOSPHATASE TEST
USING CHARM II 6000/6600 AND LUMINOMETER/LUMINATOR
[Unless otherwise stated all tolerances are ±5%]

Sample

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

   APPARATUS

2. __________ CP, items 1 - 32 (as necessary)
   1. __________ Unless otherwise stated, "shake vigorously" refers to standard microbiological mixing, i.e., 25 times in a one foot arc in seven seconds
3. __________ Incubator Block for 13 x 100 mm test tubes or 2 mL microtubes
   1. __________ Thermostatically-controlled at 35±1C
   2. __________ Temperature checked by electronic display or by thermometer in small well in block or by liquid immersion, records maintained
4. __________ Pipettors and Pipets
   1. __________ Fixed volume or electronic, 100 µL
   2. __________ Calibration checked as specified in CP item 6e, records maintained
   3. __________ Disposable, 10 mL (ASTM) pipet with 0.1 mL graduations
5. __________ Reagent Dispenser
   1. __________ Fixed volume or electronic, 1.0 mL
   2. __________ Calibration checked (CP item 6e) with 10 mL ASTM graduated cylinder, records maintained
6. __________ Test tubes or microtubes
   1. __________ Test tubes for Charm II 6600/Charm II 6000 systems, disposable borosilicate glass 13 x 100 mm, dirt and scratch free
   2. __________ Microtubes - for Luminometer/Luminator, 2 mL screw cap
7. __________ 6000/6600 or Luminometer/Luminator Analyzer
   1. __________ Operating instructions available
   2. __________ Definitions:
      1. __________ Fluid white milks - including skim through whole fat milk
      2. __________ Unflavored liquid dairy products - including half and half, buttermilk, creams (light, medium and whipping), and etc
      3. __________ Flavored liquid dairy products - Liquid products that can be accurately pipetted, containing flavor additives and/or thickening agents including flavored milks, and etc
      4. __________ Solid/semisolid dairy products - thick dairy products not able to be pipetted, solid and/ or powdered additives, including cheese, yogurt, cottage cheese, ice cream mix, ice cream milk, whey, sour cream, and heavy cream ³ 36%, and etc
8. __________ Water Bath, circulating, 34±1C and 63±1C (or 66±1C if fat > 10%), or 13 x 100 test tube dry well heater blocks acceptable (Confirmation procedure)
9. __________ Centrifuge - Charm II Heraeus (3,400 RPM), minifuge, or equivalent (1,200 - 2,000 g).
10. __________ Handling and storage
    1. __________ Kit contains Reagent AP, Stopping Solution and Alkaline Phosphatase Positive Control Tablet

       __________ Kit: Lot # _____ Rcd. Date _____ Exp Date _____

       1. __________ For solid/semisolid dairy products, Diluent AP

          __________ Diluent AP: Lot # _____ Exp. Date _____

    2. __________ Reagents stored at 0-4.4C until expiration date
    3. __________ Stopping Solution must be at 18-24C at time of use, may be stored at room temperature, expiration date marked 2 month from room temperature storage
    4. __________ Bottles labeled with receive and open dates

    CONTROLS

11. __________ Negative Control
    1. __________ Product type. Prepare at least 50 mL for calibrator and positive control preparation
       1. __________ Fluid white milk - heat a sample of product (highest fat content) to 95±1C for 1 minute with stirring
       2. __________ Flavored liquid dairy products - heat a chocolate (highest fat content) to 95±1C for 1 minute with stirring
       3. __________ Unflavored liquid dairy products - heat pasteurized light cream to 95±1C for 1 minute with stirring
       4. __________ Solid/semisolid dairy products - mix or knead 5 g of product (highest fat content) with 20 mL Diluent AP until homogeneous and heat to 95±1C for 1 min with stirring
       5. __________ Note, if using 13 x 100 test tube dry well heater block at 95C it takes 10 minutes to heat product to 95C for one minute, use temperature control
    2. __________ Cool rapidly in an ice bath and hold at 0-4.4C,
    3. __________ Kept at 0-4.4C, the Negative Control may be used for up to 48 hours
    4. __________ If desired, distribute 1 mL quantities in small tubes (Milk only),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, vials labeled with preparation and expiration dates
12. __________ Positive Control (for daily checks)
    1. __________ Reconstitute positive control (450 mU/L) with negative control, item 11, as indicated on label, or alternatively use 350 mU/L calibrator (item 13a2a)
    2. __________ Shake vigorously and let settle 10 minutes at 0-4.4C for re-suspension
       1. __________ For solid/semisolid dairy products only, add 1 mL of reconstituted positive control with 3 mL of Negative control to complete preparation of positive control
    3. __________ Shake vigorously again and use for test
    4. __________ Positive controls and calibrators held at 0-4.4C may be used for 48 hours, milk controls may be frozen at -15C or lower for up to 3 weeks, thaw in refrigerator prior to use
    5. __________ With 6600 and C2Soft, enter either the triplicate RLU average of Positive Control or triplicate RLU average of 350 mU/L calibrator as the pos avg and CP in C2Soft configuration file. Refer to C2Soft manual

    CALIBRATION

13. __________ With each new kit lot # check calibration of analyzer
    1. __________ Prepare 350mU/L, 175mU/L, 44mU/L (milk only), 88mU/L (flavored and unflavored only) calibrators using Negative Control, item 11
       1. __________ Rehydrate a calibrator tablet with 100 uL water, mix to disperse tablet, wait 1 minute and mix again
       2. __________ Add the specified volume of Negative Control to each dissolved calibrator tablet to make calibators:
          a. Add 2.5 mL to make 350mU/L
          b. Add 5 mL to make 175 mU/L
          c. Add 10 mL to make 88 mU/L (flavored and unflavored only)
          d. Add 20 mL to make 44 mU/L (fluid white milk only)
    2. __________ Calibrate instrument by testing each calibration control (350, 175, 44 (or 88) mU/L) in triplicate

       6600 with C2Soft Software

    3. __________ For fluid white milks, unflavored or flavored liquid dairy product on the 6600 system with C2Soft software, follow the Standard Curve Calibration procedure
       1. __________ Program has a separate assay line for each product group, fluid white milk, flavored and unflavored liquid dairy product
       2. __________ In calibrate mode, enter low concentration (44 or 88 mU/L) value, followed by 3 replicate counts
       3. __________ Enter medium concentration (175 mU/L) value, followed by 3 replicate counts
       4. __________ Enter high concentration (350 mU/L) value, followed by 3 replicate counts
       5. __________ Calibration successful will be prompted at end of the procedure
    4. __________ For solid/semisolid dairy products using the 6600 system with C2Soft, follow instructions for positive average or control point setup
       1. __________ Count 3 replicates of 350 mU/L control
       2. __________ Control point is equal to average of triplicate counts

       Luminometer/Luminator system

    5. __________ For fluid white milk, unflavored or flavored liquid dairy products, determine average value for each calibrator
       1. __________ Set up a separate channel and calibration for each product group, fluid white milk, flavored and flavored liquid dairy products
       2. __________ Check calibration
          1. __________ Average negative control tested in triplicate. Average must be less than 5 (less than 15 for flavored dairy products)
          2. __________ Average 44 mU/L (or 88 mU/L unflavored and flavored liquid dairy products) calibrator, must be between 35 - 53 mU/L (45 – 110 mU/L unflavored and flavored liquid dairy products)
          3. __________ Average 175 mU/L positive control, must be 145 - 205 mU/L
          4. __________ Average 350 mU/L calibrator, must be 320 - 400 mU/L
       3. __________ If conditions are not met, recalibrate according to Luminometer/Luminator calibration instructions
    6. __________ For solid/semisolid dairy products verify control point of 350 mU/kg
       1. __________ Count 3 replicates of 350 mU/kg control
       2. __________ Average 350 mU/kg positive control, must test 350±105 mU/kg
       3. __________ If conditions are not met, recalibrate according to Luminometer/Luminator calibration instructions

    DAILY PERFORMANCE CHECKS

14. __________ Daily and to verify calibration, test a Negative Control (item 11) and Positive Control (item 12), for at least one product
    1. __________ Test negative control beginning from item 15a or with solid and semi-solid dairy products from 15a2
    2. __________ Verify Negative Control calibration
       1. __________ Fluid white milk test, unflavored and flavored assay value < 5 mU/L with luminometer or < 44 mU/L (<88 mU/L flavored and unflavored) with 6600 and C2Soft
       2. __________ Solid and semi-solid dairy products < 350 mU/kg or less than or equal to the control point
    3. __________ Verify Positive Control calibration
       1. __________ Positive Control (450 mU/L) rehydrated with fluid white milk, flavored and unflavored fluid dairy products, must be 300-585 mU/L or 350mU/L calibrator must be 247-453 mU/L
       2. __________ Solid and semi-solid dairy products, within ± 30% of 350 mU/kg or the control point

    TEST PROCEDURE

15. __________ Procedure
    1. __________ Prepare sample
       1. __________ For fluid white milks, unflavored and flavored, invert filled retail container 25 times, each inversion a full cycle down and up, shake or vortex negative control
       2. __________ For solid/semisolid dairy products (not including controls, item 11b & 12) add 1 part to 4 parts Diluent AP
          1. __________ Mix or knead until homogeneous
          2. __________ Centrifuge for 3 minutes
          3. __________ Use liquid extract in item 15c
    2. __________ Dispense 100 µL of Reagent AP into test tubes or microtubes
    3. __________ Dispense 100 µL of the prepared sample (item 15a) or mixed controls (items 11d & 12) just above the Reagent AP and immediately mix
       1. __________ Use a new pipet tip for each sample, place pipet tip in sample or prepared control (no more than 1 cm), draw up and remove tip from sample/control, expel once to pre-wet tip
       2. __________ Draw sample or control into pipet tip, touch off to side of container
       3. __________ Holding pipet 90° to lab bench at eye level, dry exterior of tip (if necessary) by wiping from the pipet toward the tip, be careful not to touch end of tip
       4. __________ Dispel 100 µL sample directly above surface of Reagent AP (do not dispense down side of test tube or microtube)
       5. __________ Depress plunger several times to completely expel sample
       6. __________ Mix test tubes or microtubes with a back-and- forth motion for 10 seconds, use a vortex mixer if available
    4. __________ Place the test tube/microtube in the 35±1C incubator for 3 minutes
    5. __________ Within 10 seconds after incubation add 1 mL of room temperature (18-24C) Stopping Solution
    6. __________ Remove test tubes/microtubes from incubator, cap and shake each vigorously or vortex for 10 seconds
    7. __________ Place test tube/microtube into analyzer within 3 minutes, tubes held at room temperature (Note: stability of count may be stabilized by placing tubes/microtubes in a room temperature bath
       1. __________ 6600 with C2Soft software
          1. __________ Select appropriate assay type
          2. __________ Enter ID of sample and press enter
          3. __________ Load sample in analyzer and press enter
          4. __________ In 5 seconds RLU reading will be displayed, mU/L value will appear in results or pop-up window
          5. __________ For solid/semisolid dairy products, sample RLU will be compared to control point
       2. __________ Luminometer/Luminator
          1. __________ Select AP Assay (or customized channel)
          2. __________ Press Start or Enter
          3. __________ In 5 seconds mU/L reading will be displayed
    8. __________ Counting of all test tubes/microtubes must be completed in 3 minutes
    9. __________ Samples with = 350 mU/L (category 4) (or for solid/ semisolid dairy products, values greater than or equal to control point) of ALP activity are suspect positive containing about 0.1% (v/v) or more raw milk and must be tested for microbial, and reactivated phosphatase (items 16 & 17)

    CONFIRMATION

16. __________ Microbial Phosphatase
    1. __________ Heat 1.0 mL of suspect sample at 63±1C for 30 minutes, stirring or mixing every 10 minutes
       1. __________ For semisolid/solid dairy products dilute 1.0 g suspect sample with 4.0 mL diluent AP, mix or knead until homogeneous
       2. __________ If fat content is > 10%, heat at 66±1C for 30 minutes
    2. __________ Cool sample rapidly to 0-4.4C in an ice bath
    3. __________ Test positive and negative controls following item 15
    4. __________ Test heated sample and unheated sample (original sample) following item 15 (semisolid/solid dairy products begin at item 15b)
    5. __________ Interpretation
       1. __________ Controls test as specified in item 14
       2. __________ If heated and unheated sample have equal activity (±30%, mU/L or RLU) the sample is regarded Not Found for residual phosphatase, the activity originally measured is microbial
       3. __________ If the heated sample is more than 30% below unheated sample (mU/L or RLU), the sample contains milk phosphatase activity, either residual or reactivated
17. __________ Reactivated Phosphatase
    1. __________ Magnesium acetate solution commercially available
    2. __________ Or, prepared in laboratory
       1. __________ Dissolve 35.4 g of magnesium acetate tetra- hydrate, Mg (C₂H₃O₂)₂·4H₂0 in 25 mL MS water, warming slightly to aid dissolution
       2. __________ Pour solution into 100 mL volumetric flask, rinse original container several times and add rinses to flask
       3. __________ After cooling to room temperature, make up to 100 mL (stable for 1 year at 0-4.4C)
    3. __________ Procedure
       1. __________ Label separate test tubes as "Blank" and "Test"
       2. __________ Add a 5.0 mL aliquot of sample (unheated, original sample not prepared as in 15a) to each test tube
          1. __________ For semisolid/solid dairy products, combine 2.5 g product and 10.0 mL Diluent AP
          2. __________ Mix or knead until homogeneous, and add 5.0 mL to clean test tubes labeled "Blank" and "Test"
       3. __________ Add 0.1 mL MS water to the sample labeled "Blank", and 0.1 mL magnesium acetate solution to the sample labeled "Test"
       4. __________ Cap tubes and heat both aliquots for 1 hr at 34±1C
       5. __________ Remove samples from water bath and cool rapidly to 0-4.4C in an ice bath
       6. __________ Dilute 1 mL of sample containing magnesium acetate (Test) with 5 mL (1:6 dilution) of negative control product (item 11), label tube as "Diluted Test"
       7. __________ Test undiluted sample containing no magnesium acetate (Blank) and diluted sample containing magnesium acetate (Diluted Test) for phosphatase activity following item 15 (semisolid/solid dairy products begin at item 15b)
    4. __________ Interpretation
       1. __________ If the diluted aliquot containing magnesium acetate (Diluted Test) has equal (±30%) or greater phosphatase activity than the undiluted aliquot containing no magnesium (Blank), the sample is regarded as Not Found 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 (Diluted Test) contains less (30% below or less) activity 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

18. __________ Report as:
    1. __________ Residual phosphatase Not Found (NF)
       1. __________ Report as < 44 mU/L (< 88 mU/L unflavored and flavored liquid dairy products or < 350 mU/kg solid/semisolid dairy products)
    2. __________ Residual phosphatase Positive
       1. __________ Microbial and reactivatable phosphatase are not demonstrated
       2. __________ Suspect positives greater than or equal (±30%) to 350 mU/L, category 4 or greater than the control point must be tested for microbial and reactivated phosphatase (items 16 and 17)
       3. __________ Report mU/L values, mU/L range or greater than control point when equal to or greater than 44 mU/L fluid white milk, 88 mU/L un- flavored and flavored liquid dairy products or 350 mU/kg semisolid/solid dairy products
    3. __________ Report as Not Found for residual phosphatase if:
       1. __________ If microbial phosphatase present
       2. __________ If reactivated phosphatase present
       3. __________ If there is documentation to show that the product was treated such that reactivated phosphatase may be present