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

DETECTION OF INHIBITORY SUBSTANCES IN MILK
DELVOTEST 5 PACK/Visual & DelvoScan Reader
For Raw and Finished Cow and Goat Milk
[Unless otherwise stated all tolerances are ±5%]

Sample

1. __________ Laboratory Requirements (see CP, item 33 & 34), except
   1. __________ For Appendix N testing, see Appendix N General Requirements form, items 9-14

   APPARATUS

2. __________ Cultural Procedures, items 1-23, except
   1. __________ For Appendix N testing, see Appendix N General Requirements form, items 1-7
3. __________ Dry incubator and/or water bath 64±2C
4. __________ Heating block and/or water bath thermostatically controlled at 82±2C, for confirmation
5. __________ Fixed Volume or electronic 100 µL pipettor (_______________) with appropriate tips
6. __________ Forceps, Tablet Dispenser, or equivalent
7. __________ Test tubes, 10 mL or greater for beta-lactam confirmation (optional)
8. __________Timer
9. __________ DelvoScan Reader (optional) (approved for white milk only)
   1. __________ Software version
   2. __________ Scanner (_____________________)
      1. __________ Instrument calibrated once every 30 days
      2. __________ Latest calibration date
   3. __________ Computer with Windows operating system
   4. __________ Printer
   5. __________ Kodak Q-60 5x7" color reference photo card
   6. __________ Black/Dark cloth for scanner background

   MATERIALS

10. __________ See Cultural Procedures, items 24-32
11. __________ Delvotest P 5 Pack Kit
    1. __________ Kit: Lot # _______ Exp. Date _______
    2. __________ Bottle of nutrient tablets Lot # _______
    3. __________ Store kits at 0-15C
    4. __________ Opened bottles of nutrient tablets at room temperature Date Opened: _________
    5. __________ Run a positive control (item 13) and negative control (item 14) with each new lot of kits, give appropriate reactions, records maintained
12. __________ Beta-lactamase, 10,000,000 IU/mL (not required if beta- lactamase is not used for confirmation)
    1. __________ Stored as per manufacturer's instructions
    2. __________ Do not use beyond expiration date
       1. __________ Mfg. _______ Lot No. _______ Exp. Date ______
    3. __________ Test each lot for suitability, add beta-lactamase to 5.0 ppb positive control (item 13) and add to one (1) well, beta-lactamase neutralizes positive control; records maintained
13. __________ Commercial Standard, 5.0 ppb Penicillin G Positive Control
    1. __________ Store according to label instructions

       __________Mfg. _______ Lot # _______ Exp. Date _______

    2. __________ Rehydrate as per manufacturer's instructions
    3. __________ Test for suitability each time prepared, add to one (1) well, must produce appropriate reaction (purple) or DelvoScan positive (< POS>); records maintained
    4. __________ Store solution at 0 4.4C for no more than 2 days
    5. __________ Or, distribute sufficient amount in small containers, seal and freeze at -15C or below in non-frost-free freezer (or in a small styrofoam box, placed in center of frost-free freezer) for no more than 2 months

       __________Date prep. ________ Lab Exp. Date: ________

14. __________ Negative Control
    1. __________ Inhibitor Free Milk (fluid milk product with milkfat 0.00 to 3.5%, total solids < 13%)
       1. __________ Test for suitability each time prepared, add to one (1) well, must produce appropriate reaction (yellow) or DelvoScan negative (< NEG >); records maintained

    TECHNIQUE

15. __________ Test Procedure
    1. __________ Identify samples
    2. __________ Use one negative and one positive control with each set of samples run (£ 94 samples)
    3. __________ Depending on the number of samples to be tested, take out a sufficient number of whole multiplates and/or cut off the number of wells needed
    4. __________ Remove aluminum top foil and using forceps or tablet dispenser add one nutrient tablet to each test well
    5. __________ Mix samples/controls by shaking 25 times in 7 sec through 1 ft arc or vortex, use within 3 minutes
    6. __________ Pipetting procedure
       1. __________ With tip securely fastened to the end of the pipettor and the pipettor in a vertical position, depress the plunger to the first stop or for electronic pipettors as per manufacturer
       2. __________ With the plunger still depressed, insert tip 1 cm below surface of the sample (avoid foam)
       3. __________ Release plunger slowly allowing tip to fill (quickly releasing the plunger will cause inaccurate filling and may foul pipettor)
       4. __________ Remove tip from sample and depress plunger to empty tip back into sample
       5. __________ If blow out type pipettor used, press the plunger to the second stop to completely empty the tip
       6. __________ Press plunger to first stop and repeat 2 and 3 above
       7. __________ Touch off to a dry spot on the inside of the sample container
    7. __________ Add 100 µL of mixed sample/control to a test well
    8. __________ Change pipettor tips for each sample and control
    9. __________ Close used wells carefully with sealing sheets, provided with test kit
    10. __________ Place sealed multiplates and/or blocks in pre-warmed dry incubator or water bath and incubate at 64±2C for the time period specified by the manufacturer. Time is approximate and test is complete when controls give proper color reactions
    11. __________ Remove from dry incubator or water bath and visually read test result from the bottom side, see item 17b for interpretation
    12. __________ Optionally read multiplates with DelvoScan Reader (Multiplates from water bath must be dried off prior further handling)
        1. __________ Start DelvoScan software by double click on icon
           1. __________ Select test (Delvo P 5 pack) and press next
           2. __________ Input operator, sample and test kit data
              1. __________ Enter number of test on scan-bed
              2. __________ Enter test kit lot number
              3. __________ Enter sample data
           3. __________ Press 'SCAN' and results will be displayed
           4. __________ Press 'PRINT' to obtain hard copy result data
    13. __________ Samples with purple color on all or part of solid medium, or that read as DelvoScan positive (< POS >) must be promptly confirmed
    14. __________ Samples that are yellow after incubation or that read as DelvoScan negative (< NEG >), inhibitor not detected (Not Found)
16. __________ After each reading, remove cuvette and close door immediately
    1. __________ indent a
       1. __________ Prepare and label tubes with 5 mL of each suspect sample
       2. __________ Prepare and label a tube with 5 mL of inhibitor free milk (item 14)
       3. __________ Prepare and label a tube with 5 mL of positive control milk
       4. __________ Heat all tubes to 82±2C for 2 minutes (TC required)
       5. __________ Remove and cool rapidly in an ice bath to room temperature
       6. __________ Use of beta-lactamase (optional by State Regulatory Agency)
          1. __________ Prepare and label two tubes with 5 mL of each suspect sample and two tubes for the positive and negative controls
          2. __________ Heat all tubes to 82±2C for 2 minutes (TC required)
          3. __________ Remove and cool rapidly in an ice bath to room temperature
          4. __________ Add 200 µL (2 x 100 µL) of beta-lactamase to one tube of each sample and control
          5. __________ Agitate, shake or vortex, to thoroughly mix tubes and let stand 15 minutes at room temperature
       7. __________ Cut off enough wells for all sample and control tubes
          1. __________ Or, alternatively Delvotest P ampoules may be used (must be certified for this procedure)
       8. __________ Remove top foil and add one (1) nutrient tablet to each test well
       9. __________ Vortex tubes and add 100 µL of mixed sample/control to a test well (as per 15f1-7 above), identify samples, repeat for all samples and controls
       10. __________ Change pipettor tips for each sample and control
       11. __________ Close wells carefully with sealing strips, enclosed in test kit
       12. __________ Place sealed blocks in pre-warmed dry incubator or water bath and incubate at 64±2C for the time period specified by the manufacturer. Time is approximate and test is complete when controls give proper color reactions
       13. __________ Remove from dry incubator or water bath and visually read test result from the bottom side
       14. __________ Optionally read multiplates with DelvoScan Reader (Multiplates from water bath must be dried off prior further handling)
           1. __________ Start DelvoScan software by double click on icon
              1. __________ Select test (Delvo P 5 pack) and press next
              2. __________ Input operator, sample and test kit data
                 1. __________ Enter number of test on scan-bed
                 2. __________ Enter test kit lot number
                 3. __________ Enter sample data
              3. __________ Press 'SCAN' and results will be displayed
              4. __________ Press
       15. __________ Record the color reactions or DelvoScan test result of all samples and controls
       16. __________ Controls give appropriate reactions/colors, if not repeat test
    2. __________ Interpretation of Confirmation Tests
       1. __________ Wells that are yellow or yellow/purple or that read as DelvoScan negative (< NEG>) after incubation, inhibitor not detected (Not Found)
       2. __________ Wells that are purple or that read as DelvoScan positive (< POS>) after incubation, inhibitor present (Positive)
       3. __________ Interpretation of optional beta-lactamase test:
          1. __________ If the untreated milk sample is yellow or yellow/purple, or DelvoScan negative (< NEG>) and the corresponding beta-lactamase treated milk sample is yellow or yellow/purple, or DelvoScan negative (< NEG>), inhibitor not Detected (Not Found)
          2. __________ If the untreated milk sample is purple or DelvoScan positive (< POS>) and the corresponding beta-lactamase treated milk sample is yellow or yellow/purple, or DelvoScan negative (< NEG>), sample is Positive for beta-lactam
          3. __________ If the untreated milk sample is purple or DelvoScan positive (< POS>) and the corresponding beta-lactamase treated milk sample is also purple or DelvoScan positive (< POS>), sample is Positive for inhibitor (non-beta-lactam), report to State regulatory agency
          4. __________ If the untreated milk sample is yellow or yellow/purple or DelvoScan negative (< NEG>) and the corresponding beta-lactamase treated milk sample is purple or DelvoScan positive (< POS>), test is invalid, repeat test
    3. __________ Confirmation of Appendix N samples, see Appendix N General Requirements form item 12-13, perform confirmation as in items 15a-n above (use of beta-Lactamase required) and interpret as in item 16b3 above
17. __________ Recording and Reporting (for Appendix N also see Appendix N General Requirements form, item 14)
    1. __________ Record test performed, interpretation of unknowns (samples) and controls
    2. __________ Report presence of inhibitor only for heated milk samples
    3. __________ If inhibitor is not detected report as Not Found
    4. __________ Report presence of inhibitor as Positive (+) or Positive for beta-lactam (if confirmed with beta- lactamase or item 16c)
    5. __________ If inhibitor is present, plate counts cannot be reported