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

DETECTION OF INHIBITORY SUBSTANCES IN MILK
Bacillus stearothermophilus Disc Assay, Charm Tablet Method For Raw and Finished Cow and Goat Milk
[Unless otherwise stated all tolerances are ±5%]

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. __________See Cultural Procedures, items 1-23, except
   1. __________ For Appendix N testing, see Appendix N General Requirements form, items 1-7
3. __________Fixed volume or electronic pipettors: 90 µL and 500 µL (optionally 50 µL) with appropriate tips
4. __________ Forceps, Fine Points, Stainless Steel
5. __________Water Bath and/or heating block, Thermostatically Controlled at 64±2C, and 82±2C
6. __________ Incubator 64±2C
7. __________ Vernier, Dial or Digital Calipers, metal (readable to 0.1 mm)
8. __________Stirring hot plate/stirring bar (optional)
9. __________100 mL Class A graduate cylinder
10. __________13 x 100 mm test tubes
11. __________250 mL Erlenmeyer flasks

    MATERIALS

12. __________See Cultural Procedures, items 24-3
13. __________Filter Paper Discs, Blank, Unimpregnated, Non-sterile
    (Brand: ___________ Lot#: ___________)
    1. __________ High absorbability, diameter 12.7±0.1 mm
14. __________Charm PM Indicator Agar
    1. __________ Do Not Autoclave - (see plate preparation, item 19 below)
       
15. __________Charm Beta-lactamase tablet or liquid concentrate (not required if beta-lactamase is not used for confirmation)
    1. __________ Stored at -15C or below

       __________Lot #: ________ Exp. date ________

    2. __________ Do not use beyond expiration date

       __________ Mfg. _____ Lot #  _____  Exp. Date  _____

    3. __________ Reconstitute freeze dried concentrate as per manufacturer instructions
       1. __________ Liquid concentrate stored at -15C or below in a non- frost-free freezer or in a styrofoam box in a frost- free freezer and used within 2 Weeks
    4. __________ Test each lot for suitability, add beta-lactamase to 5.0 ppb positive control (item 16) and add to one (1) disc, beta-lactamase neutralizes zone produced by positive control; records maintained
       

       __________Zone size: ________

16. __________Charm 5.0 ppb Penicillin G Standard Positive Control
    1. Store according to label directions

       Lot#: ________ Exp. date __________________

    2. Store and rehydrate according to label instructions

       __________

    3. Test for suitability each time prepared, add to one (1) disc, must produce zone 16 - 20 mm; records maintained

       __________Avg. Zone Size: _______

    4. Use rehydrated standard within 48 hours if refrigerated
    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: ________

17. __________ Negative Control
    1. __________ Negative Control
       1. __________ Temperature checked by placing standardized thermometer in tube containing liquid (bulb submersed) in heating unit, records maintained
          

          __________Lot#: ________ Exp. date ________

       2. __________ Use rehydrated negative control within 72 hours if refrigerated
          

          __________Date prep. ________ Lab Exp. Date: ________

       3. __________ 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: ________

    2. __________ Inhibitor Free Milk (fluid milk product with milkfat 0.00 to 3.5%, total solids < 13%)
       1. __________ Test for suitability, add to one (1) disc, produces no zone; records maintained
          

          __________Zone size:

18. __________Charm Spore Tablets
    1. __________ Bacillus stearothermophilus tablets containing 100,000,000 (±10 million) spores per tablet
       

       __________Lot#: ________ Exp. date ________

    ASSAY PLATE
19. __________Preparation of Plate
    1. Prepare agar according to label, 3.2g/95 mL H 2 O, bring agar to a boil
    2. Promptly cool to 64±2C (Temperature Control [TC] used)
       1. __________ Optionally, temperature may be determined by inserting a dedicated thermometer (not used for any other purpose) directly into test agar
    3. Add 1 spore (white) tablet to 5 mL deionized water in 13 x 100 mm test tube
    4. Shake test tube 25 times through 1 foot arc in 7 seconds, or vortex for 10 seconds and let settle 1 minute
    5. __________ Repeat item d
    6. __________Decant spore mixture into agar tempered to 64±2C leaving residue on bottom of tube (avoid pouring mixture down side of flask)
    7. __________ Mix agar well for 1.5 minutes but avoid incorporation of air bubbles, optionally use stirring bar on magnetic stir plate
    8. __________Constantly mix remaining agar during preparation of plates
    9. __________Pipet 6 mL inoculated agar into plastic petri dish (15 x 100 mm, bottom plate inner diameter 86.1 - 87.0mm)
    10. __________Or, appropriate amount of agar into other size [(Dcm) 2 6/8.65 2 = V]; Dcm = inner diameter of plate in centimeters; V = volume (mL) of agar to add in dishes, records maintained
    11. __________Plates have flat bottoms and do not buckle after agar has been added, plates observed before and after preparation for suitability
    12. __________Swirl plate gently on level surface to evenly distribute agar
    13. __________Allow agar to solidify on a level surface for 15 minutes with lid ajar
    14. Use within 5 days, if stored at 0-4.4C in airtight container
        

        __________Date prep. ________ Lab Exp. Date: ________

    TECHNIQUE

20. __________ Laboratory Procedure, Screening
    1. __________Label bottom of plates prior to adding discs, use template as a guide to assure discs will be placed at least 10 mm from the petri dish wall and from other discs
    2. __________Each test plate may contain a maximum of 5 test sample discs plus a positive control and negative control disc (7 discs total as per template, for larger plates more discs may be placed, maintain comparable spacing)
    3. Mix sample/control by shaking 25 times in 7 se
    4. Samples/controls (maintained at 0-4.4C) must be tested within 3 min of agitation
    5. __________ 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. Press plunger to first stop and repeat 2 and 3 above
       6. __________ Touch off to a dry spot on the sample container
       7. Using clean, dry forceps, remove a disc from its container and place the disc (using a template as a guide) on the agar surface of the inhibitor plate
       8. Press the disc gently with the forceps to insure good contact and then fill disc immediately
       9. With the pipettor in a vertical position and the tip about 5 mm above the center of the disc depress the plunger to the first stop in such a way as to get a rapid drop-wise release of the sample
       10. __________ Sample not applied too slowly or quickly (streamed)
       11. Allow a second or two for the milk to absorb into the disc14. Repeat the above until all samples have been done
       12. __________ 12. If blow out type pipettor used, press the plunger to the second stop to completely empty the tip
       13. __________Gently touch off the tip on an area of the disc away from where the sample was deposited
       14. __________ Repeat the above until all samples have been done
    6. __________ Place a positive control disc containing 5.0 ppb penicillin G and a negative control disc on each test plate using above procedure
       1. __________ Vary the location of positive control discs in a series of test plates, i.e. center or outside of the plate
    7. __________ Invert plate(s) and incubate at 64±2C until well defined zones of inhibition are obtained (usually 2.5 - 3 hr) with the 5.0 ppb positive control(s), plate(s) should be yellow
    8. __________ Remove plates from incubator and allow to cool on a level surface for 2 minutes (do not remove lid before plates are cooled)
    9. __________ Examine positive control zone. A valid test requires a positive control zone of 16-20 mm. If zone size is < 16 or > 20 mm the test must be repeated
    10. __________ Examine plate for zones of inhibition surrounding the test discs, zones of > 12.7 mm indicates presence of inhibitory substances
    11. __________ Measure zones of inhibition by using calipers
        1. __________ Use the inside diameter points (smaller points)
        2. __________ Anchor one point in the bottom of the plate at the edge of the zone and expand calipers until the other point rests on the other edge
        3. __________ Read calipers and report zone size to the nearest 0.1 mm
    12. __________ Zones of £ 12.7 mm are read as no zone
    13. __________ Zones > 12.7 mm must be promptly confirmed to report as positive for inhibitor or beta-lactam residue
21. __________ Laboratory Procedure, Confirmation
    1. __________ Inhibitor confirmation and optional beta-lactamase confirmation
       1. __________ Heat a 0.5 mL (500 µL) portion of each suspect sample to 82±2C for 2 minutes (TC required)
       2. __________ Cool promptly in ice bath to room temperature
       3. __________Label bottom of plates prior to adding discs
       4. __________Vortex for 10 seconds, use within 3 minutes
       5. __________Add 90 µL of heated samples to a disc on plate as in item 20e
       6. __________ Use of beta-lactamase ( optional by State Regulatory Agency)
          1. __________Add one beta-lactamase (red) tablet to each of the heated samples and mix samples as in item 21a4
          2. __________ Let particulates settle for 1 minute then add 90 µL to a disc on plate (Avoid clogging pipet tip with particulates by pipetting from top of samples)
          3. __________Or, alternatively add 50 µL of beta-lactamase liquid concentrate (item 15c), mix samples, wait 1 minutes then add 90 µL to a disc on plate
       7. __________ Proceed as in items 20f-m
    2. __________ Or, use 6 inch partial immersion thermometer placed directly into small thermometer well in middle of heating unit, records maintained
       1. Inhibitor present
          
          1. __________ Zones ³ 16mm of the heat treated 21a5 sample is Positive for inhibitor
             
       2. __________ Beta-lactam present (optional)
          1. A zone around the disc containing the heat treated milk sample (21a5) but no zone around the disc containing beta-lactamase 21a6c, treated milk sample, sample is Positive for beta-lactam
          2. __________ Zones around the heat treated sample (21a5) of equal size, or < 4 mm greater, than beta-lactamase treated sample (21a6) is Positive for inhibitor
          3. __________ Zones around both the beta-lactamase treated milk sample (21a6) and the heat treated milk sample discs (21a5), and , the zone around the beta-lactamase treated milk sample disc (21a6) is ³ 4 mm smaller than the zone around the heat treated milk sample disc (21a5) [ex. beta- lactamase = 14 mm, untreated = 18 mm], sample is Positive for beta-lactam and inhibitor
    3. __________ Confirmation of Appendix N samples , see Appendix N General Requirements form item 12-13, perform confirmation as in items 21a1-7 above ( use of beta-lactamase required ) and interpret as in item 21b2 above
22. __________ Recording and Reporting (for Appendix N also see Appendix N General Requirements form, item 14)

1. __________Record numeric values for all measurable zone sizes for samples and controls (screen and confirmation), if no zone is observed record as No Zone (NZ)
2. __________Report presence of inhibitor only from heated milk samples
3. __________ Report sample as Positive for inhibitor (if heat only used 21a1-5) or Positive for beta-lactam where demonstrated (21a6 or 21c), and zone size ³ 16 mm
4. __________If a non-beta-lactam inhibitor is demonstrated (21a6 or 21c), report as Positive for inhibitor when zone size ³ 16 mm, report to State Regulatory Agency
5. __________ If both beta-lactam and non-beta-lactam inhibitors are demonstrated (21a1-7 or 21c), report test as Positive for beta-lactam and inhibitor when zone size ³ 16 mm, report to State Regulatory Agency
6. __________Report numeric values for all measurable zone sizes for samples and controls
7. __________ Report when zone size > 12.7 and < 16 mm as positive but Below Actionable Level
8. __________Report absence of inhibitor (no zone) as Not Found
9. __________If any inhibitor is present, i.e., zone > 12.7 mm, plate counts cannot be reported