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U.S. Department of Health and Human Services

Food

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M-I-05-3d: 2400b-1 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%]


 

SAMPLES
  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