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U.S. Department of Health and Human Services

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M-I-05-3: 2400h ELECTRONIC SOMATIC CELL COUNT Foss 90

 

June 2, 2005
 

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

[Unless otherwise stated all tolerances are ±5%]

  1. __________ Laboratory Requirements (see CP, item 33 & 34)
    1. __________ Un-preserved samples may be run from 24 to 72 hours after initial collection
    2. __________ Samples may be run from 8 hours up to 7 days after initial collection if preserved with 0.02% 2-bromo- 2-nitropropane-1,3-diol (BronopolTM)or 0.05% potassium dichromate (K2Cr2O7)
    3. __________ Comparative test with DMSCC
      1. __________ Performed by each analyst performing ESCC test
      2. __________ Test 4 samples (100K-200K, 300K-500K, 600K-800K and 900K-1.2M) in triplicate for both DMSCC (three separate smears each) and ESCC (three separate sub-samples each, do not read same sample three times)
      3. __________ Results must be shown to be acceptable prior to official testing by analyst performing comparison, i.e. analyst is not certified until found acceptable. (co-requisite for certification)
      4. __________ Copy of comparison and results in QC record (or easily accessible file in laboratory)
    4. __________ Analysts certified in DMSCC

      APPARATUS
  2. __________ See Cultural Procedures, items 1–5
  3. __________ Fossmatic 90 Electronic Somatic Cell Counter
  4. __________ Pipettor, fixed volume or electronic (______________________)
    1. __________ Calibrated to deliver 500 µL milk (see CP item 6e)
    2. __________ Records maintained
  5. __________ Pipettor Tips
    1. __________ Disposable, replace for each sample
    2. __________ Reusable
      1. __________ Rinse in 40C deionized (DI) or MS water
      2. __________ Rinse in sample more than 1 time
      3. __________ Do not use for more than 25 samples
  6. __________ Water Bath
    1. __________ Circulating and thermostatically controlled to 37–42C
    REAGENTS
  7. __________ Stock Dye Solution, 0.1% Ethidium Bromide (caution TOXIC, use gloves when handling and do not breath dust)
    1. __________ Dissolve 1.0g ethidium bromide (C21H20BrN3) in 1 liter DI or MS water by heating to 40-60C
    2. __________ Store in light-proof, air-tight bottle no more than 60 days
    3. __________ Date prep. ________ Exp. Date ________
  8. __________All stock dye/buffer and detergent solutions labeled with date prepared and expiration date
    1. __________ Dissolve 10 mL Triton X-100 in 1 liter DI or MS water by heating to 60C.
    2. __________ Store in air-tight container no more than 25 days
    3. __________ Date prep. ________ Exp. Date ________
  9. __________ Dye/Buffer Solution
    1. __________ Dissolve 51.0g KH phthalate and 13.75g KOH in 10 L DI or MS water by heating to 40–60C
    2. __________ Add 150 mL 1% Triton X-100 (item 8), store less than 7 days in airtight container
    3. __________ Date prep. ________ Exp. Date ________
  10. __________ Ammonium Hydroxide (NH4OH) Solution, Reagent Grade, 25%
  11. __________ All stock dye/buffer/rinsing solutions labeled with date prepared and expiration date

    WORKING SOLUTIONS
  12. __________ Working Dye Solution/Zero Control (used within 7 days)
    1. __________ Dilute 26 mL stock dye solution (item 7a) to 2.5 liter with stock buffer solution (item 9b2)
    2. __________ Date prep. ________ Exp. Date ________
  13. __________ Cell Counter
    1. __________ Add 10 mL stock rinsing solution (item 8) to 25 mL of 25% NH4OH and dilute to 10 liters with DI or MS water
    2. __________ Date prep. ________ Exp. Date ________
  14. __________ Optionally use manufacturer's reagent kits and instructions specific for each instrument

    START UP
  15. __________ All working dye and rinsing solutions labeled with date prepared and expiration date
  16. __________ Cell Counter
    1. __________ Assure adequate volume of working solutions, not used beyond expiration date(s)
    2. __________ Turn on power and cycle at least six times__________
    3. Blind count
    4. __________ Vacuum pressure setting minimum of -40 KPa
    5. __________ Dispenser filling time 4–5 seconds
    6. __________ Intake filling time 3–4 seconds
    7. __________ IF ANY ABOVE PARAMETERS ARE WRONG, CORRECT BEFORE PROCEEDING
    8. __________ Records maintained on all parameters
  17. __________ Milk Standards
    1. __________ Commercially prepared: ____________________
      Lot# ______ Date Rcd. ______
      1. __________ Four samples in ranges 100K–200K, 300K–500K, 600K– 800K and 900K–1.2M
      2. __________ Do DMSCC in triplicate on each standard in set and average counts, records maintained
      3. __________ DMSCC check performed in rotation by all certified analysts
      4. __________ Standards used within one week
    2. __________ Certified provider: ___________________
      1. __________ Four standards in ranges 100K–200K, 300K-500K, 600K– 800K and 900K–1.2M
      2. __________ Maintain copies of all provided DMSCC values
      3. __________ Measure and maintain records of temperature (0–7.2C) of standards as received
      4. __________ Maintain copies of all correspondence regarding problems
      5. __________ Standards used by manufacturer's expiration date
      __________
    3. Laboratory prepared (weekly)
      1. __________ Prepare from raw milk > 18 hours old preserved with 0.05% potassium dichromate (K2Cr2O7)
      2. __________ Or, preserved with 0.02% 2–brono-2–nitropropane- 1,3–diol (Bronopol™)
      3. __________ Standards cannot be preserved with formalin
      4. __________ Prepare 4 standards in ranges 100K–200K, 300K–500K, 600K–800K and 900K–1.2M, used within one week
        Date prep. ________ Exp. Date ________
      5. __________ Do DMSCC in triplicate on each standard prepared and average counts, records maintained
      6. __________ DMSCC check performed in rotation by all certified analysts
    4. __________ Hourly Control Sample (instrument drift check)
      1. __________ Use one of the standards (items 17a or b) in the 500–800K range, run in triplicate and determine average
      2. __________ Optionally, prepare sufficient control/sample 500–800K range, run in triplicate and determine average
    PROCEDURE
  18. __________ Testing Standards (each time instrument used)
    1. __________ Heat standards to 37–42C (using a temperature control) and read within 30 minutes of reaching temperature, used once and then discarded, i.e., do not re-use
    2. __________ Invert 10 times, pipet 500 µL into intake chamber within 3 min
    3. __________ Run standards in triplicate and average the counts for each level, records maintained
    4. __________ Each standard's average must be within 10% of the DMSCC (item 17) for that level, except within 15% for 100–200K standard, records maintained
    5. __________ Repeatability – a standard in the 300K to 800K range must have a coefficient of variation (CV) of 5% or less on 10 replicates (Refer to Operating Manual), records maintained
    6. __________ THESE PARAMETERS MUST BE ACHIEVED BEFORE PROCEEDING__________
  19. Testing Samples
    1. __________ Heat samples to 37–42C (using a temperature control) and read within 30 minutes of reaching temperature, samples must not be re-used and must be discarded after use
    2. __________ Invert 10 times, pipet 500 µL into intake chamber within 3 min
    3. __________Record number of cells counted for each sample
  20. __________ With continuous operation:
    1. __________ Run a standard or optionally a control/sample (item 17d) in the 500K to 800K range hourly, must be within 5% of the original established instrument average value (optionally, within 10% of original DMSCC average)
    2. __________ Run standard/control 3x
    3. __________ Run zero control (in item 12)
    4. __________ Maintain records
  21. __________ Routine maintenance
    1. __________ Perform as described in operating manual
    2. __________ Maintain records
    REPORTS
  22. __________ Computing and Reporting Counts
    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