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U.S. Department of Health and Human Services

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M-I-05-3b 2400h-2 Electronic Somatic Cell Count Bentley Somacount 150/300/500

 

June 2, 2005
 

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


(Unless otherwise stated all tolerances ±5%)

  1. __________ Laboratory requirements (see CP, item 33 & 34)
    1. __________ Un-preserved samples may be run up to 72 hours after initial collection
    2. __________ Samples may be run 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. __________ Cultural procedures, items 1-5
  3. __________ Electronic Somatic Cell Counter
    1. __________Bentley SomacountTM 150 _____
    2. __________ Bentley SomacountTM 300 _____
    3. __________ Bentley SomacountTM 500 _____
  4. __________ Water bath
    1. __________Circulating and thermostatically controlled to 37-42C
    REAGENTS
  5. __________ Stock Dye/Buffer Solution
    1. __________Dissolve 80g of tripotassium citrate monohydrate, (K3C6H5O7,H2O), 3.0g of citric acid monohydrate(C6H8O7,H2O), and 0.25g (1 tablet) of ethidium bromide (C21H20BrN3) in 750 mL of deionized (DI) or MS water. Heat to 40-60C and stir until totally dissolved (Caution: Ethidium bromide is TOXIC. When handling, avoid contact with skin and do not breathe dust.)
    2. __________Add 10 mL of neutral detergent, Triton X-100, and stir until totally dissolved. Adjust volume to 1 Liter with DI or MS water
    3. __________Store refrigerated (0-4.4C) in airtight, light- proof container for no longer than 90 days
    4. __________Container labeled with date prepared and expiration date

      __________ Date prep. _____ Exp. Date  _____

    WORKING SOLUTIONS
  6. __________ Dye/Buffer Solution
    1. __________ Dilute 1 part of Stock Dye/Buffer solution with 9 parts of DI or MS water
    2. __________ Protect from light and use within 21 days
    3. __________Date prep. ________ Exp. Date ________
  7. __________ Rinse Solutions
    1. __________Add 20 mL of alkaline detergent, RBS-35, per liter of DI or MS water and mix
    2. __________Use within 7 days
    3. __________Date prep. ________ Exp. Date ________
  8. __________ Optionally use manufacturer's reagent kits andinstructions
  9. __________All dye/buffer and rinsing solutions labeled with date prepared and expiration date
    START UP
  10. __________Cell Counter
    1. __________Check that the amount of dye/buffer solution (item 6)and rinse solution (item 7) in the supply containers is of sufficient volume for the number of samples to be run
    2. __________Solutions not to be used beyond expiration date(s)
    3. __________Turn on computer and instrument, wait 20 minutes before proceeding
    4. __________Laser power > 0.25 mW
    5. __________|PMT voltage| > 10 mV
    6. __________Coil temperature between 67-73C
    7. __________Run DI OR MS water at least 3 times; reading must be zero (0) on every test
    8. __________IF ANY PARAMETERS ARE OUT OF TOLERANCE, CORRECT BEFORE PROCEEDING
    9. __________Records maintained on all parameters each time instrument is used
  11. __________Milk Standards
    1. __________Commercially prepared: ____________________
      Lot# ______ Date Rcd. ______
      1. __________Four standards 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: ____________________
      Lot# ______ Exp. Date ______ Date Rcd. ______
      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 (BronopolTM)
      3. __________Standards cannot be preserved with formalin
      4. __________Prepare 4 standards in ranges 100K-200K, 300K-500K, 600K-800K and 900K-1.2M, use 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 11a 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
    PROCEDURES
  12. __________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. __________Mix by inverting at least 2x, test standards within 3 minutes
    3. __________Run the 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 11) 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
  13. __________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. __________Mix by inverting at least 2x, test samples within 3 minutes
    3. __________Samples must be tested within 10 minutes after being removed from waterbath
    4. __________Samples must not be reused and must be discarded after use
    5. __________Record number of cells counted for each sample
  14. __________With continuous operation:
    1. __________Run a standard or optionally a control/sample (item 11d) 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 control 3x
    3. __________Run zero control (item 10g) hourly
    4. __________Maintain records
  15. __________Routine maintenance
    1. __________Perform as described in operating manual
    2. __________Maintain records
    REPORTS
  16. __________Computing and Reporting of 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 six 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 second digit is odd round up, raising the second digit by 1 (odd up, 235 to 240)
      2. __________When 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 count as < 100,000 ESCC/mL