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U.S. Department of Health and Human Services

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M-I-05-3: 2400h-1 ELECTRONIC SOMATIC CELL COUNT Foss 250/300/360/400

 

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 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. __________ See Cultural Procedures, items 1-5
  3. __________ Automated Electronic Somatic Cell Counters
    1. __________ Fossomatic 250
    2. __________ Fossomatic 300
    3. __________ Fossomatic 360
    4. __________ Fossomatic 400
  4. __________ Water Bath
    1. __________ Circulating and thermostatically controlled to 37-42C
    REAGENTS
  5. __________ Stock Dye/Buffer Solution
    1. __________ Dissolve 2.5g (or number of tablets specified by manu- facturer) ethidium bromide (C21H20BrN3) in 1 liter deionized (DI) or MS water (caution TOXIC, use gloves when handling and do not breath dust), heat to 40-60C and mix to dissolve
    2. __________ Add 400g tripotassium citrate monohydrate (C6H5O7K3·H2O), 14.5g citric acid monohydrate (C6H8O7·H2O), and 4 liters DI or MS water, heat to 40-60C and mix to dissolve
    3. __________ Add dye and buffer solutions together and mix
    4. __________ Add 50 mL neutral detergent, Triton X-100 to mixture and stir until dissolved
    5. __________ Store refrigerated (0-4.4C) in airtight, light-proof container for no longer than 90 days
    6. __________ Date prep. ________ Exp. Date
  6. __________ Stock Detergent Solution
    1. __________ Dissolve 10 mL neutral detergent, Triton X-100 in 1 liter of DI or MS water and heat 40-60C to complete solution
    2. __________ Store refrigerated (0-4.4C) in airtight, container for no longer than 30 days
    3. __________ Date prep. ________ Exp. Date ________
  7. __________ Ammonium Hydroxide (NH 4 OH) Solution, Reagent Grade, 25%
  8. __________All stock dye/buffer and detergent solutions labeled with date prepared and expiration date

    WORKING SOLUTIONS
  9. __________ Dye/Buffer Solution
    1. __________ Dilute 1 L dye/buffer stock solution (item 5) with 9 L DI or MS water
    2. __________ Protect from light and use within 21 days
    3. __________ Date prep. ________ Exp. Date ________
    PERFORMANCE TESTING
  10. __________ Rinsing Solution (use within 7 days)
    1. __________ Add 10 mL of stock neutral detergent stock solution (item 6) and 25 mL of ammonium hydroxide solution (item 7) and suspend to 10 L with DI or MS water
    2. __________ Date prep. ________ Exp. Date ________
  11. __________ Optionally, use manufacturer's reagent kits and instructions specific for each instrument
  12. __________All working dye/buffer and rinsing solutions labeled with date prepared and expiration date

    START UP
  13. __________ Cell Counter
    1. __________ Check that the amount of dye/buffer solution (item 9) and rinsing (cleaning) solution (item 10) in instrument supply containers is of sufficient volume for the number of samples to be run
    2. __________ Solutions not used beyond expiration date(s)
    3. __________ Turn on power and cycle at least six times
    4. __________ Perform a zero check before starting any measurements, within acceptable limits, single counts up to 5 and mean up to 3
    5. __________ IF ANY ABOVE PARAMETERS ARE WRONG, CORRECT BEFORE PROCEEDING
    6. __________ Records maintained on all parameters each time instrument is used
    REPORTING AND RECORDS
  14. __________ 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-bromo-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 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 14a 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
  15. __________ 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, place in rack and put onto automatic track, run within 10 minutes__________
    3. Run the standards six times and average the counts for each level, records maintained
    4. __________ Each standard's average must be within 10% of the DMSCC (item 14) for that level, except within 15% for 100K-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 PROCEEDIN
  16. __________ 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, place in rack and put onto automatic track, run within 10 minutes of reaching the testing temperature__________
  17. __________ With continuous operation:
    1. __________ Run a standard or optionally a control/sample (item 14d) 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 6x
    3. __________Run zero control (item 13d)
    4. __________ Maintain records
  18. __________ Routine maintenance
    1. __________ Perform as described in operating manual
    2. __________ Maintain records
    REPORTS
  19. __________ 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