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U.S. Department of Health and Human Services

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M-I-05-3: 2400h-4 ELECTRONIC SOMATIC CELL COUNT Somascope MKII

 

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 (Bronopol™) 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. __________ Somascope MKII manual _____
    2. __________ Somascope MKII automatic _____
  4. __________ Water Bath
    1. __________ Circulating and thermostatically controlled to 37-42C
    REAGENTS
  5. __________ Staining Kits
    Warning: Before using the chemicals for making up the staining solution, read the Material Safety Data Sheet (MSDS) provided in the appendix of the manual, wear safety glasses and latex gloves when handling the chemicals
    1. __________ One liter concentrate kit, makes 5 L staining solution when diluted mixed with deionized (DI) or MS water

      __________Date Rcd _____ Exp. Date _____

      1. __________ Store at 0-5C and protected from light
    2. __________ Twenty liter powder kit, consists of three different components, mixed with 20 L DI or MS water

      __________Date Rcd _____ Exp. Date _____

      1. __________ Staining Concentrate (powder)
        1. __________ Store powder 0 - 5C and protected from light
      2. __________ Staining Detergent (liquid)
        1. __________ Store at room-temperatu¢re protected from light
      3. __________ Staining Buffer (powder)
        1. __________ Store at room-temperature protected from light
    WORKING SOLUTIONS
  6. __________ Working Staining Solutions
    1. __________ One liter Concentrate Kit
      1. __________ Add 4 L of DI or MS water
      2. __________ Mix on a magnetic stirrer at room temperature to make working stain solution
    2. __________ Twenty liter Powder Kit
      1. __________ Dissolve the staining buffer (item 5b3) in about 18 L of DI or MS water and stir until the powder is fully dissolved
      2. __________ Add the Staining Detergent (item 5b2) to about one liter of warm (35 - 45C) DI or MS water and mix well (preferably with a magnetic stirrer) to dissolve the detergent
        1. __________ The detergent must be well dissolved, no powder residue visible (optical test)
      3. __________ Add the detergent solution (item 6b2) to the 18 L of staining buffer (item 6b1) and mix
      4. __________ Dissolve the Staining Concentrate (item 5.b.1.) in 3 mL of 35-45C DI or MS water
        1. __________ Mix until the powder is dissolved (optical test)
        2. __________ Keep the concentrate (powder and solution) protected from strong light during preparation
      5. __________ Add the dissolved concentrate to the approximately 19 L of buffer and add DI or MS water until the total volume is 20 L
    3. __________ Store the working staining solutions up to 2 months at 0 - 5C protected from light

      __________ Date prep. ________ Exp. Date ________

    4. __________ Use the staining solution at room-temperature

      __________ Date filled _____

      1. __________ The contents of the 5 liter staining container can be left at room temperature
      2. __________ The contents must be used within 7 days
      3. __________ Clean container once a month as per manufacture instructions
  7. __________ Working Rinse Solution
    1. __________ Detergent Container
      1. __________ Alkaline detergent; DECON 90, Contrad 70 or RBS 50
      2. __________ Fill the black detergent reservoir with about 50 mL of undiluted detergent in the Sample Preparation Unit
      3. __________ Check reservoir visually daily for volume and add more detergent as necessary
    2. __________ Water Container
      1. __________ Measure 5 mL of Triton X-100 and place in 100 mL of DI or MS water
      2. Mix the above solution until the Triton X-100 is completely dissolved
      3. __________ Dilute the 100 mL mixture above to 5 liters with room temperature DI or MS water
      4. __________ Mix well
      5. __________ Pour the above into the water container provided with the instrument
    START UP
  8. __________ Somatic Cell Counter
    1. __________ Check to see if the amount of staining solution (about 1.6 mL per sample), detergent and rinse solution (about 1 mL per sample) is of sufficient volume for the number of samples to be run
    2. __________ Solutions not be used beyond expiration date
    3. __________ Turn on the HBO lamp, Sample Preparation Unit and wait 15 minutes
    4. __________ Turn on the internal computer of the Somascope MKII
    5. __________ Put the peristaltic pump in operation position
    6. __________ Perform a clean sequence
    7. __________ Run at least 5 times with rinse solution (item 7b), last reading must be below 5
    8. __________ IF ANY PARAMETERS ARE OUT OF TOLERANCE, CORRECT BEFORE PROCEEDING
    9. __________ Records maintained on all parameters each time instrument is used
  9. __________ Milk Standards
    1. __________ Commercially prepared: ____________________

      __________ Lot# ______ Date 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 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 (Bronopol™)
      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
    PROCEDURE
  10. __________ 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, records maintained
    6. __________ THESE PARAMETERS MUST BE ACHIEVED BEFORE PROCEEDING
  11. __________ 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
  12. __________ With continuous operation:
    1. __________ Run a standard or optionally a control/sample (item 9d) 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 8g) hourly
    4. __________ Maintain records
  13. __________ Shut down procedure
    1. __________ Perform a clean cycle twice
    2. __________ Put instrument pipette in beaker of rinse solution
    3. __________ Release peristaltic pump
    4. __________ Switch off PC, SPU, internal computer of Somascope MKII, HBO-lamp in this order
  14. __________ Routine maintenance
    1. __________ Perform as described in operating manual
    2. __________ Maintain records
    REPORTS
  15. __________ 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