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

ELECTRONIC SOMATIC CELL COUNT
Bentley Somacount 150/300/500
(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 (Bronopol^TM) 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 Somacount^TM 150 _____
   2. __________ Bentley Somacount^TM 300 _____
   3. __________ Bentley Somacount^TM 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 (Bronopol^TM)
       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