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U.S. Department of Health and Human Services

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M-I-05-3: 2400g DIRECT MICROSCOPIC SOMATIC CELL COUNT

June 2, 2005
 

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

[Unless otherwise stated all tolerances are ±5%]

SAMPLES
  1. __________Laboratory Requirements (See CP, item 33 & 34)
    APPARATUS
  2. __________See Cultural Procedures, items 1-4
    1. __________ Functional fume hood, face velocity 100 ft/min
      1. __________ Checked annually, records maintained, unit tagged
  3. __________Microscope Slides, Clean (see item 18), 2.54 x 7.62 cm
    1. __________ 11.28 mm diameter areas delineated
    2. __________ Optionally, with center marks on sides of delineated area
       
    3. __________ Optionally, 5.08 x 7.62 or 5.08 x 11.43 cm with 11.28 cm delineated areas
  4. __________ Syringe
    1. __________ Metal (____________________)
      1. __________ Suitable for rapid and convenient transfer of 0.01 mL of milk
         
      2. __________ Calibrated as specified in CP item 6e to deliver 0.0103±0.0005g (average of 10 consecutive weighings with milk)

        __________ Avg. Wt. __________ Date __________

      3. __________ Syringe etched with identification (imprinted serial number acceptable) and tagged with calibration date
    2. __________ Micropipettor, with appropriate tips (__________________)
      1. __________ Suitable for rapid and convenient transfer of 0.01 mL of milk
      2. __________ Calibrated as specified in CP item 6e to deliver 0.0103±0.0005g (average of 10 consecutive weighings with milk)

        __________ Avg. Wt. __________ Date __________

      3. __________ Syringe etched with identification (imprinted serial number acceptable) and tagged with calibration date
    3. __________ Records of syringe (metal or micro) calibration maintained
  5. __________Dissecting Needle, Bent Point
    1. __________ Suitable for spreading milk film
  6. __________ Drying Device, Slide Drier or Incubator
    1. __________ Clean, dust-free, level surface
    2. __________ Heat source regulated at 40-45C
      1. __________ Temperature monitored with thermometer
  7. __________ Forceps or Slide Holder
    1. __________ Required for dipping and holding slides
  8. __________Staining Jars or Trays
    1. __________ With tight fitting covers
    2. __________ Convenient size for holding solvents and stains
  9. __________Slide Storage
    1. __________ Clean, dust-free insect-proof boxes, cases or files
  10. __________Microscope Type: ______________________________
    1. __________ Binocular with 1.8 mm oil immersion objective, rack and pinion sub-stage, condenser with iris diaphragm
    2. __________ Oculars, 10X (12X or 12.5X), Huygenian or wide-field
    3. Optics provide a Single Strip Factor of 6070 or smaller
      1. __________ Each analyst measures field diameter and calculates SSF annually, round to three significant figures
      2. __________ Calculation of Single Strip Factor
        1. __________ Using a stage micrometer (item 11), measure field diameter (D) of oil immersion objective lens in mm D = ________ mm
        2. __________ Compute SSF with formula:

          __________ SF = 10,000/(11.28 x D) SSF is _______________

    4. __________ Mechanical Stage
      1. __________ Suitable for examination of slides, smooth action, does not drift, allows proper tracking of smears
    5. __________ Microscope Lamp, provides adequate illumination
  11. __________Stage Micrometer Ruled with 0.1 and 0.01 mm Divisions
  12. __________Hand Tally, accurate
    MATERIALS
  13. __________ Immersion Oil
    1. __________ Refractive index 1.51-1.52 at 20C
  14. __________ Levowitz-Weber Modification of the Newman-Lampert Stain
    1. __________ Slowly add 0.6 g certified methylene blue chloride to 52 mL of 95% ethyl alcohol and 44 mL of tetrachlorethane (reagent grade) in a 200 mL flask and swirl to dissolve
    2. __________ When making stain, use gloves and prepare in fume hood (tetrachlorethane is TOXIC)
    3. __________ Let stand for 12-24 hr at 4.4-7.2C
    4. __________ Filter through Whatman No. 42 filter paper or equivalent
    5. __________ Add 4 mL of glacial acetic acid
    6. __________ Store in a clean, tightly closed container (traces of water or solvent may cause problems with this stain)
    7. __________ Or, Commercially prepared (xylene or tetrachlorethane)

      __________Brand __________ Lot No __________

  15. __________ Canadian Formula Stain
    1. __________ Commercially prepared (xylene or tetrachlorethane)

      __________Brand __________ Lot No __________

  16. __________ Alternate Methylene Blue Stain
    1. __________ Prepare as in item 14 with reagents:
      1. __________ Combine: 0.5 g cert. methylene blue chloride
        56 mL 95% ethyl alcohol
        40 mL xylene
        4 mL glacial acetic acid
         
  17. __________Stirring hot plate/stirring bar (optional)
    1. __________ Carnoy's fixative
      1. Combine:
        60 mL chloroform
        20 mL glacial acetic acid
        120 mL 100% ethyl alcohol
    2. __________ Pyronin Y-methyl green stain
      1. __________ Combine:
        1.0 g Pyronin Y
        0.56 g methyl green
        196 mL water
      2. __________ Filter through Whatman No. 1 paper before use
      3. __________ Stain is light sensitive; store in brown bottle
  18. __________ Slides, Cleaning
    1. __________ Physically clean
    2. __________ New slides may be cleaned by soaking in strong cleaning solution
    3. __________ Rinse thoroughly in flowing water 10-15 sec and MS water
    4. __________ Used slides may be soaked in hot detergent or wetting agent until all residues are removed, rinsed as above
    5. __________ Air or heat dry with minimal exposure to dust, insects, etc. and store dry
    6. __________ Or, store slides in alcohol and flame just before use
    PROCEDURE
  19. __________ Slide Identification
    1. __________ Legibly and indelibly identify each sample area on margin of slide
  20. __________ Sample Agitation
    1. __________ Mix samples by shaking 25 times in 7 sec with 1 ft movement, sample removed within 3 minutes
    2. __________ Optional: Warm high fat samples to 40C for no longer than 10 minutes prior to testing (discard after testing)
  21. __________ Sample Measurement and Smear Preparation (Metal Syringe)
    1. __________ Before use and between successive samples, rinse syringe 2 - 3 times in clean, 25-35C water
    2. __________ Before transferring test portion to slide, dip tip of syringe not over 1 cm below surface (excluding foam) of milk and repeatedly rinse
    3. __________ Holding tip beneath surface, rinse syringe three times with milk, then fully depress and release plunger and withdraw test portion
    4. __________ With clean paper tissue or cloth, remove excess milk from exterior of tip (with syringe tip up, wipe downward away from tip)
    5. __________ Holding instrument vertical, place tip near center of area for smear, touch the slide with the tip and expel the test portion
      1. __________ With plunger still fully depressed, touch off once against a dry spot
      2. __________ Do not release plunger until after touching off and removing tip from slide
      3. __________ Spread milk with point of bent needle point (item 5), not hockey stick style
      4. __________ Wipe needle dry between samples on tissue or towel
    6. __________ After spreading test portion, dry smears at 40-45C within 5 min on level surface (see item 6)
    7. __________ To prevent smears from cracking and peeling from slide during staining, do not heat too rapidly
    8. __________ Protect smears and slides from damage until read
  22. __________ Metal Syringe Cleaning
    1. __________ Do not allow residues to dry on instrument
    2. __________ Immediately after use, carefully disassemble and clean syringe
    3. __________ Do not remove spring unless necessary
    4. __________ Use only soap-less detergents and/or fat solvents sparingly as needed
    5. __________ Clean all residues from measuring tube circulating detergent with bulb on delivery end
    6. __________ Clean piston with dry paper tissue or cloth
  23. __________ Sample Measurement and Smear Preparation (Micropipettor)
    1. __________ Use clean tip for each sample
    2. __________ Depress plunger and dip tip not over 1 cm below surface (excluding foam) of well-mixed milk, fully release plunger slowly, remove tip from sample and dispel back to sample, re-insert tip and fully release plunger and withdraw test
      portion, touch off to dry area of sample container
    3. __________ If necessary, remove excess milk from exterior of tip by wiping away from the tip with clean paper tissue or cloth
    4. __________ Holding instrument vertical, place tip near center of area for smear, expel test portion and touch off once to dry spot
    5. __________ Spread milk with point of bent needle point (item 5), not hockey stick style
    6. __________ Wipe needle dry between samples on tissue or towel
    7. __________ After spreading test portion, dry smears at 40-45C within 5 min on level surface (see item 6)
    8. __________ To prevent smears from cracking and peeling from slide during staining, do not heat too rapidly
    9. __________ Protect smears and slides from damage until read
  24. __________ Staining Films
    1. __________ Levowitz-Weber and Methylene Blue Stains
      1. __________ Use ventilated hood for steps 2-4
      2. __________ Submerge or flood slides with fixed, dried smears in stain for 2 min (timer used)
      3. __________ Drain off excess stain by resting edge of slide on absorbent paper
      4. __________ Dry thoroughly (air dry or use cool forced air)
      5. __________ Dip dry stained slides in 3 changes of tap water at 35-45C
      6. __________ Drain and air dry slides before examining smears
    2. __________ Pyronin Y-Methyl Green Stain ( New York Modification)
      1. __________ Slide is run through the following staining scheme
        __________ Carnoy's fixative 5 min
        50% Ethanol 1 min
        30% Ethanol 1 min
        H20 1 min Stain 6 min
        N Butyl alcohol flush briefly
        Xylene flush briefly
      2. __________ Cells stain blue or blue-green; RNA and background stain pink
  25. __________ Examination
    1. __________ Adjust microscope lamp to provide maximal optical resolution
    2. __________ Locate edge of smear to be read using low power
    3. __________ Place 1 drop immersion oil on smear
    4. __________ Carefully lower oil immersion lens
    5. __________ Focus and locate center of edge of area and begin counting cells
    6. __________ Count all cells in field wide strip across diameter of a single smear, focusing up and down as necessary
    7. __________ Identifying and counting somatic cells
      1. __________ Cells possess a nucleus stained dark blue (bovine) or blue or blue-green (caprine)
      2. __________ Cells generally 8 microns or larger (bovine; caprine may be smaller); do not count cells less than 4 microns; fragments counted only if more than 50% of nuclear material visible
      3. __________ Cluster of cells counted as one unless nuclear units are clearly separated; focus up and down to ensure that there are no bridges connecting nuclear masses
      4. __________ Count cells touching only top or bottom half of strip
      5. __________ If in doubt, do not count
    8. __________ After examination of each smear record strip count
    9. __________ Conduct monthly comparative counting between analysts (refer to SPC item 19)
  26. __________ Slide Storage
    1. __________ Remove oil by dipping in xylene (or equivalent), 15-20 sec
    2. __________ Air dry
    3. __________ Place in suitable storage (item 9)
    REPORTS
  27. __________ Records and Reporting
    1. __________ Maintain record of strip count for each smear examined
    2. __________ Compute DMSCC/mL, multiply number of cells counted (strip count) by the SSF (item 10.c.2.b.)
    3. __________ Report somatic cell counts as DMSCC/mL, record only first two left hand digits, round as necessary
      1. __________ If the third digit is 5 round the second number using the following rules
        1. __________ When the second digit is odd round up (odd up, 235 to 240)
        2. __________ When the second digit is even round down (even down, 225 to 220)