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June 2, 2005
Milk Safety References
National Conference on Interstate Milk Shipments (NCIMS)
DIRECT MICROSCOPIC SOMATIC CELL COUNT
[Unless otherwise stated all tolerances are ±5%]
SAMPLES
- __________Laboratory Requirements (See CP, item 33 & 34)
APPARATUS
- __________See Cultural Procedures, items 1-4
- __________ Functional fume hood, face velocity 100 ft/min
- __________ Checked annually, records maintained, unit tagged
- __________Microscope Slides, Clean (see item 18), 2.54 x 7.62 cm
- __________ 11.28 mm diameter areas delineated
- __________ Optionally, with center marks on sides of delineated area
- __________
Optionally, 5.08 x 7.62 or 5.08 x 11.43 cm with 11.28 cm delineated areas
- __________ Syringe
- __________ Metal (____________________)
- __________
Suitable for rapid and convenient transfer of
0.01 mL of milk
- __________
Calibrated as specified in CP item 6e to deliver
0.0103±0.0005g (average of 10 consecutive weighings with milk)
__________ Avg. Wt. __________ Date __________
- __________
Syringe etched with identification (imprinted serial
number acceptable) and tagged with calibration date
- __________ Micropipettor, with appropriate tips (__________________)
- __________
Suitable for rapid and convenient transfer of 0.01 mL of milk
- __________
Calibrated as specified in CP item 6e to deliver
0.0103±0.0005g (average of 10 consecutive
weighings with milk)
__________ Avg. Wt. __________ Date __________
- __________
Syringe etched with identification (imprinted serial
number acceptable) and tagged with calibration date
- __________
Records of syringe (metal or micro) calibration
maintained
- __________Dissecting Needle, Bent Point
- __________ Suitable for spreading milk film
- __________ Drying Device, Slide Drier or Incubator
- __________ Clean, dust-free, level surface
- __________ Heat source regulated at 40-45C
- __________ Temperature monitored with thermometer
- __________ Forceps or Slide Holder
- __________ Required for dipping and holding slides
- __________Staining Jars or Trays
- __________ With tight fitting covers
- __________ Convenient size for holding solvents and stains
- __________Slide Storage
- __________ Clean, dust-free insect-proof boxes, cases or files
- __________Microscope Type: ______________________________
- __________
Binocular with 1.8 mm oil immersion objective, rack and pinion sub-stage, condenser with iris diaphragm
- __________ Oculars, 10X (12X or 12.5X), Huygenian or wide-field
- Optics provide a Single Strip Factor of 6070 or smaller
- __________
Each analyst measures field diameter and calculates SSF annually, round to three significant figures
- __________
Calculation of Single Strip Factor
- __________
Using a stage micrometer (item 11), measure field diameter (D) of oil immersion objective lens
in mm D = ________ mm
- __________
Compute SSF with formula:
__________ SF = 10,000/(11.28 x D) SSF is _______________
- __________ Mechanical Stage
- __________
Suitable for examination of slides, smooth action, does not drift, allows proper tracking of smears
- __________ Microscope Lamp, provides adequate illumination
- __________Stage Micrometer Ruled with 0.1 and 0.01 mm Divisions
- __________Hand Tally, accurate
MATERIALS
- __________ Immersion Oil
- __________ Refractive index 1.51-1.52 at 20C
- __________ Levowitz-Weber Modification of the Newman-Lampert Stain
- __________
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
- __________
When making stain, use gloves and prepare in fume hood
(tetrachlorethane is TOXIC)
- __________ Let stand for 12-24 hr at 4.4-7.2C
- __________ Filter through Whatman No. 42 filter paper or equivalent
- __________ Add 4 mL of glacial acetic acid
- __________
Store in a clean, tightly closed container (traces of
water or solvent may cause problems with this stain)
- __________ Or, Commercially prepared (xylene or tetrachlorethane)
__________Brand __________ Lot No __________
- __________ Canadian Formula Stain
- __________ Commercially prepared (xylene or tetrachlorethane)
__________Brand __________ Lot No __________
- __________ Alternate Methylene Blue Stain
- __________ Prepare as in item 14 with reagents:
- __________
Combine: 0.5 g cert. methylene blue chloride
56 mL 95% ethyl alcohol
40 mL xylene
4 mL glacial acetic acid
- __________Stirring hot plate/stirring bar (optional)
- __________ Carnoy's fixative
-
Combine:
60 mL chloroform
20 mL glacial acetic acid
120 mL 100% ethyl alcohol
- __________ Pyronin Y-methyl green stain
- __________
Combine:
1.0 g Pyronin Y
0.56 g methyl green
196 mL water
- __________ Filter through Whatman No. 1 paper before use
- __________ Stain is light sensitive; store in brown bottle
- __________ Slides, Cleaning
- __________ Physically clean
- __________
New slides may be cleaned by soaking in strong
cleaning solution
- __________
Rinse thoroughly in flowing water 10-15 sec and
MS water
- __________
Used slides may be soaked in hot detergent or wetting
agent until all residues are removed, rinsed as above
- __________
Air or heat dry with minimal exposure to dust, insects,
etc. and store dry
- __________ Or, store slides in alcohol and flame just before use
PROCEDURE
- __________ Slide Identification
- __________
Legibly and indelibly identify each sample area on margin
of slide
- __________ Sample Agitation
- __________
Mix samples by shaking 25 times in 7 sec with 1 ft
movement, sample removed within 3 minutes
- __________
Optional: Warm high fat samples to 40C for no longer
than 10 minutes prior to testing (discard after testing)
- __________ Sample Measurement and Smear Preparation (Metal Syringe)
- __________
Before use and between successive samples, rinse
syringe 2 - 3 times in clean, 25-35C water
- __________
Before transferring test portion to slide, dip tip of syringe not over 1 cm below surface (excluding foam)
of milk and repeatedly rinse
- __________
Holding tip beneath surface, rinse syringe three times
with milk, then fully depress and release plunger and
withdraw test portion
- __________
With clean paper tissue or cloth, remove excess milk
from exterior of tip (with syringe tip up, wipe
downward away from tip)
- __________
Holding instrument vertical, place tip near center of area for smear, touch the slide with the tip and expel
the test portion
- __________
With plunger still fully depressed, touch off once
against a dry spot
- __________
Do not release plunger until after touching off
and removing tip from slide
- __________
Spread milk with point of bent needle point (item 5),
not hockey stick style
- __________
Wipe needle dry between samples on tissue or
towel
- __________
After spreading test portion, dry smears at 40-45C
within 5 min on level surface (see item 6)
- __________
To prevent smears from cracking and peeling from slide
during staining, do not heat too rapidly
- __________ Protect smears and slides from damage until read
- __________ Metal Syringe Cleaning
- __________ Do not allow residues to dry on instrument
- __________
Immediately after use, carefully disassemble and
clean syringe
- __________ Do not remove spring unless necessary
- __________
Use only soap-less detergents and/or fat solvents
sparingly as needed
- __________
Clean all residues from measuring tube circulating
detergent with bulb on delivery end
- __________ Clean piston with dry paper tissue or cloth
- __________ Sample Measurement and Smear Preparation (Micropipettor)
- __________ Use clean tip for each sample
- __________
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
- __________
If necessary, remove excess milk from exterior of tip
by wiping away from the tip with clean paper tissue
or cloth
- __________
Holding instrument vertical, place tip near center of area for smear, expel test portion and touch off once
to dry spot
- __________
Spread milk with point of bent needle point (item 5),
not hockey stick style
- __________ Wipe needle dry between samples on tissue or towel
- __________
After spreading test portion, dry smears at 40-45C
within 5 min on level surface (see item 6)
- __________
To prevent smears from cracking and peeling from slide
during staining, do not heat too rapidly
- __________ Protect smears and slides from damage until read
- __________ Staining Films
- __________ Levowitz-Weber and Methylene Blue Stains
- __________
Use ventilated hood for steps 2-4
- __________
Submerge or flood slides with fixed, dried smears
in stain for 2 min (timer used)
- __________
Drain off excess stain by resting edge of slide
on absorbent paper
- __________ Dry thoroughly (air dry or use cool forced air)
- __________
Dip dry stained slides in 3 changes of tap
water at 35-45C
- __________ Drain and air dry slides before examining smears
- __________ Pyronin Y-Methyl Green Stain ( New York Modification)
- __________ 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
- __________
Cells stain blue or blue-green; RNA and background
stain pink
- __________ Examination
- __________
Adjust microscope lamp to provide maximal optical
resolution
- __________ Locate edge of smear to be read using low power
- __________ Place 1 drop immersion oil on smear
- __________ Carefully lower oil immersion lens
- __________
Focus and locate center of edge of area and begin
counting cells
- __________
Count all cells in field wide strip across diameter
of a single smear, focusing up and down as necessary
- __________ Identifying and counting somatic cells
- __________
Cells possess a nucleus stained dark blue (bovine)
or blue or blue-green (caprine)
- __________
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
- __________
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
- __________
Count cells touching only top or bottom half of
strip
- __________ If in doubt, do not count
- __________ After examination of each smear record strip count
- __________
Conduct monthly comparative counting between analysts
(refer to SPC item 19)
- __________ Slide Storage
- __________
Remove oil by dipping in xylene (or equivalent), 15-20 sec
- __________ Air dry
- __________ Place in suitable storage (item 9)
REPORTS
- __________ Records and Reporting
- __________
Maintain record of strip count for each smear
examined
- __________
Compute DMSCC/mL, multiply number of cells counted
(strip count) by the SSF (item 10.c.2.b.)
- __________
Report somatic cell counts as DMSCC/mL, record
only first two left hand digits, round as necessary
- __________
If the third digit is 5 round the second number
using the following rules
- __________
When the second digit is odd round up
(odd up, 235 to 240)
- __________
When the second digit is even round down
(even down, 225 to 220)