Microscopic Somatic Cell Count Research Articles

Somatic Cell Reference Samples for Calibration of Milk Somatic Cell Counting Methods

Somatic cell count samples (SCCS) for use in calibration of milk somatic cell counting methods were prepared from raw bulk milk preserved with potassium dichromate. Somatic cells were separated by centrifugation, then appropriate cell dilutions were prepared in the dichromate-preserved skim milk. Somatic cell counts from SCCS stored at 4°C were stable over a 23-wk period. No bacterial contamination was detected in these samples. In a collaborative study among eight laboratories, SCCS were not affected by usual conditions by shipping. The SCCS can be used as reference standards for the direct microscopic somatic cell count and the Fossomatic and Coulter Counter somatic cell counting methods.

Comparison of Semiautomated Method with Official Optical Somatic Cell Counting Method III for Determining Somatic Cells in Milk

The new method specifying the Fossomatic-90 differs from the official method, 46.105-46.109, in that the modified instrument includes a halogen lamp; a semiconductor photoelectric detector; a less expensive, bench-top cabinet; manual injection of a larger sample, and a reduced capacity. The new instrument was compared with 2 optical somatic cell counters in routine use. On each of 3 days, 12 subsamples were prepared for each of 5 cell count levels from AM milk with half kept fresh and half preserved with 0.05% potassium dichromate. Subsamples were refrigerated and read 30+ h post-collection. Duplicate sets were read in random order on each machine daily (CV 0.77%). Two sets of slides read by 2 technicians each (strip reticle on 2 smears/slide) gave geometric mean direct microscopic somatic cell count (DMSCC) levels of 296, 526, 772, 930, and 1438 th/mL. Within-technician CV values (from day-level means) ranged from 1.68 to 2.28%. Geometric mean cells in th/mL on the new machine were significantly higher than those on the other two (674 vs 621) and were closer to the DMSCC (694). On the new machine, cell counts were 8.5% greater than on the original machines, were only 2.9% lower than the DMSCC, and showed no significant evidence of bias. Preserved samples averaged slightly greater than fresh (5.3%) but only on the original machines. Carryover by covariance analysis was insignificant. Except for cell levels, high machine precision (error CV value of 1.18%) gave differences with statistical but not practical significance, even for regulatory laboratories.

Preparation, Use and Evaluation of Standards Developed for Simultaneous Monitoring of Coulter and Fossomatic Electronic Cell Counting Instruments in Ontario

Special standards for Somatic Cell Counting (SCC) were developed at the Central Milk Testing Laboratory in Guelph for Ontario laboratories performing the tests. One laboratory used a Coulter milk counter for composite herd samples, two laboratories used five Fossomatic counters for cow samples, while the fourth used a Coulter counter for research purposes. Standards were prepared at low, medium and high count levels from fresh herd milks containing somatic cells near levels of 100,000, 500,000 and 1,000,000/ml. As the lifespan of the suspensions of somatic cells was three months, they were prepared in sufficient quantities to test daily for 3 months, and to be used throughout the day for calibration maintenance. The actual counts in the standards were established by direct microscopic somatic cell count (DMSCC) performed in two laboratories simultaneously with exchange of slides. Counts were re-confirmed every week by the same DMSCC procedures. The standards are suitable for initial calibration, daily calibration maintenance, daily instrument performance appraisal and trouble-shooting. In daily use each laboratory tests each standard five times each morning on each instrument. The same vials are used throughout the day, every 0.5 h for appraisal of continuous calibration and instrument function. The mean and standard deviations of standards are determined and compiled for each day.

Effect of Parity, Stage of Lactation, and Intramammary Infection on Concentration of Somatic Cells and Cytoplasmic Particles in Goat Milk

Aseptic foremilk samples (about 15ml) were collected biweekly for four samplings and then monthly throughout lactation from 35 does in three herds on Dairy Herd Improvement test. Bacteriology, direct microscopic somatic cell counts, and cytospin differential cell counts were performed on all samples. Milk yield was obtained from monthly Dairy Herd Improvement records. Thirty-nine percent of udder halves were infected, and coagulase negative staphylococci were the most frequent isolates (201 of 205). Somatic cell counts were influenced by lactation number, stage of lactation, and intramammary infection. Also, infection of one udder half increased somatic cells in the corresponding uninfected mammary half of the same animal. Cytoplasmic particles were not affected by stage of lactation or by intramammary infection but were greater for goats in first lactation. Milk yield was influenced by lactation number, stage of lactation, and intramammary infection.

Screening tests for abnormal milk and physical examination of the udder

Many tests have been described for use in detecting abnormal milk. These include: black plate or strip cup, catalase test, (modified) Whiteside test, serum albumin, milk chloride, electrical conductivity, California Mastitis Test (CMT), Wisconsin Mastitis Test (WMT), Direct Microscopic Somatic Cell Count (DMSCC) and Electronic Somatic Cell Counts (ESCC). In summary, the black plate is still a useful tool for the examination of lactating cows to detect clinical mastitis.
 The CMT Test is a useful cowside test for detecting subclinical infections.
 ESCC provides useful information for assessing the prevalence of subclinical infection and for regular evaluation of mastitis control practices.
 Examination of the udder may be indicated. Udder palpation is not as important in the current scheme of management of cows as it may have been in the past but could still provide useful additional indications for culling decisions.
 Teat end lesions may be reduced by avoiding chemical irritation and faulty machine function. However, teat end appearance may be related to high production and, depending on degree, is not always an indication of unusual trauma.

Reliability and Analyst Performance Limits for the Field and Single Strip Direct Microscopic Somatic Cell Count Procedure

The means and total components of variance were compared for the field and single strip direct microscopic somatic cell count (DMSCC) procedures. The field count procedure averaged 12 – 28% higher than the single strip count procedure in the 300,000 to 1,200,000 DMSCC/ml range. The sum of the components of variance in logarithm units for the field procedure was 0.01058 (1485 degrees of freedom) with a coefficient of variation of 24%, whereas the sum for the single strip procedure was 0.00834 (2834 degrees of freedom) with a coefficient of variation of 21%. This study demonstrates that the single strip procedure yields more reliable and less variable results than does the field procedure.

Differentiation and Enumeration of Somatic Cells in Goat Milk

Non-leukocytic cell-like particles commonly observed in goat milk were examined ultrastructurally and cytochemically. Transmission electron microscopy indicated that these particles were generally membrane-bound and anucleate. They contained granular material in the dilated cisternae of the endoplasmic reticulum and homogeneous electron translucent inclusions that resembled lipid. Histochemical and fluorescent staining indicated that the particles contained large amounts of protein, some lipid, but no deoxyribonucleic acid. Several methods routinely used for estimating somatic cell counts in cow milk were compared to determine which one would give accurate estimates of somatic cell counts in goat milk. No significant difference was found (P> .05) among methods which specifically measure deoxyribonucleic acid. These included Membrane Filter-DNA, direct microscopic somatic cell counts using Pyron in Y-methyl green stain, and Fossomatic cell counts. Results of the Wisconsin Mastitis Test did not differ significantly from Fossomatic cell counts. Because Coulter electronic counts and direct microscopic somatic cell counts using Levowitz-Weber stain could not differentiate between the cell-like particles and the actual leukocytes, these methods resulted in elevated cell counts that were highly variable. Results indicate that only those counting methods that are specific for deoxyribonucleic acid can distinguish cell-like particles from somatic cells, and thereby give reliable estimates of somatic cell numbers in goat milk.

Relationship of somatic cell count and cell volume analysis of goat's milk to intramammary infection with coagulase-negative staphylococci.

The prevalence of intramammary infection in 4 commercial goat herds was studied in conjunction with electronic somatic cell count and volume analysis determined using a Coutler Counter and volume analyser. Neither streptococci nor mycoplasma were isolated from any half and the prevalence of intramammary infection with Staphylococcus aureus ranged from 0 to 3% between herds. For coagulase-negative staphylococci the range from infected halves was 36-71%. There was no significant difference between the mean total microscopic somatic cell count for halves infected with coagulase-negative staphylococci and those free from infection. A similar trend was observed for electronic somatic cell counts although the mean electronic cell count was greater than the mean total microscopic count on the 2 occasions that they were compared. The correlation coefficients between the 2 cell counting methods were 0.86 and 0.94. Between herds there were significant differences in mean electronic somatic cell count, with herd means ranging from 438 x 103 to 1684 x 103 cells/ml. In 2 of the 4 herds studied, milk samples from halves infected with coagulase-negative staphylococci had a significantly higher prevalence of cell volume distributions with a modal cell volume between 65 mu 3 and 100 mu 3. This was attributed to a higher proportion of polymorphonuclear neutrophils. Use of electronic somatic cell count and cell volume analysis were considered of little value in predicting infection caused by coagulase-negative staphylococci as there was a high proportion of false negative and false positive predictions.

Milking Vacuum Influencing Indicators of Udder Health1,2

In 23 first-calf heifers we ascertained effects that milking vacuums of 34, 42.5, and 51 kPa had on udder health by percentage of quarter-samples positive for Staphylococcus aureus, total counts of bacteria on blood agar, teat end scores, California Mastitis Test, and direct microscopic somatic cell count.Measures were higher for 51 kPa than for 34 or 42.5 kPA. Samples from cows milked at 42.5 kPa had a lower percentage of samples positive for Staphylococcus aureus, lower total count, lower California Mastitis Test, and lower direct microscopic somatic cell count than samples from cows milked with 34 kPa. Teat end scores increased with increase in milking vacuum. Approximately 49% of the samples from cows milked with 51 kPa were positive for Staphylococcus aureus compared with 19.9 and 16.7% for 34 and 42.5 kPa. Milking vacuum of 51 kPa resulted in the highest percentage of cows with three (44.5%) and four (24.1%) quarters positive for Staphylococcus aureus. A milking vacuum of 51 kPa could increase mastitis in a herd having Staphylococcus aureus as the prevalent microorganism causing mastitis.

Variation in Food Microbiological Tests Used to Evaluate Analyst Performance

Procedures for evaluating analyst performance in state and federal milk and food laboratory quality assurance programs depend on the estimates of variation for the methods used. The total components of variance for a method consist of replicate error, among-analysts variance, and analyst-sample interaction components. Eleven methods were evaluated in this study. Total variance estimates of milk analytical procedures for the Standard Plate Count, plate loop count, coliform plate count, direct microscopic somatic cell count, electronic somatic cell count and Wisconsin mastitis test were 0.01045, 0.01371, 0.01590, 0.01265, 0.00504 and 4.06, respectively. Estimates of the logarithmic total variance of food analytical procedures for the aerobic plate count, coliform most probable number (MPN), Escherichia coli MPN, fecal coliform MPN and Staphylococcus aureus MPN were 0.00853, 0.10205, 0.14705, 0.14780 and 0.24245, respectively. The study showed that current analyst performance levels used by state and federal laboratory quality assurance programs are satisfactory.

Membrane Filter-Deoxyribonucleic Acid Method of Somatic Cell Counting: Collaborative Study

In the membrane filter-deoxyribonucleic acid test, somatic cells are trapped on a special filter. A chemical determination of the DNA of the trapped material gives an estimate of the number of cells. The MF-DNA was evaluated and compared with the direct microscopic somatic cell counts (DMSCCs) in 10 laboratories. Cell levels in the 10 bulk milk samples analyzed ranged from 380,000 to 1,700,000/mL. Regression analysis gave a relation of 0.181 absorbance at 490 nm, when 2.5 mL of milk was analyzed and 5.0 mL reagent was used for color development. The 95% confidence limits on the slope were 0.158-0.204. Analysis of variance showed that both procedures had significant (P less than 0.001) systematic errors, but that only the DMSCC method showed a significant laboratory X sample interaction (P less than 0.001). The repeatability standard deviation of the MD-DNA procedure was 117,000 cells/mL (14%), and the reproducibility standard deviation was 198,000/mL (23%). This compared favorably with the DMSCC method which has a repeatability of 162,000/mL (19%) and a reproducibility of 187,000/mL (34%). The error of the MF-DNA was constant at all cell counts, whereas there was a tendency for the error to increase in the DMSCC method at high cell counts. This method has been adopted as official first action.

Methods to Detect Abnormal Milk – A Review

Screening and confirmatory methods for detecting abnormal milk, mastitic milk, or milk of high somatic cell count are reviewed. Those procedures reviewed in some detail include the Catalase Test. Brabant Mastitis Reaction, pH and chlorine analysis, Ruakura Rolling Ball Viscometer method. California Mastitis Test (CMT), Wisconsin Mastitis Test (WMT), Optical Somatic Cell Count (OSCC). Direct Microscopic Somatic Cell Count (DMSCC), and Electronic Somatic Cell count (ESCC). Other detection methods are tabulated.

Comparison of the Automated with the Semi-Automatic Coulter Counter Method and the Direct Microscopic Somatic Cell Count (DMSCC) on Raw Milk Samples

The automatic Milk Cell Counter (MCC) and semi-automatic electronic cell counter (ESCC) of Coulter Electronics were compared with each other and with the direct microscopic cell count (DMSCC) on raw milk samples with various cell counts. The average DMSCC count on 241 samples of milk with Wisconsin Mastitis Test (WMT) results of 22 mm and higher was 55,000 cells/ml above the average MCC count when calibrated to a 4.4-μm minimum particle diameter. This difference is statistically significant at the 1% level. On 24 different raw milk samples of widely varying somatic cell count analyzed in replicate six times per sample, the standard deviations for replicate samples were 34,300, 34,900 and 136,000 for the MCC, ESCC and DMSCC, respectively. For these tests, the MCC had been calibrated to a 4.3-μm minimum particle diameter. The average difference between counts by the MCC and ESCC methods was only 6080/ml, but this was statistically significant at the 5% level. The average MCC count with the equipment set at 4.3-μm minimum particle diameter was 58,000 above the average DMSCC count.

Improved Automated Optical Somatic Cell Counting Method for Raw Milk: Collaborative Study

A collaborative study was conducted to compare the improved automated optical somatic cell counting procedure (OSCC II) with the direct microscopic somatic cell counting method (DMSCC) in raw milk. Samples were prefixed with formaldehyde and introduced into an Auto-Analyzer system. Dilution, clarification, and cell counting were performed automatically. Five collaborators participated in the study; they analyzed 48 samples in duplicate, using 2 different sampling rates. The results were compared with DMSCC counts reported by 3 different analysts. Statistical results show that the standard deviation for the DMSCC method is 0.0825 and for the OSCC II, at a sampling rate of 80/hr, 0.0434. When results are compared, the OSCC II procedure is as accurate as, and is significantly more precise than, the DMSCC method. The faster sampling rate of the OSCC II at 120/hr has some effect on precision but little effect on accuracy. The method has been adopted as official first action.

Fossomatic Method of Somatic Cell Counting in Milk: Collaborative Study

Abstract Fossomatic, a fully automatic, fluoro-optoelectronic somatic cell counter, was evaluated in 6 laboratories. One set of 6 duplicate milk samples, fresh and preserved, was read with instruments as routinely calibrated to each laboratory’s direct microscopic somatic cell counts (DMSCCs), i.e., local standard. A second set was read after standardizing the instrument to a 3-sample common standard (DMSCC) shipped to each collaborator. Cell levels of the unknowns ranged from 371 to 2301 thousand (th)/ml by DMSCC, with an s per sample of 14.8% and s of a level mean of 1.8%. Fossomatic values averaged 1014 compared to 1031 th cells/ml by DMSCC, had homogeneous variance for duplicates within laboratories (s = 4.0%), and had an error per observation of 6.2%. The linear coefficient of 0.9966±0.0039 (for fresh milk and common standard) for the Fossomatic values regressed on DMSCC gave an excellent and unbiased estimate of the cell concentration in a milk sample. A small bias was evident in only 5 of 24 linear regressions (fresh or preserved within each standard) where slopes differed significantly from 1. All 5 were on values derived from a local standard with maximum biases of +18% and —28% (at a 1 million level). Preserved milk had slightly lower (P < 0.01) cell concentrations than fresh milk but only by 3.6% at 1 million. Although condition X level was significant (P < 0.01), the 6 cell levels differed by a magnitude of only 27— 45 th cells/ml. Between-laboratory variation (the component was 4.6%) and laboratory X level interaction were both significant, but their contributions to error were small. The spread in laboratories at any cell level was about 12% and the error of estimate for a sample sent to 2 laboratories was 6.8%. Although interactions were significant with this precise method, they were not of such a magnitude as to invalidate cell count estimates in practice. The Fossomatic method has been adopted as official first action.

Enhancement of Chemotactic Response of Polymorphonuclear Leukocytes into the Mammary Gland and Isolation from Milk

Intramammary infusion of saline containing .1% glycogen resulted in diapedesis of numerous leukocytes (93% polymorphonuclear leukocytes) into milk. Direct microscopic somatic cell counts of primary milk 11h after infusion with 100ml of either saline containing .1% glycogen, saline, distilled water, or noninfused averaged 4.17, 1.06, .05, and .10×106 cells/ml of milk. Four times more polymorphonuclear leukocytes were isolated from residual milk (6.1×109 cells) collected 11h following infusion than from primary milk (1.5×109). The polymorphonuclear leukocytes isolated from either the primary or residual milk collected 11h after infusion of saline containing .1% glycogen phagocytosed Staphylococcus aureus with great avidity. Sixty-eight percent of a culture of heatkilled phosphorus-32-labeled Staphylococcus aureus (400×106) were phagocytosed within 60min by 25×106 polymorphonuclear leukocytes. When isolated polymorphonuclear leukocytes were incubated with live Staphylococcus aureus, 98% of the ingested organisms were killed. Isolation of polymorphonuclear leukocytes from residual milk 11h after intramammary infusion of saline containing .1% glycogen will provide large numbers of polymorphonuclear leukocytes for in vitro studies of the cellular defense of the mammary gland.

Estimating Somatic Cells in Milk Samples by the Membrane-Filter-DNA Procedure

Further developments in the membrane-filter DNA procedure for determining the somatic cell content of milk samples are described. A statistical experiment has been done comparing the membrane-filter test to the direct microscopic somatic cell count. Under conditions of the assay a relationship of 0.093 absorbance unit per million cells was obtained at a wavelength of 490 nm. The membrane filter technique is more precise than the direct microscopic count and is not subject to techician bias. A method is described for standardizing the procedure between and within laboratories based on use of lyophilized bovine somatic cells trapped on membrane filters as a calibration device.

Experience in Adaptation and Comparative Evaluation of the Coulter Counter and Direct Microscopic Somatic Cell Count (DMSCC) Method for Determining Somatic Cell Counts in Milk

A Coulter Counter, Model ZBI, was introduced into a provincial government laboratory and adapted for use in counting somatic cells in milk. The procedure followed was a modification of the International Dairy Federation method, the main difference being in the formulation of the fixative and a slight change in diluent make-up. The procedure was further modified in operating details to enhance routine analytical work. During one year of use in the ongoing provincial laboratory program, 8130 milk samples were checked for somatic cells by the Coulter Counter. Of this number, 509 were selected at random and checked by the Direct Microscopic Somatic Cell Count (DMSCC) method. The correlation coefficient for these samples for the two methods was 0.973. The mean cell counts were 841.600 and 839,900 for the DMSCC and Coulter Counter methods, respectively. The regression formula for estimating DMSCC from Coulter Counter results was determined to be ŷ= 0.8023(x) + 163.45.

Examination of problem mastitis herds in Ohio

4 problems can occur in a dairy herd which may cause the herd to be identified as a problem mastitis herd. These are (i) antibiotic residues present in bulk tank milk, (ii) high bacterial count, (iii) high leucocyte count, and (iv) increased number of serious clinical cases with deaths. Milk samples were taken from Ohio dairies during the period from Jan. to May 1975 and data are given for (i) and (ii): less than or equal to 0.45% of samples contained antibiotic residues; and less than or equal to 7.5% contained >100 000 bacteria/ml. To determine (iii), milk samples were screened using the Wisconsin Mastitis Test and if a high reading was recorded, the leucocyte count was confirmed using Direct Microscopic Somatic Cell Count. Between 1/4 and 1/3 of Ohio herds were experiencing quite high leucocyte counts. Measures are outlined to assist the dairyman in preventing (i)-(iv) which involves: checking the environment, milking machine function and milking technique; cow examination and dry cow therapy.

Effect of the Postmilking Teat Dip “Bovadine” on the Incidence of Staphylococcus aureus and Mastitis

The University milking dairy herd was divided into two groups according to breed. Diagonally opposite teats of each cow were dipped with “Bovadine” after each milking. The remaining two teats served as controls. The Wisconsin Catalase Test, Wisconsin Mastitis Test, and Direct Microscopic Somatic Cell Count were used to detect incidence of mastitis in composite samples from dipped and control teats. Samples of milk were plated on Baird-Parker Agar to detect staphlococci. The postmilking teat dip caused a 31.4% reduction in new cases of mastitis and 42.3% reduction in staphylococcal infections during the 19 months of the study. The teat dip caused a reduction in new cases of staphylococcal infections and in number of quarters infected with mastitis; however, these reductions were non-significant. The WMT had the highest correlation when compared with all the other tests used to detect mastitis. Results indicated that the WMT would be the best single test to use to detect cows with mastitis.