Microscopic Cell Counts Research Articles

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.

Secondary Growth in Needle Leaves of Pinus longaeva (Bristlecone Pine) and Other Conifers: Quantitative Data

Needle leaves of Pinus longaeva can be accurately dated and remain alive on branches for 30 or more yrs, making this species ideal to study secondary growth in leaves. Field observations and regression analysis of needle age versus mean needle length both indicate primary (elongation) growth of needles is completed in the first year. Statistical analysis of cell counts for one- to 33-yr-old needles indicate production along the entire length of needles of 1.0-1.7 cell layers per year of secondary phloem, but no secondary xylem. Microscopic measurements and cell counts reveal that with advancing needle age phloem increases radially and transfusion tissue buckles, but the number of endodermal cells and xylem width do not change. Living phloem remains constant in amount (ca. 9 layers) with advancing needle age, indicating yearly replacement of old by new phloem. For comparison to P. longaeva, needle leaves were also studied for ten other conifer taxa with maximum needle longevities ranging from 3 to 19 yrs. In needles of each taxon no secondary xylem is produced, but secondary phloem production occurs throughout the post-elongation lifespan of the needles regardless of maximum needle longevity.

SECONDARY GROWTH IN NEEDLE LEAVES OF PINUS LONGAEVA (BRISTLECONE PINE) AND OTHER CONIFERS: QUANTITATIVE DATA

Needle leaves of Pinus longaeva can be accurately dated and remain alive on branches for 30 or more yrs, making this species ideal to study secondary growth in leaves. Field observations and regression analysis of needle age versus mean needle length both indicate primary (elongation) growth of needles is completed in the first year. Statistical analysis of cell counts for one‐ to 33‐yr‐old needles indicate production along the entire length of needles of 1.0‐1.7 cell layers per year of secondary phloem, but no secondary xylem. Microscopic measurements and cell counts reveal that with advancing needle age phloem increases radially and transfusion tissue buckles, but the number of endodermal cells and xylem width do not change. Living phloem remains constant in amount (ca. 9 layers) with advancing needle age, indicating yearly replacement of old by new phloem. For comparison to P. longaeva, needle leaves were also studied for ten other conifer taxa with maximum needle longevities ranging from 3 to 19 yrs. In needles of each taxon no secondary xylem is produced, but secondary phloem production occurs throughout the post‐elongation lifespan of the needles regardless of maximum needle longevity.

A Simple Laboratory Alternative to Irreversibly Sickled Cell (ISC) Counts

Irreversibly sickled cells (ISC) are considered to be a hallmark of sickle cell disease, yet their number in peripheral blood smears varies greatly among different homozygous SS patients. This variation has suggested a role for ISC in the varying clinical manifestations of the disease. Efforts to determine the role of ISC have been complicated by the difficulty in standardizing the quantification of these cells. For this reason, we have attempted to develop an alternative method of quantification that would be less variable than the microscopic counting of cells on blood smears. Because ISC are dehydrated dense cells, a measurement based on cell density seemed an attractive alternative approach. Analysis of whole blood samples on a simple, 2-step density gradient, spun in a microhematocrit centrifuge, showed a strong correlation between the proportion of high density cells and the percentage of morphologically identified ISC. Parallel ektacytometric measurements of cell deformability, another parameter that reflects the low water content and high MCHC of ISC, were also strongly correlated with ISC counts. These findings suggest that either of these measurements, sensitive to the special physical properties of ISC, could be used as an objective substitute for the microscopic counting of ISC.

A simple laboratory alternative to irreversibly sickled cell (ISC) counts

Irreversibly sickled cells (ISC) are considered to be a hallmark of sickle cell disease, yet their number in peripheral blood smears varies greatly among different homozygous SS patients. This variation has suggested a role for ISC in the varying clinical manifestations of the disease. Efforts to determine the role of ISC have been complicated by the difficulty in standardizing the quantification of these cells. For this reason, we have attempted to develop an alternative method of quantification that would be less variable than the microscopic counting of cells on blood smears. Because ISC are dehydrated dense cells, a measurement based on cell density seemed an attractive alternative approach. Analysis of whole blood samples on a simple, 2- step density gradient, spun in a microhematocrit centrifuge, showed a strong correlation between the proportion of high density cells and the percentage of morphologically identified ISC. Parallel ektacytometric measurements of cell deformability, another parameter that reflects the low water content and high MCHC of ISC, were also strongly correlated with ISC counts. These findings suggest that either of these measurements, sensitive to the special physical properties of ISC, could be used as an objective substitute for the microscopic counting of ISC.

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.

Small-granule (neuro)endocrine cells in the infracardiac lobe of a hamster lung. Number, subtypes, and distribution.

Small-granule APUD (amine precursor uptake and decarboxylation) endocrine cells were surveyed in 600 3 microns glycol methacrylate-embedded, periodic acid-Schiff (PAS)-lead hematoxylin-stained serial sections comprising 95% of the infracardiac lobe of a hamster lung. Results were confirmed by less systematic study of other hamster lungs. Positions of endocrine cells were marked on cardboard profiles of bronchi and bronchioles for assembly into a 70 X enlarged three-dimensional model from which size and branching of the airway were determined. Records were made for computer analysis of the number and staining patterns of endocrine cells, the nature of contiguous epithelial cells, and the presence of underlying smooth muscle and blood and lymph vessels. APUD cells occurred in 95% of all airways, at a mean density of six solitary cells and 10 cell clusters (neuroepithelial bodies) per millimeter of airway length, measured along the bronchial-bronchiolar long axis. Nineteen percent of endocrine cell loci (29% of all cells) were found at bronchioloalveolar portals in all regions of peripheral lung. Twenty percent of loci (28% of all cells) occurred about the origins of lateral airway branches; these included 4% of loci on carinal points of bifurcation. Two groups of APUD cells had distinctive anatomic relationships: 1) 13% of loci (20% of cells) were related to pulmonary capillaries and venules, mainly at bronchioloalveolar portals; and 2) 39% of loci (53% of cells) overlaid peribronchial muscle, mainly in larger airways where changes in diameter might affect ventilation. In this lobe, APUD cells were not related to goblet or mast cells; 74% of loci abutted Clara and/or ciliated cells, 17% great alveolar cells. Few loci were associated with pulmonary arteries and veins. Five APUD cell types were identified by PAS-lead hematoxylin staining. Types I, II, and V, with granules approximately 0.2 micron in diameter, made up 38%, 45%, and 2% of cells, respectively. Types III and IV, 10% and 5% of cells, respectively, had larger granules. Types I, II, III, and V occurred as solitary cells as well as in neuroepithelial bodies. One-third of the neuroepithelial bodies contained a single cell type; the rest were mixed. Type IV cells, with coarse lead hematoxylin-positive granules, usually were found in large neuroepithelial bodies containing two to four cell types and were never seen occurring alone. We conclude that 1) hamster lungs contain different kinds of APUD cells; 2) those likely have a variety of functions; 3) distinctions merely between solitary and clustered cells may not be significant; 4) the histophysiology of many neuroepithelial bodies probably is more complex than previously suspected; and 5) PAS-lead hematoxylin is superior to argyrophilia and amine fluorescence for light microscopic counting and analysis of pulmonary APUD cells in this species.

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.

Optical measurements for biomass determination in limnological studies of eutrophication

An optical technique for determining phytoplankton biomass in lakes and reservoirs is described. By making measurements of light intensity over relatively short intervals of depth the absorption coefficient can be calculated. This permits a determination of the biomass. Good correspondence between values of the biomass using this method and other methods of sampling has been obtained. The procedure is rapid and yields results without the necessity of transporting water samples to a laboratory to make microscope cell counts.

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.

Enumeration of bacteriophages and host bacteria in sewage and the activated-sludge treatment process.

Bacteriophage populations in an activated-sludge sewage treatment plant were enumerated. A newly developed assay for quantitation of total phages, employing direct electron microscopic counts, was used in conjunction with the plaque assay. The total concentration of phages was significantly higher in reactor mixed liquor and effluent than in influent sewage, indicating a net production of phages within the reactor. Maximum total phage concentrations in the fluid phase of sewage, activated-sludge mixed liquor, and reactor effluent were 2.2 x 10(7), 9.5 x 10(7), and 8.4 x 10(7)/ml, respectively. Conditions were optimized for isolation of predominant heterotrophic aerobic bacteria from sewage and mixed liquor. Blending at ice water temperatures was superior to ultrasound or enzyme treatments for maximum release of viable bacteria from microbial floc. A solidified extract of mixed liquor was superior to standard media for cultivating maximum numbers of heterotrophic bacteria. The highest culture counts for sewage and mixed liquor were 1.4 x 10(7) and 1.3 x 10(9)/ml, respectively, which represented only 3 and 6.8% of the total microscopic cell counts. Only 3 out of 48 dominant bacterial isolates from either mixed liquor or sewage were hosts for phages present in the system. The sum of phage populations infecting these three hosts accounted for, at best, 3.8% (sewage) and 0.2% (mixed liquor) of the total number of phages present. Generally, specific phage titers were lower in mixed liquor than in sewage, indicating that these hosts were not responsible for the net production of phages in the reactor. This study emphasizes the limitations of the plaque assay for ecological studies of phages, and it suggests that bacteria responsible for phage production in activated-sludge mixed liquor are either minor components of the heterotrophic population, floc-producing strains, or members of other physiological groups.

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.