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Nuclepore Membrane Filters for Milk Somatic Cell Estimations in Low Count Milk
TF~CHNICAL
NOTt~S
Nuclepore Membrane Filters for Milk Somatic Cell Estimations in Low Count Milk brahe (.65~ pore size) filter originally described. Since we were concerned with somatic cells only, and not bacteria, the larger pore size permitted more rapid filtration while apparently retaining all of the somatic cells. The Nuclepore filters were selected because they are resistant to alcoholic solutions and do not retain stains, thus providing a more contrasting background for the microscopic examination of cells. One disadvantage of these membranes is that the pores are clearly visible at high magnification and may be distracting. This distraction may be minimized by careful adjustment of the microscope condenser. A technique for eliminating these pores with chloroform after staining has been described (4) but was not used routinely in our laboratory because it produced some distortion of the filter. Finally Sudan Black B (5) was used for the staining of leukocyte granules instead of the benzidinehydrogen peroxide method originally described. Technique for preparation of a specimen follows.
Abstract
A technique f o r staining and counting milk somatic cells on Nuclepore membrane filters is described. Precision snd accuracy were compared with the direct microscopic somatic cell count modified for low cell count milk. The method appears suitable for application to low count milk from normal quarters.
Introduction
Duitschaever et al. (1, 2) have previously investigated membrane filters for the differentiation and enumeration of somatic cells in milk. Use of their technique in our laboratory has confirmed that it is highly suitable ~or differential studies. I t also appeared that the technique might be adapted to increase the precision of counting cells in milk from quarters with low cell content because the method permitted the concentration on the filter of all cells from a relatively large volume of milk. Materials and Methods
!VIodifieations of the technique were made. Nuclepore 1 filters (25 mm diameter and 2~ pore size2) were used instead of the l~illipore mereAuthorized for publication as Paper 4022 on July 26, 1971 in the Journal Series of the Pennsylvania Agricultural Experiment Station. i General Electric Company, Valleeitos Atomic Laboratory, P.O. Box 946, Pleasanton, California 94566. 2 The 2~ pore size used in this work is no longer available. A 3/z pore size is available and is suitable. 3 I~ationaI Biological Stain lqA0629. A. H. Thomas Co., P.O. Box 779, Philadelphia, P a , 19105. Stain is prepared in stock solution containing .3 g in 100 ml absolute ethanol. This should be prepared several days before use to completely dissolve all stain. Buffer for preparation of working stain contains 16 g phenol, 30 ml ethanol, .3 g ~A2HPO 4 and 100 ml distilled H20. The phenol is dissolved in the alcohol, the NA2HPO 4 in water and the 2 solutions are mixed. Working stain is prepared by mixing 60 ml of stock Sudan Black B and 40 ml of buffer. Stain is filtered before use. Working stain is usable for at least one week if refrigerated but should be refiltered before each use.
379
1. To 10 ml of milk add 1 ml of preservative solution (.24 g ttgC]2 and 1.04 g K2Cr20 7 in 100 ml I t 2 0 ) . This dilution is considered in computing the factor for calculation of cell numbers. 2. Pipet .1 or .2 ml of the preserved milk into 2 ml of .25 ~ sucrose solution in a polyethylene test tube. To obtain more cells for counting from low-count milk the larger volume was used. Milk samples were pre-tested with the California Mastitis Test (CMT) to determine the probable cell level. F o r samples with CMT scores of negative or trace .2 ml of milk was used; for higher scores .1 ml was used. 3. F i l t e r cell suspension through a Nuclepore filter using 15 to 20 cm of vacuum. The filter is held, shiny side up, in a standard laboratory filter holder for membrane filters. Rinse tube with an additional 2 ml sucrose and pass this through filter. 4. While the filter is still in the filter holder, cover surface with Sudan Black B 3 and allow to stand 15 sec. Apply vacuum to remove stain. 5. With vacuum on, rinse filter twice with absolute methanol. I f stain is still visible on filter, rinse again. With vacuum on, rinse with distilled water.
380
JOURNAL OF DAIRY SCIENCE
TABLE 1. Comparison of filter and direct microscopic somatic cell count methods for counting milk somatic cells. Filter
Range
Number samples a
~ean within sample SD
(Cells/ml) 0-100,000 100,000-200,000 200,000-500,000 500,000-1,000,000 1,000,000-2,000,000
12 6 6 6 6
(Cells/nil) 4,100 ]2,300 17,500 44,000 73,000
DMSCC
l~ean within sample CV
~¢fean within sample SD
~ean within sample CV
(%) 14.8 8.7 6.2 7.1 5.2
(Cells/ml) 5,300 12,700 25,400 40,000 55,000
(%) 24.8 9.4 7.9 5.9 3.4
Six replicate counts were made on each sample by each method. 6. Remove filter and place cell side up on microscope slide. Fix filter in place with stainless steel clips4 for staining. Do not allow filter to dry before staining. 7. Stain in May-Greenwald stain5 (2 min), rinse in distilled water, stain in Giemsa stain 6 (2 min), and rinse in distilled water. 8. Allow filters to air dry before applying immersion oil. Filters may be mounted, but we have had better results by applying immersion oil directly to filter. After examining do not remove immersion oil. When stored in a dust-proof container, the filters will remain in good condition for at least several months. The technique of enumerating all cells in a diametric strip across the filter was adapted from the direct microscopic somatic cell count (DMSCC) (3). Because our chief interest was in low cell count milk, cells lying within a wide strip were counted. This strip was defined with an eyepiece reticle with a line spacing of 12 ram; this defined a strip of .131 mm width in the field of view of the oil immersion objective (magnification, 970 × ) . The strip factor was calculated as described for the DMSCC (3) taking into consideration the volmne of milk filtered and the filtration area of the filter. This area was calculated from the measured diameter of the effective filtration surface. For 4 Small stainless clips, Catalog XX6201701, Millipore Corp., Bedford, Mass., 01730. 5 Harleco Item 660. A. H. Thomas Co., ]'.O. Box 779, Philadelphia, Pa., 19105. Dilute with 1 volume distilled water. Keep covered when ~ot. in use. 6 IIarleco Item 620. A. It. Thomas Co., P.O. Box 779, Philadelphia, Pa., 19105. Dilute with 9 Volumes distilled water; filter before use. Keep covered when not ix use. JOU]~NAL OF D&IR.¥ SCIENCE Xd~OL. 55, NO. 3
each cell count, all cells in horizontal and vertical strips of one filter were counted. The strip factors for this system were 677 for .2 ml and 1,354 for .1 ml. Precision and accuracy were assessed by comparison with results from the DMSCC (3) modified for low-count milk. The wide eyepiece reticle described above was used in the same microscope that was used for the filter counts. F o r each count 4, instead of 2 milk films as described for the DMSCC, were prepared, and the horizontal and vertical strips on each of the films were counted. Thus, for one count, 8 strips were counted. The strip factor for this system was 6,925. Individual quarter samples were obtained in various cell count ranges, as shown in Table 1. Six replicate counts were made on each sample by each method. Results and Discussion
The mean standard deviations within sample and coefficients of variation in each cell count range are in Table 1. The results suggested that the filter method might be more precise at low cell counts but that the advantage is lost at higher counts. To test this, variances among observations within samples were computed. For each range, the ratio of the mean error variances for the two methods was computed and compared to tabular F values. A twotailed test with respect to F was applied since there was no reason to expect either method to yield a larger error variance. The differences in the error variances did not approach significance in the intermediate ranges but tended to be smaller ( P < 0 . 1 ) for the filter method in the lowest range and smaller ( P < 0 . 1 ) for the DMSCC in the highest range. This is probably because the larger cell numbers
TECHNICAL
NOTES
381
I.--
correlation between single counts made by ca~.h method oil individual samples would not be nearly so high. The data indicate that the method produces counts which agree with the DMSCC. The membrane technique is time-consuming and its application will be limited. H o w e v e r , it nmy be useful for research purposes where accuracy and precision are required on lowcount milk. Its utility is increased because highly acceptable differential counts may be made on the same preparation.
o
Acknowledgment
4,0 Z cr
Of
rn
UJ
3.OK DO
X
Y = .991 X
_1 ...I W
o
o~ (.9 O .J
0
I
I
I
I
lO
2.0
5.0
40
LOG SOMATIC CELLS X i()3/ml (DMSCC)
FIG. ]. Regression line through the origin showing the relationship between mean somatic cell counts obtained by the filter and direct microscopic somatic cell count methods. counted on the filter in the lower ranges minimize counting errors; in the higher ranges the advantage gained by counting more cells becomes relatively less important and other errors inherent in the filter method may weigh more heavily. To assess the accuracy of the filter method a regression line relating the log of the mean counts by each method was calculated. Logarithms were used to maximize the effects of counts in the lower ranges. The regression line through the origin and its equation are shown in F i g u r e ]. The least squares regression line had a small positive intercept (.15) on the ordinate; however, as no significant reduction in residual mean square was obtained with this line, the line calculated through the origin, which theory requires, is presented. The correlation coefficient f o r this line is high (R = .99); individual points are means and the
The author thmlks Miss Erika Zelem for expert technical assistance and Dr. Marvin L. l~isius for advice on statistics. R. J. EBERHART, Department of Veterinary Science, The Pennsylvania State University, University Park ] 6 8 0 2 References
(1) Duitschaever, C. L., and G. C. Ashton. 1968. Comparison of counts using a Breed-type smear and millipore membrane methods on fresh and preserved milk samples. J. Dairy Sci., 51 : 665. (2) Duitschaever, C. L., and A. G. Leggatt. 1967. Cells in bovine milk: Differential staining in suspension : collecting, counting and examining on millipore membranes. Stain Technol., 42: 183. (3) National Mastitis Council, Subcommittee on Screening Tests. 1968. Direct microscopic somatic cell count in ml]k. J. Milk Food Techno]., 31: 350. (4) Reynaud, A. J., and Eileen B. King. ]967. A new filter for diagnostic cytology. Acta Cytologica, 11: 289. (5) Sheehan, It. L., and G. W. Storey. 1947. An improved method of staining leukocyte granules with Sudan Black B. J. Pathol. Bact., 59 : 336.
JOURNAL OF DAIRY SCIENCE VOL. 55, NO. S
NOTt~S
Nuclepore Membrane Filters for Milk Somatic Cell Estimations in Low Count Milk brahe (.65~ pore size) filter originally described. Since we were concerned with somatic cells only, and not bacteria, the larger pore size permitted more rapid filtration while apparently retaining all of the somatic cells. The Nuclepore filters were selected because they are resistant to alcoholic solutions and do not retain stains, thus providing a more contrasting background for the microscopic examination of cells. One disadvantage of these membranes is that the pores are clearly visible at high magnification and may be distracting. This distraction may be minimized by careful adjustment of the microscope condenser. A technique for eliminating these pores with chloroform after staining has been described (4) but was not used routinely in our laboratory because it produced some distortion of the filter. Finally Sudan Black B (5) was used for the staining of leukocyte granules instead of the benzidinehydrogen peroxide method originally described. Technique for preparation of a specimen follows.
Abstract
A technique f o r staining and counting milk somatic cells on Nuclepore membrane filters is described. Precision snd accuracy were compared with the direct microscopic somatic cell count modified for low cell count milk. The method appears suitable for application to low count milk from normal quarters.
Introduction
Duitschaever et al. (1, 2) have previously investigated membrane filters for the differentiation and enumeration of somatic cells in milk. Use of their technique in our laboratory has confirmed that it is highly suitable ~or differential studies. I t also appeared that the technique might be adapted to increase the precision of counting cells in milk from quarters with low cell content because the method permitted the concentration on the filter of all cells from a relatively large volume of milk. Materials and Methods
!VIodifieations of the technique were made. Nuclepore 1 filters (25 mm diameter and 2~ pore size2) were used instead of the l~illipore mereAuthorized for publication as Paper 4022 on July 26, 1971 in the Journal Series of the Pennsylvania Agricultural Experiment Station. i General Electric Company, Valleeitos Atomic Laboratory, P.O. Box 946, Pleasanton, California 94566. 2 The 2~ pore size used in this work is no longer available. A 3/z pore size is available and is suitable. 3 I~ationaI Biological Stain lqA0629. A. H. Thomas Co., P.O. Box 779, Philadelphia, P a , 19105. Stain is prepared in stock solution containing .3 g in 100 ml absolute ethanol. This should be prepared several days before use to completely dissolve all stain. Buffer for preparation of working stain contains 16 g phenol, 30 ml ethanol, .3 g ~A2HPO 4 and 100 ml distilled H20. The phenol is dissolved in the alcohol, the NA2HPO 4 in water and the 2 solutions are mixed. Working stain is prepared by mixing 60 ml of stock Sudan Black B and 40 ml of buffer. Stain is filtered before use. Working stain is usable for at least one week if refrigerated but should be refiltered before each use.
379
1. To 10 ml of milk add 1 ml of preservative solution (.24 g ttgC]2 and 1.04 g K2Cr20 7 in 100 ml I t 2 0 ) . This dilution is considered in computing the factor for calculation of cell numbers. 2. Pipet .1 or .2 ml of the preserved milk into 2 ml of .25 ~ sucrose solution in a polyethylene test tube. To obtain more cells for counting from low-count milk the larger volume was used. Milk samples were pre-tested with the California Mastitis Test (CMT) to determine the probable cell level. F o r samples with CMT scores of negative or trace .2 ml of milk was used; for higher scores .1 ml was used. 3. F i l t e r cell suspension through a Nuclepore filter using 15 to 20 cm of vacuum. The filter is held, shiny side up, in a standard laboratory filter holder for membrane filters. Rinse tube with an additional 2 ml sucrose and pass this through filter. 4. While the filter is still in the filter holder, cover surface with Sudan Black B 3 and allow to stand 15 sec. Apply vacuum to remove stain. 5. With vacuum on, rinse filter twice with absolute methanol. I f stain is still visible on filter, rinse again. With vacuum on, rinse with distilled water.
380
JOURNAL OF DAIRY SCIENCE
TABLE 1. Comparison of filter and direct microscopic somatic cell count methods for counting milk somatic cells. Filter
Range
Number samples a
~ean within sample SD
(Cells/ml) 0-100,000 100,000-200,000 200,000-500,000 500,000-1,000,000 1,000,000-2,000,000
12 6 6 6 6
(Cells/nil) 4,100 ]2,300 17,500 44,000 73,000
DMSCC
l~ean within sample CV
~¢fean within sample SD
~ean within sample CV
(%) 14.8 8.7 6.2 7.1 5.2
(Cells/ml) 5,300 12,700 25,400 40,000 55,000
(%) 24.8 9.4 7.9 5.9 3.4
Six replicate counts were made on each sample by each method. 6. Remove filter and place cell side up on microscope slide. Fix filter in place with stainless steel clips4 for staining. Do not allow filter to dry before staining. 7. Stain in May-Greenwald stain5 (2 min), rinse in distilled water, stain in Giemsa stain 6 (2 min), and rinse in distilled water. 8. Allow filters to air dry before applying immersion oil. Filters may be mounted, but we have had better results by applying immersion oil directly to filter. After examining do not remove immersion oil. When stored in a dust-proof container, the filters will remain in good condition for at least several months. The technique of enumerating all cells in a diametric strip across the filter was adapted from the direct microscopic somatic cell count (DMSCC) (3). Because our chief interest was in low cell count milk, cells lying within a wide strip were counted. This strip was defined with an eyepiece reticle with a line spacing of 12 ram; this defined a strip of .131 mm width in the field of view of the oil immersion objective (magnification, 970 × ) . The strip factor was calculated as described for the DMSCC (3) taking into consideration the volmne of milk filtered and the filtration area of the filter. This area was calculated from the measured diameter of the effective filtration surface. For 4 Small stainless clips, Catalog XX6201701, Millipore Corp., Bedford, Mass., 01730. 5 Harleco Item 660. A. H. Thomas Co., ]'.O. Box 779, Philadelphia, Pa., 19105. Dilute with 1 volume distilled water. Keep covered when ~ot. in use. 6 IIarleco Item 620. A. It. Thomas Co., P.O. Box 779, Philadelphia, Pa., 19105. Dilute with 9 Volumes distilled water; filter before use. Keep covered when not ix use. JOU]~NAL OF D&IR.¥ SCIENCE Xd~OL. 55, NO. 3
each cell count, all cells in horizontal and vertical strips of one filter were counted. The strip factors for this system were 677 for .2 ml and 1,354 for .1 ml. Precision and accuracy were assessed by comparison with results from the DMSCC (3) modified for low-count milk. The wide eyepiece reticle described above was used in the same microscope that was used for the filter counts. F o r each count 4, instead of 2 milk films as described for the DMSCC, were prepared, and the horizontal and vertical strips on each of the films were counted. Thus, for one count, 8 strips were counted. The strip factor for this system was 6,925. Individual quarter samples were obtained in various cell count ranges, as shown in Table 1. Six replicate counts were made on each sample by each method. Results and Discussion
The mean standard deviations within sample and coefficients of variation in each cell count range are in Table 1. The results suggested that the filter method might be more precise at low cell counts but that the advantage is lost at higher counts. To test this, variances among observations within samples were computed. For each range, the ratio of the mean error variances for the two methods was computed and compared to tabular F values. A twotailed test with respect to F was applied since there was no reason to expect either method to yield a larger error variance. The differences in the error variances did not approach significance in the intermediate ranges but tended to be smaller ( P < 0 . 1 ) for the filter method in the lowest range and smaller ( P < 0 . 1 ) for the DMSCC in the highest range. This is probably because the larger cell numbers
TECHNICAL
NOTES
381
I.--
correlation between single counts made by ca~.h method oil individual samples would not be nearly so high. The data indicate that the method produces counts which agree with the DMSCC. The membrane technique is time-consuming and its application will be limited. H o w e v e r , it nmy be useful for research purposes where accuracy and precision are required on lowcount milk. Its utility is increased because highly acceptable differential counts may be made on the same preparation.
o
Acknowledgment
4,0 Z cr
Of
rn
UJ
3.OK DO
X
Y = .991 X
_1 ...I W
o
o~ (.9 O .J
0
I
I
I
I
lO
2.0
5.0
40
LOG SOMATIC CELLS X i()3/ml (DMSCC)
FIG. ]. Regression line through the origin showing the relationship between mean somatic cell counts obtained by the filter and direct microscopic somatic cell count methods. counted on the filter in the lower ranges minimize counting errors; in the higher ranges the advantage gained by counting more cells becomes relatively less important and other errors inherent in the filter method may weigh more heavily. To assess the accuracy of the filter method a regression line relating the log of the mean counts by each method was calculated. Logarithms were used to maximize the effects of counts in the lower ranges. The regression line through the origin and its equation are shown in F i g u r e ]. The least squares regression line had a small positive intercept (.15) on the ordinate; however, as no significant reduction in residual mean square was obtained with this line, the line calculated through the origin, which theory requires, is presented. The correlation coefficient f o r this line is high (R = .99); individual points are means and the
The author thmlks Miss Erika Zelem for expert technical assistance and Dr. Marvin L. l~isius for advice on statistics. R. J. EBERHART, Department of Veterinary Science, The Pennsylvania State University, University Park ] 6 8 0 2 References
(1) Duitschaever, C. L., and G. C. Ashton. 1968. Comparison of counts using a Breed-type smear and millipore membrane methods on fresh and preserved milk samples. J. Dairy Sci., 51 : 665. (2) Duitschaever, C. L., and A. G. Leggatt. 1967. Cells in bovine milk: Differential staining in suspension : collecting, counting and examining on millipore membranes. Stain Technol., 42: 183. (3) National Mastitis Council, Subcommittee on Screening Tests. 1968. Direct microscopic somatic cell count in ml]k. J. Milk Food Techno]., 31: 350. (4) Reynaud, A. J., and Eileen B. King. ]967. A new filter for diagnostic cytology. Acta Cytologica, 11: 289. (5) Sheehan, It. L., and G. W. Storey. 1947. An improved method of staining leukocyte granules with Sudan Black B. J. Pathol. Bact., 59 : 336.
JOURNAL OF DAIRY SCIENCE VOL. 55, NO. S