Fluorescence microscopy methods for the determination of somatic cell count in raw cow’s milk
P. Zajac, S. Zubricka, J. Capla, L. Zelenakovahttps://doi.org/10.17221/222/2015-VETMEDCitation:Zajac P., Zubricka S., Capla J., Zelenakova L. (2016): Fluorescence microscopy methods for the determination of somatic cell count in raw cow’s milk. Veterinarni Medicina, 61: 612-622.
The direct fluorescence microscopy method with ethidium bromide staining can be used for somatic cell counting in raw cow’s milk. However, this method has some limitations that may influence the results of the analysis. We therefore aimed at improving the procedure of somatic cell nuclei staining. We tested the hypothesis that ethidium bromide can better penetrate into the DNA of cells with degraded somatic cell walls or into dead cells. Therefore, we increased the temperature of the sample to 100 °C in order to disrupt the somatic cell wall membrane and to improve ethidium bromide penetration to somatic cell nuclei. In all, 90 samples of raw cow’s milk were analysed in this experiment. Three parallel measurements were performed using each of the microscopic methods and the routine flow cytometry method. In all, 810 microscopic smears were analysed. The somatic cells were counted using fluorescence microscopic methods and flow cytometry. The increased temperature during the sample preparation improved (P < 0.005) the penetration of ethidium bromide into the somatic cell nuclei. It is concluded that the direct fluorescence microscopy method is suitable for precise laboratory analysis of somatic cell in raw cow’s milk.Keywords:
microscopy; cow milk; fluorescence; flow cytometry; temperature; somatic cell; wall; temperature; DMSCCReferences:
Alekish MO (): The association between the somatic cell count and isolated microorganisms during subclinical mastitis in heifers in Jordan. Veterinární Medicína, 60, 71-76 https://doi.org/10.17221/7980-VETMEDANSES – French Agency for Food, Environmental and Occupational Health and Safety (2013): European Union Reference Laboratory for Milk and Milk Products. Interlaboratory proficiency testing trial – Final report, EILA/Anses/LRUE MMP/EDB/2013/01. Somatic cell counting in raw cow’s milk by EN ISO 13366-1 standard method.ATSDR – Agency for Toxic Substances and Disease Registry (1996): Toxicological Profile for 1,1,2,2-Tetrachloroethane (Update). Agency for Toxic Substances and Disease Registry. U.S. Public Health Service, U.S. Department of Health and Human Services, Atlanta.Burvenich Christian, Van Merris Val�rie, Mehrzad Jalil, Diez-Fraile Araceli, Duchateau Luc (2003): Severity of E. coli mastitis is mainly determined by cow factors. Veterinary Research, 34, 521-564 https://doi.org/10.1051/vetres:2003023Cervinkova D, Vlkova H, Borodacova I, Makovcova J, Babak V, Lorencova A, Vrtkova I, Marosevic D, Jaglic Z (2013): Prevalence of mastitis pathogens in milk from clinically healthy cows. Veterinarni Medicina 58, 567–575.Commission Regulation (EC) No. 1662/2006 of 6 November 2006 amending Regulation (EC) No. 853/2004 of the European Parliament and of the Council laying down specific hygiene rules for food of animal origin.Dworecka-Kaszak Bozena, Krutkiewicz Alicja, Szopa Daniel, Kleczkowski Miroslaw, Biegańska Malgorzata (2012): High Prevalence of Candida Yeast in Milk Samples from Cows Suffering from Mastitis in Poland. The Scientific World Journal, 2012, 1-5 https://doi.org/10.1100/2012/196347Ellison SLR, Williams A (2012) Eurachem/CITAC guide: Quantifying Uncertainty in Analytical Measurement. 3rd edn. Available: www.eurachem.org.Gallier SYFCH (2010): Understanding the structure of the bovine milk fat globule and its membrane by means of microscopic techniques and model systems. [PhD Thesis.] University of Otago, Dunedin, New Zealand.Gonzalo C., Martínez J.R., Carriedo J.A., San Primitivo F. (2003): Fossomatic Cell-Counting on Ewe Milk: Comparison with Direct Microscopy and Study of Variation Factors. Journal of Dairy Science, 86, 138-145 https://doi.org/10.3168/jds.S0022-0302(03)73593-0GRAPPIN R., JEUNET R. (1974): Premiers essais de l'appareil " Fossomatic " pour la détermination automatique du nombre de cellules du lait. Le Lait, 54, 627-644 https://doi.org/10.1051/lait:1974539-54032Harmon R.J. (1994): Physiology of Mastitis and Factors Affecting Somatic Cell Counts. Journal of Dairy Science, 77, 2103-2112 https://doi.org/10.3168/jds.S0022-0302(94)77153-8HEALD C. W., JONES G. M., NICKERSON S. C., PATTERSON W. N., VINSON W. E. (1977): Preliminary Evaluation of the Fossomatic Somatic Cell Counter for Analysis of Individual Cow Samples in a Central Testing Laboratory. Journal of Food Protection, 40, 523-526 https://doi.org/10.4315/0362-028X-40.8.523Heeschen W (1975): Determination of somatic cells in milk (technical aspect of counting). Bulletin of the International Dairy Federation 85, 79–92.Idriss Sharaf Eldeen Abu Baker, Tančin Vladimír, Foltys Vladimír, Kirchnerová Katarína, Tančinová Dana, Vršková Martina (2013): Relationship between mastitis causative pathogens and somatic cell counts in dairy cows. Potravinarstvo, 7, - https://doi.org/10.5219/304ISO 707 (2008): Milk and Milk Products – Guidance on Sampling. International Organization for Standardisation, Geneva, Switzerland.ISO 13366-1 (1997): Part 1: Microscopic method (Reference method). In: Milk – Enumeration of Somatic Cells. International Organization for Standardisation, Geneva, Switzerland.ISO 13366-1 (2008): Part 1: Microscopic method (Reference method), superseding ISO 13366-1 (1997). In: Milk – Enumeration of Somatic Cells. International Organization for Standardisation, Geneva, Switzerland.ISO 13366-1:2008/Cor 1 (2009): Part 1: Microscopic method (Reference method). In: Milk – Enumeration of Somatic Cells. International Organization for Standardisation, Geneva, Switzerland.ISO 13366-2 (2006): Part 2: Guidance on the operation of fluoro-opto-electronic counters. In: Milk – Enumeration of Somatic Cells. International Organization for Standardisation, Geneva, Switzerland.ISO/IEC 17025 (2005): General Requirements for the Competence of Testing and Calibration Laboratories. International Organization for Standardisation, Geneva, Switzerland.Unluturk Sevcan, Pelvan Merve (2015): Application of Flow Cytometry and Fluorescence Techniques in Somatic Cell Analysis of Raw Milk. International Journal of Food Processing Technology, 2, 11-16 https://doi.org/10.15379/2408-9826.2015.02.01.2Petersson K.H., Connor L.A., Petersson-Wolfe C.S., Rego K.A. (2011): Short communication: Evaluation of confirmatory stains used for direct microscopic somatic cell counting of sheep milk. Journal of Dairy Science, 94, 1908-1912 https://doi.org/10.3168/jds.2010-3574Py�r�l� Satu (2003): Indicators of inflammation in the diagnosis of mastitis. Veterinary Research, 34, 565-578 https://doi.org/10.1051/vetres:2003026Raugel PJ (ed.) (1999): Rapid Food Analysis and Hygiene Monitoring: Kits, Instruments and Systems. Springer-Verlag, Berlin. 383 p.Rysanek D, Babak V, Zouharova M (2007): Bulk tank milk somatic cell count and sources of raw milk contamination with mastitis pathogens. Veterinarni Medicina 52, 223–230.Sartori L.C.A., Santos R.C., Marin J.M. (2014): Identification of Candida species isolated from cows suffering mastitis in four Brazilian states. Arquivo Brasileiro de Medicina Veterinária e Zootecnia, 66, 1615-1617 https://doi.org/10.1590/1678-7576Madsen P. Schmidt (1975): Fluoro-opto-electronic cell-counting on milk. Journal of Dairy Research, 42, 227- https://doi.org/10.1017/S0022029900015260Schmidt-Madsen P (1979): Influence of storage and preservation of milk samples on microscopic and Fossomatic somatic cell counts. Nordisk Veterinaer Medicin 31, 449–454.Ubben EH (2004): Quality management for DMSCC from milk sample to ring trial. Lecture In: Workshop of the European National Reference Laboratories, 9–10 September 2004, BFEL, Kiel, Germany.Varzakas T (2015): Chapter 3. Centrifugation-filtration. In: Varzakas T, Tzia C (eds): Food Engineering Handbook. Food Process Engineering. CRC Press, Boca Raton. 90 p.Vasiľ Milan, Elečko Juraj, Zigo František, Farkašová Zuzana (2012): OCCURRENCE OF SOME PATHOGENITY FACTORS IN COAGULASE NEGATIVE STAPHYLOCOCCI ISOLATED FROM MASTITIS MILK IN DAIRY COWS. Potravinarstvo, 6, - https://doi.org/10.5219/186Vermunt AEM, Loeffen GJM, Van Der Voet H, Naber MAAM (1995): Development of reference samples for the calibration and quality control of somatic cell count using a Fossomatic instrument. Netherlands Milk and Dairy Journal 49, 111–123.Zajac P (2007): Optimalization of the reference method for determination of somatic cells count in raw cow milk. [PhD Thesis.] Slovak University of Agriculture in Nitra, Slovakia.Zajác Peter, Tomáška Martin, Murárová Anna, Čapla Jozef, Čurlej Jozef (2012): QUALITY AND SAFETY OF RAW COW’S MILK IN SLOVAKIA IN 2011. Potravinarstvo, 6, - https://doi.org/10.5219/189