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Portugal and Turkey

Cooperation Training
sessions in the
framework of the
National Program of
Waste Monitoring

November 3rd 2015
INIAV, UEISTSA
Polo do Lumiar

Somatic Cell Count in
cow’s raw milk by
reference
method
Cristina Aleixo
Manuela Vida
INIAV – UEISTSA
Portuguese NRL-MMP

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Quinta do
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Oeiras

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5

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Quinta da Fonte Boa - Vale de
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4
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7

INIAV’S MISSION

Applied Research
Scientific and Technical Support
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•National Reference Laboratories (NRLs)
•Gene Resources:
•Portuguese Plant Gene (BPGV)
•Portuguese Animal Gene Bank (BPGA)
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OUR NUMBERS Human resources: 620 Financial resources : Experimental Farms: Education: staff members 27 M€ 1105 ha 600 students On going Research Projects: 92 Scientific and Technical Publications: 1144 .

MAIN RESEARCH AREAS • Food Science and Technology • Food and Feed Safety • Forestry • Plant and Animal Health • Animal Microbiology and Pathology • Animal Production • Plant and Animal Breeding and Genetics • Agrarian Systems: Production and Sustainability • Viticulture and Oenology • Soil fertility and Plant mineral nutrition .

8 M€ INIAV cost: 8.1 M€ 7 22 32 2 2 6 42 Number of projects / financial source FCT PRODER 7PQ FEDER PRRN QREN COST Outros 13% 2% 1% 4% 0% 2% 32% 45% INIAV cost/ financial source .RESEARCH PROJECTS: 92 PROJECTS with outside funding (2014 data) Global budget: 36.

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Lab & Residues Analysis ex-LNIV UEI&S Food Safety & Food Technology Food Science and Technology Secretariat Food Technolog y Labs & Pilot Plant Lumiar.OUR Research Unit DTIA exINETI DTPA e EVN ex-INIA Food Microb. Oeiras Feed Feed Control Lab Lisboa Residues Analysis Residues Analysis Lab. -Lisboa Residues & Txicological Analysis Lab .Vairão Residues of Pesticides Lab.Oeiras Oenology and Viticulture Food Microbiology AgroIndustrial Labs Microbiology Lab Lumiar Lisboa Food Microbiology Lab Vairão Oenology Lab Dois Portos .

OUR Lab AGRO INDUSTRIAL MICROBIOLOGY LAB.to the definition of traditional products and new products development .for the characterization. . innovation and demonstration. experimentation. Unit Research of Food Science and Technology. MISSION: to perform research. conservation and processing of agricultural food products.food quality and safety . .

Fusion of the Food Microbiology with the Industrial Microbiology Laboratories Matrixes: Milk and milk products Meat and meat products Vegetable products Chocolates and ice-creams Sugar and honey Baby food Feedstuffs Waters Pharmaceutical products Gas oils and Biodiesel .OUR Lab AGRO INDUSTRIAL MICROBIOLOGY LAB.

Activities: Research and Development contracts projects Training training courses academic internships projects Technical assistance Laboratorial services NATIONAL REFERENCE LABORATORY .OUR Lab AGRO INDUSTRIAL MICROBIOLOGY LAB.

OUR Lab AGRO INDUSTRIAL MICROBIOLOGY LAB. Accreditation Experience from 1992 to 2007 12 methods accreditated for water analysis (Accreditation Annex nº L 024-4) from 1994 to 2008 12 methods accreditated for food analysis (Accreditation Annex nº L 009419) from 2006 to 2008 5 methods accreditated for GMO analysis (ENGL member since the beginning and NRL since 2006) (Accreditation Annex nº L 0094-19) .

STATE FUNCTIONS NATIONAL REFERENCE LABORATORIES Milk and Milk Products Coagulase Positive Staphylococcus and staphylococcal toxins Salmonella in Feed Processed Animal Protein (DNA ruminant detection) GMO detection in Food and Feed .

STATE FUNCTIONS NATIONAL REFERENCE LABORATORIES .

STATE FUNCTIONS NATIONAL REFERENCE LABORATORIES Working plan Implementation of the methods Personnel qualification Participation on proficiency tests Participation on EURLs workshops and training sessions Survey of the national situation in collaboration with the competent authority Planning and compilation of data concerning the official control Changing of lab building facilities to Oeiras Preparation for Accreditation (for next year?) .

STATE FUNCTIONS NATIONAL REFERENCE LABORATORIES .

3rd November 2015 . UEISTSA.Somatic Cells Count Portugal and Turkey Cooperation Training sessions in the framework of the National Program of Waste Monitoring INIAV. Polo do Lumiar.

from the body • Leukocytes released from the blood to combat udder infection. prevent or reduce inflammation (mastitis) Macrophage (60%) Neutrophiles (10%) • Epithelial cells – presents in low number (0-2%) Lymphocytes( 28%) . and thereby.What are Somatic Cells? From Greek “Somatikós” = body.

– in response to a physical. stress and poor nutrition.What are Somatic Cells? • Released from the blood to combat udder infection. to prevent or reduce inflammation (mastitis) • Factors that contribute to increases SCC on milk include: – – – – sub-clinical mastitis. . lactation number. • chemical or – infeccious agression – and thereby. advance in stage of lactation.

.The meaning of SCC in milk • SCC is a specific laboratory test – expresses the number of somatic cells per milliliter of milk • When analyzed individually – method of diagnosis of Subclinical Mastitis • When analyzed in the tank – indicative of the quality of raw milk.

000 500.000 48 29 Source: NMF 1996 .000 6 16 0 6 1.000 32 18 1.500.000.SCC and milk • SCC is an indicator of milk quality There is a direct relationship between – high SCC and milk composition – SCC and high rates of infection of cow’s breast quarters SCC tank (cells/mL) % breast quarters % production losses 200.

immunoglobulins and – Undesireble decrease of other components • • • • Fat. lactoferrin. casein. etc. potassium.SCC and milk • SCC is an indicator of milk quality – high value of SCC there is an increase of some milk components • sodium. calcium. . pH • serum protein. lactose. chlorides.

51 3.02 0.35 0.147 0. 2001 Main change Reduction of synthesis Passage of blood Source: Schallibaum.057 0.6 1.000 Lactose Casein 4.6 0.8 1.23 0.81 4.10 0.9 Serum protein Serum albumins Chlorides.74 2.13 0.74 3.69 3.9 2.6 2.096 0.000 >1.SCC and milk Components (g/100mL) SCC x 1.135 6.000 cell/mL < 100 < 250 500 -1.157 6.091 0.105 0.21 2.173 6. 2001 .81 0.180 6.25 0.65 4.062 0. 2001 Sodium Potassium pH.31 0.82 0.091 0.79 4.25 Fat 3.121 0.

SCC and quality in milk and milk products • Cheeses: – – – – – – decrease in industrial yield. low rate of hardening of clot and defects of texture. negative changes in the sensory properties. high solid loss in serum. increased water content in the clot. increased time for clot formation .

– reducing the period of useful life. • Butter – sensorial quality impaired: rancid and rusty flavor. • Yogurt – growth inhibition of mo used in the manufacture of the product.SCC and quality in milk and milk products • Powdered milk – change of thermal stability. • UHT milk – Reducing the time for start of gelatinization. – undesirable flavors in the finished product. .

The meaning of SCC in milk Industry's concern with the consequences of mastitis in animals reduces the concentration of milk components (casein. . mostly) reduces the industrial yield reduces shelf-life of dairy products affects product’s quality offered to consumers Economical Losses! Damage for everyone from the farmer to the consumer.

if not uniquely .conditions detailed in EC Regulation 2074/2005. • Given the workload to implement the reference methods for TF and SCC.EN ISO 4833 for TF or EN ISO 13366-1 for SCC. routine controls (own checks) are currently performed in Europe in overwhelming majority . – alternative methods .SCC methods and EU Regulation • EC Regulation 2074/2005 (modified by EC Regulation 1664/2006) defines the testing methods for raw milk to be used to check compliance with the limits for total flora and somatic cells count laid down in Regulation 853/2004 – reference methods .by .

• The EURL MMP is conducting at European level the harmonization of national conversion equations between the alternative and EN ISO 4833 reference method. for TF in raw milk.SCC methods and EU Regulation • To check compliance with legal limits of EC Regulation 853/2004 the results of the instrumental methods have to be converted and expressed into the unit of the reference method. • This requires the establishment of a conversion equation between the instrumental and the reference method. which has been identified as a critical point for the implementation of the instrumental method. .

III . III) and – for dairy products (Chapter II. • The Regulation 2074/2005 modified by Regulation 1664/2006 includes the description of testing methods for raw milk and heat-treated milk. – They include criteria on somatic cell count for raw cow’s milk.SCC methods and EU Regulation • Microbiological criteria have been fixed on Regulation 853/2004: – for raw milk in Section IX (Chapter I. • including the reference method for somatic cell count as well as conditions for the use of alternative methods (if they are validated against EN ISO 133661 in accordance with the protocol of the Standard EN .criteria for the use of raw cow’s milk for further processing).

SCC methods and EU Regulation • The EU has set the legislative limit of ≤ 400 x 103 SCC/mL • The permitted SCC limit varies internationally. e. but pressure to reduce SCC further.g. Bonus for < 200 x 103 cells/mL • It is considered by some research that milk constituents ‘abandon their physiological ranges’ at SCC >100 x 103 cells/mL and that ‘infection’ is present at SCC > 100 x 103 cells/mL .

000 cells/mL • Pathogenic micro-organisms » Corynebacterium bovis. coliforms • increase of somatic cells to 1.000.SCC in milk • “Normal" Threshold – SCC – < 100. Streptococcus agalactiae.000cells /mL • Main pathogenic micro-organisms » Staphylococus aureus. coagulase-negative staphylococci • are responsible for average scores of SCC .000 cells/mL • Threshold value to indicate the presence or absence of inflammation or mastitis – 100.000 cells /mL < SCC < 250.

SCC – the reference method • EN ISO 13366 –“Enumeration of somatic cells part 1: microscopic method“ Microscopic method (reference method) Fluoro-opto-electronic method (alternative method) Fossomatic FC (uses flow cytometry technology) .

– Is applicable for the counting of somatic cells in cow’s milk – This method is suitable for preparing standard test samples and determining reference method values .EN ISO 13366-1 “Enumeration of somatic cells part 1: microscopic method“ • Scope – EN ISO 13366 part 1 specifies a microscopic method (reference method) for the counting of somatic cells in both raw and chemically preserved milk.

to give the number of cells per millilitre .EN ISO 13366-1 “Enumeration of somatic cells part 1: microscopic method“ • Principle – A test portion of milk to be examined is spread over a slide to form a smear. . the stained cells are counted using a microscope. – The smear is dried and then the cells are stained with Newman-Lampert modified dye – Subsequently. – The number of cells counted in a defined area are multiplied by a working factor.

• In our laboratory. the samples are stored in an inverted position to avoid fatty substances adhering to the lids – Heat the test sample in a water bath set at 40 °C. store the test samples at a temperature of 4 °C ± 2 °C. – Cool the sample to the temperature at which the micropipette has been calibrated (room temperature) – If necessary dilute test samples (with an estimated somatic cell count of > 1 000 000 cells/mL) in a phosphate buffer solution to obtain a somatic cell count of .EN ISO 13366-1 “Enumeration of somatic cells part 1: microscopic method“ • Preparation of test sample – Prior to testing. – Mix the test sample carefully. – Analyse the test samples within 6 h after sampling.

We do not wash them. for each test sample.g.EN ISO 13366-1 “Enumeration of somatic cells part 1: microscopic method“ • Preparation of the smear • Prepare. – Dip the slides in ethanol ( of volume fraction 95%) – Flame and cool – Using the micropipette 10 L of the test sample (eventually diluted) – Place the mixture on a clean slide with an area of 1 cm2 • In our lab we use slides only once. a smear not damaged by the dyeing process). – Using the needle. while ensuring that . spread the test sample evenly over the entire area defined. at least two smears and count the best one (e.

Obtained solution is passed through an appropriate filter into an airtight bottle. Glacial acetic acid added and carefully mix again the solution. – Modified Newman-Lampert stain solution is composed: » » » » » Ethanol + Tetrachloroethane heated in a water bath at 65ºC. » Filter the Newman-Lampert stain solution again before use • In our lab we dip the slide 30 min.EN ISO 13366-1 “Enumeration of somatic cells part 1: microscopic method“ • Staining – Dip the dried smear on the slide in the modified Newman-Lampert stain solution for at least 15 min. We use Newman’s stain solution modified commercialy available. Methylene blue added under a fume cupboard and carefully mix Mixture cooled in a refrigerator to 4ºC. – Dry the smear at ambient temperature. – Dip the smear gently in tap water until all surplus dye is washed away. .

• In our lab the best magnification is 400 X – The training and skill of the analyst is the main crucial success factor for a proper performance of the method • frequent execution of the method and participation in interlaboratory trials . count the cell nuclei in the obtained smear of fields entirely filled with milk smear only.EN ISO 13366-1 “Enumeration of somatic cells part 1: microscopic method“ • Determination: reading optimization (1) – Using the microscope . in order to have an average maximum number of 20 cells in each field. • We advise to check microscopes once a year by an external service provider – Choose the best magnification (from 500× to 1 000×).

are listed in Table 1. Fields and strips to be counted shall be chosen so as to obtain a representative count for the . The cells generally are 8 µm or larger. unless the nuclear unit(s) is (are) clearly separated.EN ISO 13366-1 “Enumeration of somatic cells part 1: microscopic method“ • Determination: reading optimization (2) Table 1 -Minimum number (N) of cells » » » » » » » » » The cells possess a stained nucleus. it is essential that the listed minimum number of cells be counted. For a proper execution of the method. Do not count cells less than 4 µm Count fragments only if more than 50 % of nuclear material is visible. Count cell clusters as one. in order to arrive at the listed coefficient of variation. Cells in milk are distributed according to a Poisson distribution The minimum number (N) of cells to be counted in relation to the cell count level.

of 0. in vertical strips in regularly spaced fields – a) For a circular shape. adjust the field to the border of the smear.EN ISO 13366-1 “Enumeration of somatic cells part 1: microscopic method“ • Counting in sucessive fields (1) – Count nuclei in successive fields.5 mm is suitable for circular shape. A distance. In the case of an uncovered surface near the border of the template. • b) Place the upper or lower edge of the field circumference tangentially onto the internal upper or lower border of the template (the latter should not appear in the field). so as to allow counting of a minimum of 5 fields from the top of the strip. from the left side of the horizontal diameter. start from a sufficient distance. dL. dL. .

dH. move the objective with a fixed space distance. until the opposite side (bottom or top) of the strip is reached. repeat the operation in c). counting is performed after moving up the objective until the border disappears again completely from the field. of 1 mm is considered appropriate. d) From the last field counted. dH.EN ISO 13366-1 “Enumeration of somatic cells part 1: microscopic method“ • Counting in sucessive fields (2) • c) After having counted the first field. If the lower or upper border appears and it fills less than half the field surface. down or up to the next graduation in the direction of the lower or upper edge and count the new field. A distance. . which then only covers smear.

EN ISO 13366-1 “Enumeration of somatic cells part 1: microscopic method“ • Counting in sucessive fields (3) • e) Then move the objective to the right by a distance.g. • f) Repeat from b) to e) until the right side of the template is reached. by changing the starting place and/or adapting the step-bystep moving distances) so as to obtain appropriate field numbers that are representative of the entire smear. focus the microscope objective on other locations (e. dw (dw = 2 mm). and start a new strip in the opposite direction (top or bottom). supplementary fields can be counted. To do this. • g) In case an insufficient number of fields is counted (see Table 1). • h) Calculate the result. .

of the smear. c. • Df is the diameter.28 mm diameter of the smear. d = 0. expressed in number of cells per millilitre. of the smear. • Nf is the number of fields counted completely. Ws is the width. in millimetres. in millimetres.EN ISO 13366-1 “Enumeration of somatic cells part 1: microscopic method“ • Calculation of results (1) – Check the 11. in millimetres. Dc is the diameter. in millilitres. of cells by using the following equation: • • c is the total concentration. with a 1:1 dilution. d is the dilution factor used (If no dilution is required. of the microscope field.) • • . using the graduations and vernier of the microscope. • Nt is the total number of cells counted. • Vm is the volume. in millimetres. • • Ls is the length. – Calculate the total concentration. d = 1. of the smear. of the test sample smeared.5.

01 mL – Working Factor with objective 40X = 60 132 – Working Factor with objective 100X = 392 716 .EN ISO 13366-1 “Enumeration of somatic cells part 1: microscopic method“ • Calculation of results (2) – For our lab • Dc 11.28 mm • Df of microscope with objective 40 X = 0.46 mm or • Df of microscope with objective 100 X = 0.18 mm – We determine the value of the diameter of the microscope field (Df) by a calibrated micrometer • Vm = 0.

EN ISO 13366-1 “Enumeration of somatic cells part 1: microscopic method“ • Expression of results – Express the test results in whole figures of thousands • for example: express 401 586 cells/ml as 402 000 cells/ml – Precision • The values for the repeatability and reproducibility were derived from the result of an interlaboratory carried in accordance with ISO 5725-1 and study study test for for cows' cows' milk milkout involving involving 5725-2. samples A and B: 245 000 cells/ml. and Details 18 18ISO laboratories laboratories and 13 13 of this interlaboratory test are summarized countries countries in Annex A of the Standard. » The mean values for each level were respectively: » Level 1. . » Level 2. samples C and D: 455 000 cells/ml.

EN ISO 13366-1 “Enumeration of somatic cells part 1: microscopic method“ • Repeatability – The absolute difference between two independent single test results. will in not more than 5 % of cases be greater than the values given in Table 2: . obtained using the same method • • • • • • on identical test material in the same laboratory by the same operator using the same equipment within a short interval of time.

obtained – – – – – – using the same method on identical test material in different laboratories with different operators using different equipment.EN ISO 13366-1 “Enumeration of somatic cells part 1: microscopic method“ • Reproducibility • The absolute difference between two single test results. will in not more than 5 % of cases be greater than the values given in Table 3: .

it is of utmost importance to develop Certified Reference Materials (CRMs) • CRMs are needed to calibrate instrumental methods. Belgium) in cooperation with the .CRMs • Given the deficiencies of the reference microscopic method for SCC in raw milk (lack of reproducibility) and the limited number of laboratories using it.Method update . • JRC/IRMM (Geel. mostly used for routine analyses of SCC in raw milk. as to have comparable SCC analyses conducted within each European country and between different European countries.

smear staining. • A2-day session include both a presentation of the reference method EN ISO 13366-1 and a practical session: – – – – smear preparation. .Training sessions • The EURL MMP (EDB Unit) organized once a year a training session on how to count somatic cells in raw cow’s milk by the reference method EN ISO 13366-1. calculation and expression of results.Method update . slide counting.

part 2 METHOD PRINCIPLE ADVANTAGES / DISADVANTAGES reference method laborius The cells are fixed on Optical microscopy tiring (induces counting glass slide. Count cells that have minimum intensity of fluorescence due to the staining of nucleic DNA Rotine method authomatic method medium and high yield easy processing high precision high capacity analyser: 500 samples/hour .SCC in milk .EN ISO 133661 part 1 vs. stained (EN ISO 133661 – errors) and counted using the 1) requires very well trained microscope technicians little accuracy Based on flowcytometry technology . (equipament Fossomatic) (EN ISO 133661 – 2) counts and characterises particles and cells while they are flowing.

November 3tr 2015 Instituto Nacional de Investigação Agrária e Veterinária. Av.aleixo@iniav.P. UEISTSA.Thank you for your attention! Cristina Aleixo cristina.pt Portugal and Turkey Cooperation – Training sessions in the framework of the National Program of Waste Monitoring INIAV. Quinta do Marquês. Portugal Tel : (+ 351) 21 440 3500 | Fax : (+ 351) 21 440 3666 www.pt . Polo do Lumiar. I. 2780-157 Oeiras.iniav. da República.

Annexes .

EN ISO 13366-1 “Enumeration of somatic cells part 1: microscopic method“ .

EN ISO 13366-1 “Enumeration of somatic cells part 1: microscopic method“ .

EN ISO 13366-1 “Enumeration of somatic cells part 1: microscopic method“ .

EN ISO 13366-1 “Enumeration of somatic cells part 1: microscopic method“ .