SOLUCIONES INNOVADORAS EN MICROBIOLOGÍA

Transcripción

1 MERCK/SIGMA-ALDRICH ALDRICH SOLUCIONES INNOVADORAS EN MICROBIOLOGÍA Jvo Siegrist (Product Manager Global), Pedro Gutierrez (Field Sales Specialist Analitycal) XV MRAMA WORKSHOP 22/11/2016

2 Test & Kits ID Membranes Una rápida y fácil confirmación de las bacterias directamente de las placas de agar Principios: Las enzimas específicas de las bacterias son detectadas por sustratos cromogénicos y fluorogénicos específicos. Visibles como colonias coloreadas o fluorescentes. Transfer to membrane sec. 1-4 h incubation at 37 C 2

3 Test & Kits Enzyme Confirmation Cards Una confirmación rápida y fácil de las bacterias Principio: Las enzimas específicas de las bacterias son detectadas por sustratos cromogénicos y fluorogénicos específicos. Visible como color o reacción de fluorescencia. Smear colony on spot 1 drop of reagent Result (within 5-10 min.) Cat. No. Target Organism E. coli Total Coliform Fecal Coliform Salmonella Enterococcus Gram+/Gram Differentiation Neisseria gonorrhea Group A Streptococcus Staphylococcus aureus 3

4 Test & Kits FlouroSELECT Assays (Analisis de superficies) E. coli Assay Kit Gram-Negative Assay Kit MRSA Assay Kit (1000 ufc/sample, result in 5 min.) Total Coliform Assay Kit Phycocyanin test (fluorometer wavelengths 600 nm ex / 650 nm em ) sigma.-aldrich.com/fluoroselect Substrato Fluorógeno (responsable de la fluorescencia) Celula con Enzima lisis Enzima 4

5 Test & Kits Métodos inmunológicos Test de lates Identificación (confirmación) Reacción de aglutinación de anticuerpo (clumping) También proporciona sistemas combinados con métodos bioquímicos (por ejemplo, Staphylo Monotec Plus System: detecta proteína A, polisacárido capsular (serotipo 5) y coagulasa, determinación explícita para Staphylococcus aureus incluyendo MRSA) Staphylococcus aureus resistente a la meticilina 5

6 Test & Kits Técnicas de sondas genómicas: ejem. HybriScan Detección de ARN (principalmente 16S rrna) con sondas (hibridación en sándwich) y análisis fotométrico cuantitativo - Automático (económico) - altamente específico - Más simple y más barato que la PCR - Muy rápido - No detecta células muertas - Detecta organismos no cultivables - Casi ningún efecto de inhibición o matriz como con la PCR 6

7 Certified Reference Microorganisms Vitroids & LENTICULE discs Los discos contienen microorganismos viables en un numero definido de ufc certificadas Rangos estrechos que varían de '000 ufc por disco. (posible hasta 107) Cepas procedentes de CECT, NCTC y NCPF Los discos se fabrican bajo la guía ISO 34 Certificado según ISO Muy estable (1 o más años) en un estado viable (sin fase lag o tiempo de recuperación). Desviación de hasta 4% a 100 ufc Almacenamiento a -20 C (posible envío a temperatura ambiente). No requiere ultra congelador Ahorro de tiempo y costes (manejo sencillo, no mantenimiento de cultivos) Screw Cap Vitroid Disc Filter plug Desiccant 7

8 Una solución precisa y conveniente Usando discos LENTICULE y Vitroids Saque el disco con los ufc requeridos fuera del congelador Deposite sobre la placa o el medio liquido de cultivo. Espere 10 minutos! Y listo. Enumere en su medio 8 Titel der Präsentation DD.MM.YYYY

9 Certified Reference Microorganisms Aplicaciones Validación de nuevos métodos Asegurar la calidad de los resultados diarios (agua, alimentos, bebidas, etc.) Desarrollo del método Cultivos de inicio (I+D, Fermentación, ) Pruebas de rendimiento de medios cultivo acorde a ISO 11133: 2014 Formación de personal sigma-aldrich.com/mibi-crm Materiales para ensayos inter-laboratorio y ensayos de aptitud 9

10 Certified Reference Microorganism LENTICULE discs Portfolio Cat. No. Reference Material Origin Strain No. ufc Range Mean ufc Range Accreditation RMF02275L Aspergillus brasiliensis (formerly NCPF 2275 Low Level Aspergillus niger) CRM07464L Bacillus cereus NCTC 7464 Low Level CRM07464M Bacillus cereus NCTC 7464 High Level x 10^4 RM11322Q Campylobacter jejuni NCTC High Level 10^2 - RMF03255L Candida albicans NCPF 3255 Low Level RMF03255H Candida albicans NCPF 3255 High Level 2 x 10^4 - RM09750L Citrobacter freundii NCTC 9750 Low Level CRM00506L Clostridium bifermentans NCTC 506 Low Level CRM13170L Clostridium perfringens NCTC Low Level CRM13170M Clostridium perfringens NCTC High Level x 10^4 CRM11467L Cronobacter sakazakii NCTC Low Level CRM10006L Enterobacter aerogenes NCTC Low Level CRM10006M Enterobacter aerogenes NCTC High Level x 10^4 CRM00775L Enterococcus faecalis NCTC 775 Low Level CRM00775M Enterococcus faecalis NCTC 775 High Level x 10^4 CRM00775H Enterococcus faecalis NCTC 775 High Level >10^5 CRM09001L Escherichia coli NCTC 9001 Low Level CRM09001M Escherichia coli NCTC 9001 High Level x 10^4 CRM09001H Escherichia coli NCTC 9001 High Level >10^5 CRM13216L Escherichia coli NCTC Low Level CRM12900L Escherichia coli O157 (NT) NCTC Low Level Cat. No. Reference Material Origin Strain No. ufc Range Mean ufc Range Accreditation CRM08167L Klebsiella oxytoca NCTC 8167 Low Level CRM11368M Legionella bozemanii NCTC High Level x 10^4 CRM11371M Legionella micdadei NCTC High Level x 10^4 CRM12821L Legionella pneumophila NCTC Low Level CRM12821M Legionella pneumophila NCTC High Level x 10^4 CRM11288L Listeria innocua NCTC Low Level CRM11994L Listeria monocytogenes NCTC Low Level CRM11994M Listeria monocytogenes NCTC High Level x 10^4 RMBLANK0 Negative Control (0 ufc) CRM10662L Pseudomonas aeruginosa NCTC Low Level CRM10662M Pseudomonas aeruginosa NCTC High Level x 10^4 CRM09528L Raoultella planticola (formerly Klebsiella aerogenes) NCTC 9528 Low Level CRM09528M Raoultella planticola (formerly NCTC 9528 High Level x 10^4 Klebsiella aerogenes) RMF03191L Saccharomyces cerevisiae NCPF 3191 Low Level RMF03191M Saccharomyces cerevisiae NCPF 3191 High Level x 10^4 - CRM06676L Salmonella enteritidis NCTC 6676 Low Level CRM07832L Salmonella Nottingham NCTC 7832 Low Level CRM12023L Salmonella Typhimurium NCTC Low Level CRM06571L Staphylococcus aureus NCTC 6571 Low Level CRM06571M Staphylococcus aureus NCTC 6571 High Level x 10^4 CRM11047L Staphylococcus epidermidis NCTC Low Level RM11218Q Vibrio furnissi NCTC Low Level RM10903Q Vibrio parahaemolyticus NCTC Low Level RM11176L Yersinia enterocolitica NCTC Low Level

11 Certified Reference Microorganism Vitroids Portfolio Strain WDCM Cat. No. CECT ufc range - VT Acinetobacter baumanni - VT VT Aspergillus brasiliensis VT VT Bacillus subtilis VT VT Bacillus cereus VT Candida albicans VT VT Candida albicans VT Citrobacter freundii - VT VT '000-7'000 Clostridium sporogenes VT VT VT Enterobacter aerogenes VT VT VT VT VT VT Enterococcus faecalis VT VT VT VT Enterococcus faecium VT VT Strain WDCM Cat. No. CECT ufc range VT VT VT VT Escherichia coli VT Variable VT VT VT VT VT Legionella pneumophila (serogroup 1) VT Legionella pneumophila VT Fluoribacter bozemanae - VT VT Klebsiella pneumoniae VT Klebsiella pneumoniae VT Proteus hauseri - VT Proteus mirabilis - VT VT VT VT variable VT VT VT VT Pseudomonas aeruginosa VT VT VT VT VT VT VT ' '000 Salmonella enterica subsp. Enterica serovar Typhimurium VT VT Salmonella enterica subsp. Enterica serovar Abony VT Salmonella enterica subsp. Enterica serovar Enteridis - VT Staphylococcus aureus susp. Aureus VT VT VT VT VT VT

12 Microorganism Proficiency Testing Ensayos de aptitud (PT) y ISO PT s son una herramienta eficaz para ayudar a los laboratorios a asegurarse de que ofrecen resultados correctos. Principio: Muestras con contenido conocido, pero no divulgado pasan los procedimientos de rutina. Resultado: Evaluación externa independiente del cumplimiento de la prueba. Asegura los resultados del laboratorio de ensayos. Filosofía de ISO/IEC 17025: La misma muestra; en diferentes momentos, de diferentes análisis, y de muchos laboratorios diferentes, debe reflejar un resultado grato. 12

13 Microorganism Proficiency Testing Flujo de trabajo de PT Laboratorio Micribiologico Organizacion acreditata para PT (Sigma-Aldrich) Planificación de programas. Coordinación de los laboratorios Envía formularios de solicitud Preparación del kit de muestra y distribución (Vitroids homogéneos y estables) Proceso de ensayo/calibración y presenta los resultados Recopilación de resultados, estadística, análisis de resultados, preparación y envío de informe a los laboratorios participantes 13

14 Microorganism Proficiency Testing PT schemes & QC Sets 14 PT Cat# QC Cat# Description MIC020 Clostridium perfringens in Water MIC014 QCMIC014 E. coli - Sludge MIC107 QCMIC107 E. coli in Drinking & Surface Water - Quantitative - WS 3 Levels MIC207 QCMIC207 E. coli in Drinking & Surface Water - Quantitative- WS 4 Levels MIC007 QCMIC007 E. coli in Drinking and Surface Water - Quantitative - WS MIC022 E. coli in Seawater - Quantitative WP MIC122 E. coli in Seawater - Quantitative WP 3 Levels MIC003 QCMIC003 E. coli in Water - Quantitative WP MIC103 QCMIC103 E. coli in Water - Quantitative WP 3 Levels MIC009 QCMIC009 E. coli Quantitative - Soil MIC109 QCMIC109 E. coli Quantitative - Soil 3 Levels MIC209 QCMIC209 E. coli Quantitative - Soil 4 Levels MIC015 QCMIC015 Fungi and Yeast - WS MIC004 QCMIC004 Legionella in Water - WP MIC019 QCMIC019 Listeria - WP MIC008 QCMIC008 Pseudomonas aeruginosa - WS MIC021 Salmonella - P/A 5 samples MIC013 QCMIC013 Salmonella - Sludge MIC113 QCMIC113 Salmonella - Sludge 3 Levels MIC213 QCMIC213 Salmonella - Sludge 4 Levels MIC106 QCMIC106 Salmonella - WS 3 Levels MIC206 QCMIC206 Salmonella - WS 4 Levels MIC006 QCMIC006 Salmonella for Drinking/Surface Water - WS MIC012 QCMIC012 Standard Plate Count - WP MIC112 QCMIC112 Standard Plate Count - WP 3 Levels MIC212 QCMIC212 Standard Plate Count - WP 4 Levels MIC002 QCMIC002 Standard Plate Count - WS MIC102 QCMIC102 Standard Plate Count - WS 3 Levels MIC202 QCMIC202 Standard Plate Count - WS 4 Levels MIC011 QCMIC011 Streptococcus/Enterococcus - Drinking & Surface Water MIC111 QCMIC111 Streptococcus/Enterococcus - Drinking & Surface Water 3 Levels MIC211 QCMIC211 Streptococcus/Enterococcus - Drinking & Surface Water 4 Levels MIC123 Streptococcus/Enterococcus - Seawater WP 3 Levels MIC105 QCMIC105 Streptococcus/Enterococcus - WP 3 Levels MIC023 Streptococcus/Enterococcus in Seawater - WP MIC205 QCMIC205 Total & Fecal Streptococcus/Enterococcus - WP 4 Levels MIC005 QCMIC005 Total and Fecal Streptococcus/Enterococcus - WP MIC016 QCMIC016 WS-Enterococci -Sample (1-10) MIC001 QCMIC001 WS-Microbiological - Sample (1-10)

15 Herramientas Promociones Microbiology Manual 4th Edition Microbiology Focus 4 times per year emicrobiology Focus eanalytical 15

16 RAPID DETECTION OF MICROORGANISMS IN BEVERAGE USING FLUORESCENCE-BASED TECHNOLOGY Eusebi Rivera Field Sales Specialist, Biomonitoring XV MRAMA WORKSHOP 22/11/2016

17 Objective Faster microbiological method Develop a rapid method for the detection of microorganisms associated to the membrane filtration and incubation on traditional culture medium Challenge Time-to-result reduced Easy method close to traditional method Easy to validate In agreement with regulations Universal method Compatible with ID method

18 Traditional detection method vs. rapid 1) Preparation of sample according traditional method Membrane filtration Membrane transfer on culture medium 2) Incubation according procedure used Temperature - Time Traditional 3) Counting Colonies counting after incubation

19 Traditional detection method vs. rapid 1) Preparation of sample according traditional method Membrane filtration Membrane transfer on culture medium 3) Staining Membrane transfer onto Petri pad soaked with staining solution C - 30 minutes Rapid 2) Incubation according procedure used Temperature - Time

20 Principle 3) Staining Membrane transfer onto Petri pad soaked with staining solution C - 30 minutes Microorganism / microcolony before staining Membrane incubated onto pad soaked with staining solution 30 minutes C Fluorescent microorganism / micro-colony visible according to specific reader

21 Traditional detection method vs. rapid 1) Preparation of sample according traditional method Membrane filtration 3) Staining Membrane transfer onto Petri pad soaked with staining solution Membrane transfer on culture medium C - 30 minutes Rapid 4) Counting 2) Incubation according procedure used Temperature - Time Fluorescent colonies counting (Option) Re-incubation Membrane re-incubation to continue growth and identify microorganisms Yes or > limit No or < limit Traditional 3) Counting Colonies counting after incubation

22 1/3 of the traditional method time Traditional Rapid 0 Filtration Filtration Incubation Incubation Staining & Counting Re-incubation possible 5/7 DAYS Counting Counting

23 Filterability and compatibility Wide range of beverages compatible with the fluorescence technology Water Aromatized water Ice tea Apple juice Energetic drink Wine Good filterability Absence of fluorescence background Absence of fluorescence inhibition

24 Media compatibility Variety of media commonly used in the monitoring of beverages compatible with the fluorescence technology Sabouraud Dextrose Agar Tryptic Soy Agar MRS Agar Potato Dextrose Agar BAT Agar Orange Serum Broth Wallerstein Nutrient Broth Yeast and Mold Agar Yeast Extract Agar Absence of fluorescence background

25 Performances Matrix Contaminant Incubation condition Time of detection Red wine Saccharomyces cerevisiae Wallerstein 30 C 23 hours

26 Performances Matrix Contaminant Incubation condition Time of detection Coconut water Candida albicans Sabouraud Dextrose Agar - 25 C 24 hours Soya sauce Candida albicans Sabouraud Dextrose Agar - 25 C 24 hours

27 Performances Matrix Contaminant Incubation condition Time of detection Mineral water, direct well Waterborne microorganisms Yeast Extract Agar C 24 hours

28 Performances Matrix Contaminant Incubation condition Time of detection Mineral water Enterococci Slanetz-Bartley w/o TTC 37 C 24 hours

29 Study of statistical equivalence Rapid method vs. traditional method Alicyclobacillus acidoterrestris in energetic drink Traditional method Potatoes Dextrose Agar 46 C 3 days Rapid method Potatoes Dextrose Agar 46 C 20 hours

30 Study of statistical equivalence Rapid method vs. traditional method Level of contamination Replicate Rapid method after 20 hours of incubation (ufc) Fluorescent spot Colonies after re-incubation Traditional method (ufc) Low Medium High NA

31 Study of statistical equivalence Rapid method vs. traditional method Low Medium High Parameter Anderson- Darling Normality Test p-value Two Sample T- Test vs. Traditional method p- value Rapid method Rapid method Rapid method Criteria Trad. Trad. Trad. Re- Re- Re- method method method Fluo incuba- Fluo incuba- Fluo incuba- tion tion tion Kruskalp Wallis Test p value p 0,05 0,674 0,474 NA 0,188 0,128 NA 0,698 0,875 NA

32 Study of statistical equivalence Rapid method vs. traditional method Linearity of fluorescent spots Fluorescent spots (average ufc) y = 0,9649x + 7,1667 R² = 0, Traditional method (average ufc) A. acidoterrestris Lineal (A. acidoterrestris) Colonies after re-incubation (average ufc) Linearity of colonies after re-incubation y = 1,0109x + 7,1406 R² = 0, Traditional method (average ufc) A. acidoterrestris Lineal (A. acidoterrestris)

33 Conclusion Rapid method based on growth Fluorescent staining of micro-colonies on membrane Fluorescent micro-colonies visible through specific reader or associated to a camera Membranes re-incubation to continue growth and perform identification Rapid method statistically equivalent to the traditional method Rapid detection depending on microorganism and matrix Universal detection of viable microorganisms