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
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 30-60 sec. 1-4 h incubation at 37 C 2
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 75444 E. coli 77643 Total Coliform 40926 Fecal Coliform 55283 Salmonella 56305 Enterococcus 74203 Gram+/Gram Differentiation 92598 Neisseria gonorrhea 77701 Group A Streptococcus 80031 Staphylococcus aureus 3
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
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
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 http://www.sigma-aldrich.com/hybriscan 6
Certified Reference Microorganisms Vitroids & LENTICULE discs Los discos contienen microorganismos viables en un numero definido de ufc certificadas Rangos estrechos que varían de 30-100'000 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 17025 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
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
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
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 30-120 - Aspergillus niger) CRM07464L Bacillus cereus NCTC 7464 Low Level 30-120 CRM07464M Bacillus cereus NCTC 7464 High Level 500-5 x 10^4 RM11322Q Campylobacter jejuni NCTC 11322 High Level 10^2 - RMF03255L Candida albicans NCPF 3255 Low Level 30-120 - RMF03255H Candida albicans NCPF 3255 High Level 2 x 10^4 - RM09750L Citrobacter freundii NCTC 9750 Low Level 30-120 - CRM00506L Clostridium bifermentans NCTC 506 Low Level 30-120 CRM13170L Clostridium perfringens NCTC 13170 Low Level 30-120 CRM13170M Clostridium perfringens NCTC 13170 High Level 500-5 x 10^4 CRM11467L Cronobacter sakazakii NCTC 11467 Low Level 30-120 CRM10006L Enterobacter aerogenes NCTC 10006 Low Level 30-120 CRM10006M Enterobacter aerogenes NCTC 10006 High Level 500-5 x 10^4 CRM00775L Enterococcus faecalis NCTC 775 Low Level 30-120 CRM00775M Enterococcus faecalis NCTC 775 High Level 500-5 x 10^4 CRM00775H Enterococcus faecalis NCTC 775 High Level >10^5 CRM09001L Escherichia coli NCTC 9001 Low Level 30-120 CRM09001M Escherichia coli NCTC 9001 High Level 500-5 x 10^4 CRM09001H Escherichia coli NCTC 9001 High Level >10^5 CRM13216L Escherichia coli NCTC 13216 Low Level 30-120 CRM12900L Escherichia coli O157 (NT) NCTC 12900 Low Level 30-120 Cat. No. Reference Material Origin Strain No. ufc Range Mean ufc Range Accreditation CRM08167L Klebsiella oxytoca NCTC 8167 Low Level 30-120 CRM11368M Legionella bozemanii NCTC 11368 High Level 500-5 x 10^4 CRM11371M Legionella micdadei NCTC 11371 High Level 500-5 x 10^4 CRM12821L Legionella pneumophila NCTC 12821 Low Level 30-120 CRM12821M Legionella pneumophila NCTC 12821 High Level 500-5 x 10^4 CRM11288L Listeria innocua NCTC 11288 Low Level 30-120 CRM11994L Listeria monocytogenes NCTC 11994 Low Level 30-120 CRM11994M Listeria monocytogenes NCTC 11994 High Level 500-5 x 10^4 RMBLANK0 Negative Control (0 ufc) - - - - CRM10662L Pseudomonas aeruginosa NCTC 10662 Low Level 30-120 CRM10662M Pseudomonas aeruginosa NCTC 10662 High Level 500-5 x 10^4 CRM09528L Raoultella planticola (formerly Klebsiella aerogenes) NCTC 9528 Low Level 30-120 CRM09528M Raoultella planticola (formerly NCTC 9528 High Level 500-5 x 10^4 Klebsiella aerogenes) RMF03191L Saccharomyces cerevisiae NCPF 3191 Low Level 30-120 - RMF03191M Saccharomyces cerevisiae NCPF 3191 High Level 500-5 x 10^4 - CRM06676L Salmonella enteritidis NCTC 6676 Low Level 30-120 CRM07832L Salmonella Nottingham NCTC 7832 Low Level 30-120 CRM12023L Salmonella Typhimurium NCTC 12023 Low Level 30-120 CRM06571L Staphylococcus aureus NCTC 6571 Low Level 30-120 CRM06571M Staphylococcus aureus NCTC 6571 High Level 500-5 x 10^4 CRM11047L Staphylococcus epidermidis NCTC 11047 Low Level 30-120 RM11218Q Vibrio furnissi NCTC 11218 Low Level 30-120 - RM10903Q Vibrio parahaemolyticus NCTC 10903 Low Level 30-120 - RM11176L Yersinia enterocolitica NCTC 11176 Low Level 30-120 - 10
Certified Reference Microorganism Vitroids Portfolio Strain WDCM Cat. No. CECT ufc range - VT091112 911 50-80 Acinetobacter baumanni - VT091114 911 130-270 - VT091115 911 600-1 400 Aspergillus brasiliensis 00053 VT000532 2574 50-80 00053 VT000533 2574 80-120 Bacillus subtilis 00003 VT000032 356 50-80 00003 VT000036 356 3 000-7 000 Bacillus cereus 00001 VT000013 193 80-120 Candida albicans 00054 VT000543 1394 80-120 00054 VT000545 1394 600-1 400 Candida albicans 00054 VT000546 1394 3 000-7 000 Citrobacter freundii - VT004014 401 130-270 - VT004016 401 3'000-7'000 Clostridium sporogenes 00008 VT000082 485 50-80 00175 VT001752 684 50-80 00175 VT001753 684 80-120 Enterobacter aerogenes 00175 VT001754 684 130-270 00175 VT001755 684 600-1 400 00175 VT001756 684 3 000-7 000 00083 VT000834 194 130-270 00009 VT000092 481 50-80 00009 VT000093 481 80-120 Enterococcus faecalis 00009 VT000094 481 130-270 00009 VT000095 481 600-1 400 00009 VT000096 481 3 000-7 000 00010 VT000102 410 50-80 Enterococcus faecium 00010 VT000104 410 130-270 00010 VT000105 410 600-1 400 11 Strain WDCM Cat. No. CECT ufc range 00090 VT000902 515 50-80 00090 VT000904 515 130-270 00090 VT000905 515 600-1 400 00090 VT000906 515 3 000-7 000 Escherichia coli 00090 VT000909 515 Variable 00090 VT000902 515 50-80 00013 VT000133 434 80-120 00013 VT000136 434 3 000-7 000 00012 VT000122 516 50-80 00012 VT000127 516 50 000-150 000 Legionella pneumophila (serogroup 1) 00205 VT002057 8734 50 000-150 000 Legionella pneumophila 00107 VT001077 7109 50 000-150 000 Fluoribacter bozemanae - VT072766 7276 3 000-7 000 - VT072767 7276 50 000-150 000 Klebsiella pneumoniae 00097 VT000971 143 15-40 Klebsiella pneumoniae 00097 VT000975 143 600-1 400 Proteus hauseri - VT004835 484 600-1 400 Proteus mirabilis - VT000233 4168 80-120 - VT000237 4168 50 000-150 000 00024 VT000244 110 130-270 00024 VT000249 110 variable 00025 VT001142 108 50-80 00025 VT001143 108 80-120 00025 VT001145 108 600-1 400 00026 VT000261 111 15-40 Pseudomonas aeruginosa 00026 VT000262 111 50-80 00026 VT000262 111 50-80 00026 VT000263 111 80-120 00026 VT000264 111 130-270 00026 VT000265 111 600-1 400 00026 VT000266 111 3 000-7 000 00026 VT000267 111 50'000-150'000 Salmonella enterica subsp. Enterica serovar Typhimurium 00031 VT000312 4594 50-80 00031 VT000313 4594 80-120 Salmonella enterica subsp. Enterica serovar Abony 00029 VT000292 545 50-80 Salmonella enterica subsp. Enterica serovar Enteridis - VT000303 4300 80-120 Staphylococcus aureus susp. Aureus 00032 VT000322 239 50-80 00032 VT000322 239 50-80 00032 VT000323 239 80-120 00032 VT000324 239 130-270 00032 VT000325 239 600-1 400 00032 VT000326 239 3 000-7 000
Microorganism Proficiency Testing Ensayos de aptitud (PT) y ISO 17025 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
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
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)
Herramientas Promociones Microbiology Manual 4th Edition Microbiology Focus 4 times per year www.sigmaaldrich.com/microbiology emicrobiology Focus eanalytical 15
RAPID DETECTION OF MICROORGANISMS IN BEVERAGE USING FLUORESCENCE-BASED TECHNOLOGY Eusebi Rivera Field Sales Specialist, Biomonitoring XV MRAMA WORKSHOP 22/11/2016
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
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
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 30-35 C - 30 minutes Rapid 2) Incubation according procedure used Temperature - Time
Principle 3) Staining Membrane transfer onto Petri pad soaked with staining solution 30-35 C - 30 minutes Microorganism / microcolony before staining Membrane incubated onto pad soaked with staining solution 30 minutes 30-35 C Fluorescent microorganism / micro-colony visible according to specific reader
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 30-35 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
1/3 of the traditional method time Traditional Rapid 0 Filtration Filtration Incubation Incubation Staining & Counting Re-incubation possible 5/7 DAYS Counting Counting
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
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
Performances Matrix Contaminant Incubation condition Time of detection Red wine Saccharomyces cerevisiae Wallerstein 30 C 23 hours
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
Performances Matrix Contaminant Incubation condition Time of detection Mineral water, direct well Waterborne microorganisms Yeast Extract Agar 20-25 C 24 hours
Performances Matrix Contaminant Incubation condition Time of detection Mineral water Enterococci Slanetz-Bartley w/o TTC 37 C 24 hours
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
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 1 2 3 4 5 72 64 27 80 62 72 60 35 69 56 93 63 42 61 73 Medium 1 2 3 4 5 49 83 91 84 100 42 81 86 86 93 112 72 120 104 88 High 1 2 3 4 5 181 164 156 171 165 169 156 143 168 163 192 152 136 168 NA
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 0.025 Kruskalp 0.1 0.168 0.380 0.719 0.150 Wallis Test p- 0.740 0.710 0.345 0.827 value 0.434 p 0,05 0,674 0,474 NA 0,188 0,128 NA 0,698 0,875 NA
Study of statistical equivalence Rapid method vs. traditional method Linearity of fluorescent spots Fluorescent spots (average ufc) 180 160 140 120 100 80 60 40 20 0 y = 0,9649x + 7,1667 R² = 0,9796 0 50 100 150 200 Traditional method (average ufc) A. acidoterrestris Lineal (A. acidoterrestris) Colonies after re-incubation (average ufc) 200 150 100 50 0 Linearity of colonies after re-incubation y = 1,0109x + 7,1406 R² = 0,9794 0 50 100 150 200 Traditional method (average ufc) A. acidoterrestris Lineal (A. acidoterrestris)
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