Ayude a sus clientes a garantizar la seguridad del agua.

Pseudalert*

Detección de Pseudomonas aeruginosa en agua de piscina/spa y agua

embotellada en 24 horas

NUEVO

Pseudalert*

para detección de Pseudomonas aeruginosa en 24 horas

Tecnología de enzimas bacterianas

Pseudalert utiliza la tecnología de enzimas bacterianas para detectar P. aeruginosa en agua.

Los microorganismos de P. aeruginosa crecen y se reproducen rápidamente, usando el abundante suministro de aminoácidos, vitaminas y otros nutrientes presentes en el reactivo de Pseudalert. Las cepas de P. aeruginosa en crecimiento activo contienen una enzima que escinde el sustrato para producir fluorescencia azul bajo luz ultravioleta.

La presencia de P. aeruginosa en agua de piscina/spa puede causar “foliculitis de la tina”, “oído de nadador” y gran cantidad de otras infecciones, mientras que la contaminación del agua embotellada puede conducir a enfermedades en poblaciones sensibles.

La prueba IDEXX Pseudalert proporciona resultados definitivos para P. aeruginosa en la mitad de tiempo que los métodos de prueba convencionales.

Fácil• La fluorescencia de los pocillos o el recipiente

indica un resultado positivo

• La misma plataforma de uso que para las pruebas IDEXX Colilert*:

• Reactivos listos para usar, no se requiere preparación de los medios

• Práctico envase unidosis

• Sin compuestos tóxicos

• Utilice en combinación con IDEXX Quanti-Tray* o Quanti-Tray*/2000 para realizar el recuento de acuerdo con el número más probable (NMP)

Rápido• Resultados definitivos en 24 horas

• Tiempo de manipulación inferior a 1 minuto que permite optimizar el flujo de trabajo

Exacto• Riesgo reducido de falsos positivos: Pseudalert

detecta P. aeruginosa

• Resultados definitivos sin pasos de confirmación adicionales

• Lectura de resultados mediante un simple cambio de color fluorescente lo que minimiza la interpretación subjetiva de resultados

Resultados de Pseudalert vistos bajo luz ultravioleta

© 2010 IDEXX Laboratories, Inc. Reservados todos los derechos. • 09-68277-01

* Colilert, Pseudalert y Quanti-Tray son marcas comerciales o marcas comerciales registradas de IDEXX Laboratories, Inc. o de sus filiales en los Estados Unidos y/u otros países. La Política de Privacidad de IDEXX se encuentra disponible en idexx.com.

IDEXX Laboratories, Inc., One IDEXX Drive Westbrook, Maine 04092 EE.UU. • idexx.com/water

Tel: 1-207-556-4496 o 1-800-321-0207 • Fax: 1-207-556-4630

Pseudalert le ofrece la opción de cuantificar los resultados empleando el sistema IDEXX Quanti-Tray*, o le proporciona resultados de presencia/ausencia usando un recipiente.

Para obtener más información o hacer un pedido, consulte la página idexx.es/pseudalert. En Centroamérica y Latinoamérica, llame al 1-800-321-0207. En España, llame al 93 267 2673.

Agregue reactivo a la muestra de agua.

Tape el recipiente y agite para disolver (la muestra

se volverá amarilla).

Incube el recipiente durante 24-28 horas a 38 ºC ± 0.5 ºC.

Lea los resultados bajo luz ultravioleta. La fluorescencia azul indica presencia de P. aeruginosa.

Agregue al recipiente 2 gotas de Solución

Antiespumante de IDEXX. Vierta la muestra en

la Quanti-Tray* o Quanti-Tray*/2000.

Selle utilizando un Sellador para Quanti-Tray* e incube

durante 24-28 horas a 38 ºC ± 0.5 ºC.

Lea los resultados bajo luz ultravioleta, cuente

los pocillos fluorescentes azules y remítase a la

tabla de NMP.

Presencia/ausencia:

1

3

2

4

5

3

4

Cuantificación:

En primer lugar, siga estos pasos fáciles:

Guía de referencia rápida Pseudalert*

Consejos rápidos para leer los resultados de Pseudalert

1Lámpara UV, catálogo #WL160 (110 V) o #WEA160F (220 V).2Cámara de visualización UV, catálogo #WCM10.

IDEXX Laboratories, Inc.One IDEXX Drive, Westbrook, Maine 04092 EE.UU. • 1-800-321-0207 • idexx.com/water

© 2010 IDEXX Laboratories, Inc. Reservados todos los derechos • 09-68278-00*Pseudalert y Quanti-Tray son marcas comerciales o marcas comerciales registradas de IDEXX Laboratories, Inc. o de sus filiales en los Estados Unidos y/u otros países.La Política de Privacidad de IDEXX se encuentra disponible en idexx.com.

Después de 24 horas de incubación a 38 °C ± 0.5 °C (o 26 horas para muestras carbonatadas):

• Lea los resultados usando una lámpara ultravioleta1 (UV) de 6 watt y 365 nm; asegúrese de que la lámpara tenga la longitud de onda y la intensidad adecuadas.

• Utilice una cámara de visualización2 o lea los resultados en un lugar oscuro para evitar la interferencia de la luz ambiente.

• Interprete toda fluorescencia azul como POSITIVA, aun cuando la señal fluorescente sea débil.

• Si no está seguro acerca de un pocillo o recipiente que presente fluorescencia débil al cabo de 24 horas, incúbelo durante 1–4 horas más.

Consejos para la lectura de fluorescencia (vista bajo luz UV)

Lectura cuantitativa con la Quanti-Tray* Lectura de presencia/ausencia

– + +–

+

–

–

+

–

+

–

+

+

– +

Negativo Positivo

Para obtener más información o hacer un pedido, consulte la página idexx.es/pseudalert En Centroamérica y Latinoamérica, llame al 1-800-321-0207 En España, llame al 93 267 2673

North/SouthAmerica:+12075564496or+s18003210207

Europe:+0080043399111

UK:+44(0)1638676800

China:+862161279528

Japan:+81422715921

Australia:+1300443399

IDEXXLaboratories,Inc.,OneIDEXXDrive,Westbrook,Maine04092USA

idexx.com/water

06-18569-05

Pseudalert*

Introduction The Pseudalert* test detects the presence of Pseudomonas aeruginosa in water samples. The test is based on a bacterial enzyme detection technology that signals the presence of Pseudomonas aeruginosa through the hydrolysis of a substrate present in the Pseudalert reagent. Pseudomonas aeruginosa cells rapidly grow and reproduce using the rich supply of amino acids, vitamins and other nutrients present in the Pseudalert reagent. Actively growing strains of Pseudomonas aeruginosa have an enzyme that cleaves the substrate to produce a blue fluorescence under ultraviolet (UV) light. Pseudalert detects Pseudomonas aeruginosa at 1 cfu in either 100 mL or 250 mL samples within 24 hours.

StorageStore at 2–30°C away from light

Presence/Absence (P/A) Procedure1. Add contents of one appropriately sized snap pack to either a 100 mL or 250 mL sample in a sterile, transparent,

nonfluorescing vessel.2. Cap vessel and shake.3. Incubate at 38±0.5°C for 24 hours.4. Read results according to Result Interpretation table below.

Quanti-Tray* Enumeration Procedure (100 mL Samples Only)1. Add contents of one snap pack to a 100 mL water sample in a sterile vessel.2. Cap vessel and shake until dissolved.3. Add 2 drops of IDEXX Antifoam Solution1 to the sample/reagent mixture. Note: IDEXX 120 mL sample vessels2 containing antifoam are also available.4. Pour sample/reagent mixture into a Quanti-Tray* or Quanti-Tray*/2000 and seal in a Quanti-Tray* Sealer. 5. Place the sealed tray in a 38±0.5°C incubator for 24 hours.6. Read results according to the Result Interpretation table below. Count the number of positive wells and refer to the

MPN table provided with the trays to obtain a Most Probable Number.

Result Interpretation

Appearance Result

No blue fluorescence Negative for Pseudomonas aeruginosa

Blue fluorescence† Positive for Pseudomonas aeruginosa † Presence of blue fluorescence greater than the amount present in a negative control sample

•Lookforbluefluorescencewitha6-watt,365-nmUVlightheldwithin5inchesofthesampleinadarkenvironment. Face light away from your eyes and toward the sample.

•Referto–/+fluorescencereadguideonkitbox.ChartcolorsareasseenunderaUVlight.•Pseudalertresultsaredefinitiveat24–28hours.Inaddition,positivesforPseudomonas aeruginosa observed before 24

hours, and negatives observed after 28 hours are also valid.

Procedural Notes•Useonlysterile,nonbuffered,oxidant-freewaterfordilutions.•Forcomparison,anincubatedsterilewaterblankcontainingPseudalertreagent(negativecontrol)canbeusedwheninterpretingresults.•Thisinsertmaynotreflectyourlocalregulations.Forcompliancetesting,besuretofollowappropriateregulatoryprocedures.•Pseudalertisaprimarywatertest.Pseudalertperformancecharacteristicsdonotapplytosamplesalteredbypreenrichmentorconcentration.•Pseudalerthasnotbeenvalidatedforusewithflavoredbottledwater,marinewaterorcarbonatedwatersamples.•AseptictechniqueshouldalwaysbefollowedwhenusingPseudalert.DisposeofmaterialsinaccordancewithGoodLaboratoryPractices.•Thepresenceofahighmineralcontent(especiallymagnesiumand/orcalcium)cancausethePseudalertreagentmixturetobecomecloudy,

but this does not affect the outcome.

Quality Control Procedures1.OneofthefollowingqualitycontrolproceduresisrecommendedforeachlotofPseudalert: A. IDEXX-QC Pseudomonas3: Pseudomonas aeruginosa, Escherichia coli, Pseudomonas fluorescens. B. Additional Quality Control Methods.

i. For each of the American Type Culture Collection (ATCC)4 bacterial strains, (Pseudomonas aeruginosa ATCC 27853 or 10145, Escherichia coli ATCC 25922 and Pseudomonas fluorescens ATCC 13525) streak the culture onto labeled TSA or blood agar plates and incubate at 35±0.5°C for 18–24 hours.

ii. For each bacterial strain, touch a sterile 1 µL inoculating loop to a colony and use it to inoculate a labeled test tube containing 5 mL of sterile deionized water. Close cap and shake thoroughly.

iii. For each bacterial strain, take a 1 µL loop from the test tube and use it to inoculate a labeled vessel containing either 100 mL or 250 mL of sterile deionized water. These are your controls.

2. Follow the P/A Procedure or Quanti-Tray Enumeration Procedure above.3. Results should match the Result Interpretation table above.

Pseudalert* Test Kit

1. IDEXX Antifoam Solution catalog number: WAFDB2. IDEXX 120 mL Shrink-banded Vessels with Antifoam catalog number: WV120SBAF-2003. IDEXX-QC Pseudomonas, IDEXX Catalog #UN3373-WQC-PSE 4. American Type Culture Collection, 1-800-638-6597 atcc.org

*Pseudalert and Quanti-Tray are trademarks or registered trademarks of IDEXX Laboratories, Inc. or its affiliates in the United States and/or other countries.

Patent information: idexx.com/patents.

©2013 IDEXX Laboratories, Inc. All rights reserved.

Introducción El kit Pseudalert* detecta la presencia de Pseudomonas aeruginosa en muestras de agua. El análisis se basa en la detección de una enzima bacteriana de Pseudomonas aeruginosa que cataliza la hidrólisis del sustrato presente en el reactivo del kit Pseudalert. Las bacterias Pseudomonas aeruginosa crecen y se multiplican con rapidez gracias al gran aporte en aminoácidos, vitaminas y otros nutrientes del reactivo del kit Pseudalert. Las cepas en crecimiento activo de Pseudomonas aeruginosa poseen una enzima que hidroliza el sustrato del reactivo y produce una fluorescencia azul cuando se expone a la luz ultravioleta UV. Pseudalert detecta bacterias Pseudomonas aeruginosa en 24 horas a una concentración de 1 UFC en muestras de 100 ml o 250 ml.

ConservaciónAlmacenar a una temperatura entre 2–30°C protegido de la luz.

Procedimiento de presencia/ausencia (P/A) 1. Añadir el contenido de la dosis Snap adecuadamente predispensada a una muestra de 100 ó 250 ml

en un recipiente estéril transparente, no fluorescente.2. Tapar y agitar el recipiente.3. Incubar a 38±0,5°C durante 24 horas.4. Leer los resultados de acuerdo con la tabla de interpretación de resultados que figura abajo.

Procedimiento de enumeración Quanti-Tray* (sólo muestras de 100 ml)1. Añadir el reactivo a una muestra de 100 ml de agua, en un recipiente estéril.2. Tapar y agitar el recipiente hasta que el contenido se haya disuelto.3. Añadir 2 gotas de solución antiespumante de IDEXX1 a la mezcla muestra/reactivo.

Nota: IDEXX también comercializa recipientes para muestras de 120 ml que ya contienen antiespumante.2

4. Verter la mezcla muestra/reactivo en una Quanti-Tray o Quanti-Tray*/2000 y sellar utilizando un Quanti-Tray* Sealer.5. Colocar la bandeja sellada en una estufa de incubación a 38±0,5°C durante 24 horas. 6. Leer los resultados de acuerdo con la tabla de interpretación de resultados que figura abajo. Contar el número de pocillos positivos y

referirse a la tabla NMP proporcionada con las bandejas para obtener el número más probable.

Interpretación de resultados

Apariencia Resultado

Sin fluorescencia azul Muestra negativa para Pseudomonas aeruginosa

Con fluorescencia azul† Muestra positiva para Pseudomonas aeruginosa † Intensidad de la fluorescencia azul superior a la de la fluorescencia del control negativo.

•ObservarlafluorescenciaazulenunambienteoscuroyconunaluzUVde6vatiosy365nm,mantenidaaunos12cmdelamuestra. Aleje la luz de sus ojos y oriéntela hacia la muestra.

•Paralosresultadosreferirsealaguíadelectura-/+fluorescenciaqueseincluyeenlacaja.Estoscoloressonlosquesevenbajo luz UV.

•LosresultadosdePseudalertsondefinitivostranscurridas24–28horas.Además,lospositivosparaPseudomonas aeruginosa observados antes de 24 horas y los negativos observados después de 28 horas también son válidos.

Notas sobre el procedimiento•Utilizarsolamenteaguaestéril,notamponada,libredeoxidantes,paraefectuarlasdiluciones.•Parapoderrealizarlascomparacionesalahoradeinterpretarlosresultados,sepuedeutilizarcomoblancoaguaestérilincubadaconelreactivoPseudalert

(control negativo).• Esposiblequeesteprospectonoreflejelanormativalocaldesupaís.Pararealizarpruebasquelacumplan,asegúresedeseguirlosprocedimientosreglamentarios

correspondientes.•Pseudalertesfundamentalmenteunapruebaparaanalizaragua.LascaracterísticasderendimientodePseudalertnosepuedenaplicaramuestrasquehayan

sido alteradas previamente por algún tipo de enriquecimiento o concentración. •ElkitPseudalertnosehavalidadoparaserutilizadoconmuestrasdeaguaembotelladaaromatizada,conaguamarinaoaguacarbonatada.•SiempredebeaplicarseunatécnicaasépticacuandoseutilicePseudalert.Elmaterialdebedesecharsedeacuerdoconlasbuenasprácticasdelaboratorio.•LamezcladereactivodelkitPseudalertpuedevolverseturbiocuandolamuestrapresentaunaltocontenidoenminerales(especialmentemagnesioy/o

calcio).

Procedimientos de control de calidad1. Se recomienda uno de los siguientes procedimientos de control de calidad para cada lote de Pseudalert: A. IDEXX-QC Pseudomonas3: Pseudomonas aeruginosa, Escherichia coli, Pseudomonas fluorescens. B. Métodos de control de calidad adicionales: i. Para cada una de las cepas bacterianas de Colección Americana de Cultivos Tipo (ATCC)4 (Pseudomonas aeruginosa ATCC 27853 o 10145,

Escherichia coli ATCC 25922 y Pseudomonas fluroescensATCC13525),siembreenestríaselcultivosobreplacasetiquetadasdeagarsangreoTSAeincube a 35±0,5°C durante 18 a 24 horas.

ii. Para cada cepa bacteriana, recoja una muestra de la colonia con una asa de siembra estéril de 1µl e inocúlela en un tubo de ensayo marcado que contenga 5 ml de agua desionizada estéril. Cierre la tapa y agite bien.

iii. Para cada cepa bacteriana, introduzca una asa de siembra estéril de 1µl en el tubo de ensayo e inocule con ella un recipiente marcado que contenga 100 o 250 ml deaguadesionizadaestéril.Lasmuestrasasíobtenidasseránsuscontroles.

2. Siga el procedimiento P/A o el procedimiento de enumeración de Quanti-Tray que se indica más arriba.3. Los resultados deben coincidir con la tabla de interpretación de resultados que se indica más arriba.

Kit de análisis Pseudalert*

1. Referencia de la solución antiespumante de IDEXX: WAFDB2.Referenciadelosrecipientesde120mldeIDEXXconantiespumanteypelículatermoretráctil:WV120SBAF-2003. IDEXX-QC Pseudomonas, IDEXX Catalog #UN3373-WQC-PSE 4. American Type Culture Collection, 1-800-638-6597 atcc.org

*PseudalertyQuanti-TraysonmarcasomarcasregistradasdeIDEXXLaboratories,Inc.osusfilialesenEstadosUnidosdeAméricay/oenotrospaíses.

Información sobre la patente: idexx.com/patents.

©2013IDEXXLaboratories,Inc.Todoslosderechosreservados.LapolíticadeprivacidaddeIDEXXestádisponibleenIDEXX.com

Pseudalert Read Guide

v 2.0

Key Points

• Read results in the dark.• Use a 6 Watt, 365nm UV light.• Replace the fluorescent tube annually.

• Wells exhibiting blue fluorescence are positive regardless of intensity• Use a negative control for comparison.

Negative & Positive Controls

Positive Control – ATCC 27853Negative Control – DI Water

Negative well

Strong positive blue

fluorescence

Sample 1Positive wellsNegative wells

Sample 2Positive wellsNegative wells

Finished Water Samples

Using an Insert

A Quanti-Tray insert can be used to separate wells and cut down on reflection

Without Insert With Insert

Without Insert

Positive wellsNegative wells

With Insert

Positive wellsNegative wells

Strong Positives

Weak PositivesWithout Insert

Positive wellsNegative wells

With Insert

Positive wellsNegative wells

Mixed SampleWithout Insert

Positive wellsNegative wells

With Insert

Positive wellsNegative wells

Spiked Samples: ATCC 27853Spa (High Sulphur) & Finished Water

Spa Water

Positive wells

Negative wells

Finished Water

Positive wells

Negative wells

Reg. Mercantil Ctral Barcelona – Tomo 30959 – Folio 0108 – Hoja/Dup. 182204 – C.I.F.B-61740361 Tel : +34 (0) 93 414 26 08 ▪ Fax : + 34 (0) 93 414 74 78▪ www.idexx.es

IDEXX Laboratories S.L C/Plom 2, 3°

08038 BARCELONA

Barcelona a 17 Junio 2014

IDEXX confirma lo siguiente en referencia al equipo necesario para el uso de

nuestra metodología Pseudalert.

Requisitos de equipo:

a-1) Incubador: Estabilidad +/-0,5ºC.

Homogeneidad: No se tiene en cuenta. Se

recomienda sin embargo evitar abrir y cerrar la estufa con demasiada frecuencia . Recomendamos igualmente controlar la temperatura y utilizar

estufas de aire forzado.

a-2) Termómetro: Incertidumbre de medida: No se tiene en cuenta.

Requisitos de ensayo:

Durante la realización del ensayo le temperatura conseguida debe

estar comprendida en el intervalo [38,5ºC/37,5ºC] sin considerar la incertidumbre de medida ni las características del incubador.

En resumen, la estabilidad de la estufa debe de ser +/- 0.5C. Es con este

criterio que se desarrolló el método. La incertidumbre no influye mientras

sea controlada.

Aconsejamos estufas de aire forzado y que la apertura de la puerta se reduzca al mínimo necesario.

Cordialmente,

Paloma Pascual Mayoral

IDEXX Laboratorios,S.L. Paloma-pascual@idexx.com Tfno.: 637 55 12 62

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IDEXX Summary 14D Topic: Beta Trial Study report comparing Pseudalert* versus the ISO 16266 method

for the detection of Pseudomonas aeruginosa in 250 mL thermal pool and bottled water samples

Title: “Comparison of the performance of the IDEXX Pseudalert test against the ISO

16266 method at recovering confirmed Pseudomonas aeruginosa from thermal pool and bottled water samples”

Author: IDEXX Laboratories Date: March 2011 Report Highlights:

• Pseudalert was compared to the ISO 16266 method at the Nestlé Waters Quality Assurance Center in Vittel, France which regularly tests thermal pool and bottled water samples.

• Data from the completed study showed:

o Pseudalert had comparable detection of P. aeruginosa versus the ISO 16266

method from 250 mL thermal pool water samples naturally contaminated with bacteria

o Pseudalert had comparable detection of P. aeruginosa versus the ISO 16266 method from 250 mL bottled water samples spiked with reference and wild strains.

o Pseudalert was able to suppress the false detection of a P. putida strain that was spiked into 250 mL bottled water samples at concentrations exceeding 100 cfu/sample.

• Pseudalert performed as well or better than the ISO 16266 method at detecting

P. aeruginosa in 250 mL thermal pool and bottled water samples

*Pseudalert is a trademark or registered trademark of IDEXX Laboratories, Inc. or its affiliates in the United States and/or other countries.

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Technical Note

Comparison of the performance of the IDEXX Pseudalert* test against the ISO 16266 Method at recovering Pseudomonas aeruginosa from thermal pool and bottled water

samples

Scope This technical note contains data collected at the Nestlé Waters Quality Assurance Center in Vittel, France that evaluated the performance of the Pseudalert test prior to its launch in September 2010. Samples of water used in the production of bottled water products and from thermal (mineral) pools/spas served as the test matrix for this study. The microorganisms present in these water samples were from wild populations that occurred naturally in the environment and did not result from supplemental spiking activities. Additional water samples were collected that were spiked with different bacterial species to simulate a contamination event. Testing occurred over the course of two months. This study compared the relative recovery of confirmed P. aeruginosa by Pseudalert after 24 hours of incubation against the ISO 16266 method.

Procedure

1. Water samples (>500 mL) were collected for testing. Thermal pool water samples were analyzed the day of sampling and stored at room temperature. Bottled water samples were obtained from facilities located at multiple global locations. They were shipped and stored at room temperature prior to testing.

2. A 250 mL aliquot of each sample was processed and analyzed following the procedures outlined in the ISO 16266 protocol.

3. A 250 mL aliquot of each sample was processed and analyzed following the

procedures outlined in the Pseudalert package insert for 250 mL presence/absence testing using an IDEXX testing vessel. Pseudalert was incubated for 24 hours at 38±0.5ºC.

4. Presumptive P. aeruginosa positive samples from the ISO 16266 method were

subjected to the following confirmation procedures:

• No further confirmation was required if the colony was blue/green on CN agar • Confirmation in acetamide broth was performed if the colony was not blue/green but

fluorescent on CN agar • Confirmation in Oxidase, Kings B, and acetamide broth were performed if the colony

was not fluorescent but reddish/brown color on CN agar

5. Pseudalert positive samples were not confirmed.

6. The heterotrophic count of each water sample was measured following the ISO 6222 method.

* Pseudalert is a trademark or registered trademark of IDEXX Laboratories, Inc. or its affiliates in the United States and/or other countries.

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Results

Twenty three thermal pool water samples were analyzed during this study. Of these samples, four were found to be naturally contaminated with P. aeruginosa. The ability of the Pseudalert* and the ISO 16266 methods to detect these natural P. aeruginosa populations is shown below:

Recovery of natural P. aeruginosa populations by the two methods matched perfectly, which suggests that both methods have similar specificities for P. aeruginosa. Four of the samples (No.’s 1, 2, 3, and 4) tested with the ISO 16266 method had presumptive positive colonies that required secondary confirmation to exclude the presence of P. aeruginosa. Pseudalert was able to suppress these bacteria and did not require additional testing. Over

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half of the samples had heterotrophic bacterial populations ranging from 2 to 330 (average ~ 93 cfu/mL) that did not interfere with the performance of the Pseudalert* method. Sixty three bottled water samples were analyzed for this study. These samples were challenged with different concentrations of P. aeruginosa and P. putida isolates to compare the relative sensitivity & specificity of the two methods. The data are presented below:

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The recovery of the spiked P. aeruginosa strains by Pseudalert* was shown to be equivalent to the ISO 16266 method even when present a concentrations as low as 1 cfu/250mL of sample. Pseudalert was also able to suppress the false detection of the P. putida strain at all the concentrations tested. The presence of P. putida or high concentrations (>3,000 cfu/mL) of heterotrophic bacterial populations did not interfere with the performance of the Pseudalert test. The industrial water sample was found to contain natural populations of P. aeruginosa which was detected by both test methods. Two

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discrepant results were seen with samples #35, and #36 where either the Pseudalert* or ISO 16266 method failed to detect the spiked P. aeruginosa strain. This is likely due to the low concentration of bacteria (~ 1 cfu/250mL) spiked into the bottled water sample which caused one of the methods to receive no bacteria. The fact that this occurred with both methods supports the claim that they have comparable limits of detection.

Conclusions

The data presented demonstrates equivalent detection of P. aeruginosa in thermal pool and bottled water samples by Pseudalert when compared to the ISO 16266 method. Pseudalert was able to accurately recover very low concentrations of P. aeruginosa (as low as 1 cfu/250mL of sample) without interference from biological or chemical agents present in the water samples. The Pseudalert method showed comparable recovery against the ISO 16266 method with five samples (4 thermal pool and 1 industrial water) containing naturally occurring populations of P. aeruginosa as well as from thirty two bottled water samples spiked with P. aeruginosa. Pseudalert was also able to suppress the false detection of a spiked P. putida strain even at concentrations exceeding 100 cfu/sample. Based on the data collected by the Nestlé Waters Quality Assurance Center we conclude that after 24 hours of incubation Pseudalert performs at least as well as the ISO 16266 method at the specific detection of P. aeruginosa from 250 mL thermal pool and bottled water matrices. For technical questions, please contact: IDEXX Laboratories Technical Support 1 IDEXX Drive Westbrook, ME 04092 207-556-4496 / 1-800-321-0207 www.idexx.com/water Pseudalert Quick Reference Guide

About IDEXX Laboratories IDEXX Laboratories, Inc. is the global market leader in diagnostics and information technology solutions for animal health and water and milk quality. Headquartered in Maine, IDEXX employs over 4,700 people in more than 60 locations around the world. IDEXX is the world leader in microbiology testing technologies that ensure safe water. As the world's preferred provider of innovative drinking-water microbiology test kits, IDEXX is known for its breakthrough products. We provide easy, rapid, accurate and cost-effective water-testing solutions. Our sales, customer service and technical support teams serve customers in over 75 countries and our products have governmental approval or acceptance in 36 countries world-wide.

idexx.com

IDEXX Summary 14B Topic: Beta Trial Study report comparing Pseudalert* versus EN ISO 16266:20081 in pool/spa

waters for detection and enumeration of Pseudomonas aeruginosa Title: “Comparison of the performance of the IDEXX Pseudalert* test against the EN ISO

16266:2008 method at recovering confirmed Pseudomonas aeruginosa from pool/spa water samples”

Author: IDEXX Laboratories Date: October 2010 Report Highlights:

• Pseudalert was compared to EN ISO 16266:2008 at an independent laboratory that regularly tests pool/spa waters.

• Data from the completed study showed:

o Pseudalert was able to accurately detect and quantify the presence of P. aeruginosa, even in the presence of very high bacterial populations (>1,120 cfu/mL).

o Pseudalert had comparable detection and quantification of P. aeruginosa versus EN ISO 16266:2008 (p = 0.082)** from naturally contaminated pool/spa water samples. Two methods are comparable if p > 0.05.

o Pseudalert has better recovery (p = 0.009)** of P. aeruginosa versus EN ISO 16266:2008 from pool/spa water samples spiked with different strains of the bacterium. Two methods are comparable if p > 0.05.

o Pseudalert accurately recovered very low concentrations of P. aeruginosa (as low as 1 cfu/100ml of sample)

• Pseudalert performed as well or better than EN ISO 16266:2008 for detection and

quantification of P. aeruginosa in pool/spa water samples

* Pseudalert and Quanti-Tray are trademarks or registered trademarks of IDEXX Laboratories, Inc. or its affiliates in the United States and/or other countries. 1. ISO 16266: Water quality — Detection and enumeration of Pseudomonas aeruginosa — Method by membrane Filtration, Geneva. International Standards Organization; The text of ISO 16266:2006 has been approved by CEN as EN ISO 16266:2008 without any modification; COMITÉ EUROPÉEN DE NORMALISATION, Brussels, Belgium ** Based on student’s t-test (one tail; paired samples)

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Technical Note

Comparison of the performance of the IDEXX Pseudalert* test against the EN ISO 16266:20081 method at recovering confirmed Pseudomonas aeruginosa from pool/spa water

samples

Product Description

The Pseudalert test detects the presence of Pseudomonas aeruginosa in bottled, pool, and spa water samples. The test is based on a bacterial enzyme detection technology that signals the presence of Pseudomonas aeruginosa through the hydrolysis of a substrate present in the Pseudalert reagent. Pseudomonas aeruginosa rapidly grows and reproduces using the rich supply of amino acids, vitamins, and other nutrients present in the Pseudalert reagent. Actively growing strains of Pseudomonas aeruginosa have an enzyme that cleaves the substrate to produce a blue fluorescence under UV light. Pseudalert detects Pseudomonas aeruginosa at 1 cfu in either 100 mL or 250 mL samples within 24 hours for non-carbonated water samples and within 26 hours for carbonated samples.

Scope This technical note contains data collected at an independent certified laboratory located in Germany that evaluated the performance of the Pseudalert test prior to its launch in September 2010. The test matrix for this study was pool/spa water, samples of which were collected at numerous locations prone to natural P. aeruginosa contamination. The microorganisms present in these pool/spa water samples were from wild populations that occurred naturally in the environment and did not result from supplemental spiking activities. Additional pool/spa water samples were collected that were unlikely to be contaminated with naturally occurring P. aeruginosa. These samples were spiked with seven P. aeruginosa isolates that were collected from environmental sources and displayed different morphological characteristics on the EN ISO 16266:2008 media. Testing occurred over the course of several months. This study compared the relative recovery of confirmed P. aeruginosa by Pseudalert after 24 hours of incubation against the EN ISO 16266:2008 method.

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Procedure

1. Samples (>200 mL) were collected at numerous locations (maximum of five

collections per site) in accordance with ISO 194582 specifications. Storage time was recorded and care was taken to test within 12 to 18 hours of collection, however, some samples were tested within 48 hours of collection.

2. A 100 mL aliquot of each sample was processed and analyzed following the procedures outlined in EN ISO 16266:2008. Additional confirmation procedures were added (see description below) based on the morphological characteristics of the presumptive positive samples.

3. A 100 mL aliquot of each sample was processed and analyzed following the procedures outlined in the Pseudalert package insert for 100 mL quantification using the Quanti-Tray* device. Pseudalert was incubated for 24 - 28 hours at 38±0.5ºC.

4. The Heterotrophic Plate Count of each sample was determined following the HPC

method3 incubated at both 20ºC and 36ºC.

5. Presumptive P. aeruginosa positive samples from the EN ISO 16266:2008 method were subjected to confirmation procedures based on the following scenarios:

• Blue/Green colonies = oxidase & API 20NE • Fluorescent colonies = oxidase, acetamidase, growth at 42ºC, and API 20NE • Red/Brown colonies = oxidase, acetamidase, Kings B (fluorescence), growth at 42ºC, and API 20NE

6. Pseudalert positive wells were confirmed in the following way:

• Blue fluorescent wells = oxidase, acetamidase, growth at 42ºC, and API 20NE

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Results Eighty six pool water samples were included in this study. Of these samples, sixteen were found to be naturally contaminated with P. aeruginosa. The ability of Pseudalert and EN ISO 16266:2008 (shown below as ISO 16266) to detect and quantify these natural P. aeruginosa populations is shown below:

Sample name Pseudalert (MPN/100mL)

ISO 16266(cfu /100mL)

HPC 20°C (cfu / mL)

HPC 36°C (cfu / mL)

indoor whirlpool (fra), filtrate >201 >500 (pres.) 0 39indoor pool (fra2b), filtrate 74 48 1 440indoor whirlpool (fra), filtrate 19 28 0 3natural pool, MU1, swimmer 0 2 250 1120natural pool, MU1, wading pool 9 4 570 700natural pool, MU2, nonswimmer 1 0 100 120natural pool, MU3, swimmer 2 0 220 488natural pool, MU3, wading pool 34 not analyzable >300 >300open air pool (oes), pool 29 32 0 45open air pool (sto), swimmer 38 42 >300 >300natural pool, MU4, swimmer 2 2 450 580natural pool, MU4, nonswimmer 2 0 490 800natural pool, MU4, wading pool 6 0 760 1080indoor whirlpool (fra), filtrate 3 2 0 0indoor pool (fra5), filtrate 6 3 0 0indoor pool (fra6), filtrate 201 186 0 3

(pres.) = P. aeruginosa present in sample but exact concentration could not be determined.

Recovery of natural P. aeruginosa populations by both methods was analyzed statistically using the paired t-test and showed comparable (p=0.082) recovery. One sample (natural pool, MU3, wading pool) was excluded from this analysis because the EN ISO 16266:2008 method was unreadable due to non-target bacterial interference on the membrane filter. Another sample (indoor whirlpool (fra), filtrate) was also excluded from the analysis because the recovered P. aeruginosa populations exceeded the counting range of both methods.

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The majority of the pool/spa water samples had high heterotrophic bacterial contamination that did not interfere with the performance of the Pseudalert test. Sixteen pool/spa water samples, which did not contain natural P. aeruginosa contamination, were spiked with environmental isolates described in the following table:

Strainisolated from

(origin) Bacterial IDcolony color on CN agar fluorescence

Api 20 NEprofile

Api 20 NEconfirmation

101 river water (Rhine) P. aeruginosa brownish-green + 1154475 99.9%102 open air pool P. aeruginosa toxic-green ++ 1154575 99.5%103 river water (Main river) P. aeruginosa green (+) 1354575 99.9%104 carbonized water dispenser P. aeruginosa brownish-green + 1054475 99.4%105 cooling tower P. aeruginosa brownish + 1154575 99.5%106 carbonized water dispenser P. aeruginosa petrol (+) 1054555 98.4%107 underground hydrant (pipe construction) P. aeruginosa reddish-brown + 1154575 99.5%

++ = strong fluorescence + = moderate fluorescence (+) = weak fluorescence The ability of Pseudalert and EN ISO 16266:2008 (shown below as ISO 16266) to detect and quantify these spiked P. aeruginosa populations is shown below:

Sample name Pseudalert (MPN/100mL)

ISO 16266(cfu /100mL)

HPC 20°C (cfu / mL)

HPC 36°C (cfu / mL)

SpikeStrain

open air pool (lan1), pool 109 49 1 0 101indoor pool (ben1), pool 130 49 0 0 102open air pool (ben2), pool 130 16 0 1 103artificial lake (ben3), lake 38 0 n.a. n.a. 104open air pool (hep1), pool 70 1 n.a. n.a. 105lake (arguk1), lake 130 33 58 124 106indoor pool (dar1), pool 10 6 n.a. n.a. 101indoor pool (fra11) whirlpool 31 19 n.a. n.a. 102indoor pool (fra12), whirlpool 10 8 n.a. n.a. 104natural pool (MU5+), wading pool 118 100 172 504 103natural pool (MU5+), nonswimmer 50 75 376 416 104open air pool (Obe1), wading pool 78 80 0 2 105open air pool (Oes), swimmer 66 50 0 0 106open air pool (Rei), filtrate 38 65 0 0 107 n.a. = not applicable (HPC count not determined)

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Recovery of spiked P. aeruginosa populations by both methods was analyzed statistically using the paired t-test and showed a non-comparable (p = 0.009)) recovery. Pseudalert was able to consistently recover more P. aeruginosa from these spiked samples than the EN ISO 16266:2008 method.

Conclusions

The data presented above clearly demonstrates the favorable detection and quantification of P. aeruginosa by Pseudalert when compared against the EN ISO 16266:2008 method with pool/spa water samples. Pseudalert was able to accurately recover very low concentrations of P. aeruginosa (as low as 1 cfu/100mL of sample) without interference from the heterotrophic bacterial populations or chemical residues present in the pool/spa water samples. Even the presence of very high bacterial populations (in excess of 1,120 cfu/mL) did not interfere with the ability of Pseudalert to accurately detect and quantify the presence of P. aeruginosa. The EN ISO 16266:2008 method experienced significant non-target bacterial interference with one of the pool/spa water samples that did not pose a problem for Pseudalert. The Pseudalert method was also shown to recover spiked P. aeruginosa isolates better than the EN ISO 16266:2008 method. The reason for this discrepancy is unclear and unexpected since these isolates were originally recovered from pool/spa water sources using the EN ISO 16266:2008 method. Based on these data we conclude that, after 24 hours of incubation, Pseudalert performs at least as well as the EN ISO 16266:2008 method at the specific detection and quantification of P. aeruginosa from pool/spa water matrices. For technical questions, please contact: IDEXX Laboratories Technical Support 1 IDEXX Drive Westbrook, ME 04092 207-556-4496 / 1-800-321-0207 www.idexx.com/water Pseudalert Quick Reference Guide

About IDEXX Laboratories

IDEXX Laboratories, Inc. is the global market leader in diagnostics and information technology solutions for animal health and water and milk quality. Headquartered in Maine, IDEXX employs over 4,700 people in more than 60 locations around the world. IDEXX is the world leader in microbiology testing technologies that ensure safe water. As the world's preferred provider of innovative drinking-water microbiology test kits, IDEXX is

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known for its breakthrough products. We provide easy, rapid, accurate and cost-effective water-testing solutions. Our sales, customer service and technical support teams serve customers in over 75 countries and our products have governmental approval or acceptance in 36 countries world-wide.

References

1. ISO 16266: Water quality — Detection and enumeration of Pseudomonas aeruginosa — Method by membrane Filtration, Geneva. International Standards Organization; The text of ISO 16266:2006 has been approved by CEN as a EN ISO 16266:2008 without any modification; COMITÉ EUROPÉEN DE NORMALISATION, Brussels, Belgium

2. ISO 19458:2006 Water Quality – Sampling for microbiological analysis, Geneva. International Standards Organization

3. German Drinking Water Directive (Trinkwasserverordnung - TrinkwV) by May 21st 2001 (obligatory since January 1st 2003) in annex 5 number 1

* Pseudalert and Quanti-Tray are trademarks or registered trademarks of IDEXX Laboratories, Inc. or its affiliates in the United States and/or other countries.