Guía de referencia rápida Pseudalert* - Pàgines de la UAB

Guía de referencia rápida Pseudalert *
Consejos rápidos para leer los resultados de Pseudalert
Después de 24 horas de incubación a 38 °C ± 0.5 °C (o 26 horas para muestras carbonatadas):
• Lea los resultados usando una lámpara ultravioleta 1 (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 visualización 2 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
1 Lámpara UV, catálogo #WL160 (110 V) o #WEA160F (220 V).
2 Cámara de visualización UV, catálogo #WCM10.
IDEXX Laboratories, Inc.
One IDEXX Drive, Westbrook, Maine 04092 EE.UU. • 1-800-321-0207 • idexx.com/water
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© 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.
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Negativo Positivo
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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

Kit de teste Pseudalert*
Introdução
O teste Pseudalert detecta Pseudomonas aeruginosa em amostras de água engarrafada, de piscinas ou spas. A base do teste é a detecção de uma enzima bacteriana, que assinala
a presença de Pseudomonas aeruginosa ao hidrolisar o substrato presente no reagente Pseudalert. As células de Pseudomonas aeruginosa crescem rapidamente e se reproduzem
devido às elevadas concentrações de aminoácidos, vitaminas e outros nutrientes presentes no reagente para Pseudalert. Cepas de Pseudomonas aeruginosa em crescimento ativo
possuem uma enzima que cliva o substrato, produzindo fluorescência azul sob luz UV. Pseudalert detecta Pseudomonas aeruginosa na concentração de 1 ufc em amostras de
100 ml ou 250 ml dentro de 24 horas em amostras de água não carbonatada e dentro de 26 horas em amostras carbonatadas.
Conteúdo
• WPSE020I contém 20 Snap Packs para amostras de 100 ml.
• WPSE200I contém 200 Snap Packs para amostras de 100 ml.
• WPSE250-20I contém 20 Snap Packs para amostras de 250 ml.
• WPSE250-100I contém 100 Snap Packs para amostras de 250 ml.
Armazenagem
Armazenar a 2°C–30°C e ao abrigo da luz.
Procedimento para testes de presença/ausência (P/A) em amostras de água não carbonatada
1. Adicione o conteúdo de um snap pack de tamanho apropriado a uma amostra de 100 ou 250 ml em um recipiente estéril,
transparente e não fluorescente.
2. Tampe o recipiente e agite.
3. Incube a 38°C ± 0,5°C por 24 horas. Os resultados serão válidos por até 28 horas.
4. Leia os resultados conforme a tabela Interpretação de resultados apresentada abaixo.
Procedimento para testes de presença/ausência (P/A) em amostras de água carbonatada
1. Desgaseifique a amostra agitando-a vigorosamente no recipiente original até que não haja mais gás visível ou audível (solte a tampa
para permitir a saída do gás após agitar).
2. Adicione o conteúdo de um snap pack de tamanho apropriado a uma amostra de 100 ou 250 ml em um recipiente estéril,
transparente e não fluorescente.
3. Agite vigorosamente o recipiente durante 30–60 segundos para liberar o gás carbônico (afrouxe a tampa para permitir a saída do gás
depois de agitar).
4. Recoloque a tampa e coloque o recipiente em um banho-maria a 38°C ± 0,5°C por 20 minutos.
5. Agite vigorosamente o recipiente até que não haja mais gás visível ou audível (afrouxe a tampa para permitir a saída do gás depois de agitar).
6. Recoloque a tampa e coloque em banho-maria a 38°C ± 0,5°C por 26 horas. Os resultados serão válidos por até 28 horas.
7. Leia os resultados conforme a tabela Interpretação de resultados apresentada abaixo.
Procedimento de enumeração Quanti-Tray* (somente amostras de 100 ml)
Observação: Ao testar amostras de água carbonatada engarrafada, siga as etapas 1–3 do “Procedimento para testes de presença/ausência
(P/A) em amostras de água carbonatada” e, em seguida, siga a etapa 3 abaixo.
1. Adicione o conteúdo de um snap pack a uma amostra de água de 100 ml em um recipiente estéril.
2. Tampe o recipiente e agite até dissolver.
3. Adicione 2 gotas da solução antiespuma IDEXX 1 à mistura de amostra/reagente.
Observação: Também estão disponíveis recipientes de amostras IDEXX de 120 ml com antiespuma. 2
4. Coloque a mistura de amostra/reagente em uma Quanti-Tray ou Quanti-Tray/2000 e vede com um selador Quanti-Tray.
5. Coloque a placa selada em banho-maria a 38°C ± 0,5°C por 24 horas (26 horas para água carbonatada). Os resultados serão válidos por até 28 horas.
6. Leia os resultados conforme a tabela Interpretação de resultados apresentada abaixo. Conte o número de cavidades positivas e consulte a tabela de número mais provável
(NMP) fornecida com as placas para verificar o número mais provável.
Interpretação do resultado
Aspecto Resultado
Sem fluorescência azul Negativo para Pseudomonas aeruginosa
Fluorescência azul † Positivo para Pseudomonas aeruginosa
† Presença de fluorescência azul em quantidade superior à observada na amostra de controle negativo.
• Para pesquisar fluorescência azul, coloque uma lâmpada UV de 6 watts e 365 nm a aproximadamente 12 cm da amostra em um ambiente escuro. Aponte a luz para amostra
e na direção oposta a seus olhos.
• Os Pseudalert dá resultados definitivos em 24–28 horas para amostras não carbonatadas. Além disso, resultados positivos para Pseudomonas aeruginosa observados antes
de 24 horas e negativos observados após 28 horas também são válidos.
Observações sobre o procedimento
• Use apenas água estéril, não tamponada e sem oxidantes para as diluições.
• Para comparação, pode-se utilizar um branco incubado de água estéril contendo o reagente Pseudalert (controle negativo) ao interpretar os resultados.
• Estas instruções podem não ser inteiramente compatíveis com os regulamentos cabíveis em seu local de trabalho. Para manter a conformidade, verifique se os
procedimentos regulatórios cabíveis estão sendo seguidos.
• Pseudalert é um teste para água primária. As características do desempenho do Pseudalert não se aplicam a amostras alteradas por pré-enriquecimento ou concentração.
• O Pseudalert não foi validado para uso com amostras de água engarrafada aromatizada ou água do mar.
• Empregue sempre técnicas assépticas ao utilizar o Pseudalert. Descarte os materiais de acordo com as boas práticas laboratoriais.
• A presença de altas concentrações de minerais, especialmente magnésio ou cálcio, pode turvar o reagente Pseudalert.
Procedimientos de control de calidad
Os procedimentos de controle de qualidade a seguir são recomendados para cada lote de Pseudalert e devem ser realizados com mais frequência se exigido pela
regulamentação:
1. Para cada cepa bacteriana do American Type Culture Collection (ATCC) 3 listada abaixo, repique a cultura em placas TSA ou ágar-sangue identificadas e incube a 35°C ± 0,5°C
por 18–24 horas.
2. Para cada cepa bacteriana, toque a colônia com uma alça de inoculação estéril de 1 μl e utilize-a para inocular um tubo de ensaio identificado contendo 5 ml de água
deionizada estéril. Feche a tampa e agite vigorosamente.
3. Para cada cepa bacteriana, pegue uma alça de 1 μl do tubo de ensaio e use-a para inocular um recipiente identificado contendo 100 ou 250 ml de água deionizada estéril.
Esses tubos servirão como controles.
4. Para testar os controles, siga as etapas abaixo dos testes de Presença/Ausência Pseudalert ou Quanti-Tray Enumeration. Compare os resultados obtidos com os esperados a seguir.
Controle Nº. ATCC Resultado esperado
P. aeruginosa ATCC 27853 ou ATCC 10145 Fluorescência azul
E. coli ATCC 25922 Sem fluorescência azul
P. fluorescens ATCC 13525 Sem fluorescência azul
1. Número de catálogo da solução antiespuma IDEXX: WAFDB
2. Número de catálogo de recipientes IDEXX 120 ml com lacre encolhível e antiespuma: WV120SBAF-200
3. American Type Culture Collection, 1-800-638-6597
©2010 IDEXX Laboratories, Inc. Todos os direitos reservados.
*Pseudalert e Quanti-Tray são marcas comerciais ou marcas comerciais registradas da IDEXX Laboratories, Inc. ou de suas filiais nos Estados Unidos
da América e/ou em outros países.
Pseudalert* Testkit
Einleitung
Der Pseudalert-Test weist Pseudomonas aeruginosa in Wasser in verschlossenen Behältnissen, Schwimmbeckenwasser und Spa-Wasser nach. Der Test basiert auf dem
Nachweis bakterieller Enzyme und zeigt das Vorliegen von Pseudomonas aeruginosa durch Hydrolyse eines im Pseudalert-Reagenz enthaltenen Substrats an. Die Zellen von
Pseudomonas aeruginosa wachsen und reproduzieren sich rasch, indem sie den hohen Gehalt an Aminosäuren, Vitaminen und anderen Nährstoffen im Pseudalert-Reagenz
nutzen. Aktiv wachsende Stämme von Pseudomonas aeruginosa weisen ein Enzym auf, dass das Substrat des Reagenz spaltet und im UV-Licht eine blaue Fluoreszenz bewirkt.
Pseudalert weist Pseudomonas aeruginosa im Bereich von 1 CFU (koloniebildende Einheit/KBE) in 100-ml- oder 250-ml-Proben nicht kohlensäurehaltigen Wassers binnen
24 Stunden und in Proben mit kohlensäurehaltigem Wasser innerhalb von 26 Stunden nach.
Inhalt
• WPSE020I enthält 20 Snap Packs für 100-ml-Wasserproben.
• WPSE0200I enthält 200 Snap Packs für 100-ml-Wasserproben.
• WPSE250-20I enthält 20 Snap Packs für 250-ml-Wasserproben.
• WPSE250-100I enthält 100 Snap Packs für 250-ml-Wasserproben.
Lagerung
Bei 2–30 °C lichtgeschützt lagern.
Presence/Absence (P/A)-Verfahren für nicht kohlensäurehaltige Wasserproben
1. Den Inhalt eines entsprechend großen Snap Packs zu 100 ml oder 250 ml Wasserprobe geben, die sich in einem sterilen,
transparenten und nicht fluoreszierenden Gefäß befindet.
2. Gefäß verschließen und schütteln.
3. Bei 38 °C ± 0,5 °C 24 Stunden inkubieren; Ergebnisse sind bis zu einer Gesamtinkubationszeit von 28 Stunden gültig.
4. Die Testergebnisse gemäß der nachstehenden Tabelle zur Ergebnisinterpretation ablesen.
Presence/Absence (P/A)-Verfahren für kohlensäurehaltige Wasserproben
1. Kohlensäure aus der Probe durch kräftiges Schütteln des Originalbehältnisses so lange entfernen, bis kein Gas mehr zu sehen oder
zu hören ist (Verschlusskappe immer wieder öffnen, um das Gas nach dem Schütteln entweichen zu lassen).
2. Den Inhalt eines entsprechend großen Snap Packs zu 100 ml oder 250 ml Wasserprobe geben, die sich in einem sterilen,
transparenten und nicht fluoreszierenden Gefäß befindet.
3. Das verschlossene Probengefäß 30 bis 60 Sekunden lang schütteln, um die restliche Kohlensäure zu entfernen (Gefäßverschluss öffnen,
um das Gas nach dem Schütteln entweichen zu lassen).
4. Verschlusskappe schließen und Gefäß für 20 Minuten in ein Wasserbad mit einer Temperatur von 38 °C ± 0,5 °C geben.
5. Das verschlossene Probengefäß nochmals kräftig schütteln, bis keine Kohlensäure mehr zu sehen oder zu hören ist (Gefäßverschluss öffnen,
um das Gas nach dem Schütteln entweichen zu lassen).
6. Verschlusskappe schließen und das Gefäß für weitere 26 Stunden in ein Wasserbad mit 38 °C ± 0,5 °C stellen; Ergebnisse sind bis zu 28 Stunden gültig.
7. Die Testergebnisse gemäß der nachstehenden Tabelle zur Ergebnisinterpretation ablesen.
Quanti-Tray* Verfahren zur quantitativen Bakterienzählung (nur für 100-ml-Proben)
Hinweis: Zum Testen von Proben kohlensäurehaltigen Flaschenwassers den Anweisungen unter „Presence/Absence (P/A)-Verfahren für
kohlensäurehaltige Wasserproben”, Schritte 1 bis 3 (siehe oben), folgen, danach mit dem nachstehenden Schritt 3 fortfahren.
1. Den Inhalt eines Snap Pack zu einer 100-ml-Wasserprobe in einem sterilen Gefäß geben.
2. Gefäß verschließen und schütteln, bis das Reagenz vollkommen aufgelöst ist.
3. Zwei Tropfen IDEXX Antischaum-Lösung 1 zur Mischung aus Probe und Reagenz hinzufügen.
Hinweis: Ebenfalls erhältlich sind IDEXX 120-ml-Probengefäße, die bereits Antischaummittel enthalten. 2
4. Die Mischung aus Probe und Reagenz in einen Quanti-Tray oder Quanti-Tray/2000 gießen und im Quanti-Tray Sealer versiegeln.
5. Den versiegelten Quanti-Tray bei 38 °C ± 0,5 °C 24 Stunden inkubieren (26 Stunden für kohlensäurehaltige Wasserproben); Ergebnisse sind bis zu 28 Stunden gültig.
6. Die Testergebnisse anhand der nachstehenden Tabelle zur Ergebnisinterpretation ablesen. Die Anzahl der positiven Probenvertiefungen zählen und die wahrscheinlichste
Zahl (MPN/Most Probable Number) an Keimen anhand der MPN-Tabelle, die den Trays beiliegt, ermitteln.
Ergebnisinterpretation
Färbung Ergebnis
Keine blaue Fluoreszenz Negativ für Pseudomonas aeruginosa
Blaue Fluoreszenz † Positiv für Pseudomonas aeruginosa
† Vorliegen einer stärkeren blau fluoreszierenden Färbung als in der Negativkontrolle
• Untersuchung auf Fluoreszenz in dunklem Umfeld mittels einer 6-Watt-UV-Lampe (365 nm Wellenlänge), die nicht weiter als 12 cm von der Probe entfernt sein soll. Das Licht
nicht in die Augen, sondern auf die Probe richten.
• Pseudalert-Testergebnisse sind für nicht kohlensäurehaltige Proben nach 24–28 Stunden definitiv. Auch die vor Ablauf von 24 Stunden beobachteten positiven sowie die nach
Ablauf von 28 Stunden beobachteten negativen Reaktionen auf Pseudomonas aeruginosa sind gültige Ergebnisse.
Hinweise zur Testdurchführung
• Nur steriles, nicht gepuffertes, keine Oxidanzien enthaltendes Wasser zur Verdünnung verwenden.
• Zum Vergleich kann zur Ergebnisinterpretation eine inkubierte Blindprobe aus sterilem Wasser, dass das Pseudalert-Reagenz enthält (Negativkontrolle), herangezogen werden.
• Manche Angaben in dieser Packungsbeilage entsprechen möglicherweise nicht Ihren örtlichen Vorschriften. Stellen Sie sicher, dass die anwendbaren behördlichen
Vorschriften befolgt werden.
• Pseudalert ist ein primärer Wassertest. Die Leistungsmerkmale für Pseudalert gelten nicht für Proben, die durch Voranreicherung oder Konzentration modifiziert wurden.
• Pseudalert ist nicht für die Verwendung bei Wasserproben von mit Geschmack versetztem Flaschenwasser oder Meerwasser validiert.
• Bei der Verwendung von Pseudalert sollte stets auf ein aseptisches Vorgehen geachtet werden. Entsorgung der Materialien gemäß den Standard-Laborpraktiken (Gute Laborpraxis/GLP).
• Bei hohem Mineralstoffgehalt des Wassers (insbesondere von Magnesium oder Kalzium) kann sich das Pseudalert-Reagenz trüben.
Qualitätskontrollverfahren
Für jede Charge von Pseudalert (bzw. auch häufiger, je nach örtlichen Bestimmungen) müssen die folgenden Verfahren zur Qualitätskontrolle angewendet werden:
1. Für jeden Bakterienstamm der American Type Culture Collection (ATTC) 3 , der unten aufgeführt ist, die Kultur auf markierte TSA- oder Blutagarplatten ausstreichen und bei
35 °C ± 0,5 °C 18–24 Stunden inkubieren.
2. Für jeden Bakterienstamm 1 μl einer Kolonie mittels steriler Inokulationsöse in ein markiertes Teströhrchen mit 5 ml sterilen, entionisierten Wassers transferieren. Röhrchen
verschließen und kräftig schütteln.
3. Für jeden Bakterienstamm 1 μl mittels geeigneter Inokulationsöse aus dem Teströhrchen entnehmen und in ein markiertes Gefäß mit 100 ml oder 250 ml sterilen,
entionisierten Wassers überführen. Dies sind die Kontrollen.
4. Folgen Sie zum Testen dieser Kontrollen den Anweisungen der oben beschriebenen Schritte des Pseudalert Presence/Absence-Verfahrens oder des Quanti-Tray-Verfahrens
zur quantitativen Bakterienzählung. Vergleichen Sie die Testergebnisse mit den nachfolgenden erwarteten Resultaten.
Kontrolle ATCC Nr Erwartetes Resultat
P. aeruginosa ATCC 27853 oder ATCC 10145 Blaue Fluoreszenz
E. coli ATCC 25922 Keine blaue Fluoreszenz
P. fluorescens ATCC 13525 Keine blaue Fluoreszenz
1. IDEXX Antischaum-Lösung - Katalognummer: WAFDB
2. IDEXX mit Schrumpfband verschlossene Einwegbehälter (120 ml) mit Antischaum-Lösung - Katalognummer: WV120SBAF-200
3. American Type Culture Collection 1-800-638-6597
©2010 IDEXX Laboratories, Inc. Alle Rechte vorbehalten.
*Pseudalert und Quanti-Tray sind Schutzmarken oder eingetragene Schutzmarken von IDEXX Laboratories, Inc. oder eines Tochterunternehmens von IDEXX
in den Vereinigten Staaten und/oder in anderen Ländern.
Pseudalert *
06-18569-01
IDEXX Laboratories, Inc., One IDEXX Drive, Westbrook, Maine 04092 USA
North/South America: 1-207-556-4496/1-800-321-0207
For Technical Support, please call:
www.idexx.com/water
UK: +44 (0) 1638 676800
Europe: 00800 4339 9111
China: +86-21-61279528
Japan: +81 422 71 5921
Australia: 1800 655 978

Pseudalert* Test Kit
Introduction
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 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 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.
Contents
• WPSE020I contains 20 Snap Packs for 100 mL samples.
• WPSE200I contains 200 Snap Packs for 100 mL samples.
• WPSE250-20I contains 20 Snap Packs for 250 mL samples.
• WPSE250-100I contains 100 Snap Packs for 250 mL samples.
Storage
Store at 2°C–30°C away from light
Presence/Absence (P/A) Procedure for Non-carbonated Water Samples
1. 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°C ±0.5°C for 24 hours; results are valid up to 28 hours.
4. Read results according to Result Interpretation table below.
Presence/Absence (P/A) Procedure for Carbonated Water Samples
1. De-gas sample by vigorously shaking sample in original container until no gas can be seen or heard
(loosen cap to allow gas to escape after shaking).
2. Add contents of one appropriately sized snap pack to either a 100 mL or 250 mL sample in a sterile, transparent, nonfluorescing vessel.
3. Shake vessel vigorously for 30–60 seconds to release carbonation (loosen cap to allow gas to
escape after shaking).
4. Secure cap and place vessel in 38°C ±0.5°C water bath for 20 minutes.
5. Shake vessel vigorously until no gas can be seen or heard to release carbonation (loosen cap to allow gas
to escape after shaking).
6. Secure cap and place in a 38°C ±0.5°C water bath for 26 hours; results are valid up to 28 hours.
7. Read results according to Result Interpretation table below.
Quanti-Tray* Enumeration Procedure (100 mL Samples Only)
Note: If testing carbonated bottled-water samples, follow the “Presence/Absence (P/A) Procedure for
Carbonated Water Samples” steps 1–3 (above), then proceed to step 3 below.
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 Solution 1 to the sample/reagent mixture.
Note: IDEXX 120 mL sample vessels 2 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°C ±0.5°C incubator for 24 hours (26 hours for carbonated samples); results are valid up to 28 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 fluorescent color greater than the amount present in a negative control sample
• Look for fluorescence with a 6-watt, 365 nm, UV light within 5 inches of the sample in a dark environment. Face light away from your eyes and toward the sample.
• Pseudalert results are definitive at 24–28 hours for non-carbonated samples. In addition, positives for Pseudomonas aeruginosa observed before 24 hours and negatives
observed after 28 hours are also valid.
Procedural Notes
• Use only sterile, nonbuffered, oxidant-free water for dilutions.
• For comparison, an incubated sterile water blank containing Pseudalert reagent (negative control) can be used when interpreting results.
• This insert may not reflect your local regulations. For compliance testing, be sure to follow appropriate regulatory procedures.
• Pseudalert is a primary water test. Pseudalert performance characteristics do not apply to samples altered by preenrichment or concentration.
• Pseudalert has not been validated for use with flavored bottled-water or marine water samples.
• Aseptic technique should always be followed when using Pseudalert. Dispose of materials in accordance with Good Laboratory Practices.
• The presence of a high mineral content (especially magnesium and or calcium) can cause Pseudalert reagent to become cloudy.
Quality Control Procedures
The following quality control procedure is recommended for each lot of Pseudalert or more often as regulations require:
1. For each of the American Type Culture Collection (ATCC) 3 bacterial strain listed below, streak the culture onto labeled TSA or blood agar plates, and incubate at 35°C
±0.5°C for 18–24 hours.
2. 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.
3. 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.
4. Follow the Pseudalert Presence/Absence or Quanti-Tray Enumeration steps above to test these controls. Compare test results to the expected results below.
Control ATCC # Expected Result
P. aeruginosa ATCC 27853 or ATCC 10145 Blue fluorescence
E. coli ATCC 25922 No blue fluorescence
P. fluorescens ATCC 13525 No blue fluorescence
1. IDEXX Antifoam Solution catalog number: WAFDB
2. IDEXX 120 mL Shrink-banded Vessels with Antifoam catalog number: WV120SBAF-200
3. American Type Culture Collection, 1-800-638-6597
©2010 IDEXX Laboratories, Inc. All rights reserved.
*Pseudalert and Quanti-Tray are trademarks or registered trademarks of IDEXX Laboratories, Inc. or its affiliates in the United States and/or other countries.
Kit de test Pseudalert*
Introduction
Le test Pseudalert détecte la présence de Pseudomonas aeruginosa dans les échantillons d’eau en bouteille, d’eau de piscine et d’eau thermale. Le test est basé sur la
détection d’une enzyme bactérienne qui, en hydrolysant le substrat présent dans le réactif Pseudalert, met en évidence la présence de la bactérie Pseudomonas aeruginosa.
Avec le milieu enrichi en acides aminés, vitamines et autres nutriments présents dans le réactif Pseudalert, la croissance et la reproduction de Pseudomonas aeruginosa
sont rapides. Les souches de Pseudomonas aeruginosa en pleine croissance expriment une enzyme qui clive le substrat, ce qui produit une fluorescence bleue visible sous
lumière ultraviolette. Pseudalert détecte Pseudomonas aeruginosa à 1 ufc dans des échantillons de 100 ml ou de 250 ml en 24 heures pour les échantillons d’eau non
gazeuse et en 26 heures pour les échantillons d’eau gazeuse.
Contenu
• WPSE020I contient 20 sachets Snap pour des échantillons de 100 ml.
• WPSE200I contient 200 sachets Snap pour des échantillons de 100 ml.
• WPSE250-20I contient 20 sachets Snap pour des échantillons de 250 ml.
• WPSE250-100I contient 100 sachets Snap pour des échantillons de 250 ml.
Conditions de conservation
À conserver entre 2 °C et 30 °C, à l’abri de la lumière.
Procédure de présence/absence (P/A) pour les échantillons d’eau non gazeuse
1. Ajouter le contenu d’un sachet Snap de taille appropriée dans un échantillon de 100 ml ou de 250 ml placé dans un récipient
stérile, transparent et non fluorescent.
2. Fermer le récipient et agiter.
3. Incuber à 38 °C ± 0,5 °C pendant 24 heures. Les résultats peuvent être lus jusqu’à 28 heures.
4. Interpréter les résultats en se référant au tableau d’interprétation des résultats ci-dessous.
Procédure de présence/absence (P/A) pour les échantillons d’eau gazeuse
1. Dégazer l’échantillon en agitant vigoureusement le récipient dans lequel il a été prélevé jusqu’à ce qu’il soit impossible de voir ou
d’entendre le gaz (desserrer le bouchon pour permettre au gaz de s’échapper après l’agitation).
2. Ajouter le contenu d’un sachet Snap de taille appropriée dans un échantillon de 100 ml ou de 250 ml placé dans un récipient stérile,
transparent et non fluorescent.
3. Agiter le récipient vigoureusement pendant 30 à 60 secondes pour libérer la carbonatation (desserrer le bouchon pour permettre au gaz
de s’échapper après l’agitation).
4. Refermer le récipient et le placer dans un bain-marie à 38 °C ± 0,5 °C pendant 20 minutes.
5. Agiter le récipient vigoureusement pour libérer la carbonatation jusqu’à ce qu’il soit impossible de voir ou d’entendre le gaz (desserrer le
bouchon pour permettre au gaz de s’échapper après l’agitation).
6. Refermer le récipient et le placer dans un bain-marie à 38 °C ± 0,5 °C pendant 26 heures. Les résultats peuvent être lus jusqu’à 28 heures.
7. Interpréter les résultats en se référant au tableau d’interprétation des résultats ci-dessous.
Procédure de numération Quanti-Tray* (échantillons de 100 ml uniquement)
Remarque : Si l’analyse concerne des échantillons d’eau gazeuse en bouteille, suivre les étapes 1 à 3 de la « Procédure de présence/
absence (P/A) pour les échantillons d’eau gazeuse » ci-dessus, puis passer directement à l’étape 3 ci-dessous.
1. Ajouter le contenu d’un sachet Snap dans un échantillon d’eau de 100 ml placé dans un récipient stérile.
2. Fermer le récipient et agiter jusqu’à dissolution.
3. Ajouter 2 gouttes de solution anti-mousse IDEXX 1 au mélange échantillon/réactif.
Remarque : des récipients IDEXX de 120 ml contenant déjà un agent anti-mousse sont également disponibles. 2
4. Verser le mélange échantillon/réactif dans un Quanti-Tray ou Quanti-Tray/2000 et fermer hermétiquement à l’aide d’une scelleuse Quanti-Tray Sealer.
5. Placer le plateau scellé dans un incubateur à 38 °C ± 0,5 °C pendant 24 heures (26 heures pour les échantillons d’eau gazeuse). Les résultats peuvent être lus jusqu’à 28 heures.
6. Interpréter les résultats en se référant au tableau d’interprétation des résultats ci-dessous. Compter le nombre de puits positifs et se référer au tableau NPP fourni avec les
plateaux pour obtenir le Nombre le plus probable (NPP).
Interprétation des résultats
Aspect Résultat
Absence de fluorescence bleue Échantillon négatif pour Pseudomonas aeruginosa
Fluorescence bleue † Échantillon positif pour Pseudomonas aeruginosa
† Présence d’une fluorescence de couleur bleue plus importante que la fluorescence détectée dans un échantillon de contrôle négatif.
• Analyser la fluorescence à l’aide d’une lampe UV de 6 watts, émettant un rayonnement de 365 nm, située à une distance de 12 cm du flacon, dans un endroit obscur. Orienter
la lampe en direction de l’échantillon et la tenir à l’écart des yeux.
• Les résultats de Pseudalert doivent être lus entre 24 et 28 heures pour les échantillons non gazeux. Les échantillons positifs pour Pseudomonas aeruginosa observés avant
24 heures et les échantillons négatifs observés après 28 heures sont également valides.
Remarques concernant la procédure
• Utiliser uniquement de l’eau stérile, non tamponnée et sans oxydant pour les dilutions.
• Afin de réaliser une comparaison, il est possible d’utiliser de l’eau stérile incubée avec du réactif Pseudalert (contrôle négatif) au moment d’interpréter les résultats.
• Cette notice peut différer des réglementations en vigueur dans votre pays. Pour que tout test soit réalisé en conformité avec ces dernières, suivre les procédures
réglementaires appropriées.
• Pseudalert est avant tout un test de contrôle de l’eau. Les caractéristiques de performance de Pseudalert ne s’appliquent pas aux échantillons altérés par un enrichissement
préalable ou une concentration.
• Pseudalert n’a pas été validé pour une utilisation avec des échantillons d’eau en bouteille aromatisée ou d’eau de mer.
• Utiliser systématiquement des techniques aseptiques lors de l’emploi de Pseudalert. Éliminer le matériel conformément aux bonnes pratiques de laboratoire.
• Le réactif Pseudalert peut se troubler en présence d’une haute teneur en minéraux (particulièrement en magnésium et (ou) en calcium).
Contrôle qualité
Il est recommandé d’exécuter la procédure de contrôle de qualité suivante pour chaque lot de Pseudalert, ou plus souvent si la réglementation l’exige.
1. Pour chaque souche bactérienne de l’American Type Culture Collection (ATCC) 3 dont la liste est donnée ci–dessous, déposer la culture sur un milieu TSA ou une gélose au
sang dûment étiquetés, et incuber à 35 °C ± 0,5 ºC pendant 18 à 24 heures.
2. Pour chaque souche bactérienne, prélever une colonie avec une anse d’inoculation de 1 μl et l’introduire dans un tube à essai étiqueté contenant 5 ml d’eau déminéralisée
stérile. Le fermer et bien homogénéiser.
3. Pour chaque souche bactérienne, prélever 1 μl du tube à essai à l’aide d’une anse 1 μl et l’inoculer dans un récipient étiqueté contenant soit 100 ml soit 250 ml d’eau
déminéralisée stérile. Il s’agit de vos contrôles.
4. Suivre les étapes de la procédure présence/absence ou de la procédure de numération Quanti-Tray du kit Pseudalert décrites ci-dessus pour analyser ces contrôles.
Comparer les résultats du test aux résultats escomptés indiqués ci-dessous.
Contrôle Nº ATCC Résultat escompté
P. aeruginosa ATCC 27853 oder ATCC 10145 Fluorescence bleue
E. coli ATCC 25922 Pas de fluorescence bleue
P. fluorescens ATCC 13525 Pas de fluorescence bleue
1. Numéro de catalogue de la solution anti-mousse IDEXX : WAFDB
2. Numéro de catalogue des récipients IDEXX avec anti-mousse protégés par une bande thermorétractable : WV120SBAF-200
3. American Type Culture Collection : 1-800-638-6597
©2010 IDEXX Laboratories, Inc. Tous droits réservés.
*Pseudalert et Quanti-Tray sont des marques commerciales ou des marques déposées d’IDEXX Laboratories, Inc. ou de ses filiales aux États-Unis et/ou
dans d’autres pays.
Kit del test Pseudalert*
Introduzione
Il test Pseudalert rileva la presenza di Pseudomonas aeruginosa in acque imbottigliate, e di piscine e centri termali. Il test si basa sul rilevamento di un enzima batterico
presente nella Pseudomonas aeruginosa, che catalizza l’idrolisi del substrato contenuto nel reagente Pseudalert. I batteri Pseudomonas aeruginosa crescono e si riproducono
rapidamente sfruttando la ricca fonte di aminoacidi, vitamine e altri nutrienti presenti nel reagente Pseudalert. I ceppi di Pseudomonas aeruginosa che proliferano rapidamente
possiedono un enzima che idrolizza il substrato, producendo una fluorescenza blu in presenza di luce UV. Pseudalert è in grado di rilevare entro 24 ore la presenza di Pseudo-
monas aeruginosa ad una concentrazione di 1 organismo in campioni da 100 ml o 250 ml di acqua non gassata ed entro 26 ore in campioni di acqua gassata.
Contenuto
• WPSE020I contiene 20 dosi di reagente Snap per campioni da 100 ml.
• WPSE200I contiene 200 dosi di reagente Snap per campioni da 100 ml.
• WPSE250-20I contiene 20 dosi di reagente Snap per campioni da 250 ml.
• WPSE250-100I contiene 100 dosi di reagente Snap per campioni da 250 ml.
Conservazione
Conservare a 2–30 °C lontano dalla luce.
Procedura di presenza/assenza (P/A) per campioni di acqua non gassata
1. Aggiungere il contenuto di una dose di reagente Snap della dimensione appropriata ad un campione di 100 ml o 250 ml in un
recipiente sterile, trasparente e non fluorescente.
2. Chiudere il recipiente ed agitarlo.
3. Incubare a 38 °C ± 0,5 °C per 24 ore. I risultati sono validi per un periodo non superiore a 28 ore.
4. Leggere i risultati in base alla seguente tabella di interpretazione dei risultati.
Procedura di presenza/assenza (P/A) per campioni di acqua gassata
1. Degassificare il campione agitandolo vigorosamente nel contenitore originale fino a quando non è possibile osservare o udire la
presenza del gas (dopo averlo agitato, allentare il tappo per lasciare fuoriuscire il gas).
2. Aggiungere il contenuto di una dose di reagente Snap della dimensione appropriata ad un campione da 100 ml o 250 ml in un
recipiente sterile, trasparente e non fluorescente.
3. Agitare il recipiente vigorosamente per 30–60 secondi per lasciare fuoriuscire il gas (dopo averlo agitato, allentare il tappo per
lasciare fuoriuscire il gas).
4. Chiudere il recipiente e collocarlo in un bagno termostatato a 38 °C ± 0,5 °C per 20 minuti.
5. Agitare il recipiente vigorosamente fino a quando non è possibile osservare o udire la presenza del gas per lasciarlo fuoriuscire
completamente (dopo averlo agitato, allentare il tappo per lasciare fuoriuscire il gas).
6. Chiudere il recipiente e posizionarlo in un bagno termostatato a 38 °C ± 0,5 °C per 26 ore. I risultati sono validi per un periodo non
superiore a 28 ore.
7. Leggere i risultati in base alla seguente tabella di interpretazione dei risultati.
Procedura di enumerazione Quanti-Tray* (solo per campioni da 100 ml)
Nota: per l’analisi di campioni di acqua gassata, seguire i passaggi 1–3 descritti precedentemente nella sezione “Procedura di presenza/
assenza (P/A) per campioni di acqua gassata” e quindi proseguire con il passaggio 3.
1. Aggiungere il contenuto di una dose di reagente Snap ad un campione di acqua da 100 ml, in un recipiente sterile.
2. Chiudere il recipiente ed agitarlo fino alla dissoluzione.
3. Aggiungere 2 gocce di soluzione antischiuma IDEXX 1 alla miscela campione/reagente.
Nota: sono disponibili anche recipienti per campioni IDEXX da 120 ml già contenenti antischiuma. 2
4. Versare la miscela campione/reagente in un Quanti-Tray o un Quanti-Tray/2000 e sigillarlo con un sigillatore Quanti-Tray Sealer IDEXX.
5. Posizionare il contenitore sigillato in un incubatore a 38 °C ± 0,5 °C per 24 ore (26 ore per campioni di acqua gassata). I risultati sono validi per un periodo non superiore
a 28 ore.
6. Leggere i risultati in base alla seguente tabella di interpretazione dei risultati. Contare il numero dei pozzetti positivi e consultare la tabella MPN in dotazione con il
contenitore per ottenere il numero più probabile (MPN).
Interpretazione del risultato
Aspetto Risultato
Nessuna fluorescenza blu Negativo per Pseudomonas aeruginosa
Fluorescenza blu † Positivo per Pseudomonas aeruginosa
† Presenza di un livello di fluorescenza blu superiore a quello presente nel campione di controllo negativo.
• Ricercare la presenza di fluorescenza usando una lampada UV da 6 watt con lunghezza d’onda di 365 nm a circa 12 centimetri dal campione e in ambiente buio. Indirizzare la
luce verso il campione e lontano dagli occhi.
• In caso di campioni di acqua non gassata, i risultati di Pseudalert sono considerati definitivi dopo 24–28 ore. Inoltre, sono validi anche i risultati positivi per Pseudomonas
aeruginosa osservati prima delle 24 ore ed i risultati negativi osservati dopo 28 ore.
Note procedurali
• Per le diluizioni usare solo acqua sterile, non tamponata e senza ossidanti.
• Per il confronto, durante l’interpretazione dei risultati è possibile utilizzare come bianco un campione di acqua sterile incubato contenente il reagente Pseudalert (controllo negativo).
• Questo inserto informativo potrebbe non riflettere i regolamenti locali dell’utente. Per i test in conformità, assicurarsi di seguire le appropriate procedure normative.
• Pseudalert è principalmente un test per l’acqua. Le caratteristiche di prestazione di Pseudalert non sono applicabili a campioni alterati da qualsiasi pre-arricchimento
o concentrazione.
• Pseudalert non è stato validato per l’utilizzo con campioni di acqua imbottigliata aromatizzata o di acqua marina.
• Quando si usa Pseudalert è necessario impiegare tecniche asettiche. Smaltire i materiali seguendo le buone pratiche di laboratorio.
• La presenza di un elevato contenuto di minerali (in particolare di magnesio e/o calcio) può rendere torbido il reagente Pseudalert.
Procedure per il controllo di qualità
La seguente procedura di controllo di qualità deve essere eseguita su ogni lotto di Pseudalert oppure con una maggiore frequenza, in conformità a quanto previsto dalle
normative specifiche:
1. Per ciascuno dei ceppi batterici ATCC (American Type Culture Collection) 3 sotto elencati, effettuare uno striscio della coltura su piastre all’agar sangue o TSA con etichetta
e incubare a 35 °C ± 0,5 °C per 18-24 ore.
2. Per ciascun ceppo batterico, strisciare sulla colonia un’ansa da inoculazione sterile da 1 μl e usarla per inoculare una provetta etichettata contenente 5 ml di acqua
deionizzata sterile. Chiudere la provetta e agitarla accuratamente.
3. Per ciascun ceppo batterico, usare un’ansa da 1 μl, inserirla nella provetta e inoculare un recipiente etichettato contenente 100 ml o 250 ml di acqua deionizzata sterile.
Questi campioni rappresentano i controlli per il test.
4. Per analizzare i controlli, seguire i passaggi della procedura di presenza/assenza Pseudalert o di enumerazione Quanti-Tray descritti precedentemente. Confrontare i risultati
ottenuti con quelli previsti riportati di seguito.
Controllo N. ATCC Risultato previsto
P. aeruginosa ATCC 27853 o ATCC 10145 Fluorescenza blu
E. coli ATCC 25922 Nessuna fluorescenza blu
P. fluorescens ATCC 13525 Nessuna fluorescenza blu
1. Numero di catalogo della soluzione antischiuma IDEXX: WAFDB
2. Numero di catalogo dei contenitori da 120 ml con pellicola termoretraibile e antischiuma IDEXX: WV120SBAF-200
3. American Type Culture Collection 1-800-638-65977
©2010 IDEXX Laboratories, Inc. Tutti i diritti riservati.
*Pseudalert e Quanti-Tray sono marchi o marchi registrati di IDEXX Laboratories, Inc. o di suoi associati negli Stati Uniti e/o in altri Paesi.
Kit Pseudalert*
Introducción
El kit Pseudalert detecta la presencia de Pseudomonas aeruginosa en muestras de agua embotellada, de agua de piscina y de agua termal. 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 UV. Pseudalert
detecta bacterias Pseudomonas aeruginosa a una concentración de 1 UFC en muestras de 100 ml o 250 ml, en 24 horas en muestras de agua no carbonatada y en 26 horas
en muestras de agua carbonatada.
Contenido
• WPSE020I contiene 20 dosis Snap para muestras de 100 ml.
• WPSE200I contiene 200 dosis Snap para muestras de 100 ml.
• WPSE250-20I contiene 20 dosis Snap para muestras de 250 ml.
• WPSE250-100I contiene 100 dosis Snap para muestras de 250 ml.
Conservación
Almacenar a una temperatura entre 2 y 30 °C protegido de la luz.
Procedimiento de presencia/ausencia (P/A) para muestras de agua no carbonatada
1. Añadir el contenido de la dosis Snap adecuadamente predispensada a una muestra de 100 o 250 ml en un recipiente estéril
transparente, no fluorescente.
2. Tapar y agitar el recipiente.
3. Incubar a 38 °C ± 0,5 °C durante 24 horas; los resultados son válidos hasta transcurridas 28 horas.
4. Leer los resultados de acuerdo con la tabla de interpretación de resultados que figura abajo.
Procedimiento de presencia/ausencia (P/A) para muestras de agua carbonatada
1. Extraer el gas de la muestra agitándola enérgicamente dentro de su recipiente original hasta que el gas no se pueda ver ni oír
(aflojar la tapa para que pueda salir el gas después de haber agitado la muestra).
2. Añadir el contenido de la dosis Snap adecuadamente predispensada a una muestra de 100 o 250 ml en un recipiente estéril
transparente, no fluorescente.
3. Agitar el recipiente enérgicamente durante 30–60 segundos para extraer el dióxido de carbono (aflojar la tapa para que pueda salir el gas
después de haber agitado el recipiente).
4. Cerrar bien la tapa y poner el recipiente en un baño María a 38 °C ± 0,5 °C durante 20 minutos.
5. Agitar el recipiente enérgicamente para extraer el dióxido de carbono hasta que el gas no se pueda ver ni oír (aflojar la tapa para que pueda
salir el gas después de haber agitado el recipiente).
6. Cerrar bien la tapa y poner el recipiente en un baño María a 38 °C ± 0,5 °C durante 26 horas; los resultados son válidos hasta transcurridas 28 horas.
7. 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)
Nota: En caso de analizar muestras de agua embotellada carbonatada, seguir los pasos 1 a 3 del “Procedimiento de presencia/ausencia
(P/A) para muestras de agua carbonatada” descrito anteriormente y continuar con el paso 3 que figura abajo.
1. Añadir el contenido de una dosis Snap 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 IDEXX 1 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 dispositivo de sellado para Quanti-Tray.
5. Colocar la bandeja sellada en una estufa de incubación a 38 °C ± 0,5 °C durante 24 horas (26 horas para muestras de agua carbonatada). Los resultados son válidos
hasta transcurridas 28 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.
• Observar la fluorescencia en un ambiente oscuro y con una luz UV de 6 vatios y 365 nm, a unos 12 cm de la muestra. Aleje la luz de sus ojos y oriéntela hacia la muestra.
• Los resultados de Pseudalert para muestras no carbonatadas son definitivos transcurridas entre 24 y 28 horas. Además, los positivos para Pseudomonas aeruginosa observados
antes de 24 horas y los negativos observados después de 28 horas también son válidos.
Notas sobre el procedimiento
• Utilizar solamente agua estéril, no tamponada, libre de oxidantes, para efectuar las diluciones.
• Para poder realizar las comparaciones a la hora de interpretar los resultados, se puede utilizar como blanco agua estéril incubada con el reactivo Pseudalert (control negativo).
• Es posible que este prospecto no refleje la normativa local de su país. Para realizar pruebas que la cumplan, asegúrese de seguir los procedimientos reglamentarios correspondientes.
• Pseudalert es fundamentalmente una prueba para analizar agua. Las características de rendimiento de Pseudalert no se pueden aplicar a muestras que hayan sido alteradas
previamente por algún tipo de enriquecimiento o concentración.
• El kit Pseudalert no se ha validado para ser utilizado con muestras de agua embotellada aromatizada o con muestras de agua marina.
• Siempre debe aplicarse una técnica aséptica cuando se utilice Pseudalert. El material debe desecharse de acuerdo con las buenas prácticas de laboratorio.
• El reactivo del kit Pseudalert puede volverse turbio cuando la muestra presenta un alto contenido en minerales (especialmente magnesio y/o calcio).
Procedimientos de control de calidad
Se recomienda seguir el siguiente procedimiento de control de calidad con cada lote de Pseudalert o más frecuentemente si la normativa así lo exige:
1. Para cada una de las cepas bacterianas del American Type Culture Collection (ATCC) 3 que se indican a continuación, siembre el cultivo en placas de TSA o agar sangre
debidamente marcadas e incúbelas a 35 °C ± 0,5 °C durante 18 a 24 horas.
2. 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.
3. 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 de agua
desionizada estéril. Las muestras así obtenidas serán sus controles.
4. Para analizar estos controles, siga el procedimiento de presencia/ausencia de Pseudalert o los pasos del procedimiento de enumeración Quanti-Tray descritos
anteriormente. Compare los resultados del análisis con los resultados previstos que se indican a continuación.
Control N.º ATCC Resultado previsto
P. aeruginosa ATCC 27853 o ATCC 10145 Fluorescencia azul
E. coli ATCC 25922 Sin fluorescencia azul
P. fluorescens ATCC 13525 Sin fluorescencia azul
1. Referencia de la solución antiespumante de IDEXX: WAFDB
2. Referencia de los recipientes de 120 ml de IDEXX con antiespumante y película termoretráctil: WV120SBAF-200
3. American Type Culture Collection, 1-800-638-6597
©2010 IDEXX Laboratories, Inc. Todos los derechos reservados.
*Pseudalert y Quanti-Tray son marcas o marcas registradas de IDEXX Laboratories, Inc. o sus filiales en Estados Unidos de América y/o en otros países..

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
Rev 03252011
idexx.com
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.

Rev 03252011
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
idexx.com
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.

Rev 03252011
Results
idexx.com
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

Rev 03252011
idexx.com
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:

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 watertesting
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 Summary 14B
idexx.com
Topic: Beta Trial Study report comparing Pseudalert* versus EN ISO 16266:2008 1 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
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Comparison of the performance of the IDEXX Pseudalert* test against the EN ISO
16266:2008 1 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
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1. Samples (>200 mL) were collected at numerous locations (maximum of five
collections per site) in accordance with ISO 19458 2 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
method 3 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 42 º C, 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 39
indoor pool (fra2b), filtrate 74 48 1 440
indoor whirlpool (fra), filtrate 19 28 0 3
natural pool, MU1, swimmer 0 2 250 1120
natural pool, MU1, wading pool 9 4 570 700
natural pool, MU2, nonswimmer 1 0 100 120
natural pool, MU3, swimmer 2 0 220 488
natural pool, MU3, wading pool 34 not analyzable >300 >300
open air pool (oes), pool 29 32 0 45
open air pool (sto), swimmer 38 42 >300 >300
natural pool, MU4, swimmer 2 2 450 580
natural pool, MU4, nonswimmer 2 0 490 800
natural pool, MU4, wading pool 6 0 760 1080
indoor whirlpool (fra), filtrate 3 2 0 0
indoor pool (fra5), filtrate 6 3 0 0
indoor pool (fra6), filtrate 201 186 0 3
(pres.) = P. aeruginosa present in sample but exact concentration could not be determined.
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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.
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Sixteen pool/spa water samples, which did not contain natural P. aeruginosa contamination,
were spiked with environmental isolates described in the following table:
Strain
isolated from
(origin)
Bacterial ID
colony color
on CN agar
fluorescence
Api 20 NE
profile
Api 20 NE
confirmation
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)
Spike
Strain
open air pool (lan1), pool 109 49 1 0 101
indoor pool (ben1), pool 130 49 0 0 102
open air pool (ben2), pool 130 16 0 1 103
artificial lake (ben3), lake 38 0 n.a. n.a. 104
open air pool (hep1), pool 70 1 n.a. n.a. 105
lake (arguk1), lake 130 33 58 124 106
indoor pool (dar1), pool 10 6 n.a. n.a. 101
indoor pool (fra11) whirlpool 31 19 n.a. n.a. 102
indoor pool (fra12), whirlpool 10 8 n.a. n.a. 104
natural pool (MU5+), wading pool 118 100 172 504 103
natural pool (MU5+), nonswimmer 50 75 376 416 104
open air pool (Obe1), wading pool 78 80 0 2 105
open air pool (Oes), swimmer 66 50 0 0 106
open 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
Rev 11032010

idexx.com
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 21 st
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.
Rev 11032010