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The life science business of Merck operates as MilliporeSigma in the U.S. and Canada.

Data Sheet

EZ-Fluo™ Rapid Detection System A fast, non-destructive, fluorescent staining-based system for microbial detection

Easy to integrate into any laboratoryThe EZ-Fluo™ Rapid Detection System from Merck enables you to significantly reduce your time to result, address contamination events sooner, and therefore, gain better control of your manufacturing process. The EZ-Fluo™ System is a rapid florescent-based technology designed for fast quantitative detection of microorganisms over a broad range of filterable samples. This new system uses standard membrane filtration to detect viable and culturable microorganisms down to 1 CFU per sample. EZ-Fluo™ test results are easily comparable to compendial method test results, which facilitates the validation and the integration of this rapid system in any laboratory without causing disruption. Detected microbial contamination can be then identified using your current ID methodology.

Faster product releaseToday, more and more QC laboratories are switching to rapid methods for the detection of microbial contamination. This is because traditional methods require several days to provide results and therefore are of limited value when it comes to releasing product to the market faster, or to conducting quick investigations when necessary.

• Rapid technology capable of providing results in 1/3 of the time required using traditional techniques

• Flexible approach based on standard membrane filtration and reduced incubation on media plate

• Non-destructive method, compatible with any ID technique

• Compact hardware, fits easily on any laboratory bench

• Cost effective solution for both routine and investigatory use

2017 - 07532 - EZ Fluo Rapid detection System Data Sheet_MRK.indd 1 3/8/18 12:06 PM

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Simple protocol using regular membrane filtration and incubationThe EZ-Fluo™ Rapid Detection System consists of a reader and staining reagents used in combination with regular 47 mm filtration membranes and standard media. Optimal results can be obtained using Merck’s membranes and comprehensive media portfolio. The use of the camera (optional) offers traceability of results and comfort when reading.

3. Counting CFUs

Count fluorescent colonies through the window of the EZ-Fluo™ Reader or use the camera (optional) to view the colonies on your computer screen.

2. Fluorescent staining

Transfer membrane to a Petri-Pad Petri dish wetted with staining reagent and incubate for 30 minutes.

1. Sample preparation

Filtration using EZ-Fit® Filtration Unit and media plates: Simply filter your sample the same way you prepare your current samples and incubate the membrane on your regular media type.

ApplicationsThe EZ-Fluo™ system can detect and quantify microbial contamination in any filterable sample. Potential applications are numerous and include testing of the following sample types:

• Raw materials

• In-process samples

• Final product

• Environmental samples

For a perfect contact of the membrane to the agar use our ReadyPlate™ 55 agar plates


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Reincubation for microorganism identificationThe EZ-Fluo™ System is a non-destructive method that enables you to continue to grow the microorganisms after they have been stained, in order to identify them using any standard ID technology. This is a tremendous advantage compared to other rapid test systems that do not allow ID of microorganisms.

Non-destructive technologyThe EZ-Fluo™ method is based on a unique, non-destructive, fluorescent marker developed by Merck. After filtration and incubation, the staining reagent is applied, and any viable and culturable microorganisms are stained. The reagent is initially non-fluorescent, but an intracellular enzyme that is active only as part of the metabolic processes of living cells, cleaves the substrate to liberate the fluorophore.

As fluorophore accumulates inside the cells, the signal is naturally amplified. In the EZ-Fluo™ reader, the cells are exposed to the excitation wavelength of the fluorescent dye so that they can be visually counted. The fluorescence allows much smaller colonies to be seen than are visible by the naked eye.

Reincubation step

Place membrane on a prefilled agar media cassette and reincubate. Collect and isolate the microorganisms and identify using any existing ID methodology.

Principle of fluorescent staining

Bacterium before staining Membrane incubation on the staining pad (30 minute incubation)

Stained bacterium

Example: Tested sample of in-process, non-sterile water using the EZ-Fluo™ system. After detection, membrane was re-incubated for full growth and identification.

Visualize plate after staining. View of membrane in the reader.

Colonies are not visible outside the reader. After reincubation, colonies are visible to the eye.

Reagents Pad


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Support and servicesMerck provides a comprehensive range of support and services to help you save your QC resources and facilitate the implementation of your EZ-Fluo™ system into your daily testing routine. Our highly experienced service teams can assist you with the following activities:

• Feasibility study and on-site evaluation

• Method development and method development consulting

• Equipment installation

• Operator training

• On-site system qualification (IQ/OQ)

• Yearly preventive maintenance

• Service plan

For further information on our support and services offering, please contact your local sales representative.

Specifications

Reader Camera

Dimensions (nominal) Length 24.9 cm (9.8 in.) 9.7 cm (3.8 in.)

Width 14.2 cm (5.6 in.) 6.6 cm (2.6 in.)

Height 12.5 cm (4.9 in.) 11.4 cm (4.5 in.)

Weight (nominal) 4.4 kg (9.7 lbs.) 0.5 kg (1.1 lb.)

Supply Voltage Input 100 V – 240 V 50 Hz – 60 Hz

Powered by the computer

Output 24 V direct current

Power Consumption Nominal 670 mA 200 mA

Maximal 2080 mA 430 mA

Power* Nominal 16 watts 1 watt

Maximal 50 watts 2.15 watts

Environmental Operating Temperature

15°C to 40°C (59°F to 104°F)

Humidity < 90%

Altitude < 3,000 m (9,842 ft.)

Regulatory Information We certify that the EZ-Fluo™ Reader and the EZ-Fluo™ Camera were designed and manufactured in application of the following European Council directives: • Electromagnetic compatibility 2004/108/EC • Restriction of the use of certain Hazardous Substances

in electrical equipment (RoHS) 2002/95/EC

Materials of Construction

Housing Painted aluminum sheet 1050

Optical chamber 304L stainless steel

Handle 304L stainless steel

Optical filter Glass

LED protector Transparent polycarbonate

Foot Polyvinylchloride (PVC)

Labels Polyester

Screen Protector polyester

Front plate 304L stainless steel

Housing Painted aluminum alloy

Window Glass

Light guard Clear polymethyl methacrylate (PMMA)

*The reader is supplied with its power cord according to region.


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Ordering Information

Description Qty./Pk Cat. No.

EZ-Fluo™ System Kits

EZ-Fluo™ System standard kit, including 1 reader, 1 camera and installation CD 1 EZFKIT001WW*

EZ-Fluo™ Hardware

EZ-Fluo™ reader 1 EZFREAD01WW*

EZ-Fluo™ camera 1 EZFCAM001

Consumables

EZ-Fluo™ Reagent Kit 57 tests EZFREAG57

EZ-Pak® Filters 0.45 µm 47 mm black gridded 600 MSP000814

EZ-Pak® Filters 0.45 µm 47 mm white gridded 600 EZHAWG474

S-Pak® Filters 0.45 µm 47 mm black gridded 600 HABG047S6

S-Pak® Filters 0.45 µm 47 mm white gridded 600 HAWG047S6

Petri-Pad™ Petri dish, sterile, 47 mm 150 PD20047S0

Petri-Pad™ Petri dish, sterile, 47 mm 600 PD20047S5

EZ-Fluo™ Accessories

EZ-Pak® Membrane Dispenser 1 EZDISP001

EZ-Pak® Dispenser Curve 1 EZCURVE01

EZ-Pak® Dispenser Curve (AC only) 1 EZCURVEAC

EZ-Stream® Pump 1 EZSTREAM1

Silicone Hose for EZ-Stream® Pump Autoclavable Internal diameter: 9.5 mm (3/8 in.) Length: 5.0 m (196.8 in.)

1 STREAMTUB

PVC Hose for EZ-Stream® Pump Not autoclavable Internal diameter: 9.5 mm (3/8 in.) Length: 3.0 m (118.1 in.)

1 XX6700034

Filter forceps 3 XX6200006P

Traceability labels Label Printing Software: available at EMDMillipore.com

1000 TRLABEL01

EZ-Fit® Filtration Units blue base Qty./Pk Cat. No.

EZ-Fit® Filtration Unit, black membrane, 0.45 µm pore size, 100 mL funnel, bulk packaging 48 EFHAW100B

EZ-Fit® Filtration Unit, black membrane, 0.45 µm pore size, 100 mL funnel, individual packaging 48 EFHAW100I

EZ-Fit® Filtration Unit, black membrane, 0.45 µm pore size, 250 mL funnel, bulk packaging 48 EFHAW250B

EZ-Fit® Filtration Unit, black membrane, 0.45 µm pore size, 250 mL funnel, individual packaging 48 EFHAW250I

EZ-Fit® Filtration Unit, black membrane, 0.45 µm pore size, 100 mL funnel, bulk packaging 48 EFHAB100B

EZ-Fit® Filtration Unit, black membrane, 0.45 µm pore size, 100 mL funnel, individual packaging 48 EFHAB100I

EZ-Fit® Filtration Unit, black membrane, 0.45 µm pore size, 100 mL funnel, bulk packaging 48 EFHAB250B

EZ-Fit® Filtration Unit, black membrane, 0.45 µm pore size, 250 mL funnel, individual packaging 48 EFHAB250I

EZ-Fit® Filtration Units pink base Qty./Pk Cat. No.

EZ-Fit® Filtration Unit, white membrane, 0.45 µm pore size, 100 mL funnel, individual packaging, no pad 48 EFHVW10IS

EZ-Fit® Filtration Unit, white membrane, 0.45 µm pore size, 100 mL funnel, multi packaging, no pad 48 EFHAW10MS

EZ-Fit® Filtration Unit, white membrane, 0.45 µm pore size, 250 mL funnel, bulk packaging, no pad 48 EFHAW25BS

EZ-Fit® Filtration Unit, black membrane, 0.45 µm pore size, 100 mL funnel, multi packaging, no pad 48 EFHAB10MS

EZ-Fit® Filtration Unit, black membrane, 0.45 µm pore size, 250 mL funnel, bulk packaging, no pad 48 EFHAB25BS

EZ-Fit® Filtration Unit, white membrane, 0.22 µm pore size, 100 mL funnel, multi packaging, no pad 48 EFGSW10MS

EZ-Fit® Filtration Unit, white membrane, 0.22 µm pore size, 100 mL funnel, individual packaging, no pad 48 EFGSW10IS

EZ-Fit® Filtration Unit, white membrane, 0.8 µm pore size, 100 mL funnel, bulk packaging, no pad 48 EFAAW10BS

EZ-Fit® Filtration Unit, white membrane, 0.8 µm pore size, 250 mL funnel, bulk packaging, no pad 48 EFAAW25BS

EZ-Fit® Filtration Unit, black membrane, 0.8 µm pore size, 100 mL funnel, bulk packaging, no pad 48 EFAAB10BS

EZ-Fit® Filtration Unit, black membrane, 0.8 µm pore size, 250 mL funnel, bulk packaging, no pad 48 EFAAB25BS

*Country code to be defined at ordering step.

The blue EZ-Fit® Filtration Units come with pad.

The pink EZ-Fit® Filtration Units come without pad.


© 2018 Merck KGaA, Darmstadt, Germany and/or its affiliates. All Rights Reserved. Merck, the vibrant M, EZ-Fluo, EZ-Pak, EZ-Stream, Microfil, Millipore and S-Pak are trademarks of Merck KGaA, Darmstadt, Germany or its affiliates. All other trademarks are the property of their respective owners. Detailed information on trademarks is available via publicly accessible resources.

To place an order or receive technical assistanceIn Europe, please call Customer Service:France: 0825 045 645 Germany: 069 86798021 Italy: 848 845 645

Spain: 901 516 645 Option 1 Switzerland: 0848 645 645 United Kingdom: 0870 900 4645

For other countries across Europe, please call: +44 (0) 115 943 0840 Or visit: MerckMillipore.com/offices For Technical Service visit: MerckMillipore.com/techservice

MerckMillipore.com

Merck KGaA Frankfurter Strasse 250 64293 Darmstadt, Germany

Lit. No. W286961/DS5588EN00 Ver. 2.02017 - 07532

03/2018

The EZ-Fluo™ rapid detection system and accessories

We provide information and advice to our customers on application technologies and regulatory matters to the best of our knowledge and ability, but without obligation or liability. Existing laws and regulations are to be observed in all cases by our customers. This also applies in respect to any rights of third parties. Our information and advice do not relieve our customers of their own responsibility for checking the suitability of our products for the envisaged purpose.

MerckMillipore.com/EZ-Fluo


EZ-Fluo™ SystemFor rapid detection of spoilage organisms in wine

Many beverage manufacturing processes are susceptible to spoilage organisms like yeast or bacteria contamination. Contamination can alter the odor, flavor or turbidity of a beverage, resulting in customer dissatisfaction and, in some cases, in product recall. For these microorganisms, traditional monitoring methods require up to 10 days to obtain microbiological results allowing the release of the product. A rapid microbiology system that can detect potential contamination 3 times faster than traditional monitoring methods would result in a significant cost saving and preserved company reputation. The EZ-Fluo™ System uses fluorescence-based technology and is a convenient and a sensitive platform for the quantitative detection of contaminants in filterable samples. This rapid microbiological method is based on a universal enzymatic fluorescent staining of viable and culturable microorganisms. The fluorescent staining procedure is non-destructive, allowing microorganism identification following a positive result.

The EZ-Fluo™ system offers a fast and reliable alternative for the rapid detection of spoilage microorganisms in wines. This evaluation study, performed by the accreditated lab Centro de Investigación y Asistencia Técnica a la Industria (CIATI AC) in Argentina, shows that the system enables a faster response and corrective action when used during the wine manufacturing process. It improves process control, product yield and the faster release of final product to market.

Materials:EZ-Fluo™ system Reader (EZFKIT001WW) Membrane Filtration systems (EZFTIMIC01)

Equipment:EZ-Fluo™ system Reagent Kits (EZFREAG57)

Media:MRS agar + tomato juice Brettanomyces agar Carr agar YEPD agar

Matrices tested:Red Wine

Microorganisms:Lactic Acid Bacteria / Oenococcus oeni strain Brettanomyces spp. Strain Acetic acid bacteria (AAB) Yeast Counts / Saccharomyces cerevisiae strain

Application Note

The life science business of Merck operates as MilliporeSigma in the U.S. and Canada.

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Principle of detectionThe principle of the fluorescence detection is based on an enzymatic reaction. The fluorogenic substrate used is a non–fluorescent viability marker which is cleaved by non-specific ubiquitous intracellular enzymes resulting in a fluorescent product. Natural amplification of fluorescence by accumulation inside cells is an indicator of microbial metabolism. The dye is diluted in a staining buffer allowing cell membrane permeability and thus dye introduction into cells.

Note: Fluorescence detection is a non-destructive method that enables the microorganisms to continue to grow after they have been stained in order to identify them using standard ID technology.

Protocol for rapid detectionThe procedure used was a standard protocol to detect spoilage microorganisms in samples of interest with the fluorescence detection:

• A filtration unit is installed onto the filtration system

• The appropriate volume of sample is poured into the filtration unit

• After filtration, the membrane is disconnected from the device and aseptically transferred onto a media cassette

• The incubation is performed according to the specifications

• After the incubation, the membrane is stained with the fluorogenic reagent for 30 min at 32.5°C (± 2.5)

• The fluorescent micro-colonies are counted using the fluorescence reader

• After detection, the stained membrane can be re-incubated on fresh media for traditional plate count and identification if required

Definition of a rapid incubation timeAn appropriate incubation time is defined as the minimal time which allows a percentage of recovery above 70% compared to the traditional method. The calculation is based on both formulas:

• The fluorescence recovery is the fluorescent dot count compared to the traditional method count. Fluorescence recovery (%) = (average of fluorescence counts / average of traditional method count) x 100

• The viability recovery is the colony count on stained membranes after re-incubation compared to the traditional method count. Viability recovery (%) = (average of Colony-Forming Units counts after re-incubation / average of traditional method counts) x 100

An optimal incubation time should allow a sufficient fluorescent signal intensity and fluorescence and viability recoveries above 70%.

Figure 2:

The picture on the right illustrates a sufficient fluorescent signal intensity translating to an appropriate incubation time. The picture on the left shows that an accurate count is not possible if the intensity of fluorescence is too low due to an insufficient incubation time.

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CIATI AC Evaluation ResultsA series of tests on red wine were carried out to determine the performance of the rapid count system compared to traditional microbiological analysis, as established by the OIV and wine industry. Results in the tables are average counts of the performed tests.

b) Naturally contaminated samples

Results

SamplesTraditional microbiological test

(CFU/10 mL) (10 days)EZ-Fluo™ System

(CFU/10 mL) (5 days)Visible colonies at counting time with EZ-Fluo™ System (5 days)

1 49 45 0

2 570 601 0

3 1 2 0

4 2 1 0

5 55 37 0

Results

Sample detailsDays of incubation

(25 °C)Traditional microbiological test

(CFU/10 mL)EZ-Fluo™ System

(CFU/10 mL)

Sample 1, estimated N0 = 3 CFU/10 mL

3 <1 <1

5 <1 3

7 2 2

10 3 4


3 <1 6

5 <1 28

7 13 33

10 29 30

Sample 3, estimated N0 = 30 x 102 CFU/10 mL

3 <1 52

5 <1 295

7 252 283

10 304 307

Five (n = 5) samples of red wine naturally contaminated with Brettanomyces spp were analyzed.

Counts were performed using both the traditional method (filtration through a 0.45 μm membrane, and

incubation on Brettanomyces agar at 25°C (±2) for 10 days) and the EZ-Fluo™ rapid system (with incubation at 25°C (±2) for 5 days).

1) Brettanomyces spp. counts

Counts of the test yeast were performed using both the traditional method (filtration through a 0.45 μm membrane, and incubation on Brettanomyces agar at 25°C (± 2) for 10 days under aerobic conditions) and the EZ-Fluo™ rapid system. The results were read at several times during incubation.

a) Inoculated samples

Three (n = 3) 500 mL samples of red wine were inoculated with different concentrations (N0) of a Brettanomyces spp. strain previously isolated and characterized in CIATI AC microbiology laboratory.

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2) Lactic acid bacteria (LAB) counts

Results




1 144 152 9

2 3 2 0

3 623 489 21

4 105 97 6

5 18 22 0

Results




(CFU/10 mL)


3 <1 2

5 1 3

7 2 2

10 4 5


3 <1 14

5 3 21

7 23 30

10 36 31


3 <1 42

5 17 310

7 254 303

10 311 320

Counts were performed using both the traditional method (filtration through a 0.45 μm membrane, and incubation on MRS agar + tomato juice at 30°C (± 2) for 10 days under aerobic conditions) and the EZ-Fluo™ rapid system. The results were read at several times during incubation.


Three (n = 3) 500 mL samples of red wine were inoculated with different concentrations (N0) of an Oenococcus oeni strain previously isolated and characterized at the CIATI AC.


Five (n = 5) samples of red wine naturally contaminated with lactic acid bacteria were analyzed.


incubation on MRS agar + tomato juice at 30°C (± 2) for 10 days) and the EZ-Fluo™ rapid system (with incubation at 25°C (± 2) for 5 days).

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3) Acetic acid bacteria (AAB) counts


Results




1 6 9 0

2 70 101 9

3 26 18 3

4 12 16 1

5 137 145 11

Results




(CFU/10 mL)


1 <1 <1

2 <1 3

3 2 4

4 5 2


1 <1 <1

2 <1 17

3 11 25

4 27 22


1 <1 <1

2 <1 252

3 199 277

4 246 280

a) Inoculated samples:

Three (n = 3) 500 mL samples of red wine were inoculated with different concentrations (N0) of an Acetobacter spp. strain previously isolated and characterized at the CIATI AC.

Counts were performed using both the traditional method (filtration through a 0.45 μm membrane, and incubation on Carr agar at 25°C (± 2) for 4 days under aerobic conditions) and the EZ-Fluo™ rapid system. The results were read at several times during incubation.

Five (n = 5) samples of red wine naturally contaminated with acetic acid bacteria were analyzed.


incubation on Carr agar at 25°C (± 2) for 4 days) and the EZ-Fluo™ rapid system (with incubation at 25°C (± 2) for 2 days).

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Results




1 28 24 7

2 1.97 x 103 2.33 x 103 46

3 115 175 15

4 42 36 9

5 5 3 0

Results




(CFU/10 mL)


1 <1 <1

2 <1 2

3 2 3

4 3 2


1 <1 <1

2 <1 32

3 18 35

4 33 29


1 <1 <1

2 <1 289

3 125 307

4 296 301

4) Yeast counts

Counts were performed using both the traditional method (filtration through a 0.45 μm membrane, and incubation on YEPD agar at 25°C (± 2) for 4 days under aerobic conditions) and the EZ-Fluo™ rapid system. The results were read at several times during incubation.


Three (n = 3) 500 mL samples of red wine were inoculated with different concentrations (N0) of a Saccharomyces cerevisiae strain previously isolated and characterized at the CIATI AC.


Five (n = 5) samples of red wine naturally contaminated with yeasts were analyzed.


incubation on YEPD agar at 25°C (± 2) for 4 days) and the EZ-Fluo™ rapid system (with incubation at 25°C (± 2) for 2 days).

Main outcomes of the CIATI AC evaluation study• According to the results obtained, the EZ-Fluo™

rapid system performed very well in obtaining counts of wine spoilage microorganisms, since it reduced the incubation time by at least 50% in every case analyzed, in comparison to that required for traditional methods (OIV).

• The EZ-Fluo™ rapid system enabled the detection and quantification of Brettanomyces spp. and lactic acid bacteria (LAB) in as little as 5 days of incubation, and of acetic acid bacteria (AAB) and yeasts in as little as 2 days of incubation.

• The EZ-Fluo™ system is a useful tool for the rapid (and non-destructive) detection and quantification of wine spoilage microorganisms.

We provide information and advice to our customers on application technologies and regulatory matters to the best of our knowledge and ability, but without obligation or liability. Existing laws and regulations are to be observed in all cases by our customers. This also applies in respect to any rights of third parties. Our information and advice do not relieve our customers of their own responsibility for checking the suitability of our products for the envisaged purpose.

Author:

Juan Martin Oteiza Laboratorio de Microbiología de Alimentos CIATI AC-CONICET Neuquén, Argentina [email protected] www.ciati.com.ar

Copyright © 2016 Merck KGaA. All Rights Reserved. Merck and Millipore are registered trademarks and the vibrant M and EZ-Fluo are trademarks of Merck KGaA.

Lit. No. AN1259EN00 Ver. 0.1 BM-16-13523

11/2016

Find contact information for your country atwww.merckmillipore.com/offices

For Technical Service, please visitwww.merckmillipore.com/techservice

www.merckmillipore.com/biomonitoring

ConclusionUsing our fluorescence-based technology as a microbiology quality control tool reduces the time needed to detect yeast and bacterial contaminations in wine dramatically. This study demonstrated that this technology could easily replace the compendial microbiological method with a 2 to 4 times faster time to result, and a full compatibility with the standard culture media traditionally used for the detection of spoilage organisms in beverages.

Moreover, as the method is non-destructive, each fluorescent micro-colony detected will continue to grow to yield visible colonies allowing the identification of the contaminants using available identification methods.

With the EZ-Fluo™ system, beverage manufacturers can improve their quality control by detecting contaminations earlier, and implementing corrective actions faster. This early answer creates savings of raw materials and manufacturing capacities. It can also help in the root cause analysis of a process failure, giving a better knowledge and confidence in the process and an increased quality control. Most importantly the product release can be accelerated and storage time decreased, resulting in a financial saving for the manufacturer.

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