Hygiena INS-MSCEC-REVA MicroSnap Coliform and E. coli Instructions

Hygiena INS-MSCEC-REVA MicroSnap Coliform and E. coli

Product Information

Specifications

• Product Name: MicroSnap Coliform and E. coli
• Manufacturer: Hygiena
• Enrichment Options Available: Enhanced Nutrient Broth (9 mL vial), Enhanced EB Broth (9 mL vial)
• Intended Use: Enumeration of coliform and E. coli from environmental surfaces, product samples, water, and other filterable liquids

Product Usage Instructions

Step 1: Enrichment

1. Collect and prepare the sample:
   • Surface Samples: Use the pre-moistened Enrichment Device to sample a 10 x 10 cm (4 x 4 inch) square area.

Swabbing technique tips:

• * i. Apply sufficient pressure to create flex in the swab shaft.

  • ii. Swab in a crisscross pattern vertically, horizontally, and diagonally in both directions.
  • iii. Rotate the swab while collecting the sample to maximize sample collection on the swab tip.
  • iv. For irregular surfaces, ensure the swabbing technique remains consistent for each test and swab a large enough area to collect a representative sample.
• Liquid Samples: Transfer 1 mL of a liquid or water sample directly to the Enrichment Device.

Frequently Asked Questions (FAQ)

• Q: What are the required materials for using MicroSnap Coliform and E. coli?
  A: The required materials include wells for swabs and vials for INCUBATOR2 and INCUBATOR. Refer to the product manual for specific product numbers.

• Q: Who is qualified to use MicroSnap Coliform and E. coli devices?
  A: Laboratory personnel trained in standard microbiological practices are qualified to use the product.

• Q: What type of samples can be tested using MicroSnap Coliform and E. coli?
  A: The product is applicable for the enumeration of coliform and E. coli from environmental surfaces, product samples, water, and other filterable liquids

Introduction

Description and Intended Use
MicroSnap® Coliform and E. coli are rapid bioluminogenic tests for the detection and enumeration of coliforms and Escherichia coli in products and environmental samples in 6 or 8 hours. MicroSnap Coliform and E. coli consist of an Enrichment Device containing a specific growth medium and a Detection Device containing a bioluminogenic substrate in which the detection reaction is measured using a hand-held luminometer from Hygiena®.

The two-step test procedure requires a short incubation period facilitating recovery of bacteria followed by a detection step. The incubation time for a sample is determined by the level of sensitivity required. During incubation, the bacteria use available food resources in the media and produce more beta- galactosidase and beta-glucuronidase, which are the diagnostic enzymes required for the bioluminogenic reaction.

After incubation, a small amount of enriched solution from the Enrichment Device is transferred to the Detection Device, activated, incubated for 10 minutes, and then measured in an EnSURE® Touch luminometer. Specific substrates react with diagnostic enzymes to produce light. The greater the number of bacteria in the sample, the higher the biomarker concentration and the greater the output of light. Light is measured in a few seconds. The unit of measurement is colony forming units (CFUs). Light output is directly proportional to the initial starting concentration of bacterial contamination in pre-enriched samples.

Some matrices, such as opaque liquid suspensions or samples with extreme pH values, may require dilution. In these cases, we offer 9 mL vials containing proprietary broths for use in place of the Step-1 enrichment device. Use of Enhanced Broths in 9 mL vials is not included under the AOAC Research Institute (RI) Performance Tested Methods℠ (PTM℠) certification.
The Enhanced EB Broth (9 mL vial) is more selective for coliforms and E. coli in the presence of lactic acid bacteria (LAB) or Enterobacteriaceae, while the Enhanced Nutrient Broth (9 mL vial) provides better recovery of aerobic bacteria, coliforms and E. coli. Guidance for selecting a MicroSnap broth is summarized in Table 1.

Table 1. MicroSnap Enhanced Broth Selection Guide.

Intended User
Laboratory personnel trained in standard microbiological practices are qualified to use MicroSnap Coliform and E. coli devices.

Applicability
MicroSnap Coliform and E. coli is applicable for the enumeration of coliform and E. coli from environmental surfaces, product samples, water and other filterable liquids. The method was validated through AOAC RI PTM certification for a variety of foods, including meat, dairy, seafood, vegetables and beverage food groups. For details, refer to AOAC RI PTM Certificate 071302 at www.hygiena.com/documents.

Required Materials (Not Provided)

• EnSURE Touch luminometer (Product No. ETOUCH)
• Dry Block Incubator (at 37 ± 0.5 °C) (Product No. INCUBATOR or INCUBATOR2)
• Block options for incubators:
  • 35 wells for swabs for INCUBATOR2 (Product No. IB001)
  • 15 wells for 9 mL vials for INCUBATOR2 (Product No. IB002)
  • 12 wells for swabs for INCUBATOR (Product No. IB003)
  • 6 wells for 9 mL vials for INCUBATOR (Product No. IB004)

Required Materials When Testing Product Samples (Not Provided)

• Sample bags

• Homogenizing equipment

• Pipettor and tips for 1 mL

• Product sample diluent options:

  • Buffered peptone water

  • Maximum recovery diluent
    Note: Maximum recovery diluent was used for the AOAC RI PTM validation study.

  • Butterfield’s diluent

  • Sterile water

Important Tips Before Starting the Test

• For samples that may require dilution (e.g., opaque solutions; samples that may contain sanitizers, surfactants or other inhibitory compounds), use the MicroSnap Enhanced EB Broth or Nutrient Broth for enrichment (for details, see Appendix and diagrams).
• Product samples can be stored before use at 2 to 8 °C for up to 2 days but must be equilibrated to room temperature (20 to 25 °C) before testing samples with MicroSnap Coliform and E. coli.
• Enrichment devices or vials (i.e., MicroSnap Coliform and E. coli Enrichment Devices, MicroSnap Enhanced EB Broth and MicroSnap Enhanced Nutrient Broth) and detection devices (i.e., MicroSnap Coliform Detection Devices and MicroSnap E. coli Detection Devices) must be equilibrated to 20 to 25 °C before use.
• Use aseptic techniques: when collecting samples or transferring enriched samples, do not touch the swab or the inside of the enrichment device or vial with your fingers.

Test Procedure

Step 1: Enrichment
The enrichment procedure is described below and is also shown in Step 1 diagrams.

1. Collect and prepare the sample, according to sample type as noted:

   • a. Surface Samples—Use the pre-moistened Enrichment Device to sample a 10 x 10 cm (4 x 4 inch) square area.
     Important swabbing technique tips:
   1. Apply sufficient pressure to create flex in the swab shaft.
   2. Swab in a crisscross pattern vertically, horizontally and diagonally in both directions.
   3. Rotate the swab while collecting the sample to maximize sample collection on the swab tip.
   4. For irregular surfaces, ensure the swabbing technique remains consistent for each test and swab a large enough area to collect a representative sample.
   • b. Liquid Samples—Transfer 1 mL of a liquid or water sample directly to the Enrichment Device.
   • c. Solid Product Samples— Transfer 1 mL of an appropriate suspension, e.g., 10% w/v food homogenate, directly to the Enrichment Device.
   1. Food homogenate should be prepared by weighing 10 or 50 g of food matrix and adding it to a stomacher bag containing 90 mL or 450 mL of diluent, respectively.
   2. For unknown sample contamination, prepare and test 1:10 serial dilutions (i.e., 10%, 1% and 0.1%).
   3. If replicate samples are required, then another 10 g or 50 g should be removed from the bulk matrix, and the dilution series should be repeated. Replication can be achieved by drawing multiple 1 mL aliquots from either the 10%, 1% or 0.1% dilutions, depending on the CFUs achieved.
      Note: When performing comparison testing, sample assays must be started within 10 minutes
      of each other for comparable results between methods.
   • d. Re-attach the swab to the swab tube. The device should look the same as it did when first removed from the bag.

2. Activate the Enrichment Device by holding the swab tube firmly and using your thumb and forefinger to break the Snap-Valve by bending the bulb forward and backward.

3. Separate the bulb and swab tube until the swab tip is above the fluid and squeeze the bulb to flush all the media into the swab tube. Ensure most of the broth is at the bottom of the swab tube.

4. Re-attach the swab to the swab tube firmly to seal the device and shake the tube gently to mix the sample and broth.

5. Incubate at 37 ± 0.5 °C for 6 hours ± 10 minutes for quantitative results (enumeration) or 8 – 24 hours for qualitative results (absence/presence).
   Note: In the qualitative AOAC RI PTM validation studies, samples were incubated for 8 hours ± 10 minutes.

Step 2: Detection
The detection procedure is described below and is also shown in (MicroSnap Enrichment Device or MicroSnap Enhanced Broth Vials).
Before beginning Step 2, turn on the luminometer. If you have programmed your MicroSnap sample in the luminometer, open the test screen of the sample you want to test.
Remember to equilibrate the MicroSnap Coliform or E. coli Detection Device (MS2-COLIFORM or MS2-ECOLI) to room temperature (10 minutes at 20 to 25 °C) before use.

1. Shake the test device by either tapping on the palm of your hand 5 times or forcefully flicking in a downward motion once.
   Note: This is necessary to bring the liquid to the bottom of the tube, which will facilitate the mixing of the enriched sample with the extractant in the tube.

2. Aseptically transfer 0.1 mL (2 drops) of enriched sample to the Detection Device.

   • a. For MicroSnap Enrichment Devices, use the built-in dropper tip as a pipette:
   1. Squeeze and release the Enrichment Device bulb to mix and draw the sample into the bulb.
   2. Aseptically open the Enrichment Device and the Detection Device by twisting and pulling to remove the bulbs.
   3. Insert the Enrichment Device swab tip 3 cm (1 inch) into the top of the Detection Device tube and gently squeeze the Enrichment Device bulb to transfer 2 drops of the enriched sample into the tube.
      Note: A fill line is added to the tube as a reference. Inconsistent transfer volumes increase the variation of the test results.
   • b. For MicroSnap broth vials:
   1. Remove the Enhanced Broth vial from the incubator then shake or vortex for 10 seconds to disperse the sample.
   2. Aseptically uncap the vial and open the Detection Device by twisting and pulling to remove the bulb.
   3. Aseptically pipette 0.1 mL of the enriched sample directly into the Detection Device tube.
   • c. Reassemble the Enrichment Device to its original state or recap the vial and return the sample to the incubator for potential retesting.
     Note: When testing replicates from the same enriched sample, all replicates must be performed within 10 minutes of each other to obtain comparable results.

3. Activate the Detection Device by holding the tube firmly and using your thumb and forefinger to break the Snap-Valve by bending the bulb forward and backward. Squeeze the bulb 3 times to release all the liquid to the bottom of the tube.

4. Shake gently for 2 seconds to mix.

5. Incubate the Detection Device for 10 ± 0.5 minutes at 37 ± 0.5 °C.

6. Immediately insert the whole device into the luminometer, close the lid and while holding the unit upright, press the button to initiate the measurement.

7. EnSURE Touch luminometers display results in CFUs in 10 seconds.
   Note: MicroSnap samples can be programmed directly on the luminometer or by using SureTrend® software.

Additional Information

Further Tests
If a positive result is found using the MicroSnap Coliform Detection Device (MS2-COLIFORM), then confirm the presence or absence of E. coli in the sample by repeating Step 2: Detection instructions above using another aliquot from the same enriched sample and the MicroSnap E. coli Detection Device (MS2-ECOLI). If performing E. coli tests only, an additional confirmatory test should be considered, such as PCR on the Hygiena BAX® System.

Potential Limit of Detection
The limit of detection is the lowest level of viable aerobic bacteria that can be detected above a food matrix background when the assay is performed correctly and efficiently.
Table 2. Potential Dynamic Range (Limit of Detection) for the EnSURE Touch Luminometer.

Sample Type| *CFU Range (Enrichment: 6 h ± 10 min)| CFU Presence or Absence (Enrichment: 8 ± 10 min) †**
---|---|---
Surface| 10 – 10,000 CFU/swab| 0  (absence)

1  CFU (caution)

≥2 CFU (presence)

Liquid (1 mL)| 10 – 10,000 CFU/mL
Suspension of solid (10% w/v)| 100 – 10,000 CFU/g

• Additional factors, such as dilutions, incubation times and matrix types, can alter the ranges shown in this table. If sample contamination is above the ranges detailed in this table, then dilutions should be made so that the contamination is within the detectable range of the luminometer. For example:
• 1% suspension will be 1,000 – 100,000 CFU for a 6-hour incubation.
• 0.1% suspension will be 10,000 – 1,000,000 CFU for a 6-hour incubation.
• † Incubation for presence/absence results can be extended up to 24 hours. Note: The enrichment time was 8 hours ± 10 minutes for the qualitative AOAC RI PTM validation studies.

Interpretation of Results
Results on EnSURE Touch luminometers are shown in CFUs, providing qualitative (presence/absence) as well as quantitative (CFU/g or CFU/mL) results. Where several dilutions are prepared and tested for samples with unknown contamination, the CFU/g or CFU/mL is calculated by multiplying the CFU result by the corresponding dilution factor. The EnSURE Touch software does this conversion, using data generated from the AOAC RI PTM validation studies as well as additional internal testing.

AOAC RI Performance Tested Methods Certification
The detection of coliform and E. coli using the MicroSnap Coliform and E. coli System (i.e., MicroSnap Coliform and E. coli Enrichment Device with either the MicroSnap Coliform Detection Device or the MicroSnap E. coli Detection Device) and Hygiena luminometers has earned AOAC RI PTM Certification (License #071302) from the AOAC Research Institute.
Foods tested under AOAC RI PTM validation are listed in Table 3.

Table 3. Validated Matrices with MicroSnap Coliform and E. coli on Hygiena Luminometers.

Quantitative (Enrichment: 6 hours ± 10 minutes)| Qualitative (Enrichment: 8 hours ± 10 minutes)
---|---
Coliform| E. coli| Coliform| E. coli
Raw ground beef| Raw ground beef| Raw ground beef| Raw ground beef
BLT sandwich| BLT sandwich| ––| BLT sandwich
Raw cod| Raw cod| Raw cod| Raw cod
Cooked chicken| Cooked chicken| Cooked chicken| Cooked chicken
Lettuce| Lettuce| Lettuce| ––
Milk| Milk| Milk| Milk
Raw chicken| Raw chicken| Raw chicken| Raw chicken
RTE ham| RTE ham| ––| ––
Raw prawns| Raw prawns| Raw prawns| Raw prawns
Bottled water| Bottled water| Bottled water| Bottled water

Additional Hygiena Validations
In addition to the matrices included in AOAC RI PTM certification studies (Table 3), Hygiena laboratories continue to test other matrices, such as chocolate milk and pasteurized milk. Optimal testing for some matrices involved use of the Enhanced EB Broth or Enhanced Nutrient Broth vials (9 mL), which were not included under the AOAC RI PTM certification. For qualitative testing, some users have validated extended incubation times (up to 24 hours), which is not included under AOAC RI PTM certification.
Contact technical support at www.hygiena.com/support for information and assistance with additional matrices.

Limitations
Samples must be brought to room temperature (20 to 25 °C) before use in MicroSnap devices. Samples that are not brought to ambient temperature before incubation (e.g., taken directly from refrigeration at 2 to 8 °C) will under- detect due to the time lag in reaching the incubation temperature.

All media or diluents used with MicroSnap Coliform and E. coli must be sterile. Inhibitors in media and diluents are the prime reason for most unsuccessful detections. Hygiena recommends the diluents listed in the Required Materials section. The enzymatic profiles of some coliforms, such as strains of Hafnia alvei, are different from most other coliforms, so these strains will not be detected and enumerated by biochemical and chromogenic detection methods such as those used in MicroSnap Coliform testing. Organisms with similar enzymatic profiles, such as Shigella sonnei, cannot be differentiated from E. coli by biochemical or chromogenic detection methods such as those used in MicroSnap E. coli testing. In addition, the enzymatic profiles for some Shiga toxin-producing E. coli (STEC), such as E. coli O157:H7, means that they will be detected and enumerated by MicroSnap Coliform testing, but not MicroSnap E. coli testing.
For additional details, see Caution and User Responsibility or contact us.

Troubleshooting
Table 4 guides how to overcome some commonly seen sample effects. For additional protocol or matrix support, contact us at www.hygiena.com/support.

Table 4. Troubleshooting.

Uncharacteristically high CFUs with some matrices, such as leafy greens and fermented dairy products.| Some sample types naturally contain high levels of nucleotides that can increase CFU results.| Contact us for assistance with customizing the RLU-to-CFU conversion and the instrument threshold levels for your sample matrix.
Uncharacteristically low CFUs with thick, opaque or dark sample matrices, such as undiluted milk or chocolate.| Interference with light detection by the luminometer can be caused by a blanching effect from the sample matrix.| Use one of the MicroSnap Enhanced Broths in 9 mL vials for enrichment. See Appendix for details.

Calibration and Controls
It is advisable to run positive and negative controls according to Good Laboratory Practice. Hygiena offers the following calibration verification device: CalCheck LED Calibration Verification Device (Product No. CAL).

Storage and Shelf Life

• Store at 2 to 8°C (36 to 46 °F).
• Do not use past the expiration date on the label.

Disposal
Disinfect before disposal. MicroSnap devices can be disinfected by autoclaving or bleaching (soak unsealed devices in 20% bleach for 1 hour). Then, they can be placed in the trash. Alternatively, MicroSnap devices may be discarded at a biohazard waste disposal facility.

Safety and Precautions

• MicroSnap device components do not pose any health risk when used correctly. Used devices confirming positive results may be a biohazard and should be disposed of safely in compliance with Good Laboratory Practice and Health and Safety Regulations (see disposal instructions above).
• Avoid prolonged exposure to light.
• Devices are designed for a single use. Do not reuse.

Caution and User Responsibility

• MicroSnap devices have not been tested with all possible food products, food processes, testing protocols or with all possible strains of the Coliform family.

• For in vitro use only. Not for use in diagnostic procedures.

• As with any culture medium-based test, MicroSnap results do not constitute a guarantee of product quality.

• No single culture medium will recover the same strain or enumerate a particular strain in the same way as another medium. Other external factors such as sampling method, testing protocol and handling may influence recovery.

• Sampling should be done aseptically to avoid cross-contamination.

• It is the user’s responsibility when selecting a test method to evaluate a sufficient number of samples.

• Verify proper incubation temperature and time for the test application.

• The incubation time will be 6 hours ± 10 minutes for quantitative results (enumeration) or 8 – 24 hours for qualitative results (presence/absence) as specified in the above instructions unless you have been directed otherwise by Hygiena’s R&D team for custom applications that require different incubation times (or temperatures).
  Note: In the qualitative AOAC RI PTM validation studies, samples were incubated for 8 hours ± 10 minutes.

• Ensure proper sample dilution so that samples can be read within the luminometer’s dynamic range.

• When testing multiple serial dilutions, all dilutions must be prepared and tested within 10 minutes of each other to obtain linear results.

• When testing replicates from the same enriched sample, all replicates must be performed within 10 minutes of each other to obtain comparable results.

• When performing comparison testing, sample assays must be started within 10 minutes of each other for comparable results between methods.

Hygiena Liability
As with any culture medium-based test, MicroSnap Coliform and E. coli results do not constitute a guarantee of the quality of food, beverage products, or processes that are tested with these devices. Hygiena will not be liable to the user or others for any loss or damage, whether direct or indirect, incidental or consequential, from the use of these devices. If this product is proven to be defective, Hygiena’s sole obligation will be to replace the product, or at its discretion, refund the purchase price. Promptly notify Hygiena within 5 days of discovery of any suspected defect and return the product to Hygiena; contact Customer Service for a Returned Goods Authorization Number.

Contact Information
For more information, visit www.hygiena.com/contact. For technical support, visit www.hygiena.com/support.

Appendix

Enrichment of Challenging Matrices with MicroSnap Enhanced EB Broth or MicroSnap Enhanced Nutrient Broth
MicroSnap Enhanced Broths contains 9 mL of a unique liquid medium designed to grow aerobic and facultative microorganisms while enhancing the production of biomarkers and specific enzymes diagnostic of coliforms and E. coli and reducing sample interferences. The broths are intended for applications requiring the detection of bacteria in challenging food samples, such as opaque liquid suspensions (see Table 1).
MicroSnap Enhanced Nutrient Broth is a ready-to-use media compatible with MicroSnap Total (MS2-TOTAL), MicroSnap Coliform (MS2-COLIFORM) and MicroSnap E. coli (MS2-ECOLI) Detection Devices. MicroSnap Enhanced EB Broth is a ready- to-use media compatible with MicroSnap EB (MS2-EB), MicroSnap Coliform (MS2-COLIFORM) and MicroSnap E. coli (MS2-ECOLI) Detection Devices. Instructions in this insert are for enriching milk, opaque solutions and other challenging food samples for coliform and E. coli testing. For help developing a protocol for your matrix, including adjusting enrichment incubation temperatures, contact Hygiena for guidance.

Important Tips Before Starting the Test

• The use of the Enhanced Broths in 9 mL vials is not included under AOAC RI PTM certification.
• Visually inspect the liquid in the vial before use. Liquid should be clear and a light straw color, not turbid or cloudy.
• Use a permanent marker to identify the sample on the vial label.

Step 1: Enrichment with MicroSnap Enhanced Broth
The enrichment procedure is described below and is also shown in Step 1 diagrams.

1. Collect and prepare the sample using aseptic techniques:
   • a. Liquid Samples—Add 1 mL of sample directly to the vial of Enhanced Broth.
   • b. Solid Samples—Transfer 1 mL of a suitable sample dilution in sterile diluent directly to the vial of Enhanced Broth.
2. Replace and tighten the cap.
3. Shake or vortex for 10 seconds to mix contents.
4. Incubate the vial in a Hygiena Digital Dry Block Incubator for 6 or 8 hours, depending on your sample type and the sensitivity required (Table 5).
   Table 5. Incubation Time, Temperature and Potential Dynamic Range. **Incubation Time*| CFU Range| Enhanced Nutrient Broth| Enhanced EB Broth
   ---|---|---|---
   Milk| Liquid or Solid Food| Milk, Liquid or Solid Food
   32 ± 0.5 °C| 30 ± 0.5 °C| 37 ± 0.5 °C**
   6 h ± 10 min| 50 – Log 6| Enumeration| Enumeration| Enumeration
   8 h ± 10 min†| <5 – 5,000| Presence/absence| Presence/absence| Presence/absence

Step 2: Detection
Follow instructions for detection as described above.

MicroSnap® Enrichment and Detection Devices for Coliform and E. coli

Step 1: Sample Enrichment

Step 2: Detection or Measurement

MicroSnap® Enhanced Broth Vial and MicroSnap Detection Device
Note: Use of the Enhanced Nutrient Broth is not included under AOAC RI PTM certification.

Step 1: Sample Enrichment

Step 2: Detection or Measurement

Documents / Resources

| Hygiena INS-MSCEC-REVA MicroSnap Coliform and E. coli [pdf] Instructions
INS-MSCEC-REVA MicroSnap Coliform and E. coli, INS-MSCEC-REVA, MicroSnap Coliform and E. coli, Coliform and E. coli, E. coli
---|---

References

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