hygiena IB001 Micro Snap Total Instruction Manual

**hygiena IB001 Micro Snap Total Instruction Manual

**

Introduction

Description and Intended Use
MicroSnap® Total is a rapid bioluminogenic test for the detection and enumeration of the total population of viable aerobic bacteria in a sample and provides results in 7 hours. MicroSnap Total consists of an incubation device containing proprietary growth media and a detection device containing bioluminogenic  reagents in which biomarkers produced by bacteria are measured using a hand-held Hygiena® luminometer.

The two-step test procedure requires a short incubation period facilitating the growth of bacteria followed by a detection step. During the incubation, bacterial numbers increase, and sample interference is  reduced. As bacteria grow, they use the available food resources in the media and generate biomarkers. The greater the number of bacteria in the sample, the higher the biomarker concentration and the greater the output of light. An aliquot of enriched sample is transferred to the Detection Device, activated, mixed and measured in a luminometer. Light output is directly proportional to the initial starting concentration of bacterial contamination in the pre-enriched sample.

Some matrices, such as opaque liquid suspensions, may require dilution (see Important Tips section). In these cases, we offer 9 mL vials containing a proprietary Enhanced Nutrient Broth for use instead of the incubation device. Use of Enhanced Nutrient Broth in 9 mL vials is not included under the AOAC Research Institute (RI) Performance Tested Methods℠ (PTM℠) certification.

Intended User

Laboratory personnel trained in standard microbiological practices are qualified to use MicroSnap Total.

Applicability

MicroSnap Total is applicable for the enumeration of metabolically viable aerobic bacteria fromenvironmental surfaces, product samples and liquids. The method (MicroSnap Enrichment Device with MicroSnap Detection Device) was validated through the AOAC RI PTM Program for a wide range of foods, including major food groups such as meat, dairy and vegetables (Table 2). For details, refer to AOAC RI PTM Certificate 031501 at www.hygiena.com/documents.

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

It is important that all media or diluents used with MicroSnap Total are sterile. Inhibitors in media and diluents are the prime reason for most unsuccessful detections. Hygiena recommends the diluents listed in the Required Materials section.
See Caution and User Responsibility for additional details.

Product Instructions

Additional Required Materials from Hygiena (Not Provided)

• EnSURE® Touch luminometer (Product No. ETOUCH)

• Dry Block Incubator (at 30 ± 0.5 °C or 32 ± 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 and 0.1 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

  • Optional: Vortex (when using Enhanced Nutrient Broth vials)

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 Nutrient Broth for enrichment (for details, see Appendix and diagrams).
  
  Note: Use of the Enhanced Nutrient Broth is not included under AOAC RI PTM
  certification.

• Product samples can be stored prior to 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 Total.

• MicroSnap Total Enrichment Devices, MicroSnap Nutrient Broth Vials and MicroSnap Total Detection Devices must be equilibrated to room temperature (20 to 25 °C) before use.

• Use aseptic techniques: when collecting samples or transferring enriched samples, wear gloves and do not touch the swab or the inside of the Enrichment Device or Vial with your fingers.

Test Procedure

Step 1: Incubation with the MicroSnap Total Enrichment Device
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 4 x 4 inch (10 x 10 cm) square area.
   Important swabbing technique tips:

2. For irregular surfaces, ensure the swabbing technique remains consistent for each test and swab a large enough area to collect a representative sample.

3. Swab in a crisscross pattern vertically, horizontally and diagonally in both directions.

4. Rotate the swab while collecting the sample to maximize sample collection on the swab tip.

5. Apply sufficient pressure to create flex in the swab shaft.
   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.

6. 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.

7. For unknown sample contamination, prepare and test 1:10 serial dilutions (i.e., 10%, 1% and 0.1%).

8.  If replicate samples are required, then another 10 g or 50 g should be removed from the bulk matrix and the dilution series repeated. Replication can be achieved by drawing multiple 1 mL aliquots from either the 10%, 1% or 0.1% dilutions depending on relative light units (RLUs)
   achieved.
   Note: When performing comparison testing, sample assays must be started within 10 minutes of each other for comparable results between methods.

9. Re-attach the swab back into the swab tube. The device should look the same as it did when first removed from the bag.

10. 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.

11. 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.

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

13. Incubate at 30 ± 0.5 °C for 7 hours ± 10 minutes.

Step 2: Detection

The detection procedure is described below and is also shown in diagrams on pages 9 and 10 (MicroSnap  Enrichment Device or MicroSnap Enhanced Nutrient Broth Vial, respectively).
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 Total Detection Device 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.
   This is necessary to bring the liquid to the bottom of the tube, which will facilitate 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:

3. Squeeze and release the Enrichment Device bulb to mix and draw the sample into the bulb.

4. Aseptically open the Enrichment Device and the Detection Device by twisting and pulling to remove the bulbs.

5. Insert the Enrichment Device swab tip 3 cm (1 inch) into the top of the Detection Device tube and lightly 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:

6. Remove the Enhanced Nutrient Broth Vial from the incubator then shake or vortex for 10 seconds to disperse the sample.

7. Aseptically uncap the vial and open the Detection Device by twisting and pulling to remove the bulbs.

8.  Aseptically pipette 0.1 mL of the enriched sample directly into the Detection Device tube.

9. 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.

10. Shake gently for 2 seconds to mix.

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

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

Additional Information

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. The sensitivity increases as the incubation time increases. At 7 hours, the detection level approaches 10 to 100 CFU per mL of incubation media, and the dynamic range of MicroSnap Total in the EnSURE Touch luminometer is proportional to the actual range of RLU feasible in the EnSURE Touch instrument (Table 1).

Table 1. Potential Dynamic Range at a 7-hour Incubation with the EnSURE Touch Luminometer.

• Additional factors, such as dilutions, incubation times and matrix types can alter the ranges shown in Table 1.
  † For samples where contamination is above the ranges detailed in Table 1, the following
  serial dilutions must be made before reading results on the luminometer:

  • 1% suspension will give a range of 1,000 – 500,000 CFU
  • 0.1% suspension will give a range of 10,000 – 5,000,000 CFU

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

AOAC RI Performance Tested Methods℠ Certification
The detection of aerobic heterotrophic bacteria using the MicroSnap Total System (i.e., MicroSnap Enrichment Device and the MicroSnap Detection Device) with the EnSURE Touch luminometer has earned AOAC RI PTM Certification (License #031501) from the AOAC Research Institute.

Food matrices (Table 2) were tested in their natural state; no spiking of bacteria wasperformed, and all samples rendered some form of countable range. Hence, the use of true negatives is difficult to perform with real food samples due to bacteria always being present even at low levels. The use of a lower limit of detection of 100 CFU per gram is acceptable when using MicroSnap Total at 7-hour incubations.

*Table 2. Validated Matrices with MicroSnap Enrichment and Detection Devices.**

Quantitative (50 g with 7 hour ± 10 Minute Enrichment Time)

Fresh ground beef (<20% fat)
Raw chicken
Raw cow’s milk
Fresh cream cake (17% fat)
Pre-packaged iceberg leaves

• MicroSnap Enrichment Device (1 mL enrichment volume) Add tional Hygiena Validations

In addition to the matrices included in AOAC certification studies (Table 2), Hygiena laboratories continue to test other matrices. Optimal testing for some matrices involved use of the Enhanced Nutrient Broth vials (9 mL), which were not included under the AOAC PTM certification.

Contact technical support at www.hygiena.com/support for information and assistance with additional matrices.

Interpretation of Results
Results on EnSURE Touch luminometers are shown in CFUs, providing quantitative results as CFU/g or CFU/mL.

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 Validation Studies as well as additional internal testing.

Troubleshooting
Table 3 provides guidance on how to overcome some commonly seen sample effects. For additional protocol or matrix support, contact us at www.hygiena.com/support.
Table 3. Troubleshooting

sample types naturally contain high levels of nucleotides that can increase CFU results.| Contact us for assistance with customizing theRLU-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 the MicroSnap Enhanced Nutrient Broth 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.

Dis osal
Disinfect before disposal. MicroSnap devices can be disinfected by autoclaving or by soakingunsealed 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 risks when used correctly. Used devices confirming positive results may be a biohazard and should be disposed of safely in compliance with Good Laboratory Practices and Health and Safety Regulations (see disposal instructions above).

• Devices and vials are designed for single use. Do not reuse.

• Sampling should be done aseptically to avoid cross-contamination.
  Avoid prolonged exposure to light.

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

• The incubation time for quantitative (enumeration) results is 7 hours ± 10 minutes 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).

• 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. Caution and User Responsibility

• MicroSnap devices have not been tested with all possible food products, food processes, testing protocols or with all possible microorganism strains.

• Must not be used in diagnostic procedures.

• 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.

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

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

• Personnel must be trained in proper testing techniques and standard microbiological practices.

Hygiena Liability
As with any culture medium-based test, MicroSnap Total results do not constitute a guarantee  of 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 use of these devices. If this product is proven to be defective, Hygiena’s sole obligation will be to replace 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

Nutrient Broth

• MicroSnap Enhanced Nutrient Broth 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 broth is primarily intended for applications requiring the detection of bacteria in opaque liquid suspensions (e.g., coliforms in pasteurized milk). 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. Instructions in this insert are for enriching milk, opaque solutions and other challenging food samples. 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 Nutrient Broth in 9 mL vials is not included under AOAC RI PTM certification.
• Visually inspect the liquid in the vial before use. The liquid should be clear and 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 Nutrient Broth
The enrichment procedure is described below and is also shown in Step 1 diagrams on page 10.

1. Collect and prepare the sample using aseptic techniques:
   a. Liquid Samples—Add 1 mL of sample directly to the vial of Enhanced Nutrient Broth.
   b. Solid Samples—Transfer 1 mL of a suitable sample dilution in sterile diluent directly to the vial of Enhanced Nutrient 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 7 hours ± 10 minutes at the appropriate temperature for your sample type (Table 4).

Table 4. Temperature and Potential Dynamic Range for a 7-hour Incubation.

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

MicroSnap® Total Enrichment and Detection Devices

Step 1: Sample Enrichment

**Surface

* 1a Surface: Swab a 10 x 10 cm area with room temperature (RT) Enrichment Device.

**Liquids

** 1b Liquids: Add 1 mL of liquid food, beverage or water directly to RT Enrichment Device.

1. **Solid Samples

Solid Samples: Add 1 mL of appropriate dilution of solid sample directly to RT Enrichment Device.
**

2. Re-insert Snap-Valve bulb into swab tube.

3. Activate Device. Bend bulb, breaking Snap-Valve

4. Lift bulb up (3 – 5 cm) and squeeze to release liquid into bottom of tube.

5. Replace bulb into tube and shake tube gently to mix sample in liquid.

6. Incubate at 30 ± 0.5 °C for 7 hours ± 10 minutes. Proceed to Step 2.

Step 2: Detection or Measurement

1. Equilibrate Detection Device to room temperature. Shake to bring liquid to bottom.

2. Aseptically transfer  2 drops (0.1 mL) of sample from Enrichment Device to Detection Device.

3. Activate Detection Device (Test) by breaking Snap-Valve. Squeeze bulb to release liquid into tube.

4. Shake tube gently to mix sample in liquid.

5. Insert device into EnSURE® Touch. In MicroSnap® application: If sample is programed, select sample; otherwise, select Quick Test. Then, Run Test.
   

6. EnSURE Touch automatically saves results. Register and sync luminometer wirelessly to the SureTrend® software to see reports and datasets.
   

MicroSnap® Enhanced Nutrient 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

1. Equilibrate Detection Device to 20 to 25 °C. Shake to bring liquid to bottom.
   

2. Shake or vortex for 10 seconds.
   

3. Aseptically transfer 0.1 mL of enriched sample to the Detection Device.

4. Reassemble Detection Device to original state.

5. Activate device by breaking Snap-Valve. Squeeze bulb to release liquid into tube.
   

6. Shake tube gently to mix sample in liquid.

7. Insert device into EnSURE® Touch. In MicroSnap® application: If sample is programed, select sample; otherwise, select Quick Test. Then, press Run Test.

8. EnSURE Touch automatically saves results. Register and sync luminometer wirelessly to the SureTrend® software to see reports and datasets.

References

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