BACTERIAL AQUATIC KIT FOR PERISTALTIC PUMPS

For use in all countries

• All kit users must familiarize themselves with data requirements set out in the Sample Manifest, prior to sampling.
  
• Please ensure all information sent to NatureMetrics is in English.
  
• Datasheets must be completed using a pencil only.
• Kits should be used within one year.

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KIT BACKGROUND

THIS KIT CAN BE USED WITH A RANGE OF PERISTALTIC PUMPS.

This protocol is based around the Vampire sampler, which can be hired from NatureMetrics. The Vampire sampler is a transportable, peristaltic hose pump for liquids, that works independent of mains power and can sample directly from the water, sample container, or sampling device. The pump head creates a vacuum which draws the water sample through the tube and pushes the water through the eDNA filter attached to the end of the tube. Using a pump, instead of a manually operated syringe, reduces the labor and time required to filter each sample, which is particularly important when sample volumes are high.

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YOU WILL NEED

• Device to capture GPS coordinates (lat / long) (e.g. GPS capable smartphone or standalone GPS device)
• Pencil (to complete the datasheet)
• Measuring jug / beaker (for measuring water filtered)
• Sterile ladles (for collecting subsamples)

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More than one person using the kit? Please purchase extra gloves.
To minimize waste we provide one pair of gloves per kit. If two or more people will be handling the contents of the kit and collecting an eDNA sample, you will need to purchase additional pairs of gloves – enough for one pair of gloves for each individual handling the kit. We recommend purchasing medical-grade nitrile gloves.

Sampling from a boat, or in waterbodies with inaccessible shorelines?
Sterile ladles to assist with sample collection can be purchased from NatureMetrics. If you need to acquire some, please contact us.

TIPS FOR GETTING THE BEST RESULTS

Avoiding Inconclusive Results:

To get the best results possible, to avoid inconclusive results, and to avoid situations where data cannot be reported from a sample, please read our HOW TO AVOID INCONCLUSIVE RESULTS guide before undertaking fieldwork.

In summary, when inconclusive results occur, it is because the target DNA was not detected, DNA was degraded, or PCR (a crucial part of the lab process) was inhibited.

Key steps you can take to help avoid inconclusive results: do not leave your samples exposed to sunlight or heat, and return samples to NatureMetrics as soon as possible after sampling.

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Avoiding contamination:

To increase confidence in results, avoiding contamination of samples is highly important. Sealed kits are sterile until opened; do not open more than one kit bag at a time, keep kit contents inside the bag before use, and put on the provided gloves before touching other kit components. Whilst wearing gloves, avoid touching anything not necessary to sample collection to minimize introducing external DNA into the sample. Change gloves between each kit. If any reusable tools are used, these should be decontaminated, using a disinfectant wipe or a bleach solution, between each sample.

USER PROTOCOL

PART 1: COLLECTING THE WATER

Ideal sampling strategies will vary among habitats and monitoring contexts. We recommend that you speak to a member of our team to plan your survey. If planning your own survey, it is important to follow the guidance outlined in the Advice Note, as suitable sampling strategies differ for different waterbodies.

We also recommend the inclusion of field negative controls (blanks), as part of your sampling strategy, to detect possible contamination introduced during sampling. These are purified water (i.e. mineral, deionised, or MilliQ water) processed in the same way as water samples (see Advice Note for details). NatureMetrics provide a free kit to be used as a field blank – one for every 20 kits purchased.

Once a suitable sampling strategy has been decided and planned:

1. Open the kit bag. Kits should be used one at a time to minimize contamination between samples. Do not open more than one kit at a time, especially if kits are to be used at different sampling locations. Put on the gloves provided, and minimize contact with the water, to avoid introducing DNA (from yourself, soil, or other waterbodies) into the water and prevent spread of disease or invasive species.

2. Collect subsamples from multiple locations around the waterbody/area you are sampling, using either the sampling bag provided to scoop up a small amount of water, or using a sterile ladle or empty mineral water bottle to transfer water into the bag.

For each sample, taking subsamples is an important part of the water collection process and typically increases detection rates. Ladles are a useful tool where the shoreline is difficult to access, and also allow equal amounts of water to be collected from each subsampling point. Sterile ladles can be purchased from NatureMetrics (contact us). In the absence of ladles, new mineral water bottles, with the water emptied, can be used in the same way.

3. Once a suitable amount of water has been collected (we recommend 1L for freshwater environments and 1-5L for marine environments), roll down the top of the sampling bag and shake for 20-30 seconds to thoroughly mix the subsamples.

4. Rest the bag in a sturdy place, either in a container to prevent the bag from tipping over, or with support from another person.

PART 2: SETTING UP THE PUMP

If operating the pump alone, it is recommended to set up the hose with the drive unit detached. If working in a team, one person can hold the drive unit, while another mounts the hose.

1. To remove the drive unit, turn the locking ring towards the unlock sign and remove the pump attachment, setting the drive unit safely aside.

2. Open the pump head by pulling up the white part at the top. Insert the silicone hose into the pump head as shown in image below, making sure there is at least 5 cm of hose between where it leaves the pump head and the hose adapter.

3. Close the pump head by pressing down the white part so that the silicone hose is securely fixed in place.

4. If it has been removed, re-mount the pump head to the drive unit and turn the locking ring towards the lock sign until it clicks.

5. Check that the drive unit is set to pump water towards the filter. As you hold the drive unit with the pump head facing away from you, water will be pumped from left to right when the orange button is pushed in on the right-hand side (forward arrow). When the orange button on the left-hand side is pushed in (backwards arrow), water will be pumped from right to left.

6. Set the drive unit to gear 1 (orange slider on top of unit).

PART 3: FILTERING THE WATER AND PRESERVING THE DNA

1. Depending on the sample collection method you have used, hang the end of the hose directly in the sampling bag or container, or attach the end of the hose to the outlet of the sampling device (contact NatureMetrics for advice on different sampling devices). If there is a high sediment load, then leave the sample for a minute to allow the sediment to settle before drawing water up into the tubing. This should increase the amount of water that you can filter.

2. Run the pump slowly by pressing the drive unit trigger slowly to fill the hose with water.

3. Once the hose is filled, attach the filter inlet (narrow end) to the hose adapter. They should twist together easily.

4. Run the pump slowly to begin with, while holding the hose securely just behind the adapter. You can increase the speed once water is flowing freely.

5. When the flow of water leaving the filter outlet (wide end) slows, decrease pump speed to reduce the build-up of pressure. If too much pressure builds up, it will make removing the filter harder or eject the adapter and filter from the hose.

6. Pass as much water through the filter as possible (see Advice Note for recommended volumes in different environments). Once all water has passed through the filter, or the filter is fully clogged, remove the hose from the sampling bag and run the pump until the hose is cleared of any water. Continue to run the pump until no more water exits from the filter to expel as much water as possible. It helps to gently shake the filter as you do this.

7. Detach the filter from the hose by unscrewing it slowly. Record the volume of water filtered on the sampling datasheet. The volume of water filtered can be measured by collecting the water that passes through the filter in a measuring jug/beaker.

8. The preservative solution may precipitate (i.e. become cloudy) when kits are stored or used in cold temperatures. While precipitation will not adversely affect the preserving action, it is recommended to warm the small syringe in gloved hands for 1-2 minutes and then to gently shake to ensure proper mixing. Uncap the small syringe (already filled with preservative solution) and twist it onto the filter inlet. Do not discard the Luer Lock cap – hold the cap in one hand as it will be needed in step 9. The solution is non-hazardous to aquatic life but release into the environment should be avoided. We advise performing this step away from the water’s edge and avoiding spills.

9. Hold the filter so that outlet points upwards. We advise you to do this step away from anyone’s eyes. Carefully and slowly press the plunger to push the preservative solution into the filter. This must be done slowly to allow the preservative solution to spread out over the filter surface. Stop when the first drop can be seen emerging from the filter outlet, but do not remove the small syringe with preservative solution. Cap the filter outlet using the Luer Lock cap that was on the small syringe in step 8.

10. Invert the filter so that the filter outlet points down, and slowly press the plunger to expel the rest of the preservative solution. The entire volume of preservative solution should be added to the filter and the small syringe should be empty. Detach the small syringe whilst keeping the plunger depressed and cap the filter inlet with the separate Luer Lock cap. The preservative solution may foam or overspill a little – don’t be alarmed.

11. Place the filter into the specimen bag and seal. If storing samples at ambient temperatures, samples should be received by the lab within 2 weeks of sampling. If this is not possible, we recommend that samples are frozen as soon as possible after sampling to enhance DNA preservation until samples are sent back to us. Cold shipping is not necessary for frozen samples. It should be noted that although freezing is recommended as best practice for optimal storage, we understand that it is not always feasible. Storing at ambient temperatures is a tried and tested method that yields excellent data. The addition of freezing is to further safeguard the samples against potential DNA degradation, which can affect species detection when samples are stored at high temperatures for long periods. Please contact NatureMetrics if return within 2 weeks or long-term freezing is not possible.

12. You should use a new bag/ladle/bottle and new silicone hose with a new pair of gloves for each sample to avoid cross-contamination. After you have processed each filter, use the disinfectant wipe provided in the kit to decontaminate the vampire sampler by wiping the exterior of the drive unit as well as the interior and exterior of the pump head. Wear gloves when handling the disinfectant wipe.

PART 4: RETURNING YOUR SAMPLES

Once you have collected and filtered your sample, follow our guidelines below to safely transport your sample to the NatureMetrics laboratory.

1. Complete the sampling datasheet using a pencil (do not use pen as ink can easily run, to the point where it becomes unreadable).

2. Place the bagged filter and sampling datasheet inside the bag that the kit contents arrived in.

3. Complete the sample manifest, which was attached to the email confirming shipping of your kits. Please record your sampling data on paper and on the sample manifest so there is a physical and electronic copy of your data.

4. For sample returns, visit our customer support desk here. Use the Logistics Request form to upload your completed sample manifest and book your sample return. NatureMetrics is fully compliant with current legislation on the transport of biological material and our operations team are on hand to ensure that all return shipments meet the required specification. We are only able to analyze samples that are returned using the correct NatureMetrics logistics procedure.

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PACKAGING NOTES AND QUICK LINKS

• Return your samples using the box that kits were sent in, where possible.
• If using an alternative box, ensure that it is in good condition and remove any old labels.
• Avoid excess spacing by adding cushioning material (e.g. bubble wrap, newspaper) to avoid the product shifting inside the box.
• Use good quality sealing tape and seal along box edges in a H pattern.
• The only essential thing to send back to us is the specimen bag containing the filter and the sampling datasheet. However, all kit contents can be sent back to NatureMetrics for recycling.

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CLIENT RESPONSIBILITIES

Important considerations for your sampling

• The absence of a detection does not necessarily imply the absence of a taxon from a location.
• Samples from some locations might have compounds that inhibit analyses resulting in reduced data generation – while we routinely test for inhibition, it is not always possible to overcome.
• It is the responsibility of the client to ensure that all efforts have been made to avoid contamination from external sources and between samples.
• Handling samples without gloves can increase the content of human DNA, reducing data generation.
• After sampling, movement of samples across borders without permission is not allowed as they are classed as biological samples.

Important considerations for interpreting your results

It should be noted that DNA can enter an ecosystem via many routes (e.g. wastewater from commercial and domestic sources). While DNA from a given taxon may be present and detectable, it is not possible to discern the source of the DNA.

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Disclaimer: Safe sample collection is the responsibility of the Client. NatureMetrics accepts no liability associated with the use of the kits and sample collection. The Client is solely responsible for the quality of the samples and the representativeness of the samples received by NatureMetrics. The information contained within the Final Report provided by NatureMetrics to the Client is not intended to be advisory, it is informational. Interpretation and decisions are the sole responsibility of the Client. NatureMetrics does not accept any liability whatsoever for any reliance placed on any information contained within, or any use that may be made of, the Final Report by the Client. Please read the full limitation of liability statement in the Terms and Conditions.

Health & Safety information for this kit can be viewed here.

See the following advice note for sampling tips on surveying ponds, lakes, rivers, streams, estuaries, seas, and oceans, as well as information on control samples and avoiding contamination.

View Advice Note

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