How to use the E. coli testkit

Monitoring purpose

Monitoring bacteria helps communities answer one of the most important questions people ask about their water: can I swim and play in the water here?

E. coli is an indicator bacterium. It is found in the gut of warm-blooded animals, and it occurs naturally in many fresh and marine waters, so finding some is normal. Finding a lot of it suggests a waterbody has been contaminated with sewage, stormwater, or run-off from livestock or wildlife like waterfowl (did you know that Canada geese are a big E. coli polluter?).

Water Rangers has developed a comprehensive sampling kit and protocol to guide volunteer testers in obtaining consistently reliable results for E. coli using R-cards from Roth Biosciences. This test is a coarse method. It is good for spotting patterns at your site over a season, and for telling low from medium from high. It is not a substitute for certified lab testing, and it cannot be used for official bathing water monitoring or to issue an advisory.

Before you use your equipment, make sure to familiarize yourself with the Water Rangers protocol. This will tell you how to pick the best spots to monitor, and will also tell you how often you should go testing.

Designing your sampling program

Selecting your site

Your site depends on your monitoring purpose. For E. coli, common places are:

• Where folks will be swimming or paddling
• Before and after a suspected contamination site (e.g. outflow). Do not sample directly from the outflow but in the receiving waterbody. Read more at waterrangers.com/purpose

How often to test

This depends on your monitoring purpose! Many choose to sample weekly at the same time. Others may choose to measure daily, or around event schedules. Because E. coli levels are related to weather, some choose to measure after storms. Still others may use it as part of monthly baseline monitoring. Learn more about setting a schedule.

Get to know your equipment

All of our E. coli testkits contain:

• 5x packs of R-Cards. Each pack includes 3 R-cards and a sterile syringe. Choose between 1mL or 3 mL varieties.
  • Choose the 1mL R-Cards for higher concentrations of E.coli. 
  • Choose 3 mL R-cards for lower concentrations of E.coli. 
• 10x pairs of protective nitrile gloves.
• A sampling cup or WhirlPak bags to collect a sterile sample.
• A ‘reacher’ stick to assist in collecting the sample.
• A ‘personal’ incubator to be used at home.
• A cooler bag to keep samples cool during transportation.
• Conductivity meter (tests for conductivity and water temperature): This measures the water’s ability to conduct electricity! You can read more about what this means and why it’s important here.
• A sampling clipboard to allow the cards to be laid out and kept level – important whilst the sample is being applied to the R-Cards.
• A notepad to record qualitative observations and E. coli results.
• A small towel.

About R-Cards™

R-Cards are a simple card with a special gel on them that grows when exposed to E. coli colonies. Groups in Canada, USA, and the UK are using this method for their community monitoring program.

We offer both a 1 mL and a 3 mL testkits and resupplies. The main difference is sensitivity: the 3 mL card uses a larger sample, so it’s better at picking up E. coli when concentrations are low. The 1 mL card works well at sites where E. coli levels are higher, and is less likely to produce an uncountable result when contamination is significant.

If you’re not sure where to start, we recommend the 1 mL card as it’s easier to use. If you consistently get very low counts (0 to 2 colonies), that’s a good sign your site may benefit from the 3 mL card instead. On the other hand, if your 3 mL card has more than 5 colonies, your site likely has high E. coli levels and the 1 mL format is the right fit. Neither card is more “correct” than the other. The goal is to match the card to your site so you get results you can actually count and use.

• They should be kept dry and out of highly humid environments. If they become wet, they will
  become unusable.
• For best performance, cards should be kept frozen until required for sampling.
• It is preferable that the cards not be subjected to high intensity light or heat.
  With these things in mind, it is best to keep R-CARDS in the sealed bags in which they were delivered. That does not mean that they cannot be exposed to normal conditions for days or weeks where temperatures do not exceed 40 degrees Celsius or where humidity is normally less than 100%.

Learn how to use your kit

Before initiating testing, ensure that you have prepared your kit, identified your testing location, and established the timing for both testing and incubation.

Quick start guide

1. Sanitation is critical for this test. Follow the sanitation checklist.
2. Before you leave, prepare your testkit. If you are processing in the field, bring the R-cards with you in your cooler bag.
3. Record your qualitative observations (weather, water colour etc.) and capture photos. Qualitative data is critical to provide context for your results.
4. Collect your sterile sample. Wearing gloves, use your sterilized vessel (Whirl-Pak bag, sanitized sample cup) to collect the water sample.
5. Record conductivity, water temperature and other test parameters.
6. Transportation. Keep the water sample in your cooler bag with an ice pack until you get home <12 deg C. You must process your sample within 3 hours of collecting it!
7. Process the R-cards. Dispense the water sample onto the card using the sterile syringe (1 mL or 3 mL). Let the plastic film fall gently and spread the water. Do NOT press on the card. Let it sit for 1-2 minutes to gel. Repeat for all three cards.
8. Incubate the cards for 24 hours at 35 °C. 15 cards can be stacked in the incubator.
9. Count colonies and convert results. Remove the cards, count the colonies (dots) on each card, multiply the counts by a conversion factor (1 mL cards x100 ; 3 mL cards x33) and report results in CFU/100mL.
10. Tidy up!

Measuring E. coli with Water Rangers

All of these instructions can be found within the field guide that comes with your kit, too! We definitely recommend reading it from cover to cover.

Planning

Sanitation checklist

• Keep your R-CARDs in the freezer when unused to prevent any contamination or growth.
• Sanitize your incubator and the inside of your testkit between uses.
• Wear clean gloves (avoid grabbing the fingertips of the gloves).
• If you’re not using a Whirl-pak bag, sanitize your collection vessel (cup, etc). Put both the cup and lid in boiling water for one minute. Do not use chemical disinfectant, as this can kill the E.coli. Place the cup and lid in a Ziploc bag to keep it sterile.
• Use sterile syringes to dispense water onto the R-CARDs (avoid touching the tip of the syringe) and use a new syringe for each site and sampling date (the same syringe can be used for all three replicates).
• Avoid touching the inside of the sterile sample collection vessel.
• Put an ice pack in your insulated cooler bag.
• Replace any item that becomes contaminated.

We strongly recommend collecting the water sample in the field and processing it at home. If you choose to process in the field, keep the R-CARDS cool while you’re out (under 12°C but not on ice) and make sure you incubate within 4 hours.

You can turn on your incubator before you leave, or wait until you are back home. Warm your incubator to 35°C. This takes about 20 minutes.

Sampling

Option A: Whirl-Pak bag

• Label the Whirl-Pak with date, time and sample location.
• If you’re not going into the water, attach the Whirl-Pak bag to the reacher stick or bucket by slotting two tabs into the white attachment.
• Tear off the perforated top. Grip the paper pull tabs on both sides and pull them apart. Once the bag is open, do not touch the inside!
• Take a sample at least 15cm below the surface. Hold the bag in the water and let it fill. Leave a few inches of space at the top of the Whirl-Pak so there’s room to seal it. If you don’t leave enough room, your sample may leak!
• To seal the Whirl-Pak, hold it by the flat wire ends at the top. Spin the bag forward three full rotations to close it tight (hold away from your body so no drops go on your face).
• Bring the two wire ends together, twist them, and bend them against the bag.
• Turn the sealed Whirl-Pak upside down for a moment to check for leaks. We recommend you stand up the Whirl-Pak in your sample cup in your cooler bag!

Option B: Sterilized sample cup

• Rinse your sample cup 3 times in the water you will be sampling.
• Take a sample 15cm below the surface (without disturbing the sediment).
• Secure the lid and place the sample cup into the cooler.

Processing

• Warm your incubator to 35°C. This takes about 20 minutes.
• Sanitize your workspace with a disinfectant and set up a flat working surface.
• Put on fresh gloves.
• Take an R-card pack out of the freezer. Each pack has three R-cards per site.
• Label the card with site ID/Name, date, time and a number 1 through 3. (e.g. Lorraine Bay 1/3, Lorraine Bay 2/3, Lorraine Bay 3/3)
• Place the first R-card on a flat, level surface. You can use the clipboard provided. If the cards are not level, the water can drip or roll off.
• Gently shake your water sample. This is an important step if you are processing at home, as the E. coli may settle.
• Unwrap the syringe, handling only the plunger and barrel. Avoid touching the tip. Note for 1 mL cards, use a 1 mL syringe and 3 mL syringe for the 3 mL cards.
• Put the syringe’s tip into your water sample.
• Pull up the plunger until there’s 1 mL (or 3 mL) of water in the syringe.
• Tap the syringe to remove any bubbles, then adjust the plunger or add more water to reach the exact volume needed.
• Peel open the corner of the R-CARD’s plastic cover.
• Slowly dispense the sample onto the center of the card.*
  • * If you’re using 3 mL cards, we recommend you add 1 mL at a time, letting the plastic cover fall and the sample spread and then adding more until a full 3mL of water is dispensed.
  • You do not need to cover the whole grid. Keep the sample flat to avoid spilling.
• Let the plastic cover gently fall down and spread out the sample.
• Wait 1–2 minutes for the liquid to gel.
• Repeat for all 3 cards. Use the same water sample and syringe for all three.
• Place the cards into the incubator.

Incubating

As part of the R-Card test kit, we offer a ‘home incubator’ which is a small USB-C powered unit originally designed for home incubation of small eggs. This allows a controlled and consistent incubation temperature.

• Set the incubator temperature to 35 deg C (it defaults to 37.8 deg), and allow it to warm up to that temperature before putting in the R-Cards. It takes approximately 20 minutes for the incubator to warm up
• Confirm the incubator is maintaining 35°C using a secondary thermometer. Record the incubation start time on your data sheet.
• Place all cards in the incubator, printed side up. Up to 15 cards can be stacked.
• Incubate for 24 hours.

Analyzing

• After 24 hours but before 36 hours, remove the R-cards from the incubator.
• Turn off your incubator.
• Note the incubation end time on your field sheet.
• Count the blue dots on each card. Each dot is an E. coli colony.
• For each 1mL card, multiply results by 100. On 3mL cards, multiply results by 33.
• Record the calculated value for each card on the data platform. You can also average your results for your own records.
• Take photos of each card to verify later.

The standard for E. coli concentration is to report it as CFU per 100mL, where CFU is colony-forming units. Each dot is one colony. Because you only used 1mL or 3mL of water per card, we must multiply the # of colonies by 100 or 33 to get to the 100mL concentration. Record the converted results on your datasheet.

We then take an average of the three cards to get a more accurate estimate of the E. coli concentration. Note: If you upload your results to the Water Rangers data platform, it will average the results for you.

Interpreting your results

A single E. coli sample is a snapshot, and levels can change day to day, so regular testing is recommended. Most regions follow similar thresholds to judge whether there’s a risk of illness during swimming and recreation.

NOTE: R-CARDs are a coarse method for assessing potential E. coli risk; it is not intended to inform regulatory actions (e.g. beach closures).

Canada 

In Canada, the federal government guidelines, Guidelines for Canadian Recreational Water Quality, for E.coli are the following:

E.coli Guidelines for Primary-Contact Recreation∗ 

• Geometric mean concentration (minimum 5 samples) ≤ 200 E. coli /100 mL 
• Single sample maximum concentration ≤ 400 E. coli /100 mL

Countries, states, and provinces have their own standards and protocols, but most are very similar when it comes to quantifying E.coli at the beach.

England

The thresholds for E. coli in English bathing waters:

• Excellent: E. coli threshold is ≤500 Colony Forming Units per 100mL (CFU/100 mL)
• Good: E. coli threshold is ≤1000 CFU/100 mL
• Sufficient: E. coli threshold is ≤900 CFU/100 mL
• Poor: E. coli values are worse than the sufficient threshold 

Scotland

In Scotland the values are:

• Excellent: E. coli threshold is ≤250 CFU/100 mL
• Good: E. coli threshold is ≤500 CFU/100 mL

Clean-up

• Used R-CARDS should be placed back into the resealable bag, then disposed of in your regular garbage. We like to give a quick spray of cleaning solution in each bag. Put unused R-Cards back in the freezer.
• Your water samples can be poured down your sink drain.
• Cut the top off the WhirlPak bag. The bottom portion can be recycled, while the top is garbage. Used syringes and gloves may be recyclable. Check local rules!
• Wipe down all surfaces, your testing equipment, bag, and incubator. Put your ice pack back in the freezer and wash your hands thoroughly.
• Store all items in a dry temperature-controlled space.

Replenishing your kit

Feedback? We want to hear from you.

We’d love to hear about your experience and get feedback on your experience with the E. coli testkit. Your responses will help us improve the kit and our training process. All responses are confidential and will be used solely for program improvement.

Click here to provide feedback on the E.coli testkit