Recreational Water Facilities and Public Pools Spas

Conformément au Règlement de l’Ontario 671/92 de la Loi sur les services en français, les renseignements d’analyses de laboratoire liés à la présente page ne sont offerts qu’en anglais parce qu’ils sont de nature scientifique ou technique et destinés uniquement à l’usage des fournisseurs de soins de santé qualifiés et non aux membres du public.

Background
This page provides information on public recreational water facilities samples collected from public pools, spas, wading pools, splash pads/spray pads and water slide receiving basins and submitted to the Public Health Ontario (PHO) laboratory by a public health unit or another agency when authorized for testing.

The target organisms of the test are Total Coliforms, Escherichia coli (E. coli), P. aeruginosa, and S. aureus. A heterotrophic plate count (HPC) analysis is also performed and used to estimate the number of viable, heterotrophic bacteria in the water sample.

The following testing options are available for recreational water facilities: Membrane filtration for Total Coliforms, E. coli, P. aeruginosa, S. aureus, and HPC by spread plate.

For information regarding other water testing options, refer to the Test Information Index. For general inquiries related to water sample collection, submission and testing, please contact PHO’s laboratory Customer Service Centre.

Updates

Added Submitters Responsibilities section
Updated the following sections: Submission and Collection Notes (check bottle expiry date prior to collection), Storage and Transport (store samples in the dark and prevent direct contact with ice packs) and Testing Methods (added assay performance and limitations)

* Copie d'impression non contrôlée. Valide uniquement le jour de l'impression : 08 août 2025.

Testing Indications

Acceptance/Rejection Criteria

Refer to the Public Health Inspector’s Guide to Environmental Microbiology Laboratory Testing.¹

Submitter's Responsibility

Microbiological sampling may be conducted to evaluate and monitor cleaning and sanitizing procedures and to ensure that a health hazard does not exist. Refer to the Ministry of Health’s Recreational Water Protocol² and Operational Approaches for Recreational Water Guideline.³

By submitting the water sample for testing, the submitter accepts Public Health Ontario’s methodology, and represents and warrants that the water sample was taken from the Location of Water Source indicated in the test requisition and that the information provided is true in all material respects at the time of submission. The Ontario Agency for Health Protection and Promotion assumes no responsibility for the accuracy of the information provided, the manner in which the sample was collected or the mode by which it was transported to the laboratory.

For legal samples, the chain of custody/requisition must be complete and accurate. To maintain the chain of custody of the sample, the “Relinquished by” section must be completed AND a Legal seal with all the required information applied over the cap when the sample is received at the laboratory. Refer to Instructions For Official Agencies Submitting Water Samples to the Public Health Ontario Laboratory.⁴

Specimen Collection and Handling

Specimen Requirements

Test Requested	Required Requisition(s)	Specimen Type	Minimum Volume	Collection Kit
Total Coliform, E. coli, P. aeruginosa, S. aureus and HPC		Water collected from a public recreational water facility	200 mL (to the fill line on the bottle). If possible, send two bottles each filled to the fill line and taped together as one submission	Sterile Water bottles - 250 mL capacity (Case of 250 bottles) Item #: 300013

Submission and Collection Notes

Prior to sampling, check to make sure the sodium thiosulfate preservative in the collection bottle has not expired. The expiry date is located on the exterior label of the case of bottles.

Complete the following fields of the requisition:

“Official Agency Address” – include the sub office if this office submitted the sample
“Sample Information - Non-Potable” section
“Identification of Collection Site & Time Collected” section
Please note these sampling tips:
- In order to provide an accurate representation of the water in the pool, select appropriate locations for sampling (e.g., if equipped with a filter, collect from sampling ports provided in the filter’s return and discharge lines).
- For pools equipped with a filter, samples may be collected from sampling ports provided in the filter’s return and discharge lines.
“For Regulated Drinking Water or Legal Samples” section – as applicable (refer to Instructions For Official Agencies Submitting Water Samples to the Public Health Ontario Laboratory⁴

Note: The sample may not be tested if the required information is incomplete and/or inaccurate when the sample is received at the laboratory.

Remove one barcode from the bottle and apply it to the top copy of the requisition in the “Barcode” field. Remove a second barcode and apply it in the corresponding location on the second copy of the form. Retain a copy of the completed requisition that includes the barcodes.

Note: Samples may not be processed if the barcodes are not affixed to the requisition. If the barcode is not able to be removed, write the barcode number in the “Barcode” box on the form.

Examine the lid on the bottle. If the tamper evident ring has separated from the cap use another bottle.

Keep the bottle closed until just before collection. Remove the cap. Do not touch the bottle mouth and neck as well as inside of the lid or bottle with hands or other surfaces, do not put the lid down, and do not ingest the sodium thiosulfate in the bottle. For accidental exposure get medical attention.

Collect samples during periods of maximum bather load and note the number of bathers as this may be helpful when interpreting the laboratory results. Hold the bottle near the base and plunge the bottle, neck downward, at a 45 degree angle to 30 cm and collect water within one meter of the water surface. Fill the bottle at that depth with a slow sweeping forward motion with the mouth of the bottle always ahead of the hand. Bring the bottle up toward the water’s surface and make sure the bottle is kept away from the non-sampling hand, body parts and avoid floating debris.

Collect the sample to the 200mL fill line. If possible, fill another bottle to the fill line. Do not overfill the bottle(s). If overfilled, remove some of the water so the bottle is just filled to 200 mL line.

Tighten the cap on the collection bottle. Leaking samples will not be processed; a new sample and requisition will be required.

Refer to Instructions For Official Agencies Submitting Water Samples to the Public Health Ontario Laboratory⁴ for step by step instructions for completing the documentation and submitting/relinquishing the sample.

Timing of Specimen Collection

Ensure adequate time to collect and transport the sample to the laboratory. All recreational water facility samples must be shipped within 24 hours of collection and tested within one calendar day of collection.

Routine recreational water facility samples are accepted Monday to Thursday 8:30 a.m. to 4:30 p.m. excluding statutory holidays. If the statutory holiday falls on a weekday, samples will not be accepted the day before the holiday as well.

Please contact PHO’s laboratory Customer Service Centre prior to the submission of samples that will be received outside of regular operating hours. Samples of an urgent nature (e.g., STAT will be processed and read with no undue delay).

Limitations

Samples will not be tested if the acceptance criteria are not met. Refer to the Public Health Inspector's Guide to Environmental Microbiology Laboratory Testing.¹

Storage and Transport

Keep specimens in the dark, stored at 2 – 8 °C following collection and ship them to PHO’s laboratory inside insulated containers or in a cooler with frozen ice packs (Note: Do not include medical samples in the cooler). Arrange the samples so they do not tip and avoid direct contact with ice packs. Frozen samples are not accepted.

Submit samples as soon as possible; they must be tested within one day of collection.

Special Instructions

Due to unforeseen circumstances, it may be necessary to refer samples to another laboratory for testing, other than the laboratory to which sample was initially submitted.

Refer to the Ministry of Health’s Recreational Water Protocol² and Operational Approaches for Recreational Water Guideline³ for more information about public recreational water facilities.

Requisitions and Kit Ordering

Test Frequency and Turnaround Time (TAT)

Samples are routinely accepted at the laboratory Monday – Thursday except holidays during regular operating hours 8:30 a.m. to 4:30 p.m. For submission around statutory holidays: Refer to PHO laboratory holiday schedule.

Please contact PHO’s laboratory prior to the submission of samples that will be received outside of these hours. The turnaround time (TAT) is up to the following number of business days from receipt at PHO’s laboratory: Four days for Total Coliforms and E. coli, up to 5 days for HPC and P. aeruginosa and up to 6 days for S. aureus.

STAT and Critical Samples Testing

Please contact PHO’s laboratory Customer Service Centre prior to the submission of urgent samples or those that will be received outside of regular operating hours. If outside of Customer Service’s hours, contact the PHO laboratory Duty Officer at (416) 605-3113.

STAT samples must be identified with “STAT” on the requisition. STAT samples and samples submitted under a drinking water regulation will be processed and read with no undue delay.

Test Methods

Specimens are tested for the microbial indicators:

Total Coliforms and Escherichia coli (E. coli): Membrane filter method modified from MECP E3407: Membrane Filtration Method Using DC Agar for the Simultaneous Detection and Enumeration of Total Coliforms and Escherichia coli in Drinking Water⁵,
Staphylococcus species (including S. aureus): Membrane filter method modified from Standard Methods for the Examination of Water and Wastewater⁶ 9213B,
Pseudomonas aeruginosa: Membrane filter method modified from Standard Methods for the Examination of Water and Wastewater69213E.
Heterotrophic Plate Count (HPC) test: Spread Plate method modified from MECP E3408: The Spread Plate Method for the Enumeration of Aerobic, Heterotrophic Bacteria in Drinking Water.⁷

Assay performance and limitations:

Total Coliforms and E. coli assay performance:

A multi laboratory study that included the PHO laboratory, determined the sensitivity and specificity of the method for Total Coliforms and E. coli on DC agar to be 93% and 91% respectively.⁸
In-house studies show that more than 90% of E. coli strains present in water can be detected using this procedure, however, false positives and false negatives can occur. Three percent (3%) of E. coli isolates are β-glucuronide – negative.⁹ This method does not allow for the determination of toxigenic species of E. coli (e.g., E. coli 0157:H7).
Some strains of Aeromonas, can be misidentified as coliforms on DC agar due to their ability to ferment lactose.¹⁰ Conversely, there are some organisms that meet the definition of a β-glucuronide negative coliform that are non-lactose fermenters. They would be considered false negatives.

Pseudomonas aeruginosa assay performance:

A study involving the Public Health Ontario laboratory demonstrated that ninety-nine percent of the typical colonies on mPA-C were confirmed as P. aeruginosa, whereas only 3% of other colony types were verified, indicating that the specificity of mPA-C for P. aeruginosa exceeded the recommendation of 90% verification of typical colonies and less than 10% confirmation of atypical colonies.¹¹

Staphylococci assay performance:

In a study investigating the performance of Baird Parker agar, the media’s specificity and sensitivity for Staphylococcus aureus was 100% and 86.6% respectively.¹²

Membrane filtration assay limitations:

Sample composition – Material that is larger than the pore size of the membrane filter (e.g., particulate matter or algae) can clog the membrane filter and interfere with target colony detection
Stressed cells – Bacterial cells that have been stressed, e.g., exposed to adverse environmental conditions, may not survive the filtration process or may be negatively affected by the chemicals used as selective agents in the medium.
Toxins – Some non-target bacteria may release bacteriocins, proteins that can inhibit the growth of target bacteria. High levels of toxic metals or organic compounds may become concentrated in the membrane filter and inhibit colony growth

Spread plate assay limitations:

Media composition – Standard methods agar does not provide the complex nutritional requirements necessary for the growth of all heterotrophs.
Sample composition – The heterogeneous nature of some samples can interfere with enumeration or there is the potential for underestimation due to viable but not culturable organisms.
Cell distribution – If cells are in close proximity on the agar surface only one colony may form resulting in an under-estimation of bacterial cell density.
Obscured plates – Spreading bacterial colonies or fungal growth may obscure other bacterial colonies and result in only an estimate of the number of bacterial colonies present.

Interpretation

Refer to the Public Health Inspector’s Guide to Environmental Microbiology Laboratory Testing.¹

Reporting

Results are reported to the submitter as indicated on the requisition and as per PHO laboratory’s reporting protocol.

References

Ontario Agency for Health Protection and Promotion (Public Health Ontario). Public health inspector's (PHI) guide to environmental microbiology laboratory testing. Toronto, ON: Queen’s Printer for Ontario; 2021 [cited 2023 Sep 21]. Available from: https://www.publichealthontario.ca/en/Laboratory-Services/Public-Health-Inspectors-Guide
Ontario. Ministry of Health. Recreational water protocol, 2019 [Internet]. Toronto, ON: Queen’s Printer for Ontario; 2019 [cited 2023 Sep 21]. Available from: https://www.health.gov.on.ca/en/pro/programs/publichealth/oph_standards/docs/protocols_guidelines/Recreational_Water%20Protocol_2019_en.pdf
Ontario. Ministry of Health. Operational approaches for recreational water guideline, 2018 [Internet]. Toronto, ON: Queen’s Printer for Ontario; 2018 [cited 2023 Sep 21]. Available from: https://www.health.gov.on.ca/en/pro/programs/publichealth/oph_standards/docs/protocols_guidelines/Operational_Approaches_to_Rec_Water_Guideline_2018_en.pdf
Ontario Agency for Health Protection and Promotion (Public Health Ontario). Instructions for official agencies submitting water samples to the Public Health Ontario laboratory [Internet]. Toronto, ON: Queen’s Printer for Ontario; 2012 [cited 2023 Sep 21]. Available from: https://www.publichealthontario.ca/-/media/Documents/Lab/water-submission-instructions.pdf
Ontario. Ministry of the Environment, Conservation and Parks. Membrane filtration method using DC agar for the simultaneous detection and enumeration of total Coliforms and Escherichia coli in drinking water and ground water. E3407 Revision. Toronto, ON : Queen’s Printer for Ontario; 2022.
Lipps WC, Braun-Howland EB, Baxter TE, editors. Standard Methods for the Examination of Water and Wastewater24th ed. Washington, DC: APHA Press; 2023
Ontario. Ministry of the Environment, Conservation and Parks. The spread plate method for the enumeration of aerobic, heterotrophic bacteria in drinking water. E3408 Revision. Toronto, ON : Queen’s Printer for Ontario; 2021.
Schop RN, et al. Comparison of the relative recovery of Escherichia coli and Total Coliforms by Differential Coliform and Chromocult Coliform agars. Poster presented at: CALA Catalyst Conference. 2014 June 2-4; Toronto, ON.
Kilian M, Bűlow P. Rapid identification of Enterobacteriaceae. II. Use of a β-glucuronidase detecting agar medium (PGUA agar) for the identification of E. coli in primary cultures of urine samples. Acta Pathol Microbiol Scand B. 1979;87(5):271-6. Available from: https://pubmed.ncbi.nlm.nih.gov/393074/
Martin NH, Trmčić A, Hsieh T-H, Boor KJ, Wiedmann M. The evolving role of coliforms as indicators of unhygienic processing conditions in dairy foods. Front Microbiol. 2016;7:1549. Available from: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5043024/
Brodsky MH, BW Ciebin. Improved medium for recovery and enumeration of Pseudomonas aeruginosa from water using membrane filters. Appl Environ Microbiol. 1978;36(1):36-42. Available from: https://pubmed.ncbi.nlm.nih.gov/100052/
Oh M-H, Kang S-I, Hong S-P, Oh SW. Comparison of four different isolation media for Staphylococcus aureus. J Korean Soc Food Sci Nutr. 2009. 2009;38(5):606-11. Available from: https://www.researchgate.net/publication/250273127_Comparison_of_Four_Different_Isolation_Media_for_Staphylococcus_aureus