Drinking Water Testing – Official Agencies
Consistent with O. Reg. 671/92 of the French Language Services Act, laboratory testing information on this page is only available in English because it is scientific or technical in nature and is for use only by qualified health care providers and not by members of the public.
This page provides water testing information for routine drinking water samples submitted to the Public Health Ontario (PHO) laboratory by an official agency (i.e., public health units, Ministry of Natural Resources and Forestry Ontario Parks, and other agencies when authorized for testing).
The target organisms of the test are Total Coliforms and Escherichia coli (E. coli). A heterotrophic plate count (HPC) can be requested to estimate the number of viable, heterotrophic bacteria in a treated water sample. The following testing options are available for official agency drinking water: Membrane filtration for E. coli, Total Coliforms and spread plate for HPC.
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.
- 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)
Refer to the Ministry of Health Safe Drinking Water and Fluoride Monitoring Protocol.5
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 samples submitted under an Ontario drinking water regulation that requires specific notification of test results as outlined in the Safe Drinking Water Act, 2002, S.O. 2002, c.32,3 the submitter must follow the instructions provided by the laboratory for: sampling labeling, chain of custody, requirements for submission, sample transport and temperature conditions, and holding time.
Refer to “Important Information Related to Drinking water Analysis” in the Public Health Inspector's (PHI) Guide to Environmental Microbiology Laboratory Testing.4
For samples submitted under an Ontario drinking water regulation, O. Reg. 170/036 or O. Reg. 319/087, the chain of custody/requisition must be complete and accurate. To maintain the chain of custody of the sample, either the “Relinquished by” section is completed or a Regulated Water seal with all the required information applied over the cap of the sample prior to submission of the sample to the laboratory. Refer to Instructions For Official Agencies Submitting Water Samples to the Public Health Ontario Laboratory.8
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.8
|Test Requested||Required Requisition(s)||Specimen Type||Minimum Volume||Collection Kit|
Total coliforms and E. coli; HPC upon request
200 mL (to the fill line on the bottle)
Sterile Water bottles - 250 mL capacity (Case of 250 bottles)
Submission and Collection Notes
Prior to sampling disinfected water sources, 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
- “Owner of the Water Supply” – Mandatory for regulated drinking water system samples
- “Sample Information – Drinking Water” section
- “Source of Drinking Water” section
- “Type of Drinking Water Systems” section
- “Reason for Sampling” section
- “Identification of Collection Site & Time Collected” section
- “Free or combined chlorine residual” – If determined
- Tick off “HPC Requested” – If required (for treated drinking water only)
- “For Regulated Drinking Water or Legal Samples” section – as applicable (refer to Instructions For Official Agencies Submitting Water Samples to the Public Health Ontario Laboratory)8
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 of the barcodes 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.
Examine the lid of the bottle. If the tamper evident ring has separated from the cap use another bottle.
Follow the water sampling instructions.
For bottled water, the water can be aseptically decanted into a PHO laboratory collection bottle or submitted in the original packaging as long as the seal is broken. Sealed bottled water products are not accepted. Contact the Canadian Food Inspection Agency if analysis of a sealed bottled water product is required.
Refer to Instructions for Official Agencies Submitting Water Samples to the Public Health Ontario Laboratory8 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 drinking water must be tested within 48 hours of collection for the results to be suitable for the purposes of the Safe Drinking Water Act, 2002, S.O. 2002, c. 32.3
Routine drinking water samples are accepted Monday to Friday 8:30 a.m. to 4:30 p.m. excluding statutory holidays. Samples with an HPC request are accepted Monday to Thursday 8:30 a.m. to 4:30 p.m. excluding statutory holidays.
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).
Samples are only accepted when submitted by a public health unit, MNRF Ontario Park or other agencies when authorized for testing.
Samples will not be tested if the acceptance criteria are not met. Refer to the Public Health Inspector's (PHI) Guide to Environmental Microbiology Laboratory Testing4 for details.
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 48 hours of collection and the sample temperature needs to be below 25 °C when received at the laboratory.
Due to unforeseen circumstances, it may be necessary to refer samples to another licensed laboratory for testing, other than the laboratory to which sample was initially submitted. For information about PHO laboratory drinking water testing licenses and accreditation visit Accreditation and Licensing.
Refer to the Ministry of Health Safe Drinking Water and Fluoride Monitoring Protocol5 for information related to drinking water protocols.
Test Frequency and Turnaround Time (TAT)
Samples are routinely accepted and tested at the laboratory Monday – Friday during regular operating hours 8:30 a.m. to 4:30 p.m. except for routine samples that require an HPC test or bottled water. These types of samples are accepted between Monday and Thursday. For submission around statutory holidays: Refer to the PHO laboratory holiday schedule. Please contact PHO’s laboratory Customer Service Centre 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 Specimens 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.
Each Public Health Ontario laboratory location holds a drinking water testing license from the Ministry of the Environment, Conservation and Parks (MECP), and drinking water tests limited to the individual laboratory’s scope of testing are accredited by the Canadian Association for Laboratory Accreditation Inc. (CALA) to the current version of ISO 17025.
Drinking water samples are tested for the presence of the microbiological indicators Total Coliforms and Escherichia coli (E. coli) by the Membrane Filtration 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 Water9. When requested, the Heterotrophic Plate Count (HPC) is tested using a modified version of the MECP’s E3408: The Spread Plate Method for Enumeration of Aerobic, Heterotrophic Bacteria in Drinking Water10(for distribution systems samples or other treated drinking water only).
Bottled water is tested for the following microbiological indicators/methods:
- 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 Water,9
- Staphylococcus species (including S. aureus): Membrane filter method modified from Standard Methods for the Examination of Water and Wastewater11 9213B,
- Pseudomonas aeruginosa: Membrane filter method modified from Standard Methods for the Examination of Water and Wastewater119213E.
- 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.10
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% respectively12
- 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.13 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.14 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 colonies15
Staphylococci assay performance:
- A study by Oh M-H et al.16 determined the sensitivity and specificity of the S. aureus isolation media Baird Parker agar to be 86.6 and 100.0 respectively
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
Samples are not tested for other contaminants, including chemical contaminants, and therefore may be unsafe to drink even when there is no significant evidence of bacterial contamination.
Results are reported to the submitter as indicated on the requisition and as per PHO laboratory’s reporting protocol. Drinking water submitted under an Ontario drinking water regulation is reported as per the Safe Drinking Water Act, 2002, S.O. 2002, c.32.3
- Health Canada. Canadian drinking water guidelines [Internet]. Ottawa, ON: Government of Canada; 2022 [modified 2022 Nov 28; cited 2023 Sep 21]. Available from: https://www.canada.ca/en/health-canada/services/environmental-workplace-health/water-quality/drinking-water/canadian-drinking-water-guidelines.html
- Ontario Drinking Water Quality Standards, O Reg 169/03. Available from: https://www.ontario.ca/laws/regulation/030169
- Safe Drinking Water Act, 2002, SO 2002, c 32. Available from: https://www.ontario.ca/laws/statute/02s32
- 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. Safe drinking water and fluoride monitoring 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/Safe_Water_Fluoride_Protocol_2019_en.pdf
- Drinking Water Systems, O Reg 170/03. Available from: https://www.ontario.ca/laws/regulation/030170
- Small Drinking Water Systems, O Reg 319/08. Available from: https://www.ontario.ca/laws/regulation/080319
- 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.
- 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.
- Lipps WC, Braun-Howland EB, Baxter TE, editors. Standard Methods for the Examination of Water and Wastewater. 24th ed. Washington, DC: APHA Press; 2023
- 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, Ciebin BW. 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;38(5):606-11. Available from: https://www.researchgate.net/publication/250273127_Comparison_of_Four_Different_Isolation_Media_for_Staphylococcus_aureus