Sewage

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.

Background
This page provides water testing information for water suspected of being contaminated by sewage and submitted to the Public Health Ontario (PHO) laboratory by a public health unit or another agency when authorized for testing.

The target organism of the test is Escherichia coli (E. coli).

The following testing options are available for suspected sewage contaminated water: Membrane filtration for E. coli.

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 Testing Methods section (added assay performance and limitations)

*Uncontrolled print copy. Valid only on day of print: 01 May 2024.

Testing Indications

Acceptance/Rejection Criteria

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

Submitter's Responsibility

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.

Samples must be transported in a Laboratory Sewage Transportation bag (#TC 125-B) without any other types of environmental or medical specimens. The requisition must not be placed in the portion of the clear biohazard bag with the sample; it is put in the side slot of that bag.

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.²

Specimen Collection and Handling

Specimen Requirements

Test Requested	Required Requisition(s)	Specimen Type	Minimum Volume	Collection Kit
E. coli	Bacteriological Analysis of Water: Multiple Sample Requisition for Official Agencies	Suspected Sewage Contamination Water	200 mL (to the fill line on the bottle)	Sewage Samples Collection Kit (TC-125-1B) labelled with a “SEWAGE SAMPLES ONLY” tag.

Submission and Collection Notes

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
“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)²

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.

Wear clean, disposable rubber/latex gloves and keep the bottle closed until just before collection. Remove the cap and ensure it is kept away from sources of contamination.

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 the sample to the 200mL fill line. Do not overfill the bottle. 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.

Wipe down the exterior of collection bottle with disinfectant. A freshly prepared 1% solution of sodium hypochlorite; i.e., 1 part household bleach to 10 parts water, is recommended.

Seal the sewage sample bottle in the plastic bag supplied.

Remove and discard the gloves. Do not put the used gloves in the plastic bag along with sewage sample. Wash/sanitize your hands.

Put the completed requisition in the side pocket of the plastic bag containing the sewage sample. Do not place the requisition inside the plastic bag with the sample.

Refer to Instructions for Official Agencies Submitting Water Samples to the Public Health Ontario Laboratory² 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 suspected sewage contaminated samples must be shipped within 24 hours of collection and tested within one calendar day of collection.

Routine Suspected Sewage Contamination water samples are accepted Monday to Friday 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 (Monday to Friday 8:30 a.m. to 4:30 p.m. excluding statutory holidays). Samples of an urgent nature (e.g., STAT will be processed and read with no undue delay).

Limitations

Samples are only accepted when submitted by a public health unit or other agencies when authorized for testing.

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

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.

Storage and Transport

Place the packaged suspected sewage sample in the hard transport case containing absorbent material and a frozen ice pack within the blue laboratory sewage transport bag (TC-125-1B). Arrange the samples so they do not tip and avoid direct contact with ice packs. Keep specimens at 2-8 °C following collection. Frozen samples are not accepted.
Note: Samples identified as Suspected Sewage Contamination will not be accepted unless received in the Sewage Sample Collection Kit. Do not include other environmental or medical samples with these samples.

Submit samples as soon as possible. The transport bag must be shipped to the local PHO Laboratory within 24 hours of collection and tested within one day of collection. Samples should be below 25 °C when received at the laboratory.

Wash/sanitize your hands after handling/packaging the transport bag. Used gloves may be bagged and disposed of in regular trash receptacles.

Special Instructions

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

Requisitions and Kit Ordering

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. 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.

Turnaround time (TAT) is up to 4 business days from receipt at PHO’s laboratory.

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

The suspected sewage contaminated water test for E. coli is performed by the Membrane Filtration method modified from the American Society for Microbiology’s Bacteriological Examination of Waters: Membrane Filtration Protocol.3

Assay performance and limitations:

E. coli assay performance:

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.4 This method does not allow for the determination of toxigenic species of E. coli (e.g., E. coli 0157:H7)

Membrane filtration assay limitations:

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
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.
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

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.

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 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
Arango Pinedo C, Foster B. Bacteriological examination of waters: membrane filtration protocol [Internet]. Houston, TX: American Society for Microbiology; 2015 [cited 2023 Sep 21]. Available from: https://asm.org/ASM/media/Protocol-Images/Bacteriological-Examination-of-Waters-Membrane-Filtration-Protocol.pdf?ext=.pdf
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/