Ice – Treated

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 testing information for treated ice samples submitted to the Public Health Ontario (PHO) laboratory for public health units 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) test is also performed to estimate the number of viable, heterotrophic bacteria in the sample.

The following testing options are available for treated ice: 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.

Updates

Added a Submitters Responsibilities section
Updated 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.

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

Total Coliform, E. coli and HPC

Bacteriological Analysis of Water: Multiple Sample Requisition for Official Agencies

Treated ice

200 mL

Sterile plastic bag with wire closure

Item # 300240

Record “Food Bag PT# 300240 B /250” on the comment line of the Requisition for Specimen Containers and Supplies form.

Submission and Collection Notes

Prior to sampling, ensure all materials required for sample collection are available including sterile plastic sample bags with round wire closure, test requisition and sterile collection device (if required).

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” – optional
“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
“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

Retain a copy of the completed requisition that includes the sample identifier.

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

Label sample bag(s) with a unique identifier that matches the identified on the requisition (i.e., Information in the “Identification of Collection Site” section of the form).

Wash hands with soap and water and dry them thoroughly prior to taking the sample.

Collect representative ice samples using either the collection device located in the freezer unit or another sterile collection device. Collect enough ice to fill a 200 mL collection bottle when the sample is melted.

Note: Use aseptic technique throughout the sample collection process. Failure to do so may compromise the test results. Do not submit melted ice. The laboratory will melt the ice in a controlled manner when preparing it for testing.

Ensure the sample bag is properly secured to prevent sample leakage.

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. The ice sample must be tested within 48 hours of collection.

Routine treated ice samples 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).

Limitations

Samples are only accepted when submitted by a public health unit 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 Testing¹ 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).

Submit samples as soon as possible; they must be tested within 48 hours 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.

To order collection kits or other PHO laboratory supplies, complete the Requisition for Specimen Containers and Supplies. Record “Food Bag PT# 300240 B /250” on the comment line of the Requisition for Specimen Containers and Supplies form for sterile sampling bags with wire closure.

Fax the form to the PHO Toronto laboratory fax number listed on the Requisition for Specimen Containers and Supplies or your local PHO laboratory.

Requisitions and Kit Ordering

Test Frequency and Turnaround Time (TAT)

Samples are routinely accepted at the laboratory Monday – Thursday 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.

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 and up to 5 days for HPC.

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

Ice 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 Water.3 The Heterotrophic Plate Count (HPC) test is also performed using a modified version of the MECP’s E3408: The Spread Plate Method for Enumeration of Aerobic, Heterotrophic Bacteria in Drinking Water.4

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.5
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.6 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.7 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.

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 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.
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/