SOP Number: 023, Revision 10
Effective Date 04/12/10
Page 1 of 13
TOTAL COLIFORMS AND E. COLI BY COLILERT
Method SM 9223B (Enzyme Substrate Test, Presence-Absence)
I.Purpose – Scope and Application
1.This method is applicable to drinking and source water.
2.Coliform bacteria, or rather their absence, is a good index of the degree of bacteriological safety of a water supply. In general, bacteria can be divided into a fecal and a non-fecal group. The coliform group consists of several genera of bacteria belonging to the family Enterobacteriaceae.
3.The CWTP Laboratory performs Colilerts analysis for three different functions.
a)Distribution Samples – Samples taken and analyzed for compliance with the Safe Drinking Water Act. Arizona Department of Environmental Quality (ADEQ) forms are filled out for these samples.
b)Offsite Samples – Two sets of samples must pass the Colilert test before a new main may be put into service. These samples involve a special informal type of reporting that is explained later.
c)Special Samples – These samples come from a variety of sources. These samples may be part of an investigation of a customer compliant, or they could be from a newly repaired well site, etc. Reports for these samples may be informal or involve an official Laboratory report.
II.General Discussion
A.Summary
1.Colilert provides simultaneous detection, specific identification and confirmation of total coliforms and E. coli in water. Colilert provides a specific indicator for the target microbes, total coliforms and E. coli. As the media’s nutrients are metabolized, yellow color and fluorescence are released, confirming the presence of total coliforms and E. coli respectively.
2.Total coliform: The chromogenic substrate ortho-nitrophenyl--D-galactopyranoside (ONPG) is used to detect the enzyme -D-galactosidase, which is produced by total coliform bacteria. The -D-galactosidase enzyme hydrolyzes the substrate and produces a color change from clear to yellow. A yellow color indicates a positive test for total coliforms when it appears between 24 to 28 hours of incubation.
3.Escherichia coli: A fluorogenic substrate 4-methylumbelliferyl--D-glucuronide (MUG) is used to detect the enzyme -glucuronidase, which is produced by E. coli. The -glucuronidase enzyme hydrolyzes the substrate and produces a fluorescent product when viewed under long-wavelength (365-nm) ultraviolet (UV) light. The presence of fluorescence indicates a positive test for E. coli.
B.Definitions
1.mL: milliliter
2.E. coli: Escherichia coli
3.UV: Ultraviolet
4.MUG: 4-methylumbelliferyl--D-glucuronide
5.ONPG: Ortho-nitrophenyl--D-galactopyranoside
6.ADEQ: Arizona Department of Environmental Quality
C.Deviations from Method
1.None
D.Responsibilities
1.It is the responsibility of the analyst to perform the analysis according to this Standard Operating Procedure and complete all documentation required for review.
2.Analysis and interpretation of the results are performed by personnel in the laboratory who have demonstrated the ability to generate acceptable results utilizing this method. This demonstration is in the form of supervisory/training review, precision and accuracy tests, or the successful completion of an unknown proficiency evaluation test.
3.Final review and sign-off of the data is performed by the Senior Chemist or designee. Signing and dating the file indicates that this review for precision, accuracy, completeness, and reasonableness is complete and satisfactory. Any errors that are found require corrective action, which includes notification to the technician/chemist who performed the work and documentation of measures taken to remediate the data.
4.It is the responsibility of all personnel who work with samples involving this method to note any anomalies or out-of-control events associated with the analysis of the samples. Any discrepancies must be noted and corrective action taken and documented.
III.Sample Preservation and Storage
1.100 mL of sample is collected in a sterile non-fluorescent collection bottle containing sodium thiosulfate. When the sample is collected, fill to 100-mL fill line on the bottle. When sample is collected, leave ample air space in the bottle (at least 2.5-cm).
2.Initiate analysis as soon as possible after collection to minimize changes in bacterial population. If analysis is not to be done immediately, maintain the sample at a temperature 4ºC or lower, but do not freeze.
3.The maximum elapsed time between collection and analysis of samples is 24 hours according to ADEQ Drinking Water Sampling Guide (30 hours according to EPA). Analyses must be performed within 2 hours of laboratory receipt.
IV.Apparatus
1.Pre-Sterilized containers with sodium thiosulfate
2.Long wavelength ultraviolet lamp
3.Color and fluorescence comparator
4.Incubator - Set at 35.0°C ± 0.5°C.
V.Interferences
1.Noncoliform bacteria, such as Aeromonas and Pseudomonas species may produce small amounts of the enzyme -D-galactosidase, but are suppressed and generally will not produce a positive response within the incubation time unless more than 104 colony-forming units (CFU)/mL (106 CFU/100 mL) are present.
2.Some strains of Shigella spp. may produce a positive fluorescence response. Because Shigella spp. are overt human pathogens, this is not considered a detriment for testing the sanitary quality of water.
3.Water samples containing humic or other material may be colored. If there is background color, compare inoculated vessels to a control vessel containing only water sample
4.In certain waters, high calcium salt content can cause precipitation but this should not affect the reaction.
VI.Reagents
1.Snap packs each containing Colilert reagent – Purchased.
2.Colilert Comparator - Purchased.
VII.Procedure
A.Quality Control Procedure
Perform the following quality control checks on each new batch of media prior to the media being used in sample analysis.
1.Auto-fluorescence check
a)Open a sealed pre-sterilized bottle of buffer water and aseptically transfer the contents to a colilert vessel.
b)Carefully separate one Snap Pack from the strip taking care not to accidentally open an adjacent pack.
c)Tap the Snap Pack to ensure that all of the Colilert powder is in the bottom part of the pack.
d)Open one pack by snapping back the top at the scoreline.
e)Add the medium to one of the vessels without allowing contact between the medium pack and the vessel.
f)Aseptically cap and seal the vessel.
g)Shake the vessel at least 25 times to dissolve the reagent.
h)Check for auto-fluorescence by placing the 6-Watt 365-nm UV light within five inches of the sample in a dark environment. Be sure the light is facing away from your eyes and towards the vessel. If fluorescence is present discard that batch of media. Notify IDEXX of the problem and get a replacement for the defective media.
i)Record the results in the media Quality Control Log.
2.Quality Control Check:
a)Open a sealed pre-sterilized bottle of buffer water and aseptically transfer the contents to a colilert vessel. Repeat the procedure three more times for a total of four vessels.
b)Inoculate three of the vessels with the following Kwik Stik cultures (refer to Kwik Stik SOP for instructions):
(1)E. Coli
(2)Klebsiella pneumoniae
(3)Pseudomonas aeruginosa
c)Carefully separate one Snap Pack from the strip taking care not to accidentally open an adjacent pack.
d)Tap the Snap Pack to ensure that all of the Colilert powder is in the bottom part of the pack.
e)Open one pack by snapping back the top at the scoreline.
f)Add the medium to one of the vessels without allowing contact between the medium pack and the vessel.
g)Aseptically cap and seal the vessel.
h)Shake the vessel at least 25 times to dissolve the reagent.
i)Repeat steps 4 to 9 for the other three vessels.
j)Record the test date and time in the Media Quality Control Log.
k)Incubate the vessels at 35 C for 24 – 28 hours.
l)After the incubation time is completed, remove the vessels from the incubator. In the Media Quality Control Log, record the time of the reading.
m)Compare each result against the color comparator.
n)If no yellow color is observed, the test is negative (absent).
o)If the sample has a yellow color equal to or greater than the comparator, the presence of total coliforms is confirmed (present). If the color is not uniform, mix by inversion then recheck.
p)If the sample is yellow, but lighter than the comparator, it may be incubated an additional 4 hours (but no more than a total of 28 hours). If it does not intensify, the sample is negative.
q)If yellow is observed, check the sample for fluorescence by placing the 6-Watt 365-nm UV light within five inches of the sample in a dark environment. Be sure the light is facing away from your eyes and towards the vessel. If fluorescence is greater or equal to the fluorescence of the comparator, the presence of E. coli is confirmed.
r)Record the results in the media Quality Control Log.
s)If the Quality Control fails, repeat the test before using the media for sample analysis. If the Quality Control fails a second time, discard that batch of media. Notify IDEXX of the problem and get a replacement for the defective media.
B.Sample Analysis
1.On the bench sheet in the appropriate spaces, record the sample type (compliance, offsite, special) and location of sample (distribution, well name, offsite location, address, etc.). Write the sample ID numbers going across the first row of boxes.
2.Carefully separate one Snap Pack from the strip taking care not to accidentally open an adjacent pack.
3.Tap the Snap Pack to ensure that all of the Colilert powder is in the bottom part of the pack.
4.Open one pack by snapping back the top at the scoreline.
5.Add the reagent to the 100-mL water sample that has been collected in a sterile transparent-nonfluorescent vessel. Do not allow contact between the medium pack and the vessel while adding the reagent.
6.Aseptically cap and seal the vessel.
7.Shake the vessel at least 25 times to dissolve the reagent.
8.Incubate the vessels at 35 C for 24 – 28 hours.
9.Immediately after placing the vessels in the incubator stamp the date and time in the ‘DATE & TIME OF SET-UP / INITIAL’ area of the bench sheet.
10.After the incubation time has been completed, remove the vessel(s) from the incubator. Stamp the date and time in the ‘DATE & TIME OF TEST READ / FINAL’ area of the bench sheet
11.Compare each result against the color comparator.
a)If no yellow color is observed, the test is negative (absent).
b)If the sample has a yellow color equal to or greater than the comparator, the presence of total coliforms is confirmed (present). If the color is not uniform, mix by inversion then recheck.
c)If the sample is yellow, but lighter than the comparator, it may be incubated an additional 4 hours (but no more than a total of 28 hours).
(1)If the color does not intensify to the level of the comparator, the sample is negative for total coliform.
(2)If the yellow color intensifies to the level of the comparator, the sample is positive for total coliform.
12.On the bench sheet in the Total Coliform Reaction (ONPG) column and under each sample ID number, record:
a)0 for a negative result
or
b)1 for a positive Total Coliform result
13.If yellow is observed, check the sample for fluorescence by placing the 6-Watt 365-nm UV light within five inches of the sample in a dark environment. Be sure the light is facing away from your eyes and towards the vessel. If fluorescence is greater or equal to the fluorescence of the comparator, the presence of E. coli is confirmed.
14.If fluorescence is questionable, incubate for an additional 4 hours (but not for more than a total of 28 hours).
a)If fluorescence intensifies to the level of the comparator, the reaction is positive for E. coli.
b)If the fluorescence does not intensify to the level of the comparator, then the reaction is negative for E. coli.
15.On the bench sheet in the E. coli (MUG) column and under each sample ID number, record:
a)0 for a negative result
or
b)1 for a positive E. coli result
16.Then, record below:
a)A (Absent) for a negative result
or
b)P (Present) for a positive total coliform result
17.Sign off below the results as analyst.
C.Use of the Date/Time Stamp
1.In the event that the use of the date/time stamp was forgotten, a supervisor must be informed and their approval given before the date and time is written in.
2.In the event that the incorrect date/time was stamped unto the bench sheet, a supervisor must approve any changes.
3.If a supervisor is not present at the time of an error, the direct supervisor must be informed at the earliest reasonable time possible.
4.The supervisor must approve any changes by initialing along side the analysis initials.
D.Reporting Results, Distribution Samples for Compliance
1.If all the samples are negative, no immediate action is necessary.
2.If one or more samples are positive:
a)Immediately notify the Senior Chemist at phone: 782-3655, or cell phone: 480-686-5300. The Senior Chemist in turn will notify the Water Quality Superintendent, Water Operations Compliance Specialist, and Utility Systems Manager via email.
b)Notify the sampler, a repeat of the original sample, a sample upstream, and a sample downstream from the sampling site, as well as, all the ground water wells on-line at the time of original sample collection, will need to be taken within 24 hours of the completion of the positive test.
3.Enter the chain of custody information along with the sample results in the computer. The spreadsheet can be found at f:\water\labshare\lab\data\PDQDATA.
4.The Senior Chemist or designee will upload the data in the “electronic Drinking Water Regulatory Database” (eDWaRD) to generate results on ADEQ forms.
E.Reporting Results for Offsite Samples
1.Record the results of the test on the Chain of Custody. Give the chain of custody to whoever is responsible for making e-mail notifications. Notify the sampler of the results when time permits.
2.Report the results to Water Quality Technicians via Lotus Notes database.
a)Open Water Lab Production database from Lotus Notes.
b)Click on the “Offsite Bacteriological – pending” tab for a list of offsite projects pending reporting.
c)Open the appropriate offsite project.
d)Enter the Laboratory ID #.
e)Enter the results for both Total Coliform and E. Coli by clicking on + or – tabs for each sample. Click + if Total Coliform and / or E. Coli are present in the sample and – if Total Coliform and / or E. Coli are absent in the sample.
f)Click on “Send Results Back to Water Quality” tab to send the results to Water Quality Technicians.
3.Alternatively, an e-mail can also be sent to report the offsite sample results. When composing the e-mail include:
a)The date the samples were taken
b)The result for each sample
c)The Project name or location
d)The offsite inspector or project manager responsible for the project.
F.Reporting Results for Special Samples
1.If a special sample is positive:
a)Notify the sampler, or notify according to any special instructions given at the time of receipt of the sample(s).
2.If a special sample is negative:
a)E-mail or phone the sampler or responsible party. Follow any request given for those samples.
3.Sometimes formal laboratory reports are necessary for special samples. If that is the case, give the completed results to the Chemist who will type a report out for the Senior Chemist to review and sign off.
VIII.Maintenance
1.Thermometers are placed on each shelf of the incubator. Check and record temperature twice daily, morning and afternoon, at least 4 hours apart. Thermometer should be in increments of 0.5°C
2.Disconnect the ultraviolet lamp monthly and clean the bulb with a soft cloth moistened with ethanol.
IX.Method Performance and Quality Control
A.Duplicates
1.Analyze duplicates at a 10% (one duplicate in every 10 samples) frequency. The matrix duplicate is a second aliquot taken from a sample and then brought through the whole sample preparation and analytical process in company with the original aliquot.
B.Acceptance Criteria and Corrective Action:
CONTROL ITEM / FREQUENCY / ACCEPTANCE CRITERIA / CORRECTIVE ACTIONBlank / Each batch of medium / Clear, no fluorescent / Repeat test to confirm. If it still doesn’t meet criteria, the replace the media
Auto-fluorescence / Each batch of medium / No fluorescent / Discard the lot of medium. Notify Idexx.
E. coli / Each batch of medium / Yellow, fluorescent / Repeat test, if doesn’t meet criteria the second time discard the lot of medium. Notify Idexx.
Klebsiella pneumoniae / Each batch of medium / Yellow, no fluorescent / Repeat test, if doesn’t meet criteria second time discard the lot of medium. Notify Idexx.
Pseudomonas aeruginosa / Each batch of medium of media / Clear, no fluorescent / Repeat test, if doesn’t meet criteria second time discard the lot of medium. Notify Idexx.
Duplicates / 10% of Samples / Agreeable results / Resample and repeat analysis for all samples in the group of samples with the failed duplicated result
C.Buffered Dilution Water
1.Before use, check each batch of buffered dilution water for sterility by adding 50 mL water to 50 mL of double strength Tryptic Soy Broth. Incubate at 35 ± 0.5°C for 24 hours. After incubation, check for growth. If any contamination is indicated, determine the cause and/or reject the batch. If it is determined that the broth was contaminated by something other than the water, re-test with another bottle.