Kennebec Water District - Laboratory Microbiological Test SOPDate: 09/20/2010
Revision No: 04
Supersedes: 06/03/10
Page 1 of 11
KENNEBEC WATER DISTRICT WATER TREATMENT PLANT
462 MAIN STREET
VASSALBORO, ME 04989
STANDARD OPERATING PROCEDURE
Standard Operating Procedure
for
Detection of Presence or Absence
OfColiform Bacteria and Escherichia ColiBacteriain Water
Using Enzymatic Substrate Test Method
Prepared for: STATE OF MAINE DRINKING WATER PROGRAM LABORATORY CERTIFICATION
Prepared by: ______James Hart,KWD Laboratory Director______Date: ___09/21/10______
Reviewed by: ______James Epstein, KWD Operator _____Date: ___05/10/10______
______Tim Wade, KWD Operator _____ Date: ___09/20/10______
Approved by: ______James Hart, KWD QA Officer ______Date: ___09/21/10______
Approved by: ______James Hart,KWD Laboratory Director______Date: ____09/21/10______
1.0 Scope and Application
1.1Kennebec Water District uses this test method for resultsdeterminationfor monitoring programs as required under the Safe Drinking Water Act.
1.2 Colisure® is a selective and differential medium for determining thepresence or absence of total coliforms and Escherichia Coliin finished water. It is a one step, ready-to-use, dehydrated, and granulated culture medium supplied in agamma-irradiated snap packs. Each snap pack unit is added to a 100 ml water sample.
1.3This method tests for coliform bacteria and/or Escherichia Colibacteria within 24 hours.
1.4 The detection limit of Colisure® is one (1) colony forming unit (CFU) of coliform bacteria or one (1) Escherichia Coli per 100 ml of sample.
2.0 Summary of Method
2.1 The color of the medium determines the presence or absence of coliform bacteria and Escherichia Coliin finished water. The content of one blister package is added to a 100 ml sample of water followed by incubation at 35°C± 0.5°C for 24 hours. If coliform bacteria are present, the medium changes color from yellow to magenta. In addition, if Escherichia Coliis present, the medium will emit a bright blue fluorescence when subjected to a longwave (366 nm) ultraviolet (UV) light.
2.2 The method is based on the detection of two enzymes (ß-galactosidase
and ß-glucuronidase) that are characteristic of the coliform bacteria group and Escherichia Coli,respectively (1,2). Identification of these enzymes is accomplished by resuscitationof the target organisms with a combination of tryptose, salts, phosphate buffers, andcarbohydrates, followed by hydrolysis of the chromogenic and/or fluorogenicenzyme substrates. At the same time, lauryl sulfate sodium salt selectively inhibitsthe growth of interfering bacteria.
2.3 Colisure® contains the chromogenic enzyme substrate 5-bromo-4-
chloro-3-indolyl-ß-D-galactopyranoside (or X-GAL) for the detection of ß-galactosidase(an enzyme indicative of the coliform bacteria group). Upon hydrolysis by ß-D -galactosidase,X-GAL releases a chromogenic compound (indigo-blue) that turnsthe medium from slightly yellow to a magenta color. The endpoint of this reactionis distinct and is not subject to interference in water samples discoloredby humic acids, turbidity, or other materials.
2.4 Colisure® also contains the fluorogenic enzyme substrate 4-methylumbelliferyl-ß-D-glucuronide (or MUG) for the detection of ß-glucuronidase (anenzyme specific to Escherichia Coli). Upon hydrolysis by ß-glucuronidase, MUG releases 4-methylumbelliferone that fluoresces when exposed to ultraviolet light. Thefluorescence differentiates the presence of Escherichia Colifrom the rest of the coliform groupas required by the EPA’s Total Coliform Rule in the analysis of drinking water(3).
3.0 Definitions
3.1Defined Substrate - An enzyme substrate that has a specific known chemicalstructure.
3.2 Chromogenic Enzyme Substrate - A substrate that releases a chromogenic
compound upon hydrolysis by an enzyme.
3.3 Fluorogenic Enzyme Substrate - A substrate that releases a fluorogenic compoundupon hydrolysis by an enzyme.
3.4 Specific Enzymes - Enzymes which react with only one particular substrate or veryclosely related compounds, e.g., ß-D-galactosidase is a specific enzyme that reactswith lactose or lactose analogs such as X-GAL. In addition, specific enzymes canalso refer to those associated with a certain species or group of microorganisms.
3.5 Hydrolysis - Enzyme catalyzed decomposition of a substrate by addition of a watermolecule.
3.6 5-bromo-4-chloro-3-indolyl-ß-D-galactopyranoside (X-GAL). A definedsubstratethat changes color from colorless to magenta after hydrolysis by ß-Dgalactosidase.
3.7 4-Methylumbelliferyl-ß-D-glucuronide (MUG) - A defined enzyme substrate thatfluoresces under long wave 366 nm UV light after hydrolysis by ß-D-glucuronidase.
3.8 Coliform Bacteria (Total Coliforms) - Bacteria that possess the enzyme ß-D-galactosidase. In Colisure, this enzyme is capable of producing a magentacolor following a 24 hour incubation period.
3.9Escherichia coli - Bacteria that possess the enzymes ß-D-galactosidase, ß-glucuronidase, and tryptophanase. In Colisure, these enzymes arecapable of producing a magenta color and fluorescence under 366 nm UV light following a 24 hour incubation period.
3.10 Positive Control - Addition of a known organism to medium that produces a positivereaction.
3.11 Negative Control - Addition of a known organism to medium that produces anegative (no) reaction.
4.0 Interferences
4.1 Chemical / Physical - There are no known interferences contained in drinking orpotential source water that interfere with the distinct color change or development offluorescence in Colisure.
5.0 Safety
5.1The analyst must know and practice normal safety procedures for working in amicrobiology laboratory while handling potentially biohazardous cultures.
5.2 Solid and liquid waste containing viable microorganisms are discarded by flushing toa sanitary drain into a subsurface wastewater treatment system.
5.3 Colisure is not a listed or suspected carcinogen. Contact with the skinor eyes may cause irritation or redness.
6.0 Equipment and Supplies
6.1 Equipment
6.1.1 Incubator at 35± 0.50°Celsius.
6.1.2 Long wave UV Light - 366 nanometer wavelength
6.2 Glassware and Supplies - All glassware and plastic vessels used for determining thepresence of coliform bacteria or Escherichia Coliaresterile and handled aseptically.
6.2.1 Sterile Graduated Plastic Sample Collection bottles with sodium thioslfate-120 ml capacity for 100 ml water sample. Obtained commercially pre-sterilized.
6.2.2 Biohazardous Waste Container.
6.3 Sterile Inoculating Loop or Needle.
6.4 Alcohol lamp and ignition system.
7.0 Reagents and Standards
7.1Colisureis provided as ready-to-use, pre-measured, dehydrated,granulated culture medium in a blister package for testing 100 ml water samples. Ifstored at room temperature (2 to 25 °C), the product has a shelf life of 12 months. This media is commercially available from The Idexx Corporation, Westbrook, Maine.
7.2Quanti-Cult is a set of ready to use bacterial cultures for quality control testing or performance evaluation standards. Each set includes three bacteria specimens: Escherichia Coli, klebsiella pneumonia, and pseudomonas aeruginosa. Each set consists of 1-50 bacterial cells in the colorless cap of a plastic vial. Quanti-Cult is delivered ina set of individually capped vials in foil packs. If stored at room temperature (2 to 8 °C), this product has a shelf life of 18 months.This media is purchased commercially from The Idexx Corporation of Westbrook, Maine.
8.0 Sample Collection, De-chlorination, Preservation, Shipment and Storage
8.1 Water Sample Collection.
8.1.1Collection Bottle – Sample containers are commercially obtained pre-sterilized polystyrene bottles – EMD 1.120SB.0102 or equivalent. Containers are non-fluorescingunder long wave (366nm) UV light.
8.1.2 Flushing Procedure – Remove faucet attachments such as screen or splash guard. Open tap fully and let water run to waste for 2-3 minutes. Reduce water flow.
8.1.3 Aseptically fill bottle to the 100 mL level without splashing or flushing out preservative.
8.2De-chlorination - Chlorinated drinking water is collected into a sterile
collection bottle containing sufficient sodium thiosulfate (Na2S203) to neutralize thechlorine present. For a 100 ml water sample, either 0.1 ml of a 3% Na2S203solution, or an equivalent amount of Na2S203 in tablet form (the pre-sterilized sample containers used by KWD contain sterile Na2S203tablets) is used.
8.3 Preservation – Watersamples are held at less than 10°Celsius or refrigerated (if not tested within 30 minutes of arrival to lab) prior to testing and are tested within 30 hours of collection.
9.0 Quality Control
9.1 The Colisure testing procedure is verified with positive and negative
controls according to Chapter V, 5.1.7.4 Manual for the Certification of LaboratoriesAnalyzing Drinking Water: Criteria and Procedures Quality Assurance – 5th Edition.U.S. EPA. Office of Ground Water and Drinking Water, EPA-815-B-2005-001. The schedule of Q. C. testing performed by KWD follows:
9.1.1Annually: Completion of independent proficiency testing as mandated for laboratory certification requirements (a series of 10 unknown samples)
NOTE: Each proficiency test batch performed is treated as an individual sample(s) with one analyst performing test.
9.1.2Weekly (or 4 X per month): Testing of a duplicate sample spiked with a Escherichia Coli control bacterial culture (See details below).
9.1.3Each new batch (dissimilar lot numbers) of test bottlesand mediaare checked for:
Fluorescence- using a long wave 366nm UV Lamp, held 5 inches in front of a bottle, to verify ‘no fluorescence’. If either the bottles or the media test positive (i.e. fluoresce), then the entire lot number of supplies is rejected and replaced with non-fluorescing materials.Results must be recorded in the Micro Biological Log Book.
Sterility - Check sample containers with 25mL of tryptic soy broth and media by incubating dilution water and media.Results must be recorded in the Micro Biological Log Book.
9.1.4Each new batch (dissimilar lot numbers) of reagent wateris checked for:
Sterility–Check reagent water with 50mL of water and 50mL of double strength tryptic soy broth.
9.20To prepare a quality control sample, (for instance, for a total coliform positive and Escherichia Coli. Negative test) fill a sample bottle to the 100mL mark with sterile water for a standard if desired, or fill duplicate sample bottle if a matrix spike is desired. Aseptically transfer a sub-sample from the test tube containing the Klebsiella pneumoniae slant tube culture with a sterile wire loop or needle:
Flame sterilize the inoculation loop, remove the loop from the flame and air cool for 10 seconds. Drag the loop along the surface of the slant tube agar, then, stir the loop into the sample to inoculate the sample water. Test this inoculated sample by the same procedure as a regular sample. When done clean and sterilize the inoculation loop or needle and store in appropriate drawer.
9.21Active test tubecultures of the following bacteria organisms, as furnished in the QuantiCult Kit,are used to evaluate the following Colisure tests:
Total Coliform test:Negative ResultPseudomonas aeruginosa
Total Coliform test:Positive ResultKlebsiella pneumonia
Escherichia Coli test:Negative resultKlebsiella pneumoniae
Escherichia Colitest:Positive resultEscherichia Coli
9.22Prepare the coliform quality control cultures by following the instructions supplied with theQuanti-Cult vials.
9.3Preparation of active organism slant tube for verifying reference cultures:
9.3.1KWD prepares an active slant tube for QC stock cultures with a single strain of organisms traceable to the American Type Culture Collection (ATCC). Currently, KWD uses reference Stock cultures from MicroBioLogics, 217 Osseo Ave. N.St. Cloud, MN 56303 (800-599-2847).
9.3.2KWD purchases sterile nutrient agar in prepared tubes. To prepare, heat agar tube in boiling water until fully melted. Place on a 45 degree angle. Allow to cool at room temperature.
9.3.3Remove the MicroBioLogics Lyfocults from the samples inthe Laboratory refrigerator and allow to equilibrate to room temperature.
9.3.4When the vial is at room temperature, carefully unscrew cap and aseptically add approximately 0.25ml purified water at room temperature.
9.3.5Recap the vial and mix gently. DO NOT INVERT. The resulting suspension should be homogeneous and must be used within 30 minutes of rehydration.
9.3.6With sterilized inoculation loop immerse in the suspension and inoculate with sweeping motion the agar surface of an appropriately labeled sterile slant tube. The culture thus produced can be used after 24 hours for inoculation of blanks and matrix spikes for bacterial QC testing. Store in Thelco-Precision Model 2 incubator @ 2 to 35 degrees C.
9.3.7Quanti-Cult Control Organism Reactions–Quality Control Verification of Media Using Organisms thatyield Indole Color and Fluorescence.
9.3.8Quanti-Cult Control Organism Reaction Results Interpretation
Table 1
Quality Control Organisms and Colisure Reaction Results
Utilizing Quanti-Cult Bacteria Reagents
Control Type / Bacteria Specie / Color Fluorescence Indole / ResultE. Coli Control / Eschericha coli (ATCC 25922) / Magenta + UV Fluoresce / Positive
Coliform Control / Klebsiella pneumoniae / Magenta + No UV Fluoresce / Negative
Negative Control / Pseudomonas aeruginosa / Yellowish + No UV Fluoresce / Negative
No Control / None / Yellowish + No UV Fluoresce / Negative
9.5Routine laboratory QA/QC procedures for applicable glassware, incubators, andother equipment should conform to: 1) Chapter V, 3.1- 3.16 Manual for theCertification of Laboratories Analyzing Drinking Water: Criteria and ProceduresQuality Assurance – 4th Edition. U.S. EPA. Office of Ground Water and DrinkingWater, EPA-815-B-97-001. and/or 2) Section 9020. Standard Methods for the Examination of Water and Wastewater – 20th Edition – 1998. American PublicHealth Association, American Water Works Association, and Water EnvironmentFederation.
10.0 Calibration and Standardization
10.1 Acceptable performance of the Colisure method by the user is
determined by routine quality control using known cultures and is described in
Section 9.0.
10.2Equipment: All equipment described in Section 6.0 should be maintained andcalibrated according to the manufacturers' recommendations and the general QCguidelines in. 1) Chapter V, 3.1- 3.16 Manual for the Certification of LaboratoriesAnalyzing Drinking Water: Criteria and Procedures Quality Assurance – 5th Edition.U.S. EPA. Office of Ground Water and Drinking Water, EPA-815-B-2005-001.
11.0 Procedure
11.1 Test Procedure
11.1.1 Add 100 ml water sample into a sterile, transparent vessel with at least 120 mL capacity. (Refer to Section 8.0 – Sample Collection).
11.1.2 Take one (1) Colisure snap package, tap the pack to ensure the
granulated media does not stick to the bottom, and bend the upper part of the
package until it breaks open. Do not touch the opening.
11.1.3 Add the contents of the package to the water sample vessel bottle. Seal the vessel and shake contents to completelydissolve the granules.
11.1.4 Incubation: 24 ± 1 h at 35 ± 0.50°C. (Temperature variation from Ensure that the specified incubation period isfollowed in air-type incubators according to Chapter V, 5.6.8 Manual for theCertification of Laboratories Analyzing Drinking Water: Criteria and ProceduresQuality Assurance – 4th Edition. U.S. EPA. Office of Ground Water and DrinkingWater, EPA-815-B-97-001
11.2Sample Interpretation
11.2.1Visually check each vessel for a magenta color. If the sample is magenta,
coliform bacteria are present in the test sample.
11.2.2 If the solution is magenta, examine for fluorescence using a long wave UV
lamp. If fluorescence is present, the solution will glow a uniform, bright, light-bluethroughout. The presence of fluorescence indicates that Escherichia Coli is presentin the sample.
11.2.3 If no magenta color is observed, the sample is negative for coliform bacteria. (See Section 12 below for further detail regarding interpretation of Test Results).
12.0 Data Analysis, Interpretation, Reporting, and Atypical Results
12.1 Coliform bacteria determination
12.1.1A change of color from slight yellow to magenta indicates the presence of atleast 1 CFU of coliform bacteria. Report as a positive Presence/Absence Test
result. An absence of a change of color should be reported as a negativePresence/ Absence Test result. No further data analysis or calculation is required.
12.1.2 A slight pink or orange color sample should be re-incubated for 24 hours.
A sample that remains pink or orange after 24 hours is considered a negative result for Coliform bacteria and Escherichia Coli.
12.2Escherichia Coli bacteria determination
12.2.1 A fluorescence observed under long wave UV light, in combination with a
positive indole reaction (i.e. magenta), indicates a positive result for the presence of at least 1CFU of Escherichia Coli. Report as a positive Presence/Absence Test result.
12.3Atypical Sample disposition
12.3.1Samples thatturn anunexpected- or alternate- color (that is yellow is absent at commencing of test), or which change to magenta colorprior to incubation,are invalid.
12.3.2Invalid samples will be re-sampled from the same location as the invalidate sample and sent to the Maine Health and Environmental Test Laboratory for analysis.
13.0 Method Performance Characteristics
13.1 Specificity - the specificity of Colisure was assessed for recovery of
total coliforms and Escherichia Coli. Both positive and negative results were evaluatedaccording to the EPA’s ATP(5) format for the presence or absence of totalcoliforms and Escherichia Colias compared to the reference methods.
Discrepanciesbetween Colisure and reference method results in this study wereresolved with identification to species by a clinically approved commercial kit(#4345000 BBL Crystal Enteric/Nonfermenter). Both unadjusted (identification-to-speciesdata not considered) and adjusted (identification-to-species dataconsidered) results of that study appear below:
13.1.1 Total Coliforms (unadjusted) - False positive error was 7.00%. False negativeerror was 5.10%.
13.1.2 Escherichia Coli(unadjusted) - False positive error was 5.00%. False negative error was6.86%.
13.1.3 Total Coliforms (adjusted) - False positive error was 3.0%. False negative errorwas 3.96%.
13.1.4 Escherichia Coli(adjusted) - False positive error was 0.00%. False negative error was6.86%.
13.2 Comparability - the comparability of Colisure was assessed forrecovery of total coliforms and Escherichia Coli. Both positive and negative results wereevaluated according to the EPA’s ATP(5) format for the presence or absence oftotal coliforms and Escherichia Colias compared to the reference methods. Chi-square,Cochran-Mantel-Haenszel, and Breslow-Day statistics were calculated on 16sets of data for total coliforms and 15 sets of data for Escherichia Coli. See report from Montana EnvironmentalLaboratoryfor further details.
13.3 Precision and Bias - This method is a qualitative test, and as such, precision andbias statements cannot be provided.
14.0 Pollution Prevention
14.1 Wherever possible, it is recommended that Kennebec Water DistrictLaboratory personnel utilize pollutioncontrol techniques to minimize waste generation. When waste cannot bereduced at the source, recycling is recommended.
15.0 Waste Management
15.1 All wastes generated by the testing of drinking water for Total Coliforms and Escherichia Coli by enzymatic substrate method are disposed of by pouring down the facility toilets as influent to the subsurface wastewater disposal system (i.e. septic tank). This includes wastes associated with spike positive samples (i.e. FDA strain Seattle 1946) which is rated as BioSafety Level (BSL) designation #1 by the Center for Disease Control, as well as Quality Control reagents utilized periodically such as Idexx Laboratory’s QuantiCult Lyfocult specimens. BSL 1 specimens are not deemed hazardous biological waste as they are not disease causing bacteria and they do not need to be autoclaved or incinerated as such. Biological treatment using subsurface wastewater disposal systems is an approved safe method of disposition of enzymatic substrate wastes. In order to protect the air, water, and land environments, it is the responsibility of Kennebec Water DistrictLaboratory Personnel to comply with all federal, state and localregulations pertaining to waste management disposition, particularly hazardous waste identification and land disposal restrictions.
Compliance regulations also apply toany subsurface wastewater discharges and related permits(6). For further information about waste management regulations, Federal, State or local agenciesshould be contacted.