AUTHORITY: Implementing section 1401(1)(D) of the Safe Drinking Water Act (42 USC 300f(1)(D)), subpart C of the National Primary Drinking Water Regulations (40 CFR 141.21 through 141.30 (2014)), the Illinois Environmental Protection Act [415 ILCS 5] and the Civil Administrative Code of Illinois [20 ILCS 5], and authorized by Section 4(o) and (p) of the Illinois Environmental Protection Act and Sections 2310-575, 2310-580, and 2310-30 of the Civil Administrative Code of Illinois [20 ILCS 2310].
SOURCE: Adopted at 22 Ill. Reg. 14294, effective July 15, 1998; amended at 35 Ill. Reg. 14494, effective August 12, 2011; amended at 38 Ill. Reg. 16240, effective July 15, 2014; amended at 39 Ill. Reg. 14586, effective October 23, 2015; amended at 46 Ill. Reg. 19150, effective November 17, 2022.
SUBPART A: GENERAL PROVISIONS
Section 465.100 Authority (Repealed)
(Source: Repealed at 35 Ill. Reg. 14494, effective August 12, 2011)
Section 465.110 Scope and Applicability
a) This Subpart A establishes general provisions applicable to the certification program for environmental laboratories administered under this Part.
b) Nothing in this Part shall prevent uncertified laboratories from performing any quality control or other tests when the State has not required such tests to be performed by a certified laboratory.
c) Principal State Laboratory is exempt from administration under this Part.
(Source: Amended at 46 Ill. Reg. 19150, effective November 17, 2022)
Section 465.120 Definitions
For purposes of this Part, unless otherwise specifically defined or the context clearly requires a different meaning:
"Act" means Section 4(o) and (p) of the Environmental Protection Act.
"Analyst" means any person who performs analyses for certain or all parameters on samples submitted to the environmental laboratory and who meets the qualifications set forth in Section 465.310(c).
"Annual" means a twelve-month interval.
"ASTM International" or "ASTM" means a not-for-profit, voluntary standards development organization (American Society for Testing and Materials) located at 100 Barr Harbor Drive, P.O. Box C700, West Conshohocken, PA.
"Certification" means a status of approval granted to an environmental laboratory that meets the criteria established by this Part or in accordance with a reciprocity agreement entered into pursuant to Section 465.240. Certification is not a guarantee of the validity of the data generated.
"Certification Officer" means any person who is designated by the Department to inspect and evaluate environmental laboratories for compliance in meeting the criteria set forth in this Part and who meets the educational and experience qualifications set forth in Section 465.310(j).
"Day" means calendar day.
"Department" means the Illinois Department of Public Health.
"Deficiency" means a failure of an environmental laboratory to meet any requirement of this Part.
"Director" means the Director or the designee of the Director of the Department of Public Health.
"Environmental Laboratory" means any facility that performs analyses on environmental samples to determine the quality of food, milk, public water supplies, surface water, ground water, recreational waters, wastewater, air or land.
"Laboratory Pure Water" means water meeting the standards set forth in Section 465.380.
"Laboratory Supervisor" means a person who supervises the performance of the analytical procedures within an environmental laboratory and who meets the qualifications set forth in Section 465.310(a).
"Major Remodeling" means any remodeling of the laboratory facility that requires the acquisition of a local building permit.
"Principal State Laboratory" means the designated State facility that performs analyses on environmental samples to determine the quality of public water supplies in compliance with the Safe Drinking Water Act and National Primary Drinking Water Regulations (40 CFR 141). Principal State Laboratory is exempt from this Part and is certified by the regional USEPA administrator.
"Proficiency Testing Samples" or "PTs" means samples provided to a laboratory for the purpose of demonstrating that the laboratory can successfully analyze the sample within acceptance limits specified in National Standards for Water Proficiency Testing Studies Criteria Document, US EPA, January 31, 2001. The composition of the reference material is unknown to the laboratory at the time of the analysis.
"Program Certification Manager" means a person responsible for managing the drinking water laboratory certification activities within the Department. The Manager establishes one or more teams of Certification Officers to audit drinking water laboratories. A Program Certification Manager shall meet the educational and experience qualifications set forth in Section 465.310(k) or must have served or be serving as a program certification manager prior to November 1, 2022.
"Provisional Certification" means a certification status granted to an environmental laboratory to allow time for the correction of serious deficiencies. Failure to correct a deficiency during the provisional certification period allows the Department to revoke certification as specified in Section 465.180. While on provisional certification, an environmental laboratory remains approved for the analyses covered by its certification.
"Public Water Supply" means all mains, pipes and structures through which water is obtained and distributed to the public, including wells and well structures, intakes and cribs, pumping stations, treatment plants, reservoirs, storage tanks and appurtenances, collectively or severally, actually used or intended for use for the purpose of furnishing water for drinking or general domestic use and that serve at least 15 service connections or that regularly serve at least 25 persons at least 60 days per year.
"Quality Assurance" means an integrated system of management activities involving planning, quality control, quality assessment, reporting and quality improvement to ensure that a product or service meets defined standards of quality with a stated level of confidence.
"Quality Assurance Plan" means a comprehensive plan detailing the aspects of quality assurance needed to adequately fulfill the data needs of a program. This document is required before the laboratory is certified.
"Quality Control" means the overall system of technical activities whose purpose is to measure and control the quality of a product or service so that it meets the needs of the users; operational techniques and activities that are used to fulfill requirements for quality.
"Quarterly" means a three-month interval.
"Readily Accessible" means that the referenced item is located upon the premises.
"Semi-annual" means a six-month interval.
"Standard Operating Procedure" means a written document that details the method of an operation, analysis or action, the techniques and procedures of which are thoroughly prescribed and that is officially approved as the method for performing certain routine or repetitive tasks.
"The NELAC Institute" or "TNI" is an organization (National Environmental Laboratory Accreditation Program) that recognizes associations that offer accreditation of microbiology drinking water proficiency testing providers, located at P.O. Box 2439, Weatherford TX 76086, 817-598-1624.
"Too Numerous to Count" or "TNTC" means greater than 200 colonies on the membrane filter in the absence of detectable coliforms when analyzing drinking water for total coliforms.
(Source: Amended at 46 Ill. Reg. 19150, effective November 17, 2022)
Section 465.125 Incorporated and Referenced Materials
a) The following publications and federal regulations are incorporated by reference:
1) "Readycult Coliforms 100 Presence/Absence Test for Detection and Identification of Coliform Bacteria and Escherichia coli in Finished Waters", Version 1.1 January 2007; available from Millipore Sigma Corporation (an affiliate of Merck KGaA, Darmstadt, Germany), 400 Summit Drive, Burlington, MA 01803, 800-645-5476, www.sigmaaldrich.com.
2) "IDEXX SimPlateTM HPC Test Method for Heterotrophs in Water", November 2000, IDEXX Laboratories, Inc., One IDEXX Drive, Westbrook ME 04092, 800-321-0207.
3) "Simultaneous Detection and Enumeration of Total Coliforms and Escherichia coli using m-ColiBlue24 Membrane Filtration Medium", USEPA Approved Hach Method 10029, Revision 2, August 17, 1999; available from Hach Company, 5600 Lindbergh Drive, Loveland CO 80538, 800-604-3493.
4) Method 1604: Total Coliforms and Escherichia coli in Water by Membrane Filtration Using a Simultaneous Detection Technique (MI Medium), September 2002, known as EPA 821-R-02-024; available from the U.S. Environmental Protection Agency, Office of Water (4303T), 1200 Pennsylvania Avenue, NW, Washington DC 20460, 202-566-1075.
5) Method 1623 Cryptosporidium and Giardia in Water by Filtration/IMS/FA, December 2005, known as EPA 815-R-05-002; available from the U.S. Environmental Protection Agency, Office of Water (4607), 1200 Pennsylvania Avenue, NW, Washington DC 20460, Technical Support Center for subject matter (in Cincinnati, OH) 505-569-7919.
6) EPA Method 1623.1. "Cryptosporidium and Giardia in Water by Filtration/IMS/FA," 2012. EPA-816-R-12-001. Available at the National Service Center for Environmental Publications (www.epa.gov/nscep). Search "816R12001".
7) "Charm E*Colite™ Presence/Absence Test for Detection and Identification of Coliform Bacteria and Escherichia coli in Drinking Water" (E*Colite*®), January 9, 1998; available from Charm Sciences, Inc., 659 Andover Street, Lawrence MA 01843-1032, 800-343-2170.
8) Version 2.0. "Modified Colitag™ Test Method for the Simultaneous Determination of Total Coliforms and E. coli in Water." June 2020. Neogen Corporation, 620 Lesher Place, Lansing, MI 48912, 800-243-5333.
9) Manual for the Certification of Laboratories Analyzing Drinking Water", USEPA 570/9-90/008A, 5th Edition (January 2005). A copy of this manual can be obtained by contacting the U.S. Environmental Protection Agency, Ariel Rios Building, 1200 Pennsylvania Avenue, NW, Washington DC 20460, 202-272-0167.
10) Supplement 1 to the Fifth Edition of the Manual for the Certification of Laboratories Analyzing Drinking Water, June 2008, known as EPA 815‑F-08-006; available from the U.S. Environmental Protection Agency, Ariel Rios Building, 1200 Pennsylvania Avenue, NW, Washington DC 20460, 202-272-0167.
11) Supplement 2 to the Fifth Edition of the Manual for the Certification of Laboratories Analyzing Drinking Water, November 2012, known as EPA 815-F-12-006; available from the U.S. Environmental Protection Agency, Ariel Rios Building, 1200 Pennsylvania Avenue, NW, Washington DC 20460, 202-272-0167.
12) 40 CFR 141, National Primary Drinking Water Regulations (October 12, 2022).
13) 29 CFR 1910, Occupational Safety and Health Standards (March 7, 2018).
14) 40 CFR 142, National Primary Drinking Water Regulations (February 26, 2014).
15) 40 CFR 141, Subpart S, Ground Water Rule (November 8, 2006).
16) Long Term 2 Enhanced Surface Water Treatment Rule Documents (April 2009), available at: https://www.epa.gov/dwreginfo/long-term-2-enhanced-surface-water-treatment-rule-documents.
17) NEPIS US EPA Good Automated Laboratory Practices, known as EPA 2185, Office of Information Management, Research Triangle Park NC 27711, August 10, 1995.
18) Standard Methods for the Examination of Water and Wastewater, 21st Edition, 2005; 22nd Edition, 2012; or 23rd Edition, 2017; available from the American Public Health Association, 800 I Street, NW, Washington DC 20001-3710.
19) ASTM E617-13, Standard Specification for Laboratory Weights and Precision Mass Standards; available from ASTM International; 100 Barr Harbor Drive, West Conshohocken PA 19428-2959, www.astm.org.
20) NIST Handbook 150-2G, National Voluntary Laboratory Accreditation Program, Calibration Laboratories, Technical Guide for Mechanical Measurements, March 2004; available from National Voluntary Laboratory Accreditation Program, National Institute of Standards and Technology, 100 Bureau Drive, Stop 2140, Gaithersburg MD 20899-2140, E-mail: nvlap@nist.gov NVLAP Website: http://www.nist.gov/nvla.
21) TECTA EC/TC "TectaTM EC/TC Medium and the TectaTM Instrument: A Presence/Absence Method for the Simultaneous Detection of Total Coliforms and Escherichia coli (E. coli) in Drinking Water, " version 2.0, February 2017. Available from Pathogen Detection Systems, Inc., 382 King Street East, Kingston, Ontario, Canada, K7K 2Y2.
b) These incorporations by reference refer to the edition of the document on the date specified and do not include any subsequent amendments or editions.
c) The following laws and rules are referenced in this Part:
1) Safe Drinking Water Act (42 U.S.C. 300f(1)(D))
2) Civil Administrative Code of Illinois [20 ILCS 5]
3) Illinois Environmental Protection Act [415 ILCS 5]
4) Illinois Plumbing Code, Illinois Department of Public Health (77 Ill. Adm. Code 890)
5) Primary Drinking Water Standards, Pollution Control Board (35 Ill. Adm. Code 611)
6) Uniform Electronic Transactions Act [815 ILCS 333]
7) Local Records Act [50 ILCS 205]
(Source: Amended at 46 Ill. Reg. 19150, effective November 17, 2022)
Section 465.130 Certification Procedure
a) An environmental laboratory that meets or exceeds the minimum criteria for certification may receive certification from the Department for any microbiological parameter for which a methodology has been specified in this Part or for which an alternative methodology has been approved in accordance with the provisions of this Part.
b) The operational aspects of an environmental laboratory that will be evaluated in considering a request for certification are:
1) laboratory facilities,
2) personnel,
3) methodology and instrumentation,
4) data handling, and
5) quality assurance program.
c) In seeking initial certification, whether a new laboratory or a certified laboratory requesting to analyze additional or newly regulated contaminants, the petitioning environmental laboratory shall:
1) Submit a formal request for certification to the Department;
2) File with the Department on the applicable administrative questionnaires furnished by the Department, if available, or otherwise in a form approved by the Department, complete information on the five categories listed in subsection (b);
3) Analyze all proficiency testing samples (PTs) required in accordance with the applicable Sections of this Part and report the results of those analyses to the Department; and
4) Permit and cooperate in an on-site visit by Department-authorized certification officers. Certification officers shall provide the environmental laboratory with official identification and credentials. The initial visit will be arranged at the mutual convenience of both parties. The Department reserves the right to make subsequent visits without prior notice during regular working hours.
d) Initial approval or denial of certification may be made only after the procedure described in subsection (c) has been completed. If all requirements of subsection (c) are satisfactory, initial approval will be granted followed by a written narrative report. Denial of certification shall be in the form of a written narrative, giving information as to how deficiencies may be corrected, along with a completed survey form on which all deficiencies are clearly identified.
e) Biennial audit approval or denial of certification may be made only after the requirements described in subsection (b) have been assessed. If all requirements of subsection (b) are satisfactory, approval will be granted followed by a written narrative report. Denial of certification shall be in the form of a written narrative, giving information as to how deficiencies may be corrected, along with a completed survey form on which all deficiencies are clearly identified.
f) Environmental laboratories in jurisdictions not having reciprocal agreements with the Department under Section 465.240 may receive certification from the Department under this Part and shall pay all of the expenses to be incurred by the Department, including travel expenses.
(Source: Amended at 46 Ill. Reg. 19150, effective November 17, 2022)
Section 465.140 Conditions Governing the Use of Certificates
a) Certification of environmental laboratories shall be effective for a two-year period from the date of issue, unless modified or revoked by the Department. Renewal certification will be granted after successfully passing a biennial audit.
b) Certification shall be limited to those parameters for which an environmental laboratory has been approved and that are listed on the certificate of approval.
c) The certificate of approval shall be posted or displayed in a prominent place in the laboratory facility.
d) Information related to the certification of an environmental laboratory shall be accurately represented if used in any advertising and shall prominently include the statement that, "Certification by the State of Illinois is not an endorsement or a guarantee of the validity of the data generated." This information shall also specify the parameters for which the environmental laboratory has been certified. The advertising shall not include any representation that the environmental laboratory is certified to perform a type of analysis for which it lacks proper certification.
e) An environmental laboratory may surrender its certification voluntarily by notifying the Department in writing.
(Source: Amended at 46 Ill. Reg. 19150, effective November 17, 2022)
Section 465.150 Provisional Certification
a) Whenever a deficiency is found, a certified environmental laboratory may be placed on provisional certification. Provisional certification may be imposed for the following periods:
1) From 7 to 30 days if the deficiency could compromise the quality of analytical data generated by the environmental laboratory; or
2) From 90 days to one year in the case of any other type of deficiency.
b) A provisionally certified laboratory may continue to analyze samples for compliance purposes, but shall notify its clients of its provisionally certified status by providing that information in writing, as soon as practicable. In no event shall written notice be given later than 3 days after the imposition of provisionally certified status and shall also include such information on any report of any analysis performed during the period of provisional certification.
(Source: Amended at 46 Ill. Reg. 19150, effective November 17, 2022)
Section 465.170 Changes in Ownership or Operations
a) Certification shall not be transferable. In the event of a change of ownership, director, supervisor, or analyst, or relocation or major remodeling of the physical plant of an environmental laboratory, the Department shall be notified in writing within 15 days.
b) After receiving notification of any of the changes listed in subsection (a), unless otherwise specified in this Part for a specific parameter, the Department may, as applicable, require the analysis of PTs by any new analyst, or make an on-site visit. However, the Department may waive any of these actions if it finds the actions to be unwarranted in a specific case. Examples of when the waivers would be appropriate include the following circumstances:
1) Waiver of submittal of a summary of education and experience when personnel transferring from one certified laboratory to another are responsible for dealing with the same analytical methods and equivalent equipment; and
2) Waiver of an on-site visit if the pertinent test procedures involve simple techniques and equipment. Supervisor must provide training documentation approved by the Department to be eligible for the waiver.
(Source: Amended at 46 Ill. Reg. 19150, effective November 17, 2022)
Section 465.180 Revocation of Certification
a) The Department may revoke all or any part of an environmental laboratory's certification. Any of the following shall be cause for partial or total revocation of certification:
1) Expiration of a period of provisional certification, provided the laboratory has not corrected the deficiencies after being placed on provisional certification in accordance with the provisions of Section 465.150;
2) Unsatisfactory analyses of PTs as specified in Section 465.200;
3) Failure to notify the Department within 15 days after any of the changes listed in Section 465.170 have occurred;
4) Failure to comply with the requirements regarding advertising as specified in Section 465.140(d);
5) Failure to use the analytical methodology specified in this Part or approved in accordance with this Part;
6) Failure to provide notice in accordance with Section 465.150(b) of its status as a provisionally certified environmental laboratory;
7) Falsification of results of testing PTs or any other information material to the certification; or
8) When conducting PTs analysis in accordance with Section 465.200, failure to provide results proving satisfactory precision and accuracy in two successive samples shall be cause for revocation of certification for the parameter or method that is not within satisfactory limits.
b) The Department shall take the following factors into account in determining what action should be taken against a certified environmental laboratory for failing to comply with the requirements of this Section:
1) The length of time during which the failure has existed;
2) The laboratory's prior record of failures and response in correcting failures noted by the Department;
3) Whether the laboratory knowingly caused or allowed the failure; and
4) The potential effect of the failure on the quality of analytical data generated by the laboratory.
(Source: Amended at 35 Ill. Reg. 14494, effective August 12, 2011)
Section 465.190 Subcontracting by Certified Laboratories
a) The name of the laboratory actually performing the analysis shall be specified on all reports of analytical results.
b) For those tests that are required to be performed under certification, any laboratory with which a certified environmental laboratory subcontracts shall also be a certified environmental laboratory.
Section 465.200 Proficiency Testing Samples (PTs)
a) An environmental laboratory is required to participate in proficiency testing samples (PTs) analyses for each analytical parameter or method for which it seeks or wishes to maintain certification in accordance with the certification procedures of Section 465.130(c), the certification renewal procedures of Section 465.140(a), and the quality assurance requirements contained in Subpart B of this Part.
b) Heterotrophic plate count and coliform Microbiological Water Supply (WS) PT samples shall be analyzed annually (every 12 months). Cryptosporidium PT samples shall be analyzed every four to six months. PT samples shall be analyzed in the same manner as routine samples. The laboratory shall document that the analyst analyzing any PT sample is a laboratory employee who routinely analyzes drinking water compliance samples.
c) Laboratories shall acquire the PT sample from a provider acceptable to TNI.
d) For methods used to test the presence or absence of an organism in a sample, each set shall contain 10 samples, all shipped at the same time in a lyophilized, dehydrated or aqueous state. The set shall include samples, in various combinations, that contain total coliforms, fecal coliforms, E. coli, non-coliforms, and at least one blank. Each set shall be used only with a single analytical method. For a PT result to be acceptable, the laboratory shall have no false negative results and no more than one false positive result for each set.
e) For quantitative methods, each set shall contain one sample. For a PT to be acceptable, the laboratory result shall be statistically acceptable as determined by the PT provider.
f) PT samples must be analyzed and reported within the PT provider's deadline. The PT provider shall submit the laboratory's results and acceptable ranges to the Department. No fee shall be charged to the Department for the analyses.
g) In the event of a failed or unacceptable PT set as defined in 465.200(d), the laboratory must request a quick turn-around PT from the provider.
(Source: Amended at 46 Ill. Reg. 19150, effective November 17, 2022)
Section 465.210 Authority of Certification Officers
Certification officers shall have all of the following authority with regard to environmental laboratories:
a) To inspect such laboratories in scheduled on-site visits and unannounced on-site visits or remote audits in emergency circumstances. Remote audits will be followed up with an on-site visit at a later date;
b) To require the laboratory to provide information regarding the technical operation of the laboratory relevant to certification;
c) To inspect quality assurance records and any other records pertinent to certification;
d) To observe and question analysts at work on parameters or methods for which certification is sought; and
e) To grant or deny certification based upon the completion of the evaluation process.
(Source: Amended at 46 Ill. Reg. 19150, effective November 17, 2022)
Section 465.220 Hearing, Decision and Appeal
The following procedures are established for Department certification actions which are required by law to be preceded by notice and opportunity for hearing:
a) Prior to revocation or partial revocation, the Department shall give written notice to the laboratory director or owner. This notice shall include a description of the proposed action, the facts or conduct upon which the Department will rely to support its proposed action, and the procedures for requesting a hearing.
b) Notice given under subsection (a) above and any hearing requested following issuance of such notice shall be in accordance with the Department's "Rules of Practice and Procedure in Administrative Hearings" (77 Ill. Adm. Code 100).
c) If, however, the Department finds that an emergency situation warrants immediate action, summary suspension as provided for by Section 10-65(d) of the Illinois Administrative Procedure Act [5 ILCS 100/10-65(d)] may be ordered pending revocation proceedings. An emergency situation warrants immediate action if there is substantial risk to public health, safety, or welfare resulting from laboratory deficiencies that are compromising or are likely to compromise the analytical results obtained.
d) A final decision of the Director is appealable to the Circuit Courts under the Illinois Administrative Review Law [735 ILCS 5/Art. III].
Section 465.230 Liability
Representatives of the Department shall not waive the right to seek recovery for injuries incurred while inspecting an environmental laboratory facility.
Section 465.240 Reciprocity Agreements
Notwithstanding any other provision of this Part, the Certification Program Manager of the Department may elect to enter into agreements with the governments of other states or with federal governmental units for recognition of their environmental laboratory inspections and certifications if such certification program uses equivalent controls over sample collection, data handling, quality control, analytical methods, and personnel as required of environmental laboratories within Illinois.
(Source: Amended at 46 Ill. Reg. 19150, effective November 17, 2022)
SUBPART B: MICROBIOLOGICAL ANALYSES OF PUBLIC WATER SUPPLY SAMPLES
Section 465.300 Scope and Applicability
This Subpart B establishes standards applicable to environmental laboratories involved in microbiological analyses of samples of water from public water supplies and their sources.
Section 465.310 Personnel Requirements
a) The microbiology laboratory supervisor shall have a minimum of a bachelor's degree in microbiology, biology, chemistry, or related natural or physical science field, shall have completed a training course conducted or approved by the Department, and shall have received Department approval to serve as laboratory supervisor. In addition, the laboratory supervisor shall have had a minimum of 80 hours of on-the-job training in water microbiology at a certified laboratory. The supervisor shall demonstrate the ability to properly perform representative test procedures under the supervisor's supervision while under observation by the certification officer. A laboratory supervisor shall be a full-time employee who is on-site at the certified laboratory. If the laboratory supervisor position becomes vacant, then a replacement supervisor shall be in place within 60 days.
b) The parasitology principal analyst/supervisor shall have a minimum of a bachelor's degree in microbiology or a closely related field, shall have a minimum of one year of bench experience with Cryptosporidium and immunofluorescence assay (FA) microscopy, have a minimum of six months experience using Method 1623 or 1623.1, and have analyzed a minimum of 100 samples using Method 1623 or 1623.1. The principal analyst/supervisor shall participate in a monthly analyst verification, shall supervise and verify the processing and microscopy in the laboratory, and may perform the same duties as an analyst. The principal analyst/supervisor shall ensure that all laboratory personnel are able to perform the analyses to which they are assigned and that all data reported by the laboratory meet the required quality assurance and regulatory criteria.
c) A microbiology analyst performs microbiological analyses on water, shall have a minimum of a high school diploma and shall have a minimum of 30 days of on-the-job training in drinking water microbiology under an experienced analyst. In addition, an analyst shall be able to perform representative test procedures with which the analyst is involved while under the observation of the certification officer. Analysts shall be under the direct supervision of the laboratory supervisor. Before analyzing compliance samples, the analyst shall demonstrate acceptable results on samples spiked with known culture controls.
d) A parasitology analyst establishes Kohler illumination for the microscope, may perform the same duties as a technician, and is able to examine samples using the microscope. An analyst shall have a minimum of two years of college with courses in microbiology or a closely related field, a minimum of six months of bench experience with Cryptosporidium and FA microscopy, and a minimum of three months of experience using Method 1623 or 1623.1. The analyst shall participate in a monthly analyst verification.
e) A parasitology technician filters samples, performs centrifugation, elution, concentration, and purification using immunomagnetic separation (IMS), and prepares purified samples on slides for microscopic examination, but does not perform microscopic protozoan identification. A technician shall have a minimum of three months of experience in filter extraction and processing of protozoa samples by Method 1623 or 1623.1 and have analyzed a minimum of 50 samples using Method 1623 or 1623.1 for the specific procedures that he or she will be using.
f) The Department may waive the need for the academic training required by this Section, on a case-by-case basis, for highly experienced analysts who hold a high school equivalency certificate.
g) The Department may waive the need for the college education and training required by this Section, on a case-by-case basis, for supervisors of microbiology laboratories that analyze samples from drinking water systems with which the laboratory is associated. The supervisor shall have a minimum of 10 years experience in water microbiology and shall have demonstrated a working knowledge of Quality Assurance activities as justification for the waiver.
h) The Department may waive college education in lieu of experience for a parasitology supervisor or analyst who has greater than 10 years experience of protozoan identification duties.
i) If a waiver is granted, the Department will prepare a written and signed justification for the waiver.
j) The Certification Officer shall have a minimum of a bachelor's degree in microbiology, biology, chemistry, or related natural or physical science field. The Certification Officer will have had a minimum of 80 hours of on-the-job training in water microbiology at a certified laboratory. Certification Officer shall have successfully completed the appropriate USEPA Laboratory Certification Officer Course and thereafter, audit the course every 5 years. The Certification Officer shall receive periodic training regarding newly promulgated regulations, newly adopted certification criteria, and new methods. This could be done by auditing the EPA Certification Officer Course or attending Regional or State Certification Officer Meeting. Certification Officer shall be a full-time employee of the Department.
k) The Program Certification Manager shall have a minimum of a bachelor's degree in microbiology, biology, chemistry, or related natural or physical science field, in addition will have had a minimum of 80 hours of on-the-job training in water microbiology at a certified laboratory. Program Certification Manager shall audit the appropriate USEPA Laboratory Certification Officer Course and thereafter, audit the course every 5 years. The Certification Program Manager will ensure a mechanism for Certification Officers to receive periodic training regarding newly promulgated regulations, newly adopted certification criteria and new methods. This could be done by auditing the EPA Certification Officer Course or attending Regional or State Certification Officer Meeting. Program Certification Manager shall be a full-time employee of the Department.
(Source: Amended at 46 Ill. Reg. 19150, effective November 17, 2022)
Section 465.320 Laboratory Facilities
The laboratory's physical facilities shall meet the following specifications:
a) A minimum of 150 square feet of floor space shall be provided for each analyst.
b) Floors shall be covered with asphalt tile, vinyl, concrete, or other impervious, washable surface that can be easily maintained.
c) Floor space shall be available for stationary equipment such as autoclaves, incubators, and hot-air sterilization ovens. Storage space that is free of dust and insects shall be provided for the protection of glassware, media, and portable equipment.
d) Laboratories analyzing potable water, non-potable source water and recreation water, and sewage by microbiological methods shall have at least two separate rooms (a room for potable water, non-potable source water and recreation water, and a room for sewage).
e) A separate bench for preparation and sterilization of media, glassware, and equipment shall be provided.
f) Walls shall be covered with waterproof paint, enamel, ceramic tile, or other surface material that provides a smooth finish that is easily cleaned and disinfected. Ceilings shall be maintained in good condition.
g) A minimum of 6 linear feet of useable bench space, free of equipment, shall be provided for each analyst.
h) Bench tops shall be stainless steel, epoxy plastic, or other smooth, impervious material that is inert, is corrosion resistant, has a minimum number of seams, and is level.
i) Laboratory lighting shall be even and provide a minimum of 100 footcandle light intensity at all working surfaces.
j) The laboratory shall include a sink with hot and cold running water. All water supply outlets shall be protected by a backflow prevention device as specified in the Illinois Plumbing Code (77 Ill. Adm. Code 890).
k) Laboratories shall be well ventilated and free of dusts, drafts, and extreme temperature changes. Central air-conditioning is recommended to reduce contamination, permit more stable operation of incubators, and decrease moisture problems with media and analytical balances. The temperature within the laboratory shall be maintained at between 60º and 80º F.
l) An adequate electrical supply for operation of instruments and mechanical needs shall be provided. The certification officer may require verification from an official inspector or other qualified person that the laboratory meets local and national electrical codes.
m) All electrical outlets shall be properly grounded.
n) Instruments shall be properly grounded with an internal or external regulated power supply available to each instrument.
o) All plumbing shall comply with the Illinois Plumbing Code or any local plumbing code that is more stringent than the Illinois Plumbing Code. The certification officer may require verification from an official inspector or other qualified person that the laboratory meets such codes.
p) The laboratory shall include a vacuum source for use in membrane filter procedures.
q) The laboratory shall be located in an area sufficiently free from noise and vibrations to prevent interference with its functions.
r) The laboratory shall have a readily available source of laboratory pure water.
s) The laboratory shall not be located within a structure that is used as a residence.
t) No mobile laboratories shall be allowed.
u) The laboratory shall have provisions for the disposal of microbiological waste in accordance with Department standards.
v) No food or drink for consumption shall be located in laboratory area, refrigerators, freezers or other equipment.
(Source: Amended at 46 Ill. Reg. 19150, effective November 17, 2022)
Section 465.330 Laboratory Equipment
Only those instruments that are needed to analyze for the parameters for which the laboratory is being certified are required, but those instruments shall meet the following minimum specifications. A laboratory performing all of the analyses described in Section 465.360 shall have, or have access to, within the same building, all of the equipment listed in this Section with the minimum specifications cited.
a) A top loading or trip pan balance shall be clean, not corroded.
1) A torsion or trip pan balance used for weighing materials of 2 grams or more shall detect 100 mg of weight accurately at a 150 gram load.
2) An analytical balance used for weighing quantities of less than 2 grams shall be sensitive to 1 mg at a 10 gram load.
b) A magnetic stirrer shall be capable of achieving variable speeds and shall be used with a Teflon-coated stirring bar. The magnetic stirrer may be equipped with a heating element.
c) A pH meter shall have an accuracy of at least ± 0.1 units and a scale readability of at least ± 0.1 units. The pH meter may be either line/bench or battery/portable operated. pH meter must be able to calculate slope.
d) A conductivity meter and cell combination, suitable for checking laboratory pure water quality, shall be readable in ohms or mhos, and have a range capable of determining the conductivity or resistivity of laboratory pure water as described in Section 465.380(a). The conductivity meter may be either line/bench or battery/portable operated.
e) An autoclave shall be horizontal-chambered and shall meet all of the following specifications:
1) When observed during the operational cycle or when time-temperature charts are read, the autoclave shall be in good operating condition;
2) An operating safety valve shall be included;
3) Separate temperature and pressure gauges shall be located on the exhaust side;
4) The autoclave shall reach and maintain a temperature of 121º ± 1º C during the sterilization cycle, and no more than 45 minutes shall be required for a complete cycle of carbohydrate media;
5) Depressurization shall not produce gas bubbles in fermentation media; and
6) Pressure cookers shall not be used.
f) A hot-air sterilization oven shall operate at a minimum of 175º C, shall be equipped with a thermometer inserted through the top porthole or be equipped with a temperature-recording device, and shall be equipped with a thermostatic control that will not allow the temperature to deviate by more than ± 5º C from the temperature setting.
g) An incubation unit shall maintain an internal temperature of 35º ± 0.5º C or 44.5º ± 0.2º C and shall be of the following type: air or water jacketed incubator, incubator room, water bath, or aluminum block incubator. Incubation units of the aluminum block type shall have culture dishes and tubes that are snug fitting in the block. Water baths shall be circulating with covers. Laboratories that use the enzyme substrate tests with air-type incubators shall note the product incubation details indicated in Section 465.360(j)(7).
h) An ultraviolet (UV) sterilizer shall be free from radiation leaks and shall be UV efficiency tested quarterly as described in "Standard Methods for the Examination of Water and Wastewater." Proper eye protection shall be available for users of the ultraviolet sterilizer. The ultraviolet sterilizer shall not be used as a substitute for an autoclave. The unit shall be disconnected monthly and the lamps cleaned by wiping with a soft cloth moistened with ethanol.
i) A refrigerator shall maintain a temperature of between 1º and 5º C and shall be equipped with a thermometer located on the top shelf. The thermometer shall be graduated in not greater than 1º C increments, and the thermometer bulb shall be immersed in liquid unless otherwise specified by the manufacturer of the temperature monitoring system.
j) An agar tempering water bath shall be of appropriate size for holding melted medium and shall be thermostatically controlled at 45º ± 1º C.
k) The following standards shall apply to temperature-monitoring devices:
1) Glass or electronic thermometers shall be graduated in not greater than 0.5º C units for use in 35º C incubators.
2) Glass or electronic thermometers shall be graduated in not greater than 0.2º C units for use in 44.5º C water baths or aluminum block type incubators.
3) Glass or electronic thermometers shall be graduated in not greater than 1.0º C units for use in spore incubators required for autoclave quality control.
4) Electronic thermometers with thermocouplings and continuous temperature-recording devices shall be sensitive to not greater than 0.5º C when used in 35° C incubators, shall be sensitive to not greater than 0.2º C when used for 44.5º C water baths or aluminum block type incubators, and shall be sensitive to not greater than 1º C when used in spore incubators required for autoclave quality control.
5) An NIST certified thermometer, or one of equivalent accuracy graduated in 0.2º C or less, shall be available for calibration use and shall be accompanied by its certification papers and procedures for use. All thermometers and temperature-recording devices shall be calibrated annually at temperature of use against the certified thermometer to within ± 1.0º C.
6) Each laboratory operating an autoclave shall have either a maximum registering thermometer, an autoclave with an internal digital temperature record, or a datalogger in the range of 80º to 200º C graduated in increments no greater than 1º C.
7) Each laboratory shall use separate thermometers for determining the temperatures of water baths, ovens, autoclaves, samples, refrigerators, storage areas, etc.
8) The liquid column of glass thermometers shall have no separations.
9) Dial and infrared thermometers are not permitted.
l) Optical counting equipment shall include a low-power magnification device of the dissecting or stereomicroscope type with a magnification power of 10 to 15 diameters, and an external daylight fluorescent light source for sheen discernment at an angle of 60º to 80º above the colonies.
m) A mechanical hand tally shall be available for counting colonies on membrane filters or agar pour plates.
n) Where metal inoculation loops are used, loops shall be of 22 to 24 gauge chrome, or platinum-iridium wire, with loop diameters of at least 3 mm. Hot-air sterilized wooden applicator sticks, pre-sterilized cotton swabs or pre-sterilized plastic loops may be used.
o) Membrane filter equipment shall be non-leaking, uncorroded, and made of stainless steel, glass, or autoclavable plastic. Disposable single-use equipment made of plastic is also acceptable. Metal plating on membrane filter equipment shall not be worn so as to expose base metal.
p) Membrane filters shall be white, grid marked, 47 mm diameter, with 0.45 micron pore size, and made from cellulose ester materials. Another pore size may be used if the manufacturer gives performance data equal to or better than the 0.45 micron membrane filter. Membrane filters shall be autoclavable or presterilized.
q) Absorbent pads shall be of uniform thickness to permit 1.8 to 2.2 mL media absorption and shall be autoclavable or presterilized. Filter paper shall be free from growth-inhibiting substances.
r) Forceps used to handle membrane filters and absorbent pads shall have a round tip without corrugations.
(Source: Amended at 46 Ill. Reg. 19150, effective November 17, 2022)
Section 465.340 Laboratory Glassware, Plastic Ware and Metal Utensils
a) Except for disposable plastic ware, items shall be resistant to effects of corrosion, high temperature, and vigorous cleaning operations. Metal utensils made of stainless steel are preferred. Plastic items shall be of inert, non-toxic material and shall retain accurate graduations or calibration marks after repeated autoclaving. Glassware that is used for purposes that may subject it to damage from heat or chemicals shall be of borosilicate glass. All glassware shall be free of chips, cracks, or excessive etching.
b) Graduated cylinders for measurement of sample volumes and precalibrated sample containers shall have a tolerance of 2.5% or less.
c) Media-preparation utensils shall be of borosilicate glass or stainless steel, and shall be clean and free from foreign residues or dried medium.
d) Micropipettors (also referred to as Mechanical Pipettors or Pipettors) shall meet the specifications set forth in "Standard Methods for the Examination of Water and Wastewater." Pipets delivering volumes of 10 mL or less shall be accurate to within a 2.5% tolerance. Micropipettors shall be fixed volume and calibrated. Micropipettors shall be used with tips that are sterile. Containers for glass pipets shall be of either stainless steel or aluminum. Opened packages of sterile disposable pipets shall be securely resealed between uses. A pipet aid shall be used when using pipets; mouth pipetting is prohibited. The pipet shall be clean and dry. Pipet aids used to pipet outside of the certified water microbiology testing laboratory shall not be used.
e) Culture dishes shall be sterile and shall be of the tight-lid or loose-lid plastic or loose-lid glass type. In addition, culture dishes shall be of 100 mm x 15 mm (for Plate Count), 50mm x 12 mm, 60 mm x 15 mm, or other appropriate size (for membrane filter methods), and shall be clear, flat bottomed, and free from bubbles and scratches. To maintain sterility, containers for glass culture dishes shall be of aluminum or stainless steel, or glass culture dishes shall be wrapped in heavy aluminum foil or char-resistant paper. Open packages of sterile disposable culture dishes shall be securely resealed between uses. Loose-lid dishes shall be incubated in a tight-fitting container, e.g., a plastic vegetable crisper containing a moistened paper towel, to prevent dehydration of membrane filter and medium.
f) Culture tubes shall be of borosilicate glass or other corrosion-resistant glass, and shall be of sufficient size to contain culture medium, as well as the sample portions employed, without being more than three-fourths full. Culture tube closures shall be loose-fitting stainless steel, or plastic caps, or aluminum caps, or plastic screw caps with non-toxic liners. Cotton plugs and foam plugs shall not be used.
g) Dilution bottles shall be of borosilicate glass or other corrosion-resistant glass or autoclavable plastic and shall be free of chips and cracks at the lip. A graduation level shall be distinctly marked on the side of dilution bottles at 99 mL. Dilution bottle closures shall be plastic screw caps with leak-proof liners and shall not produce toxic substances during the sterilization process.
h) Sample bottles shall be sterile, of plastic or hard glass, and wide mouthed, and shall have a capacity of at least 120 mL (4 oz.) to allow at least a 1-inch head space. Reusable sample bottle closures shall be glass stoppers or screw caps (metal or plastic), capable of withstanding repeated sterilization, with leak-proof liners, and shall not produce toxic substances during the sterilization process. Glass-stoppered bottle closures shall be covered with aluminum foil or char‑resistant paper for sterilization. Metal caps with exposed bare metal on the inside shall not be used. Presterilized containers including bags, with or without a dechlorinating reagent, may be used.
(Source: Amended at 46 Ill. Reg. 19150, effective November 17, 2022)
Section 465.350 General Laboratory Practices
a) The following requirements shall apply to sterilization procedures:
1) Autoclaving of the following items shall be carried out at 121º ± 1º C for the durations specified below:
|
Item |
|
Minimum duration of autoclaving at 121° ± 1° C |
|
Membrane filters and pads |
|
10 minutes |
|
|
|
|
|
Carbohydrate-containing media (lauryl tryptose, brilliant green lactose bile broth, etc.) |
|
12-15 minutes |
|
|
|
|
|
Contaminated materials and discarded tests |
|
30 minutes |
|
|
|
|
|
Membrane filter assemblies (wrapped), sample collection bottles (empty), and individual glassware items |
|
15 minutes |
|
|
|
|
|
Rinse water volumes of 500 mL to 1000 mL |
|
45 minutes |
|
|
|
|
|
Rinse water volumes in excess of 1000 mL |
|
Time adjusted for volume; check for sterility |
|
|
|
|
|
Dilution water blanks |
|
15 minutes |
2) Membrane filters and pads and all media shall be removed from the autoclave immediately after completion of the sterilization cycle.
3) The maximum elapsed time for exposure of carbohydrate-containing media to any heat (from the time of closing the loaded autoclave to unloading) shall be 45 minutes.
4) Membrane filter assemblies shall be autoclaved between each sample filtration series. A UV sterilizer or boiling water may be used on membrane filter assemblies for at least two minutes to prevent bacterial carryover between sample filtrations, but shall not be used as a substitute for autoclaving between sample filtration series.
5) Dried glassware to be sterilized in a hot-air sterilizing oven shall be kept at 175º ± 5º C for at least 2 hours.
6) Empty sample containers shall be moistened with several drops of distilled water before autoclaving to prevent an "airlock" sterilization failure.
b) Laboratory pure water, which may be distilled or deionized, or other processed water shall meet the standards set forth in Section 465.380. Only water determined to be laboratory pure water shall be used for performing bacteriological analyses.
c) Rinse and dilution water shall be prepared in the following manner:
1) A stock phosphate buffer solution of potassium dihydrogen phosphate (KH2PO4) and a magnesium chloride solution shall be prepared as specified in "Standard Methods for the Examination of Water and Wastewater." The pH of stock phosphate buffer solution is 7.2 ± 0.5.
2) The phosphate buffer solution and magnesium chloride solution shall be autoclaved or filter sterilized, labeled, dated, and stored at 1º to 5º C.
3) The stored stock phosphate buffer solution and magnesium chloride solution shall be free of turbidity.
4) Rinse and dilution water shall be prepared by adding 1.25 mL of stock phosphate buffer solution and 5.0 mL of magnesium chloride solution per liter of laboratory pure water.
5) Alternatively, commercially prepared phosphate buffer and magnesium chloride solution may be used when preparing rinse and dilution water. The date received, expiration date, proof of sterility, and pH of phosphate buffer shall be recorded.
d) The following minimum requirements shall be met for storing and preparing media:
1) Laboratories shall use commercial dehydrated media or commercially manufactured prepared media for routine bacteriological procedures.
2) All media shall be prepared according to the media specifications of "Standard Methods for the Examination of Water and Wastewater."
3) Dehydrated media containers shall be kept tightly closed and stored in a cool, dry location. Discolored or caked dehydrated media shall not be used.
4) All water used shall be laboratory pure water.
5) Dissolution of the media shall be completed before dispensing to culture tubes or bottles.
6) Multiple Tube Fermentation (MTF) media, when prepared in tubes with loose-fitting caps, shall be used within one week after preparation. If MTF media are refrigerated after sterilization, they shall be incubated overnight at 35º C to confirm usability. Tubes of MTF media showing growth or gas bubbles shall be discarded. Refrigerated M Endo agar shall be used within two weeks after refrigeration.
7) MTF media in screw cap containers may be held up to three months, provided that the media are stored in the dark and evaporation does not exceed 1.0 mL per 10 mL total volume.
(Source: Amended at 38 Ill. Reg. 16240, effective July 15, 2014)
Section 465.360 Methodology
A laboratory shall be certified for all analytical methods listed in subsection (a) that it uses for compliance purposes. At a minimum, the laboratory shall be certified for one total coliform method and one fecal coliform/E. coli method. In addition, for laboratories that may enumerate heterotrophic bacteria (as measured by the Heterotrophic Plate Count) for compliance with the Surface Water Treatment Rule (SWTR), the laboratory shall be certified for either the Pour Plate Method or the SimPlate method for heterotrophic bacteria.
a) The following methodology with the exception of 9221D and Chromocult, as specified in the listed references, shall be followed for individual parameters:
1) Analytical Methods Approved for Compliance Monitoring under the Revised Total Coliform Rule – 40 CFR 141.852(a)(5). https://www.epa.gov/sites/default/files/2017-02/documents/rtcr_approved_methods.pdf
2) Analytical Methods Approved for Compliance Monitoring under the Surface Water Treatment Coliform Rule – 40 CFR 141.74(a)(1). https://nepis.epa.gov/Exe/ZyPDF.cgi?Dockey=P100WD7G.txt
3) Analytical Methods Approved for Compliance Monitoring under the Ground Water Rule – 40 CFR 141.402(c)(2).
https://nepis.epa.gov/Exe/ZyPDF.cgi?Dockey=P100WD6V.txt
4) Appendix A to Subpart C of Part 141. https://ecfr.io/Title-40/Part-141/Appendix-A#40:25.0.1.1.3.3.16.10.5
b) Water samples shall be shaken vigorously at least 25 times in a complete up and down or back and forth movement.
c) Sample volume analyzed for total coliforms in drinking water shall be 100 mL.
d) Aseptic practices shall be used for all microbiological procedures.
e) All samples shall be handled as though they are positive and have the potential to contaminate other samples if handled improperly. All spills shall be promptly disinfected.
f) Fermentation broth methods: The water level of the water bath shall be above the upper level of the medium in the culture tubes.
g) Multiple tube fermentation technique (for detecting total coliforms in drinking water and enumerating total coliforms in source water):
1) For drinking water samples: Various testing configurations can be used (Standard Methods 9221B), as long as a total sample volume of 100 mL is examined for each test.
2) For source water samples: Laboratories shall use at least three series of five tubes each with appropriate sample dilutions of source water (e.g., 0.1 mL, 0.01 mL, 0.001 mL).
h) Media
1) Lauryl tryptose broth (LTB) (also known as lauryl sulfate broth) shall be used in the presumptive test and 2% brilliant green lactose bile broth (BGLBB) in the confirmed test. Lactose broth (LB) may be used in lieu of LTB (40 CFR 141.21(O)(3)) if the laboratory conducts at least 25 parallel tests between this medium and LTB using the waters normally tested, and if this comparison demonstrates that the false positive rate and false negative rate for total coliforms, using LB, is less than 10%. This comparison shall be documented and the records retained. The final pH shall be 6.8 ± 0.2 for LTB, and 7.2 ± 0.2 for 2% BGLBB.
2) The test medium concentration shall be adjusted to compensate for the sample volume so that the resulting medium after sample addition is single strength. If a single 100-mL sample volume is used, the inverted vial shall be replaced with an acid indicator (bromcresol purple) to prevent problems associated with gas bubbles in large inverted tubes. The media shall be autoclaved at 121° C for 12 to 15 minutes.
3) Sterile media in tubes shall be examined to ensure that the inverted vials, if used, are free of air bubbles and are at least one-half to two-thirds covered after the water sample is added.
4) After the medium is inoculated, it shall be incubated at 35° ± 0.5° C for 24 ± 2 hours. If no gas or acid is detected, it shall be incubated for another 24 hours (total incubation time 48 ± 3 hours).
5) Each 24- and 48-hour tube that contains growth, acid, or gas shall be confirmed using 2% BGLBB. A completed test is not required.
6) For drinking water samples: Each total coliform positive sample shall be tested for the presence of E. coli.
i) Invalidation of total coliform-negative samples
1) For drinking water samples: All samples that produce a turbid culture (i.e., heavy growth) in the absence of gas/acid production, in LTB or LB, shall be invalidated. The laboratory shall collect, or request that the system collect, another sample from the same location as the original invalidated sample within 24 hours. Before invalidation, the laboratory may perform a confirmed E. coli test on the total coliform‑negative culture to check for coliform suppression. If the confirmed test is coliform positive or E. coli is detected, the sample shall be reported as such. An E. coli-positive result is considered a total coliform positive, E. coli‑positive sample, even if the presumptive or confirmed total coliform test is negative. If the follow-up test or tests are negative, the sample shall be invalidated because high levels of non-coliform bacteria in the presumptive tubes may have injured, killed, or suppressed the growth of any coliforms in the sample.
2) For source water samples: All samples that produce a turbid culture (i.e., heavy growth) in the absence of gas/acid production, in LTB or LB, shall be invalidated. The laboratory shall collect, or request that the system collect, another sample from the same location as the original invalidated sample. Before invalidation, the laboratory may perform a confirmed test on the total coliform-negative culture. If the confirmed test is total coliform positive, the most probable number shall be reported. If the test is total coliform negative, the sample shall be invalidated.
j) Enzyme (chromogenic/fluorogenic) substrate tests
1) For detecting total coliforms and E. coli in drinking water samples, a laboratory may use the MMO-MUG test (Colilert), Colisure test, E*Colite test, Readycult Coliforms 100 Presence/Absence Test, or Modified ColitagTM test. These tests, known as enzyme substrate tests, may be available in various configurations. For enumerating total coliforms in source water, a laboratory may use the Colilert test. If a laboratory uses a fermentation method to detect total coliforms in drinking water, and the sample is total coliform positive, the laboratory may transfer the positive culture to the EC+MUG test to detect E. coli, but not to any other enzyme substrate test medium in this Section.
2) Media shall not be prepared from basic ingredients, but rather from a commercially available source.
3) Media shall be protected from light.
4) Some lots of enzyme substrate media have been known to fluoresce. Each lot of medium shall be checked before use with a 365-366 nm ultraviolet (UV) light with a 6-watt bulb. For checking Colilert, Colilert-18, Colisure, Readycult, and Modified ColitagTM media, a packet of medium shall be dissolved in sterile water in a non-fluorescing vessel. If the medium exhibits faint fluorescence, the laboratory shall use another lot that does not fluoresce.
5) If the samples plus the medium exhibit an inappropriate color change before incubation, they shall be discarded and another lot of medium used. The laboratory shall notify the medium vendor and request another water sample from the water system. Before incubation, Colilert, Colilert-18, and Modified ColitagTM shall appear colorless to a slight tinge of color, while Colisure and E*Colite are yellow and Readycult shall appear slightly yellow.
6) Glass and plastic sample bottles and test tubes shall be tested before use with a 365-366 nm UV light source with a 6-watt bulb to ensure that they do not fluoresce. If they fluoresce, another lot of containers that do not fluoresce shall be used.
7) Air-type incubators may not bring a cold 100 mL water sample or samples to the specified incubation temperature for several hours. The problem may cause false negative results with the enzyme substrate tests and possibly other tests as well. Laboratories with air-type incubators shall observe the following instructions for chromogenic/fluorogenic substrate test:
|
Test |
Pre-incubation sample instructions 1,2 |
|
Colilert (Presence/Absence) |
Specified 24-hour incubation time includes time it takes to bring sample temperature up to 35° ± 0.5° C 1 |
|
Colilert Quanti-Tray |
Specified 24-hour incubation time includes time it takes to bring sample temperature up to 35° ± 0.5° C |
|
Colilert-18 (Presence/Absence) |
Prewarm sample in 35° ± 0.5° C water bath for 20 minutes or 44.5° C for 7-10 minutes |
|
Colilert-18 Quanti-Tray |
Allow sample to equilibrate to room temperature (20-30° C) before beginning 18-hour incubation time |
|
Colisure |
Allow sample to equilibrate to room temperature (20-30° C) before beginning 24-hour incubation time |
|
Readycult Coliforms |
Specified 24-hour incubation time includes time it takes to bring sample temperature up to 35° ± 0.5° C |
|
Modified ColitagTM |
If results are to be read before 22 hours, sample must be prewarmed in a 44.5° C water bath for 7-10 minutes |
1 Samples shall be brought to room temperature before incubation.
2 Information based on manufacturer's instructions.
8) If a water bath is used, the water level shall be above the upper level of the medium.
9) For E. coli testing, the laboratory shall place all total coliform-positive samples under an ultraviolet lamp (365-366 nm, 6-watt) in a darkened area. If E. coli is present, the medium will emit a blue fluorescence.
10) The enzyme substrate tests shall not be used to confirm a presumptive total coliform-positive result that was obtained in fermentation broth (e.g., LTB, LB) or on a membrane filter.
11) Any sample that produces an atypical color change (e.g., greenish black or black) shall be invalidated.
12) Any reference comparator provided by the manufacturer shall be discarded by the manufacturer's expiration date.
13) For the Colilert test, samples shall be incubated at 35° ± 0.5° C for 24 hours. A yellow color in the medium equal to or greater than the reference comparator indicates that the sample is total coliform positive. If the sample is yellow, but lighter than the comparator, it shall be incubated for another four hours. If the color is still lighter than the reference comparator at 28 hours, the sample shall be reported as negative. After 28 hours negative results are still considered valid, but positive results are not. A coliform-positive sample that fluoresces under an ultraviolet (UV) light indicates the presence of E. coli. Laboratories that use the Colilert-18 test shall incubate samples for 18 hours (up to 22 hours if the sample after 18 hours is yellow, but is lighter than the comparator). After 22 hours negative results are still considered valid, but positive results are not.
14) For enumerating total coliforms in source water with the Colilert, Colilert 18 test, Quanti-Tray, or Quanti-Tray 2000 may be used for each sample tested. Dilution water (if used) may be sterile deionized or sterile distilled water, but not buffered water.
15) If the Quanti-Tray or Quanti-Tray 2000 test is used, the sealer shall be checked monthly by adding a dye (e.g., bromcresol purple) to the water. If dye is observed outside the wells, maintenance shall be performed or another sealer shall be used.
16) For the Colisure test, samples shall be incubated at 35° ± 0.5° C for 24 hours. If an examination of the results at 24 hours is not convenient, then results may be examined at any time up to 48 hours. After 48 hours, negative or positive results are considered invalid. If the medium changes from a yellow color to a red/magenta color, the sample is total coliform positive. A coliform positive sample that fluoresces under a UV light indicates the presence of E. coli.
17) For the E*Colite test, samples shall be incubated at 35° ± 0.5° C for 28 hours. If total coliforms are present, the medium changes from a yellow color to a blue or blue-green color, or a blue color in the corners of the bag. If E. coli is present, the medium will fluoresce under a UV light. If no fluorescence is observed, the sample shall be re-incubated for an additional 20 hours (for a total incubation time of 48 hours) and again checked for fluorescence. If the medium becomes red, it shall be assumed that a faulty seal has allowed the bactericide (in the third compartment of the bag) to leak into the compartment containing the medium. In this case, the sample shall be discarded and another sample shall be requested.
18) For the Readycult Coliforms 100 Presence/Absence test, the contents of a snap pack shall be added to a 100-mL water sample, followed by incubation at 35° ± 0.5° C for 24 ± 1 hours. If coliforms are present, the medium changes color from a slightly yellow color to blue-green. In addition, if E. coli is present, the medium will emit a bright light-blue fluorescence when subjected to a long wave (365-366 nm) UV light. If confirmation of E. coli is desired, Kovac's indole reagent shall be added to the broth; the immediate formation of a red ring confirms the presence of E. coli.
19) For the Modified ColitagTM test, samples shall be incubated at 35° ± 0.5° C for 16-48 hours. If results are to be read before 22 hours, the sample must be prewarmed in a 44.5° C water bath for 7-10 minutes. During incubation, trimethylamine-N-oxide in the Modified ColitagTM medium causes the pH of the medium to increase from 6.2 to 6.8-7.2. A yellow color in the medium indicates the presence of total coliforms. A coliform-positive sample that fluoresces under a UV light indicates the presence of E. coli.
k) Membrane filter (MF) methods
1) For source water samples (SWTR): To optimize counting, appropriate sample dilutions shall be used to yield 20 to 80 total coliform colonies or 20 to 60 fecal coliform colonies for at least one dilution or volume.
2) At least one membrane filter and filtration unit sterility check shall be conducted at the beginning and the end of each filtration series (unless using single use disposable funnels) by filtering 20 to 30 mL of dilution water through the membrane filter and testing for growth. If the control indicates contamination, all data from affected samples shall be rejected and an immediate resampling shall be requested. A filtration series ends when 30 minutes or more elapse between sample filtrations.
3) Each filtration funnel shall be rinsed after each sample filtration with two or three 20 to 30 mL portions of sterile rinse water to ensure that the entire sample is rinsed off the funnel before the filter is removed. After the filter is removed, the funnel may be rinsed again with two or three 20 to 30 mL portions of sterile rinse water or exposed to UV light with a 254-nm wavelength for at least two minutes to prevent carryover between samples, especially for surface water samples.
4) Absorbent pads shall be saturated with a liquid medium (at least 2 mL of broth) and excess medium removed by decanting the plate.
5) Membrane filters shall be handled with sterile forceps that are sterilized before each use by dipping in 95% ethyl or absolute methyl alcohol and flaming. The membrane filters shall be grasped outside the effective filtration area.
l) Media used for detecting total coliforms and E. coli in drinking water, enumerating total coliforms or fecal coliforms in source water, and detecting E. coli in ground water.
1) Using M-Endo medium agar or broth (also known as M-Endo broth MF and M-Coliform broth) or LES Endo agar (also known as M-Endo agar LES) for detecting total coliforms in drinking water or enumerating total coliforms in source water: Medium may be used in the single step or enrichment techniques. Ethanol used in the rehydration procedure shall not be denatured. Medium shall be prepared in a sterile flask and brought just to the boiling point with a boiling water bath or, if constantly attended, a hot plate with a stir bar. The medium shall not be boiled. Final pH shall be 7.2 ± 0.2 for M-Endo Agar LES and Endo medium.
2) Using m-ColiBlue24 medium for detecting total coliforms and E. coli in drinking water: Purchase this medium from a commercial vendor, it cannot be prepared from basic ingredients. Ampules of broth shall be inverted two to three times to mix contents before breaking. Then, contents shall be poured evenly over absorbent pad. Unopened refrigerated ampules may be stored in the dark until the expiration date, but shall be discarded earlier if growth is observed. The final pH of the medium shall be 7.0 ± 0.2.
3) Using MI medium (with or without agar) for detecting total coliforms and E. coli in drinking water or enumerating total coliforms in source water: Commercially made pre-sterilized bottled MI agar or broth shall not be autoclaved. Bottled agar shall be melted in a boiling water bath or by other processes recommended by the manufacturer. As soon as complete melting has occurred, the medium shall be cooled slightly and immediately poured into sterile plates. Care shall be taken to prevent overheating the agar, as excessive heat destroys the effectiveness of the antibiotic cefsulodin. If dehydrated culture medium is used, it shall be prepared and autoclaved according to the manufacturer's instructions. The agar shall be cooled, freshly prepared filter-sterilized cefsulodin shall be added, and the mixture shall be immediately poured into sterile plates. The final pH of MI agar shall be 6.95 ± 0.2; the final pH of MI broth shall be 7.05 ± 0.2. The preparation and use of MI agar and MI broth are referenced in Section 465.125(a)(4). EPA Method 1604, which can be found online at www.epa.gov/microbes, is identical.
4) m-FC broth (with or without agar) for enumerating fecal coliforms in source water shall not be autoclaved. The medium shall be brought just to the boiling point. The final pH shall be 7.4 ± 0.2.
5) When stored, prepared medium shall be refrigerated. Petri dishes containing medium shall be stored in a plastic bag or tightly closed container, and used within two weeks. Before use, refrigerated sterilized medium shall be brought to room temperature. Plates with laboratory‑prepared broth medium shall be discarded after 96 hours, poured MF agar plates discarded after two weeks, and ampules of M-Endo broth and other prepared media discarded in accordance with the manufacturer's expiration date. Broth, plates, or ampules shall be discarded earlier if growth or (for M-Endo agar) surface sheen is observed. The date and time prepared shall be recorded.
6) Incubation conditions and colony color of inoculated medium
|
Medium |
Incubation |
Total coliforms |
E. coli |
|
M-Endo medium or M-Endo agar LES |
35° ± 0.5° C for 22-24 hrs |
Metallic (green-golden) sheen colonies (presumptive) |
N/A |
|
m-ColiBlue24 |
35° ± 0.5° C for 24 hrs |
Count all red and blue to purple colonies under normal/ambient light and record as the total coliform result. |
Count only blue to purple colonies and record as E. coli result. |
|
MI1 |
35° ± 0.5° C for 24 ± 2 hrs |
Fluorescent colonies under UV light |
Blue colonies under normal light |
|
m-FC |
44.5° ± 0.2° C for 24 ± 2 hrs |
N/A |
Blue colonies (fecal coliforms) |
1 If any blue, non-fluorescent colonies are found on the same plate, add their total to the Total Coliform count.
m) Invalidation of a total coliform-negative drinking water sample: All samples resulting in confluent or TNTC (too numerous to count) growth shall be invalidated unless total coliforms are detected. If no total coliforms are detected, the sample shall be recorded as "confluent growth" or "TNTC" and an additional sample shall be requested from the same sampling site. Confluent growth is defined as a continuous bacterial growth covering the entire membrane filter without evidence of total coliform type colonies. TNTC is defined as greater than 200 colonies on the membrane filter in the absence of detectable coliforms. Laboratories shall not invalidate samples when the membrane filter contains at least one coliform type colony (i.e., sheen colony for M-Endo medium, red or blue colony for m-ColiBlue24 agar, fluorescent or blue colony for MI agar. Before invalidation, the laboratory shall perform a verification test on the total coliform negative culture, i.e., on confluent or TNTC growth, and an E. coli test. If the verification test is total coliform positive, the sample shall be reported as total coliform positive. If the test is total coliform negative, the sample shall be invalidated. An E. coli positive result is considered a total coliform-positive, E. coli positive sample, even if the sample tests negative for total coliform.
n) Invalidation of source water samples (SWTR): Laboratories shall invalidate any sample that results in confluent growth or TNTC, even when total coliform or fecal coliform colonies are present.
o) For drinking water samples (to verify colonies on Endo-type medium): The entire surface of the membrane filter shall be wiped with a sterile cotton swab and the verification media (LTB, then BGLBB) shall be inoculated. Alternatively, at least five typical sheen colonies and five nontypical colonies shall be verified using either single strength lactose broth (LB) or lauryl tryptose broth (LTB) and then single strength 2% brilliant green lactose bile broth (BGLBB). In addition, sheen colonies may be verified rapidly using a cytochrome oxidase and b-galactosidase procedure. Individual colonies can be transferred with a sterile needle or loop, or applicator stick. If no sheen colonies are observed, up to five red questionable sheen colonies and up to five red non-sheen colonies representing different morphological types shall be verified.
p) For drinking water samples: Total coliform-positive colonies shall be tested for E. coli. The membrane filter tests approved by USEPA do not require additional media for such a test, except for those using Endo-type medium (M-Endo medium or M-Endo agar LES). USEPA has approved several options for testing a total coliform-positive colony on Endo-type medium for E. coli. When coliforms or EC Medium-MUG is used, the colonies shall be transferred by employing one of the options specified by the Total Coliform Rule at 40 CFR 141.21(f)(5) (see Appendix G of the USEPA Manual for the Certification of Laboratories Analyzing Drinking Water). For the swab technique, a single swab can be used to inoculate a presumptive total coliform-positive culture into three different media, EC-MUG Medium, LTB, and BGLBB, in that order. If Nutrient Agar-MUG is used, the Nutrient Agar-MUG section shall be followed.
q) For source water samples: Initial total coliform counts shall be adjusted based upon verified data, as in Standard Methods, Section 9222B(5).
r) Nutrient Agar-MUG Test (for detection of E. coli in drinking water or ground water)
1) Medium shall be autoclaved at 121° ± 1° C for 15 minutes. MUG may be added to Nutrient Agar before autoclaving. Nutrient Agar-MUG is also available commercially. The final MUG concentration shall be 100 µg/mL. The final pH shall be 6.8 ± 0.2.
2) Positive and negative controls shall be tested as stated in Section 465.400(p). Control cultures shall be filtered or spot-inoculated onto a membrane filter on M-Endo agar LES or M-Endo broth or agar, and shall be incubated at 35° ± 0.5° C for 22 to 24 hours. The filter shall then be transferred to Nutrient Agar-MUG and incubated at 35° ± 0.5° C for another four hours. The results shall be read and recorded.
3) The membrane filter containing a coliform colony or colonies shall be transferred from the total coliform medium to the surface of Nutrient Agar-MUG medium. Each sheen colony shall be marked with a permanent marker on the lid. Also, the lid and the base shall be marked with a line to realign the lid if it is removed. A portion of the colony may be transferred with a needle to the total coliform verification test before transfer to Nutrient Agar-MUG or after the 4-hour incubation time. Another method is to swab the entire membrane filter surface with a sterile cotton swab after the 4-hour incubation time on Nutrient Agar-MUG medium, and transfer to a total coliform verification test.
4) The fluorescence shall be checked using an ultraviolet lamp (365-366 nm) with a 6-watt bulb in a darkened area. Any amount of fluorescence in a halo around a sheen colony shall be considered positive for E. coli.
s) Heterotrophic Plate Count (for enumerating heterotrophic bacteria in drinking water)
1) The Pour Plate Method (Standard Methods 9215B) or the SimPlate (Standard Methods 9215E) Method shall be used for determining compliance with 40 CFR 141.74(a)(l) and shall also be used for testing reagent grade water.
2) Media
|
Method |
Medium |
Final pH |
|
Pour Plate |
Plate count agar, also known as tryptone glucose yeast agar |
7.0 ± 0.2 |
|
SimPlate |
Multiple enzyme substrate |
7.0 ± 0.3 |
3) (For Pour Plate Method) Melted agar shall be tempered at 44°-46° C in a water bath before pouring. Agar temperature control accompanies media from tempering through use. Melted agar shall be held no longer than three hours. Sterile agar medium shall not be melted more than once. The center of media in containers shall be no greater than 2.5 cm from some surface.
4) Refrigerated medium may be stored in bottles or in screw-capped tubes for up to three months, or in petri dishes for up to two weeks.
5) For most potable water samples, countable plates can be obtained by plating 1.0 mL and/or 0.1 mL volumes of the undiluted sample (dilutions may not be necessary for SimPlate, which has a counting range up to 738/mL). At least duplicate plates per dilution shall be used. Duplicate dilutions are not required for SimPlate.
6) (For Pour Plate Method) The sample shall be aseptically pipetted onto the bottom of a sterile petri dish. Then at least 10-12 mL of tempered melted (44°-46° C) agar shall be added to each petri dish. The sample and melted agar shall be mixed carefully to avoid spillage. After agar plates have solidified on a level surface, the plates shall be inverted and incubated at 35° ± 0.5° C for 48 ± 3 hours. Plates shall be stacked no more than four high and shall be arranged in the incubator to allow proper air circulation and to maintain uniform incubation temperature. Excessive humidity in the incubator shall be avoided to reduce the possibility of spreader formation on the agar medium. Excessive drying of the agar medium shall also be avoided; agar medium in plates shall not lose more than 15% by weight during 48 ± 3 hours of incubation. Agar weight loss shall be determined quarterly.
7) (For SimPlate Method) Unit Dose (for a single sample): A 10.0 mL volume of test sample shall be added to a test tube containing dehydrated SimPlate medium. Then the dissolved medium shall be poured onto the center of a plate containing 84 small wells (provided by the manufacturer, IDEXX Laboratories, Inc.). Alternatively, 9.0 mL of sterile diluent (D.I. water, distilled water, or buffered water (Standard Methods, 9050C, 1 a)) can be added to the tube, followed by a 1.0 mL sample. Then the procedure indicated for the 10.0 mL sample shall be followed. The mixture shall be distributed evenly to the 84 wells on the plate, and the excess liquid shall be drained into an absorbent pad on the plate. The plate shall then be inverted (the fluid in each well is held in place by surface tension), and incubated for 48 ± 3 hours at 35° ± 0.5° C. Bacterial density is determined by counting the number of wells that fluoresce under a 365‑366 nm UV light, and converting this value to a Most Probable Number using the Unit Dose MPN table provided by the manufacturer. If a 10.0 mL sample is used, the Unit Dose MPN/mL shall be read directly. If a 1.0 mL sample is used, then the MPN/mL value shall be corrected by multiplying it by 10.
8) (For SimPlate Method) Multiple Dose (for 10 samples of 1.0 mL each): A 100-mL sterile diluent shall be added to the dehydrated SimPlate medium to reconstitute and shaken to dissolve. Then a 1.0 mL test sample shall be pipetted to the center of a plate containing 84 small wells, followed by 9.0 mL of the reconstituted medium. The plate shall be gently swirled to mix the sample and medium, and the mixture shall be distributed evenly to the 84 wells on the plate. Then the procedure indicated in subsection (s)(7) shall be followed, except that the Multi-Dose table supplied by the manufacturer shall be used to determine the MPN/mL. If a dilution is made during sample preparation, then the MPN/mL value shall be multiplied by the dilution factor.
9) (For Pour Plate Methods) Colonies shall be counted manually using a dark-field colony counter. In determining sample count, laboratories shall count only plates having 30 to 300 colonies. For plates inoculated with 1.0 mL of undiluted sample, counts less than 30 are acceptable. Fully automatic colony counters are not suitable because of the size and small number of colonies observed when potable water is analyzed for heterotrophic bacteria.
10) Each batch or flask of agar shall be checked for sterility by pouring a final control plate. Data shall be rejected if control is contaminated.
(Source: Amended at 46 Ill. Reg. 19150, effective November 17, 2022)
Section 465.370 Sample Collection, Handling and Preservation
When the laboratory has been delegated responsibility for sample collection, handling, and preservation, there shall be strict adherence to correct sampling procedures, complete identification of the sample, and prompt transfer of the sample to the laboratory as specified in "Standard Methods for the Examination of Water and Wastewater." In addition, the following standards for sample collection, handling, and preservation of potable water samples shall be met:
a) For the sample to be representative of the potable water system, the sampling program shall include examination of the finished water at selected sites that systematically cover the distribution network.
b) Minimum sampling frequency shall be as specified in Revised Total Coliform Rule, 40 CFR 141.
c) Water shall be sampled from cold water taps that are free of aerators, strainers, hose attachments, and water purification devices. Prior to sampling, a steady flow of water shall be maintained from the tap for two to three minutes to clear the service line.
d) The sample bottle shall be filled allowing at least 1 inch of air space from the top to provide space for mixing. A minimum sample volume of 100 mL shall be collected. If a sample bottle is filled too full to allow for proper mixing, rather than pouring off and discarding a portion of the sample, the entire sample shall be poured into a larger sterile container and mixed properly, and the analysis shall proceed.
e) The sample report form shall be completed in indelible ink immediately after collecting the sample and shall contain the following information: name of system (public water system site identification number, if available); sample identification (if any); date and time of collection; sample site location; sample collector's name and organization (if not the water system); persons transporting the samples from the system to the laboratory (if not the sampler); sample type (e.g., routine, repeat); and total/free chlorine residual (if applicable).
f) When sample containers are prepared within the laboratory, the dechlorinating agent, 0.1 mL of a 3% solution of sodium thiosulfate shall be added to a 120 mL bottle to neutralize up to 5 mg/L. Volume added to larger bottles shall be adjusted to provide the same level of neutralization. Stock sodium thiosulfate solution shall be free of turbidity.
g) When the sample is delivered to the laboratory:
1) The following information shall be added to the sample report form:
A) Date and time of sample arrival; and
B) Name of the person receiving the sample for the laboratory; and
2) Each sample shall be assigned a unique laboratory number. If the sample is a repeat or replacement sample, the number assigned to the original sample shall also be recorded.
h) Records necessary to establish chain-of-custody of the samples shall be maintained.
i) For the analysis of total coliform in drinking water, the time between sample collection and the placement of the sample in the incubator shall not exceed 30 hours.
j) The time from sample collection to placement of sample in the incubator (i.e., the holding time) for total coliforms and fecal coliforms in surface water sources and heterotrophic bacteria in drinking water shall not exceed eight hours for samples being analyzed in compliance with the Surface Water Treatment Rule (40 CFR 141.74(a)(1)).
k) Samples of potable water for heterotrophic plate count analysis shall be refrigerated and delivered to the laboratory within six hours after collection, and analyzed within two hours after receipt in the laboratory.
l) Source water samples shall be received and tested at <10º C, verifying with a temperature control. Time of initiation of analyses shall not exceed eight hours from time of collection.
(Source: Amended at 46 Ill. Reg. 19150, effective November 17, 2022)
Section 465.380 Standards for Laboratory Pure Water
The following standards shall apply to all laboratory pure water:
a) Laboratory pure water shall have these characteristics:
|
Parameter |
Limits |
Frequency |
|
Conductivity |
>0.5 megohms resistance or <2 micromhos/cm at 25° C |
Monthly |
|
Cd, Cr, Cu, Pb, Ni, Zn |
Not greater than 0.05 mg/L per contaminant. Collectively, no greater than 0.1 mg/L |
Annually |
|
Total Chlorine Residual 1 |
<0.1 mg/L |
Monthly |
|
Heterotrophic Plate Count 2 |
<500 CFU/mL |
Monthly |
|
Bacteriological Quality of Reagent Water 3 |
Ratio of growth rate 0.8 to 3.0 |
Annually |
1 DPD Method shall be used. Not required if source water is not chlorinated.
2 Pour Plate Method. See Standard Methods 9215E. SimPlate method.
3 See Standard Methods, Section 9020B, under Laboratory Supplies. This bacteriological quality test is not needed for type II water or better, as defined in Standard Methods. The bacteriological quality test is not needed for water with a conductivity <1 micromhos/cm at 25° C or resistivity >1 megohms. Users of purchased bottled water are not exempt from the suitability test.
b) Laboratory pure water shall be analyzed initially and annually (every 12 months) thereafter by the test for bacteriological quality of distilled water as specified in "Standard Methods for the Examination of Water and Wastewater." Purchased laboratory pure water shall be sampled in-house; manufacturer's test results shall not be used to establish compliance. Only satisfactorily tested water shall be used in preparing media, reagents, rinse, and dilution water. If the water tested does not meet the testing requirements, the water shall not be used until corrective action has been taken and retesting determines that the testing requirements have been met.
c) Laboratory pure water shall be analyzed monthly for conductance, chlorine residual, and heterotrophic plate count. Heterotrophic plate counts shall be performed as specified in "Standard Methods for the Examination of Water and Wastewater." If the water tested exceeds requirements for these properties, the water shall not be used until corrective action has been taken and retesting determines that the testing requirements have been met.
d) Laboratory pure water shall not be in contact with heavy metals, and shall be analyzed initially and annually (every 12 months) thereafter for trace metals (especially Pb, Cd, Cr, Cu, Ni, and Zn) in the quantities specified in subsection (a) of this Section. If the water tested exceeds requirements for trace metals, the water shall not be used until corrective action has been taken and retesting determines that the testing requirements have been met.
(Source: Amended at 46 Ill. Reg. 19150, effective November 17, 2022)
Section 465.390 General Quality Assurance Procedures
a) A written description of the current laboratory quality control and quality assurance program shall be maintained and made available to analysts in an area of the laboratory where analytical work takes place. The quality assurance plan shall address all of the items listed in the Manual for the Certification of Laboratories Analyzing Drinking Water. The quality assurance plan shall be reviewed annually and updated as necessary. A record of analytical quality control tests and quality control checks on media, materials, and equipment shall be prepared and retained for five years.
b) Standard operating procedures for each parameter for which the laboratory is certified and for all required quality control procedures shall be maintained and made available to analysts in an area of the laboratory where analytical work takes place.
c) The following minimum requirements shall apply to analytical quality control tests for general laboratory practices and methodology:
1) Each laboratory shall successfully analyze at least one set of proficiency testing (PT) samples once every 12 months, for each method (For example, Colilert (18 hour) and Colilert (24 hour) would require a PT for each assay.) for which it is certified. When PT sample results indicate technical error, the Department will provide appropriate technical assistance to determine the cause and make suggestions for correction of the problem.
2) Each analyst approved for the total coliform count procedure by the membrane filter technique for source water samples (SWTR) shall verify monthly 10 colonies, including each type of atypical colony observed. Counts shall be adjusted based on percent verification.
3) Each analyst approved for the fecal coliform procedure by the membrane filter technique for source water samples (SWTR) shall verify a positive water sample monthly. At least 10 isolated colonies shall be chosen from membranes containing typical blue colonies and, if present, atypical colonies of different morphological types and shall be transferred to lauryl tryptose broth. Positive tubes shall be transferred to EC medium. The lauryl tryptose broth shall be incubated at 35.0º ± 0.5º C for 24 to 48 hours. The EC medium shall be incubated at 44.5º ± 0.2º C for 24 hours + 2 hours. Turbid growth with gas production indicates a positive result. Counts shall be adjusted based on percent verification.
4) If there is more than one analyst in the laboratory, at least once each month each analyst shall count the same heterotrophic plate count plate, total coliform membrane, and fecal coliform membrane (per certified method used to test source water samples under the SWTR). Colony counts between analysts shall agree within 10 percent. This requirement does not apply to SimPlate.
5) The standards for laboratory pure water specified in Section 465.380 shall be met.
d) The following quality control tests for heterotrophic plate count shall be used:
1) Sterility controls shall be poured for each bottle of sterile melted, tempered medium used. These controls shall be the last plate poured from each bottle used.
2) Pipets shall be checked for sterility during each series of samples plated. All affected samples shall be marked "laboratory accident", and results shall not be reported when the sterility check indicates that the pipets used within the series were not sterile.
3) Microbial density of the air during plating procedures shall be determined for each series of samples plated. The air control plate shall be the first plate set up and shall be located so that it is within the area of the plating activity. The agar shall be exposed to the air for 15 minutes as determined by the laboratory timer. The inside of the plate lid shall not be exposed. When 15 or more colonies appear on an exposed plate after a 15-minute exposure period and 48 hours of incubation at 35º C, corrective action shall be taken.
4) The sterility of dilution water, if used, shall be determined. All affected samples shall be marked "laboratory accident", and results shall not be reported when the sterility check indicates that the dilution water used within the series was not sterile.
5) Records of all sterility test results shall be maintained.
(Source: Amended at 46 Ill. Reg. 19150, effective November 17, 2022)
Section 465.400 Quality Assurance for Media, Equipment and Supplies
The following minimum requirements shall apply to quality assurance checks of laboratory media, equipment, and supplies:
a) The pH meter or meters shall be standardized before each use period with pH 7.0 and either pH 4.0 or pH 10.0 standard buffers, whichever range covers the desired pH of the media or reagent. A record of the standardization, including the percent slope, shall be maintained. Percent slope shall be 95 to 105%. Each buffer aliquot shall be used only once. Commercial buffer solutions shall be dated and shall not be used past the expiration date. Electrodes shall be maintained according to manufacturer's recommendations.
b) Balances shall be calibrated monthly using NIST standardized Echelon I or II, or equivalent ASTM 1, 2, or 3 weights. A minimum of three weights that bracket the weighing requirements of the laboratory shall be used, and these weights shall be recertified every five years. A certificate shall accompany the weights. A certificate shall state either that the weights are compliant with the requirements of ASTM E1617-13 class 1, 2 or 3 tolerances or that they are compliant with the NIST Handbook 150-2G. The certificate shall list corrective data. Electronic balances shall be calibrated annually by a qualified service representative who is not affiliated with the laboratory. A certificate of calibration from the service representative shall be available for inspection.
c) Glass and electronic thermometers and temperature-recording devices including data loggers shall be calibrated annually at temperature of use against an NIST certified thermometer to within ± 1.0° C. Mercury NIST-certified thermometers shall be checked at the ice point annually and recalibrated at least every five years at each temperature of use. Digital NIST-certified thermometers shall be checked at the ice point annually and recalibrated at least every five years to demonstrate linearity. If datalogger or autoclave digital temperature record is used, datalogger or autoclave digital temperature record shall be calibrated annually by a qualified service representative who is not affiliated with the laboratory. A certificate of calibration from the service representative shall be available for inspection. Digital thermometer probe and meter shall be calibrated as a unit. The calibration factor, date calibrated, temperature of calibration, and analyst's initials shall be tagged on each thermometer. In addition, the laboratory shall record the following information in a Quality Assurance (QA) record book:
1) Serial number or unique identifier of laboratory thermometer;
2) Serial number of NIST-traceable thermometer;
3) Temperature of laboratory thermometer;
4) Temperature of NIST-traceable thermometer;
5) Correction (or calibration) factor;
6) Date of calibration; and
7) Analyst's initials.
d) Temperature in incubation equipment shall be recorded continuously by a temperature-recording device or recorded twice daily (at times separated by at least four hours) from in-place thermometers immersed in liquid unless otherwise specified by the manufacturer of a temperature monitoring system and placed on the top and bottom shelves of the use area. Documentation shall include the date and time of reading, temperature (as determined using the correction factor of the thermometer in use), and analyst's initials. Temperature readings from walk-in incubators with a continuous temperature reading device shall be supplemented by readings from in-place thermometers placed on various shelves other than where the recorder probe is located.
e) Date, contents, sterilization time and temperature, total time in autoclave, and analyst's initials shall be recorded each time the autoclave is used. Charts, if used, are to accompany written records.
f) Hot air ovens shall be equipped with a thermometer registering up to at least 180º C, or with a temperature-recording device. The oven thermometer shall be graduated in 10º C increments or less, with the bulb placed in sand during use. Date, contents, sterilization time and temperature, total time in oven, and analyst's initials shall be recorded each time the hot air oven is used.
g) Only membrane filters recommended for water analysis by the manufacturer shall be used. Manufacturer data sheets containing information as to lot number, ink toxicity (gridline inhibition), recovery, retention, and absence of growth-promoting substances for membrane filters shall be entered into the laboratory's record system. The lot numbers of membrane filters and date received shall be recorded. Membrane filters shall not be brittle or distorted, and the manufacturer's specification/certification sheet shall be available. Positive control shall be run on each new lot of membrane filters. Any gridline inhibition shall be recorded as unacceptable. Unacceptable membranes shall not be used.
h) Washing processes shall provide clean glassware with no stains or spotting. Distilled or deionized water shall be used for final rinse. Laboratory glassware shall be washed with a detergent designed for laboratory use. A glassware inhibitory residue test (Standard Methods, Section 9020B, under Laboratory Supplies) shall be performed, and acceptable results obtained, before the initial use of a detergent and whenever a different formulation or washing procedure is used. Results shall be recorded and maintained until specific formulation of detergent is no longer in use.
i) A representative piece of each type of glassware or plastic ware from each batch of clean, dried glassware or plastic ware shall be tested for residual alkaline or acid residue using bromothymol blue indicator. If the result of the indicator test is not green, corrective action shall be taken by re-rinsing, then air drying and retesting.
j) At least one bottle per lot or batch of sterilized sample bottles, whirlpaks or QuantiTray envelopes shall be checked before first use for sterility by adding approximately 25 ml of sterile non-selective broth media to each bottle or whirlpak and 100 ml to Quantitray envelope. The bottle shall be capped and rotated so that the broth comes in contact with all surfaces and shall be incubated at 35º + 0.5º C and checked after 24 and 48 hours for growth. Sample bottles shall not be used unless satisfactory results are obtained.
k) At least one bottle per lot or batch of sterilized sample bottles prepared with sodium thiosulfate shall be checked for a sufficient amount of the dechlorinating reagent by collecting a potable sample at the laboratory tap, then checking for residual chlorine. Corrective action shall be taken if there is any residual chlorine, and bottles checked shall not be used until corrective action has been completed.
l) At least one bottle per lot of precalibrated sample containers shall be checked before first use by measuring the volume with a Class A graduated cylinder. Tolerance shall be ± 2.5%.
m) Current service contracts or in-house protocols shall be maintained on balances, autoclaves, hot-air sterilization ovens, water stills, deionizers, reverse osmosis apparatuses, water baths, incubators, etc. Service records on the equipment shall include the date, name of the servicing person, and a description of the service provided.
n) Records shall be available for inspection on all batches of sterilized media showing the type of medium, lot numbers, date, sterilization time and temperatures, final pH, and the names/unique initials of the persons responsible for all or any part of the recorded data. The final pH of the medium at 25° C shall be:
|
Media |
|
pH |
|
|
|
|
|
M-Endo broth |
|
7.2 ± 0.2 |
|
M-Endo agar |
|
7.2 ± 0.2 |
|
M-Endo LES agar |
|
7.2 ± 0.2 |
|
brilliant green |
|
7.2 ± 0.2 |
|
lactose bile broth |
|
|
|
|
|
|
|
EC Medium |
|
6.9 ± 0.2 |
|
EC-MUG |
|
6.9 ± 0.2 |
|
plate count agar |
|
7.0 ± 0.2 |
|
M-FC broth/agar |
|
7.4 ± 0.2 |
|
lauryl tryptose broth |
|
|
|
single strength |
|
6.8 ± 0.2 |
|
double strength |
|
6.8 ± 0.2 |
|
triple strength |
|
6.8 ± 0.2 |
|
Nutrient agar with MUG |
|
6.8 ± 0.2 |
|
SimPlate |
|
7.0 ± 0.3 |
|
Colilert |
|
7.3 ± 0.3 |
|
Colilert 18 |
|
7.3 ± 0.3 |
|
Colisure |
|
7.3 ± 0.3 |
|
E*Colite |
|
6.9 ± 0.2 |
|
Readycult |
|
6.8 ± 0.2 |
|
Modified Colitag |
|
6.8 ± 0.2 |
|
m-Coliblue 24 |
|
7.0 ± 0.2 |
|
MI agar |
|
6.95 ± 0.2 |
|
MI broth |
|
7.05 ± 0.2 |
|
Non-selective broth |
|
Per manufacturers instructions |
o) The laboratory using commercially manufactured prepared media shall record the date received, type of medium, lot number, sample performance when checked against cultures known to give positive and negative results, and pH verification per subsection (n). Media shall be used or discarded by the manufacturer's expiration date.
p) Each new lot of prepared commercial medium and each batch of laboratory prepared medium shall be checked before use with positive and negative culture controls. Additionally, each batch of prepared media (whether commercially prepared or laboratory prepared) shall be checked for sterility. Control organisms (e.g., total coliform, fecal coliform, and E. coli) shall be either known stock cultures (periodically checked for purity) or commercially available cultures impregnated with the organism. Results shall be recorded. The following table identifies a few positive and negative culture controls that laboratories might consider.
|
Group |
Positive Culture Control |
Negative Culture Control |
|
Total Coliforms |
Escherichia coli Klebsiella aerogenes |
Staphylococcus aureus Proteus vulgaris Pseudomonas aeruginosa |
|
Fecal Coliforms |
Escherichia coli Klebsiella pneumoniae (thermotolerant) |
Klebsiella aerogenes |
|
E. coli |
Escherichia coli (MUG-positive strain) |
Klebsiella aerogenes Klebsiella pneumoniae (thermotolerant) |
|
Enterococci |
Enterococcus faecalis Enterococcus faecium
|
Staphylococcus aureus Escherichia coli Serratia marcesens |
q) Examples of appropriate American Type Culture Collection (ATCC) strains include the following:
Enterococcus faecalis ATCC 11700
Enterococcus faecium ATCC 6057
Klebsiella aerogenes ATCC 13048
Escherichia coli ATCC 8739 or 25922
Klebsiella pneumoniae (thermotolerant) ATCC 13883
Proteus vulgaris ATCC 29905
Pseudomonas aeruginosa ATCC 27853
Serratia marcesenes ATCC 14756
Staphylococcus aureus ATCC 6538
r) Lactose broth may be used in lieu of LTB if the laboratory conducts at least 25 parallel tests between this medium and LTB using water normally tested and this comparison demonstrates that the false-positive rate and false-negative rate for total coliforms, using lactose broth, is less than 10%.
s) A maximum registering thermometer or data logger shall be used during each autoclave and hot air oven cycle to verify sterilization temperatures. An exception to this rule would be an autoclave that has a printout of temperature that has been calibrated annually by an outside service with a NIST thermometer. The oven maximum registering thermometer shall be placed in sand. The autoclave maximum registering thermometer or data logger shall be placed in a container of water, unless otherwise specified by manufacturer’s instructions. Spore strips or ampules shall be used monthly, when in use, to confirm sterilization of the autoclave. Spore strips shall be used monthly to confirm sterilization for the hot air oven. Ampules shall not be used in the hot air oven because they may explode or melt. Strips or ampules that have not been placed in the autoclave or hot air oven shall be used as positive controls each time the sterilization is checked. A record of these results shall be maintained to include the date, material sterilized, and the initials of the analyst involved. Automatic timing mechanisms on autoclaves shall be checked quarterly with a stopwatch. For a 15-minute sterilization period, the autoclave time shall be within 60 seconds of the stopwatch time. An exception to this rule would be a data logger or an autoclave that has a printout of time that has been calibrated annually by an outside service.
t) When a media-dispensing apparatus is used, the media preparer shall check and maintain a record of the accuracy of the dispenser with a graduated cylinder at the start of each volume change and periodically throughout extended runs.
u) Micropipettors shall be calibrated annually and replaced if the precision or accuracy is greater than 2.5% tolerance. Micropipettors shall be calibrated with 10 consecutive weighings annually (using a separate tip for each weighing), and the average of all 10 weighings shall be ± 2.5% of specified delivery volume. For volumes ≥ 1.0 mL, volume shall be checked by using a Class A graduated cylinder.
v) The refrigerator temperature shall be determined daily by an accurate thermometer (thermometer immersed in liquid, data logger, or other automated approved method) placed on the top shelf. The refrigerator unit shall be visibly clean. Outdated materials in the refrigerator and freezer compartments shall be discarded.
w) Ultraviolet sterilization lamps shall be tested quarterly by exposing agar spread plates containing 200 to 250 microorganisms to the light for two minutes. If the irradiation does not reduce the count of control plates by 99 percent, the lamps shall be replaced. Alternatively, lamps shall be replaced if they emit less than 70% of the initial output. Cleaning of ultraviolet sterilization lamps shall be cleaned at least monthly by disconnecting the unit and cleaning the lamps with a soft cloth moistened with ethanol. Protective eye wear shall be used when checking the operation of a 254 nm lamp.
x) Water baths shall be cleaned at least monthly. The use of distilled or deionized water for water baths is recommended.
y) Media shall be used on a first in, first out basis. Records shall be kept of the kind, amount, date received, and date opened for media. The date opened and the date received shall be written on each bottle/container/box as appropriate. Dehydrated media shall be used within six months after opening, except media stored in a desiccator which shall be used by the manufacturer's expiration date. All media shall be discarded if visible deterioration is observed (e.g., clumping, color change). It is recommended that media be ordered in quantities to last no longer than one year, and that media be ordered in quarter pound multiples rather than one pound bottles to keep the supply sealed and protected as long as possible. Any media that have passed the manufacturer's expiration date shall be discarded.
z) The conductivity meter shall be calibrated at least monthly, following the manufacturer's recommendations, using a certified and traceable low level standard of 20 micromhos or less. The meter reading shall be within 2% of the value of the standard. If an in-line unit cannot be calibrated, it shall not be used to check reagent-grade water.
aa) A spectrophotometer or colorimeter (if used) shall have wavelengths in the visible range. A calibration standard and method specific blank shall be analyzed every day that the instrument is used prior to sample analysis. The calibration standard shall give a reading in the desired absorbance range and shall be obtained from an outside source.
bb) Each batch of prepared or each lot of commercial dilution/rinse water shall be checked for sterility by adding 50 mL of water to 50 mL of double-strength, nonselective broth. The batch shall be incubated at 35° ± 0.5° C and checked for growth after 24 and 48 hours. The batch shall be discarded if growth is detected.
cc) Each batch of prepared or each lot of commercial dilution water blanks shall be checked for pH; pH shall be 7.2 ± 0.2.
dd) The accuracy of dilution blank volumes shall be verified by checking one bottle for every 25 prepared or purchased using a Class A graduated cylinder. Volume shall be 99 mL ± 2 mL. Purchased dilution blanks shall be used by manufacturer's expiration date.
ee) Each lot of purchased single use membrane filtration equipment shall be checked before use with a Class A graduated cylinder, and a record shall be maintained. Tolerance shall be ± 2.5%. Sterility check shall be performed before and after analysis per Section 465.360(k)(2).
ff) Membrane filter equipment calibration shall be checked before first use with Class A graduated cylinders, and a record shall be maintained. Tolerance shall be ± 2.5%.
(Source: Amended at 46 Ill. Reg. 19150, effective November 17, 2022)
Section 465.410 Data Handling
a) All records shall be initialed or signed by the person or persons responsible for recording all or any part of the data, or performing the various tests.
b) Either each unit shall be responsible for maintaining its own records, or all records shall be maintained in a general laboratory log book.
c) The laboratory shall record arrival time and date received in the laboratory, time and date of analysis, direct count, membrane filtration verified count, MTF completed count, analyst's name/unique initials, and other special information on each sample report form.
d) A careful check shall be made to verify that each result is entered accurately from the bench sheet onto the sample report form. The sample report form shall be initialed or signed by the person who verified the entry of information from the bench sheet.
e) All forms used in the laboratory for both sample reporting and quality control shall be approved by the certification officer to ensure that data is recorded in a format that is easily interpreted and that contains all necessary information.
(Source: Amended at 46 Ill. Reg. 19150, effective November 17, 2022)
Section 465.420 Record Maintenance
a) All records that the laboratory is required to maintain shall be recorded in indelible ink with any changes lined through so that the original entry is visible. Changes shall be initialed and dated. Documentation supporting all corrections on records shall be maintained. Electronic records and signatures are allowed. See Uniform Electronic Transactions Act [815 ILCS 333].
b) A copy of the sample report form shall be maintained by the laboratory for at least 5 years. A change in ownership, merger, or closure of a laboratory does not negate this requirement. As results are entered into computer storage system, data shall be verified against bench sheets. Electronic records shall be made available in hard copy for on-site evaluation. Electronic data shall always be backed up by protected tape, disk, or hard copy. If the laboratory changes its computer hardware or software, it shall make a provision for transferring old data to the new system so that it remains retrievable within the time frames specified. See Good Automated Laboratory Practices, EPA 2185, Office of Information Management, Research Triangle Park NC 27711, August 10, 1995.
c) Records of bacteriological analyses shall be kept for at least 5 years. Actual laboratory reports may be kept. However, data may be transferred to tabular summaries, which shall include the following information:
1) Date, place, and time of sampling;
2) Name of person who collected the sample;
3) Identification of the sample origin, such as routine distribution sample, resample, construction sample, raw or process water sample, surface or ground water sample, or other special purpose sample;
4) Date and time of receipt of sample in the laboratory;
5) Records necessary to establish chain-of-custody of the sample;
6) Date and time of sample analysis (including start and completion);
7) Name or unique initials of the persons and designation of the laboratory responsible for performing the analysis;
8) Designation of the analytical techniques or methods used; and
9) Results of the analysis.
d) The disposal of all records subject to the Local Records Act [50 ILCS 205] shall be in accordance with the provisions of that Act and Section 465.430.
(Source: Amended at 46 Ill. Reg. 19150, effective November 17, 2022)
Section 465.430 Action Response to Laboratory Results
a) For laboratory results concerning samples from public water supplies and their sources, if any sample is total coliform positive or E. coli-positive, the laboratory shall notify the State regulatory agency that has jurisdiction over the public water supply and the public water supply by the end of the day.
b) Presumptive positive microbiological test results are to be reported by the end of the day to State regulatory agency that has jurisdiction over the public water supply as preliminary without waiting for membrane filter verification or multiple tube fermentation (MTF) completion. After membrane filter verification or MTF completion or both, the verified results shall be reported. The State regulatory agency and the public water supply shall be notified when results indicate that non-coliforms may have interfered with the total coliform analysis, as defined in 40 CFR 141.21(c)(2).
c) A total coliform-positive result is based on the following:
1) The confirmed phase if the MTF is used; or
2) The verified test for the Membrane Filter Technique if M-Endo medium or LES Endo agar is used; or
3) The required color change - No requirement exists to confirm a total coliform-positive results using Colilert, Colisure, MI agar, E*Colite, m‑ColiBlue24, Readycult, or Colitag test.
d) If a sample is found to be E. coli-positive, the sample is to be reported as total coliform-positive, even when total coliform confirmation is negative.
(Source: Amended at 46 Ill. Reg. 19150, effective November 17, 2022)
Section 465.APPENDIX A Colisure P/A and Colisure Multiple Tube P/A (Repealed)
(Source: Repealed at 38 Ill. Reg. 16240, effective July 15, 2014)