TITLE 35: ENVIRONMENTAL PROTECTION
SUBTITLE F: PUBLIC WATER SUPPLIES
CHAPTER I: POLLUTION CONTROL BOARD
PART 611 PRIMARY DRINKING WATER STANDARDS
SECTION 611.526 ANALYTICAL METHODOLOGY
Section 611.526 Analytical Methodology
a) The standard sample volume required for total coliform analysis, regardless of analytical method used, is 100 mℓ.
b) Suppliers need only determine the presence or absence of total coliforms; a determination of total coliform density is not required.
c) Suppliers must conduct total coliform analyses in accordance with one of the following analytical methods, incorporated by reference in Section 611.102, or in accordance with an alternative method approved by the Agency pursuant to Section 611.480 (the time from sample collection to initiation of analysis may not exceed 30 hours, and the supplier is encouraged but not required to hold samples below 10° C during transit):
1) Total Coliform Fermentation Technique, as set forth in Standard Methods, 18th, 19th, 20th, or 21st ed., Methods 9221 A and B, as follows:
A) Lactose broth, as commercially available, may be used in lieu of lauryl tryptose broth if the supplier conducts at least 25 parallel tests between this medium and lauryl tryptose broth using the 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 percent;
B) If inverted tubes are used to detect gas production, the media should cover these tubes at least one-half to two-thirds after the sample is added; and
C) No requirement exists to run the completed phase on 10 percent of all total coliform-positive confirmed tubes.
2) Total Coliform Membrane Filter Technique, as set forth in Standard Methods, 18th, 19th, 20th, or 21st ed., Methods 9222 A, B, and C.
3) Presence-Absence (P-A) Coliform Test, as set forth in: Standard Methods, 18th, 19th, 20th, or 21st ed., Method 9221 D, as follows:
A) No requirement exists to run the completed phase on 10 percent of all total coliform-positive confirmed tubes; and
B) Six-times formulation strength may be used if the medium is filter-sterilized rather than autoclaved.
4) ONPG-MUG test: Standard Methods, 18th, 19th, 20th, or 21st ed., Method 9223. (The ONPG-MUG test is also known as the Autoanalysis Colilert System.)
5) Colisure Test (Autoanalysis Colilert System). (The Colisure Test may be read after an incubation time of 24 hours.)
BOARD NOTE: USEPA included the P-A Coliform and Colisure Tests for testing finished water under the coliform rule, but did not include them for the purposes of the surface water treatment rule, under Section 611.531, for which quantitation of total coliforms is necessary. For these reasons, USEPA included Standard Methods, Method 9221 C for the surface water treatment rule, but did not include it for the purposes of the total coliform rule, under this Section.
6) E*Colite® Test (Charm Sciences, Inc.).
7) m-ColiBlue24® Test (Hatch Company).
8) Readycult® 2000.
9) Chromocult® Method.
10) Colitag® Test.
11) Modified Colitag™ Method.
BOARD NOTE: USEPA added Standard Methods, 21st ed., Methods 9221 A, B, and D; 9222 A, B, and C; and 9223 as approved alternative methods in appendix A to subpart C of 40 CFR 141 on June 3, 2008 (at 73 Fed. Reg. 31616). USEPA added Modified Colitag™ Method as an approved alternative method in appendix A to subpart C of 40 CFR 141 on November 10, 2009 (at 74 Fed. Reg. 57908).
d) This subsection corresponds with 40 CFR 141.21(f)(4), which USEPA has marked "reserved." This statement maintains structural consistency with the federal regulations.
e) Suppliers must conduct fecal coliform analysis in accordance with the following procedure:
1) When the MTF Technique or P-A Coliform Test is used to test for total coliforms, shake the lactose-positive presumptive tube or P-A vigorously and transfer the growth with a sterile 3-mm loop or sterile applicator stick into brilliant green lactose bile broth and EC medium, defined below, to determine the presence of total and fecal coliforms, respectively.
2) For approved methods that use a membrane filter, transfer the total coliform-positive culture by one of the following methods: remove the membrane containing the total coliform colonies from the substrate with sterile forceps and carefully curl and insert the membrane into a tube of EC medium; (the laboratory may first remove a small portion of selected colonies for verification); swab the entire membrane filter surface with a sterile cotton swab and transfer the inoculum to EC medium (do not leave the cotton swab in the EC medium); or inoculate individual total coliform-positive colonies into EC medium. Gently shake the inoculated tubes of EC medium to insure adequate mixing and incubate in a waterbath at 44.5 ±0.2° C for 24 ±2 hours. Gas production of any amount in the inner fermentation tube of the EC medium indicates a positive fecal coliform test.
3) EC medium is described in Standard Methods, 18th ed., 19th ed., and 20th ed., Method 9221E.
4) Suppliers need only determine the presence or absence of fecal coliforms; a determination of fecal coliform density is not required.
f) Suppliers must conduct analysis of E. coli in accordance with one of the following analytical methods, incorporated by reference in Section 611.102:
1) EC medium supplemented with 50 μg/ℓ of MUG (final concentration). EC medium is as described in subsection (e) of this Section. MUG may be added to EC medium before autoclaving. EC medium supplemented with 50 μg/ℓ MUG is commercially available. At least 10 mℓ of EC medium supplemented with MUG must be used. The inner inverted fermentation tube may be omitted. The procedure for transferring a total coliform-positive culture to EC medium supplemented with MUG is as in subsection (e) of this Section for transferring a total coliform-positive culture to EC medium. Observe fluorescence with an ultraviolet light (366 nm) in the dark after incubating tube at 44.5 ±2° C for 24 ±2 hours; or
2) Nutrient agar supplemented with 100 μg/ℓ MUG (final concentration), as described in Standard Methods, 19th ed. and 20th ed., Method 9222 G. This test is used to determine if a total coliform-positive sample, as determined by the MF technique, contains E. coli. Alternatively, Standard Methods, 18th ed., Method 9221 B may be used if the membrane filter containing a total coliform-positive colony or colonies is transferred to nutrient agar, as described in Method 9221 B (paragraph 3), supplemented with 100 μg/ℓ MUG . If Method 9221 B is used, incubate the agar plate at 35° Celsius for four hours, then observe the colony or colonies under ultraviolet light (366-nm) in the dark for fluorescence. If fluorescence is visible, E. coli are present.
3) Minimal Medium ONPG-MUG (MMO-MUG) Test, as set forth in Appendix D of this Part. (The Autoanalysis Colilert System is a MMO-MUG test.) If the MMO-MUG test is total coliform positive after a 24-hour incubation, test the medium for fluorescence with a 366-nm ultraviolet light (preferably with a six-watt lamp) in the dark. If fluorescence is observed, the sample is E. coli-positive. If fluorescence is questionable (cannot be definitively read) after 24 hours incubation, incubate the culture for an additional four hours (but not to exceed 28 hours total), and again test the medium for fluorescence. The MMO-MUG test with hepes buffer is the only approved formulation for the detection of E. coli.
4) The Colisure Test (Autoanalysis Colilert System).
5) The membrane filter method with MI agar.
6) The E*Colite® Test.
7) The m-ColiBlue24® Test.
8) Readycult® 2000.
9) Chromocult® Method.
10) Colitag® Test.
11) ONPG-MUG Test: Standard Methods, 20th or 21st ed., Method 9223 B.
12) Modified Colitag™ Method.
BOARD NOTE: USEPA added Stadard Methods, 20th or 21st ed., Method 9223 B and Standard Methods Online, Method 9223 B-97 as approved alternative methods for E. coli in appendix A to subpart C of 40 CFR 141 on November 10, 2009 (at 74 Fed. Reg. 57908).
g) As an option to the method set forth in subsection (f)(3) of this Section, a supplier with a total coliform-positive, MUG-negative, MMO-MUG test may further analyze the culture for the presence of E. coli by transferring a 0.1 mℓ, 28-hour MMO-MUG culture to EC medium + MUG with a pipet. The formulation and incubation conditions of the EC medium + MUG, and observation of the results, are described in subsection (f)(1) of this Section.
h) This subsection corresponds with 40 CFR 141.21(f)(8), a central listing of all documents incorporated by reference into the federal microbiological analytical methods. The corresponding Illinois incorporations by reference are located at Section 611.102. This statement maintains structural parity with USEPA regulations.
BOARD NOTE: Derived from 40 CFR 141.21(f) and appendix A to 40 CFR 141 (2009).
(Source: Amended at 34 Ill. Reg. 19848, effective December 7, 2010)