Section 327IAC8-2-8.4. Analytical methods for microbiological contaminants

  4. (a) A public water system shall analyze for microbiological contaminants as follows:

(1) The standard sample volume required for total coliform analysis, regardless of analytical method used, is one hundred (100) milliliters.

(2) Public water systems need only determine the presence or absence of total coliforms, and a determination of total coliform density is not required.

(3) Public water systems must conduct total coliform analyses in accordance with one (1) of the following analytical methods or with the alternative methods listed in Appendix A to Subpart C of 40 CFR 141:

(A) Total coliform fermentation technique^{1, 2, 3} as set forth in Method 9221A* and Method 9221B*.

(B) Total coliform membrane filter technique⁴ as set forth in Method 9222A*, Method 9222B*, and Method 9222C*.

(C) Presence-absence (P-A) coliform test^{3, 5} as set forth in Method 9221D*.

(D) ONPG-MUG test⁶ as set forth in Method 9223*.

(E) Colisure test*⁷.

(F) E*Colite^® test*.

(G) m-ColiBlue24^® test*.

(4) Public water systems must conduct fecal coliform analysis in accordance with the procedure in this subdivision. When the MTF technique or presence-absence (P-A) coliform test is used to test for total coliforms, shake the lactose-positive presumptive tube or P-A bottle vigorously and transfer the growth with a sterile three (3) millimeter loop or sterile applicator stick into brilliant green lactose bile broth and EC medium to determine the presence of total and fecal coliforms, respectively. For EPA-approved analytical methods which use a membrane filter, transfer the total coliform-positive culture by one (1) of the following methods:

(A) Remove the membrane containing the total coliform colonies from the substrate with a 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.)

(B) Alternately, the laboratory may 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 EC tubes to ensure adequate mixing and incubate in a water bath at forty-four and one-half (44.5) degrees Celsius, plus or minus two-tenths (0.2) degrees Celsius, for twenty-four (24) hours, plus or minus two (2) hours. Gas production of any amount in the inner fermentation tube of the EC medium indicates a positive fecal coliform test. The preparation of EC medium is described in Method 9221E, paragraph 1(a)*. Public water systems need only determine the presence or absence of fecal coliforms; a determination of fecal coliform density is not required.

(5) Public water systems must conduct analysis of Escherichia coli in accordance with one (1) of the following analytical methods or with the alternative methods listed in Appendix A to Subpart C of 40 CFR 141:

(A) EC medium supplemented with fifty (50) micrograms per milliliter of 4-methylumbelliferyl-beta-D-glucuronide (MUG) (final concentration). EC medium is described in Method 9221E, paragraph 1(a)*. MUG may be added to EC medium before autoclaving. EC medium supplemented with fifty (50) micrograms per milliliter of MUG is commercially available. At least ten (10) milliliters 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 shall be as specified in subdivision (4) for transferring a total coliform-positive culture to EC medium. Observe fluorescence with an ultraviolet light three hundred sixty-six (366) nanometers (preferably with a six (6) watt lamp) in the dark after incubating tube at forty-four and one-half (44.5) degrees Celsius, plus or minus two-tenths (0.2) degrees Celsius for twenty-four (24) hours, plus or minus two (2) hours.

(B) Nutrient agar supplemented with one hundred (100) micrograms per milliliter of MUG (final concentration). Nutrient agar is described in Method 9221E*. This test is used to determine if a total coliform-positive sample, as determined by the membrane filter technique or any other method in which a membrane filter is used contains E. coli. Transfer the membrane filter containing a total coliform colony or colonies to nutrient agar supplemented with one hundred (100) micrograms per milliliter (final concentration) of MUG. After incubating the agar plate at thirty-five (35) degrees Celsius for four (4) hours, observe the colony or colonies under ultraviolet light three hundred sixty-six (366) nanometers (preferably with a six (6) watt lamp) in the dark for fluorescence. If fluorescence is visible, E. coli are present.

(C) Minimal medium ONPG-MUG (MMO-MUG) test as described in the article "National Field Evaluation of a Defined Substrate Methods for the Simultaneous Detection of Total Coliforms and Escherichia coli from Drinking Water: Comparison with Presence-Absence Techniques*". If the MMO-MUG test is total coliform-positive after a twenty-four (24) hour incubation, test the medium for fluorescence with a three hundred sixty-six (366) nanometer ultraviolet light (preferably with a six (6) watt lamp) in the dark. If fluorescence is observed, the sample is E. coli-positive. If fluorescence is questionable (cannot be definitively read) after twenty-four (24) hours incubation, incubate the culture for an additional four (4) hours, but not to exceed twenty-eight (28) hours total, and again test the medium for fluorescence. The MMO-MUG test with hepes buffer in lieu of phosphate buffer is the only approved formulation for the detection of E. coli.

(D) The Colisure test*.

(E) The Membrane Filter Method with MI agar*.

(F) E*Colite^® test*.

(G) m-ColiBlue24^® test*.

(6) As an option to subdivision (5)(C), a system with a total coliform-positive, MUG-negative, MMO-MUG test may further analyze the culture for the presence of E. coli by transferring a one-tenth (0.1) milliliter, twenty-eight (28) hour MMO-MUG culture to EC medium plus MUG with a pipet. The formulation and incubation conditions of EC medium plus MUG and observation of the results are described in subdivision (5)(A).

  (b) Response to a violation shall be as follows:

(1) A public water system which has exceeded the MCL for total coliforms in section 7 of this rule must report the violation to the commissioner no later than the end of the next business day after it learns of the violation and notify the public in accordance with 327 IAC 8-2.1-7 through 327 IAC 8-2.1-16.

(2) A public water system which has failed to comply with a coliform monitoring requirement, including the sanitary survey requirement, must report the monitoring violation to the commissioner within ten (10) days after the system discovers the violation, and notify the public in accordance with 327 IAC 8-2.1-7 through 327 IAC 8-2.1-16.

  (c) The time from sample collection to initiation of analysis cannot exceed thirty (30) hours. Systems are encouraged but not required to hold samples below ten (10) degrees Celsius during transit.

  (d) The agency strongly recommends that laboratories evaluate the false-positive and negative rates for the method or methods they use for monitoring total coliforms. The agency also encourages laboratories to establish false-positive and negative rates within their own laboratory and sample matrix (drinking water or source water or both) with the intent that if the method they choose has an unacceptable false-positive or negative rate, another method can be used. The agency suggests that laboratories perform these studies on a minimum of five percent (5%) of all total coliform-positive samples, except for those methods where verification or confirmation or both is already required, for example, the M-Endo and LES Endo Membrane Filter Tests, Standard Total Coliform Fermentation Technique, and Presence-Absence Coliform Test. Methods for establishing false-positive and negative-rates may be based on lactose fermentation, the rapid test for β-galactosidase and cytochrome oxidase, multi-test identification systems, or equivalent confirmation tests. False-positive and false-negative information is often available in published studies or from the manufacturer, or both.

¹Lactose broth, as commercially available, may be used in lieu of lauryl tryptose broth, if the system conducts at least twenty-five (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 coliform, using lactose broth, is less than ten percent (10%).

²If inverted tubes are used to detect gas production, the media should cover these tubes at least one-half (1/2) to two-thirds (2/3) after the sample is added.

³No requirement exists to run the completed phase on ten percent (10%) of all total coliform-positive confirmed tubes.

⁴MI agar may also be used*.

⁵Six-times formulation strength may be used if the medium is filter-sterilized rather than autoclaved.

⁶The OPNG-MUG test is also known as the Autoanalysis Colilert System.

⁷The Colisure Test may be read after an incubation time of twenty-four (24) hours.

  *The methods referenced in this section may be obtained as follows:

(1) Methods 9221A, 9221B, 9222A, 9222B, 9222C, 9221D, 9223, and 9221E may be found in "Standard Methods for the Examination of Water and Wastewater", 1992, American Public Health Association, et al., 18th edition, or "Standard Methods for the Examination of Water and Wastewater", 1995, American Public Health Association, et al., 19th edition, available from the American Public Health Association, et al., 1015 Fifteenth Street N.W., Washington, D.C. 20005.

(2) A description of the Colisure test may be obtained from IDEXX Laboratories, Inc., One IDEXX Drive, Westbrook, Maine 04092.

(3) The minimal medium ONPG-MUG test may be found in "National Field Evaluation of a Defined Substrate Method for the Simultaneous Detection of Total Coliforms and Escherichia coli from Drinking Water: Comparison with Presence-Absence Techniques", (Edberg, et al.), Applied and Environmental Microbiology, Volume 55, pages 1003─1008, April 1989.

(4) Preparation and use of MI agar is set forth in the article, "New Medium for the Simultaneous Detection of Total Coliforms and Escherichia coli in Water" by Brenner, K.P., et al., 1993, Applied and Environmental Microbiology, 59:3534-3544, and errata published in Applied and Environmental Microbiology, 59:4378. Also available from the Office of Water Resource Center (RC-4100), 401 M. Street S.W., Washington, D.C. 20460, EPA/600/J-99/225.

(5) A description of the E*Colite^® test, "Presence/Absence for Coliforms and E. coli in Water", December 24, 1997, is available from Charm Sciences, Inc., 36 Franklin Street, Malden, Massachusetts 02148-4120.

(6) A description of the m-ColiBlue24^® test, August 17, 1999, is available from the Hach Company, 100 Dayton Avenue, Ames, Iowa 50010.

These methods are available for copying at the Indiana Department of Environmental Management, Office of Water Quality, 100 North Senate Avenue, Room N1255, Indianapolis, Indiana 46204. (Water Pollution Control Division; 327 IAC 8-2-8.4; filed Dec 28, 1990, 5:10 p.m.: 14 IR 1023; errata filed Jan 9, 1991, 2:30 p.m.: 14 IR 1070; filed Apr 12, 1993, 11:00 a.m.: 16 IR 2158; filed Aug 25, 1997, 8:00 a.m.: 21 IR 51; errata filed Dec 10, 1997, 3:45 p.m.: 21 IR 1348; filed Jul 23, 2001, 1:02 p.m.: 24 IR 3968; errata filed Jul 25, 2001, 3:25 p.m.: 24 IR 3991; filed Nov 20, 2001, 10:20 a.m.: 25 IR 1092; errata filed Feb 22, 2002, 2:01 p.m.: 25 IR 2254; errata filed Feb 6, 2006, 11:15 a.m.: 29 IR 1937; filed Feb 25, 2013, 8:36 a.m.: 20130327-IR-327110667FRA)