Indiana Administrative Code › Title 327 - WATER POLLUTION CONTROL DIVISION › Article 8 - PUBLIC WATER SUPPLY › Rule 2 - Drinking Water Standards › Section 2-8.4 - Analytical methods for microbiological contaminants

Current through July 10, 2024
Authority: IC 13-13-5; IC
				  13-14-8-2;
				  IC
				  13-14-8-7; IC
				  13-18-3-1; IC
				  13-18-3-2;
				  IC
				  13-18-16-8; IC
				  13-18-16-9
Affected: IC 13-18-2; IC 13-18-16
Sec. 8.4.
(a) The
				provisions of this subsection are applicable to a PWS until all required repeat
				monitoring under section 8.1 of this rule and fecal coliform or E. coli testing
				required under section 8.3 of this rule that was initiated by a total coliform
				positive sample taken before April 1, 2016, is completed as well as analytical
				method, reporting, record keeping, public notification, and consumer confidence
				report requirements associated with that monitoring and testing. A PWS 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) A PWS needs
				only to determine the presence or absence of total coliforms, and a
				determination of total coliform density is not required.
(3) A PWS 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
				technique1,2,3 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
				test3,5 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) A PWS 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 U.S.
				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)*. A
				  PWS needs only to determine the presence or absence of fecal coliforms; a
				  determination of fecal coliform density is not required.
(5) A PWS 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 is 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 PWS
				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).