Section 465

Section 465.360 Methodology

A laboratory shall be certified for all analytical methods listed below that it uses for compliance purposes. At a minimum, the laboratory shall be certified for one total coliform method and one fecal coliform or 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, as specified in the listed references, shall be followed for individual parameters:

Method References

Approved Methods	Media	Method¹ Citation	TCR²(Detect)	SWTR²(Count)	New Main Construction² (Detect)	GWR² (Detect)
Total Coliforms
Fermentation broth method	LTB→BGLB Broth	SM¹ 9221B,C	X	X	X
Fermentation broth method	P-A Broth → BGLB Broth	SM¹ 9221D	X
Enzyme substrate method	Colilert^®, Colilert-18^®	SM¹ 9223	X	X
	Colisure^®	SM² 9223	X
	Readycult^® or Fluorocult LMX^®		X
	E*Colite^®		X
	Colitag^®		X
Membrane filter method	M-Endo or LES-Endo → LTB, BGLB Broth	SM¹ 9222B,C	X	X	X
	MI Medium	EPA Method 1604	X	X
	m-ColiBlue24^®		X
	Chromocult^®		X
	Coliscan^®		X	X
Fecal Coliforms
Fermentation broth method	LTB or P/A broth →EC broth	(SM¹ 9221B,D) SM¹ 9221E	X	X
Fermentation broth method	A-1 broth	SM¹ 9221E		X
Membrane filter method	M-Endo medium → EC broth	(SM¹ 9222B) SM¹ 9221E	X	X
Membrane filter method	mFC	SM¹ 9222D		X
Escherichia coli
Enzyme substrate method	Colilert^® or Colilert-18^®	SM¹ 9223	X			X
	Colisure^®	SM² 9223	X			X
	E*Colite^®		X			X
	Readycult^® or Fluorocult LMX^®		X			X
	LTB, P/A broth, M-Endo → EC-MUG	(SM¹ 9221B,D; SM¹ 9222B) SM¹ 9221F	X			X
	Colitag^®		X			X
Membrane filter method	MI Medium	EPA Method 1604	X			X
	m-ColiBlue24^®		X			X
	Chromocult^®		X			X
	Coliscan^®		X
	M-Endo or LES Endo → NA-MUG	(SM¹ 9222B) SM¹ 9222G	X			X
Heterotrophic Bacteria
Pour plate method	Plate count agar	SM¹ 9215B		X
Multiple enzyme substrate	SimPlate^®			X
Pour plate, spread plate, or membrane filter methods	R2A		X³

¹SM = Standard Methods for the Examination of Water and Wastewater, 18^th, 19^th or 20^th edition.

MC = "Manual for the Certification of Laboratories Analyzing Drinking Water," USEPA 570/9-90/008A, 5^th Edition (January 2005). A copy of this manual can be obtained by contacting the U.S. Environmental Protection Agency, Washington DC 20465. This manual as published and dated is exclusive of subsequent amendments or editions.

²TCR = Total Coliform Rule (40 CFR 141.21(f)), SWTR=Surface Water Treatment Rule (40 CFR 141.74(a)), New Main Construction (see 35 Ill. Adm. Code 652.203(b)). GWR = Ground Water Rule (40 CFR 141.402).

³For possible use if system operates under a variance to the TCR.

b) Laboratories shall perform parallel testing between a newly approved test and another EPA-approved procedure for enumerating total coliforms. The laboratory shall conduct at least 25 parallel tests between methods using waters normally tested. Results between methods shall vary by less than 10%.

c) Water samples shall be shaken vigorously at least 25 times in a complete up and down or back and forth movement.

d) Sample volume analyzed for total coliforms in drinking water shall be 100 mL.

e) Fermentation broth methods. The water level of the water bath shall be above the upper level of the medium in the culture tubes.

f) 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).

g) 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. Optionally, 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 medium 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 either fecal coliforms or E. coli.

h) 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 test and/or a fecal coliform/E. coli test on the total coliform-negative culture to check for coliform suppression. If the confirmed test is coliform positive or fecal coliforms/E. coli are detected, the sample shall be reported as such. A fecal coliform/E. coli-positive result is considered a total coliform positive, fecal coliform/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 MPN shall be reported. If the test is total coliform negative, the sample shall be invalidated.)

i) 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 Fluorocult LMX Broth test), or Colitag 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/Fluorocult LMX, and Colitag 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 Colitag shall appear colorless to a slight tinge of color, while Colisure and E*Colite are yellow and Readycult/Fluorocult 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) Incubators, especially small low-wattage 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/ Fluorocult LMX	Specified 24-hour incubation time includes time it takes to bring sample temperature up to 35° ± 0.5° C or 36° ± 1° C
Modified Colitag	Specified 24-hour incubation time includes time it takes to bring sample temperature up to 35° ± 0.5° C

¹If the laboratory plans to put a large load into a small incubator, samples shall be brought to room temperature before incubation.

²Information 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) in the absence of a yellow color 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 (do not incubate more than 28 hours total). If the color is still lighter than the reference comparator at 28 hours, the sample shall be reported as negative. 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 sample after 18 hours is yellow, but is lighter than the comparator).

14) For enumerating total coliforms in source water with the Colilert test, a 5-or 10-tube configuration, Quanti-Tray, or Quanti-Tray 2000 may be used for each sample dilution 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. 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, 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 medium becomes red in color, 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 or 36° ± 1° 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) Fluorocult LMX broth is identical to Readycult, except that it is a dehydrated culture medium in granulated form packed primarily in a 500 g plastic bottle. For testing a 100-mL water sample, 34 g of Fluorocult LMX shall be suspended in 1 L purified water and boiled to dissolve completely. Transfer 100-mL aliquots to 250-mL bottles and autoclave for 15 minutes at 121° C. Cool to room temperature, add the 100-mL water sample, and incubate. Do not add E. coli/Coliform Supplement to the medium.

20) For the Colitag test, samples shall be incubated at 35° ± 0.5° C for 24 ± 2 hours. During incubation, trimethylamine-N-oxide in the Colitag 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.

j) 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 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.

k) Media used for total coliforms, fecal coliforms, and E. coli by MF method 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. Ensure that ethanol used in the rehydration procedure is not 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 7.2 ± 0.1 for M-Endo medium.

2) Using m-ColiBlue24 medium for detecting total coliforms and E. coli in drinking water: Ampules of broth shall be inverted 2 to 3 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: Do not autoclave commercially-made pre-sterilized bottled MI agar or broth. Melt bottled agar in a boiling water bath (or by other processes recommended by the manufacturer). As soon as complete melting has occurred, cool slightly and pour immediately 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. Cool the agar, add freshly prepared filter-sterilized cefsulodin, and pour immediately 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) Using Chromocult^® Coliform agar for detecting total coliforms and E. coli in drinking water: Do not autoclave or overheat. The final pH shall be 6.8 ± 0.2. If a heavy background of heterotrophic bacteria is expected (especially Pseudomonas and Aeromonas species), add cefsulodin solution to the cooled (45° to 50° C) medium (dissolve 10 mg cefsulodin in 2 mL deionized or distilled water, and add solution to 1 L of medium).

5) Using Coliscan^® for detecting total coliforms and E. coli in drinking water or enumerating total coliforms in source water: Coliscan is available as a dry powder agar mix or as a presterilized bottled agar. For reconstitution and antibiotic addition, follow the protocol of the manufacturer (Micrology Laboratories, LLC). Do not overheat the antibiotic cefsulodin. The final pH of Coliscan agar shall be 7.00 ± 0.2.

6) Using m-FC broth (with or without agar) for enumerating fecal coliforms in source water: Do not autoclave. Bring medium just to the boiling point. The final pH shall be 7.4 ± 0.2.

7) 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 ampuled 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. Record date and time prepared.

8) Incubation conditions and colony color of inoculated medium

Medium	Incubation	Total coliforms¹	E. coli
M-Endo medium or M-Endo agar LES	35° ± 0.5° C for 22-24 hrs	Metallic (golden) sheen colonies (presumptive)	N/A
m-ColiBlue24	35° ± 0.5° C for 24 hrs	Red colonies	Blue to purple colonies
MI	35° ± 0.5° C for 24 ± 2 hrs	Fluorescent colonies under UV light	Blue colonies under normal light
Chromocult	36° ± 1° C for 24 ± 1 hrs	Salmon to red colonies	Dark-blue to violet colonies²
Coliscan	32°-37° C for 24-28 hrs	Pink to magenta colonies	Purple-blue colonies
m-FC	44.5° ± 0.2° C for 24 ± 2 hrs	N/A	Blue colonies (fecal coliforms)

¹Without the presence of E. coli. If an E. coli colony is present, as indicated by the last column, it shall be counted as a total coliform-positive colony.

²If confirmation of E. coli is desired, add one drop of Kovac's reagent to each dark blue to violet colony; the formation of a cherry-red color within seconds confirms the presence of E. coli.

l) 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, record as "confluent growth" or "TNTC" and request an additional sample 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, salmon to red or dark blue to violet colonies for Chromocult Coliform agar, pink-magenta or blue-purple colony for Coliscan). (Before invalidation, the laboratory may perform a verification test on the total coliform negative culture, i.e., on confluent or TNTC growth, and a fecal coliform/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. A fecal coliform/E. coli positive result is considered a total coliform-positive, fecal coliform/E. coli positive sample, even if the initial and/or verification total coliform test is negative.)

m) 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, because coliform density shall be determined.

n) For drinking water samples (to verify colonies on Endo-type medium): 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). Alternatively, sheen colonies may be verified 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, verify up to five red questionable sheen colonies and/or red non-sheen colonies representing different morphological types. Alternatively, wipe the entire surface of the membrane filter with a sterile cotton swab, and inoculate the verification media (LTB, then BGLBB).

o) For drinking water samples: Total coliform-positive colonies shall be tested for E. coli or fecal coliforms. 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). EPA has approved several options for testing a total coliform-positive colony on Endo-type medium for E. coli or fecal coliforms. When EC Medium (for fecal coliforms) or EC Medium + MUG (for E. coli) 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 up to three different media (e.g., EC or EC-MUG Medium, LTB, and BGLBB, in that order). If Nutrient Agar + MUG is used, refer to Nutrient Agar + MUG section.

p) For source water samples: Initial total coliform counts shall be adjusted based upon verified data, as in Standard Methods, Section 9222B(5).

q) For source water samples (SWTR): If two or more analysts are certified, each analyst shall count total coliforms or fecal coliform colonies on the same membrane monthly. Colony counts shall agree within 10%.

r) Nutrient Agar + MUG Test (for detection of E. coli in drinking water or ground water)

1) Medium shall be autoclaved at 121° 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 465.350(d)(9). Filter or spot-inoculate control cultures onto a membrane filter on M-Endo agar LES or M-Endo broth or agar, and incubate at 35° ± 0.5° C for 24 hours. Then transfer the filter to Nutrient Agar + MUG and incubate at 35° C for another 4 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 should it be 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) Inoculated medium shall be incubated at 35° ± 0.5 C° for 4 hours.

5) Check the fluorescence 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) MF method for detecting enterococci/fecal streptococci in ground water

1) For mE agar (SM 9230C) for the detection of enterococci: Prepare basal mE agar. Then autoclave and cool in a 44°-46° C water bath. Dissolve 0.48 g nalidixic acid and 0.4 mL 10 N NaOH into 10 mL of reagent-grade distilled water and mix. Filter-sterilize the solution, and add 5.2 mL per liter of basal mE agar. For triphenyl tetrazolium chloride (TTC), add 0.25 g of TTC to 25 mL of reagent-grade water, and warm to dissolve. Filter sterilize the solution, and add 15 mL per liter of basal mE agar. Final pH shall be 7.1 ± 0.2.

2) For m-Enterococcus agar (SM 9230C) for the detection of fecal streptococci (not enterococci): Heat to dissolve ingredients, but do not autoclave. Dispense into sterile petri plates (9 X 50 mm) (about 4 mL) and allow to solidify. Final pH shall be 7.2 ± 0.2.

3) For mEI agar (EPA Method 1600) for the detection of enterococci: Add 0.75 g indoxyl-b-D-glucoside to 1L of basal mE agar, and proceed according to subsection (s)(1), except that the preparation of TTC is as follows: Add 0.1 g of TTC to 10 mL of reagent-grade distilled water, and warm to dissolve. Filter-sterilize the solution, and add 2 mL per liter of medium. Final pH shall be 7.1 ± 0.2.

4) After filtering a 100-mL sample, place membrane in a petri dish on one of the agar media listed in subsection (s)(1), (s)(2) or (s)(3). Serial dilutions should not normally be necessary for detecting enterococci in ground water.

5) If m-Enterococcus agar is used, incubate inverted plate at 35° ± 0.5° C for 48 hours and, using magnification and a fluorescent lamp, count all light and dark red colonies as fecal streptococci.

6) If mE agar is used, incubate inverted plate for 48 hours at 41° ± 0.5° C, and then transfer filter to EIA medium. Incubate at 41° ± 0.5° C for 20-30 minutes and, using magnification and a fluorescent lamp, examine the colonies. Pink to red colonies on mE agar with a black or reddish brown precipitate on the underside of filter on EIA indicates the presence of enterococci.

7) If mEI agar is used, incubate inverted plate for 24 hours at 41° ± 0.5° C. Using magnification and a small fluorescent lamp, examine both the top and bottom of the plate for colonies with a blue halo. A colony with a blue halo, regardless of colony color, indicates the presence of enterococci.

t) Heterotrophic Plate Count (for enumerating heterotrophic bacteria in drinking water)

1) The Pour Plate Method (Standard Methods 9215B) or the SimPlate Method shall be used for determining compliance with 40 CFR 141.74(a)(l) and shall also be used for testing reagent grade water. For systems that have been granted a variance from the Total Coliform Rule's maximum contaminant level (see variance criteria in 40 CFR 141.4), any method in Standard Methods, Section 9215, Heterotrophic Plate Count, may be used with R2A medium for enumerating heterotrophic bacteria in drinking water.

2) Media

Method	Medium	Final pH
Pour Plate	Plate count agar, also known as tryptone glucose yeast agar	7.0 ± 0.2
Pour Plate	R2A agar	7.2 ± 0.2
Spread Plate	R2A agar	7.2 ± 0.2
Membrane Filter	R2A agar	7.2 ± 0.2
SimPlate	Multiple enzyme substrate	7.2 ± 0.2

3) (For Pour Plate Method) Melted agar shall be tempered at 44°-46° C in a water bath before pouring. Melted agar shall be held no longer than three hours. Sterile agar medium shall not be melted more than once.

4) (For Spread Plate Method) 15 mL of R2A agar medium (or other medium) shall be poured into a petri dish (100 x 15 mm or 90 x 15 mm) and allowed to solidify.

5) 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. Prepared petri dishes with R2A medium may be stored for up to one week.

6) 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.

7) (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 arranged in the incubator to allow proper air circulation and to maintain uniform incubation temperature. Avoid excessive humidity in the incubator to reduce the possibility of spreader formation on the agar medium. Also avoid excessive drying of the agar medium; agar medium in plates should not lose more than 15% by weight during 48 hours of incubation.

8) (For Spread Plate Method) 0.1 or 0.5 mL of the sample (or dilution) shall be pipetted onto the surface of the pre-dried agar in the plate, and then spread over the entire surface of the agar using a sterile bent-glass rod. The inoculum shall be absorbed completely by the agar before the plate is inverted and incubated. The plate shall be incubated at 20°-28° C for 5-7 days.

9) (For Membrane Filter Technique) The volume to be filtered shall yield between 20-200 colonies. The filter is transferred to a petri dish containing 5 mL of solidified R2A medium, and incubated at 20°-28° C for 5-7 days. If plates with loose-fitting lids are used, plates shall be placed in a plastic box with a close fitting lid containing moistened paper towels. Paper towels shall be rewetted as necessary to maintain moisture. Colonies shall be counted using a stereoscopic microscope at 10-15X magnification.

10) (For SimPlate Method) Unit Dose (for a single sample): A 10-mL volume of test sample is 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 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-mL sample. Then follow the procedure as indicated above for the 10-mL sample. The mixture shall be distributed evenly to the 84 wells on the plate, and the excess liquid 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 45-72 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-mL sample is used, read the Unit Dose MPN/mL directly. If a 1-mL sample is used, then correct the MPN/mL value by multiplying it by 10.

11) (For SimPlate Method) Multiple Dose (for 10 samples of 1 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 mL of the reconstituted medium. Gently swirl plate to mix the sample and medium, and distribute the mixture evenly to the 84 wells on the plate. Then continue with the procedure indicated in subsection (t)(10), 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 multiply the MPN/mL value by the dilution factor.

12) (For Pour Plate and Spread Plate Techniques) 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, except for plates inoculated with 1.0 mL of undiluted sample. Counts less than 30 for such plates 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.)

13) 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 35 Ill. Reg. 14494, effective August 12, 2011)