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 Colitag^TM 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 Colitag^TM 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 Colitag^TM 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 Colitag^TM 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 Colitag^TM 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)