Modified ColitagTM Test Method for the Simultaneous Detection of 

E. coli and other Total Coliforms in Water 

(ATP D05-0035) 

August 28, 2009 

1 Rev 08/28/2009 
 

ATP D05-0035 

Modified ColitagTM Test Detection of E. coli and other Total 

Coliforms in Water as required in National Primary Drinking Water 

Regulations
 

1.0 Scope and Application 

1.1 Modified ColitagTM is a selective and differential medium for the 

simultaneous determination of the presence or absence or enumeration of E. 

coli and other total coliforms in water.1,2,3 Modified ColitagTM is for use in 

the Environmental Protection Agency’s survey and monitoring programs 

under the Safe Drinking Water Act. This method is for use in accordance 

with the National Primary Drinking Water Regulations at 40 CFR Part 141.4 

1.2. This method allows for the detection of coliform bacteria and/or E. coli 

from 16 - 48 hours and does not require further confirmation or verification 

steps. 

1.3 Modified ColitagTM is US EPA approved for compliance monitoring of 

public water systems as required by the Total Coliform Rule. This method 

can be used as a Presence/Absence test or for quantification of bacteria in a 

most probable number format (MPN) format. 

1.4 Modified ColitagTM is capable of detecting 1 colony forming unit (CFU) of 

chlorine-injured E. coli or other coliform bacteria per 100-mL of water 

sample. There is no upper limit of detection in the MPN format as 

dilutions are used to detect and enumerate bacteria. 

2.0 Summary of Method 

2.1 Modified ColitagTM is a one-step, ready-to-use, dehydrated medium for 

analysis of water samples. For presence/absence testing, one packet of 

medium is mixed with a 100-mL sample of water and incubated at 

35±0.5°C for 16 to 48 hours. If results are intended to be read before 22 

hours incubation, samples must be pre-warmed for 7-10 minutes in a 44.5 

±0.2°C water bath. If yellow color is observed, coliforms are present. If 

blue fluorescence is observed under 365 nm ultraviolet (UV) light, E. coli 

are present. Complete directions for use are available from CPI 

International, 5580 Skylane Blvd., Santa Rosa, CA, 95403, (800) 878-7654, 

http://www.colitag.com/colitag-instructions.pdf. 

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2.2 For enumeration of E. coli and other coliforms, Modified ColitagTM is 

suitable in a variety of MPN formats. In one format, a 100-mL of water 

sample would be mixed with one packet of Modified ColitagTM and divided 

into ten 10-mL volumes before incubation. Based on the number of 

positive tubes, an MPN value for this number of tubes can be looked up in 

Table 9221:III of Section 9221C Estimation of Bacterial Density in the 

Standard Methods for the Examination of Water and Wastewater.5 Other 

most probable number formats suitable with Modified ColitagTM can be 

found in the same reference and further information is available from CPI 

International, 5580 Skylane Blvd., Santa Rosa, CA, 95403, (800) 878-7654, 

www.colitag.com. 

2.3 Modified ColitagTM is based on the detection of two enzymes, ßglucuronidase and ß-galactosidase, which are characteristic of E. coli and 

the coliform group respectively.5,6 For detection of ß-galactosidase, which 

is an enzyme indicative of the coliform group, Modified ColitagTM utilizes 

the chromogenic substrate, ortho-nitrophenyl-ß-D-galactopyranoside 

(ONPG). Upon hydrolysis of ONPG by ß-galactosidase, a distinctly yellowcolored compound, ortho-nitrophenol, is released indicating the presence of 

coliforms. For detection of ß-glucuronidase, which is the enzyme specific 

to E. coli, Modified ColitagTM utilizes the fluorogenic enzyme substrate, 4­

methylumbelliferyl-ß-D-glucuronide (MUG). Upon hydrolysis of MUG by 

ß-glucuronidase, 4-methylumbelliferone is released, a compound which 

fluoresces when exposed to ultraviolet light. The ß-glucuronidase enzyme is 

specific to E. coli and observation of this fluorescence differentiates this 

organism from other members of the coliform group. 

2.4 Quality of the detection process is assured through resuscitative 

components in the medium that promote recovery of chlorine-injured E. coli 

and other coliforms. 

3.0 Definitions 

3.1	 The definition and purposes below are specific to this method but conform 

to common usage as much as possible. 

Hydrolyzable Substrate: A chemical substrate (i.e. ONPG, MUG) that is 

capable of being hydrolyzed by bacteria such as coliforms and E. coli. 

Chromogenic Enzyme Substrate: A substrate which can be hydrolyzed by 

an enzyme releasing a colored (or chromogenic) compound. 

Fluorogenic Enzyme Substrate: A substrate which can be hydrolyzed by 

an enzyme releasing a fluorescent compound. 

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ATP D05-0035 

Ortho-nitrophenyl-ß-D-galactopyranoside (ONPG): A chemical 

substrate which is hydrolyzed by ß-D-galactosidase activity in coliforms. 

When a drinking water sample containing coliform bacteria is added to 

Modified ColitagTM and subjected to incubation, enzymatic hydrolysis of 

ONPG causes production of ortho-nitrophenol, a yellow colored compound 

indicating the presence of coliforms. 

4-Methylumbelliferyl-ß-D-glucuronide (MUG): A chemical substrate 

which is hydrolyzed by ß-glucuronidase activity in E. coli. When a 

drinking water sample containing E. coli bacteria is added to Modified 

ColitagTM and subjected to incubation, enzymatic hydrolysis causes 

production of 

4-methylumbelliferon, which is a compound that fluoresces when exposed 

to long wavelength ultra violet light (365 nm). Fluorescence by 4­

methylumbelliferone indicates the presence of E. coli. 

Proton Gradient Resuscitation: Modified ColitagTM utilizes a patented 

method of resuscitating chlorine-injured bacteria, whereby a slightly acidic 

medium allows injured coliforms to more easily maintain a proton gradient 

across the cell membrane3. 

Coliform bacteria: Bacteria, which hydrolyze ONPG at 35+0.5 degrees C 

after 24 + 2 h of incubation. 

E. coli: Bacteria, which hydrolyze MUG at 35+0.5 degrees C after 24 + 2 h 

of incubation. 

4.0 Interferences 

4.1	 Chemical/Physical: There are no known chemical interferences that would 

be found in drinking water or source water that would inhibit or interfere 

with the development of color or the production of fluorescence when one 

uses Modified ColitagTM medium. If a water sample has background color 

prior to analyses, a control blank (same water sample not inoculated with 

Modified ColitagTM medium) should be run and used as the control blank. 

4.2 Biological: Heterotrophic bacteria greater than 104/ml can yield positive 

coliform reactions7. 

5.0 Safety 

5.1 None of the components used in Modified ColitagTM are listed as a 

carcinogen or suspected carcinogen. Reference should be made to the 

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Material Safety Data Sheet provided with the product for specific 

information. 

5.2 The analyst should follow the recommended safety guidelines described in 

Standard Methods for the Examination of Water and Waste Water 

(American Public Health Association 2005), 21st edition, section 1090).8 

5.3 When working with Modified ColitagTM it is important to know and practice 

normal safety procedures for working in a microbiology laboratory. 

Routine biosafety procedures should be followed when handling this 

medium and related samples. 

6.0 Instrumentation, Equipment and Supplies 

6.1 Incubator or water bath set to 35±0.5°C 

6.2 Circulating water bath set to 44.5 ±0.2°C 

6.3 Long wavelength 6-watt UV light (365 nanometers) 

6.4 Non-fluorescing sterile glass or plastic sample collection vessels (120-mL 

or larger bottles or bags) with thiosulfate. All glassware and plastic vessels 

used for determining the presence of coliform bacteria or E. coli should be 

handled according to recommended guidelines.5 All glassware and 

containers should be washed with a suitable laboratory detergent, rinsed 

thoroughly with tap water followed by rinsing with reagent water9 and 

sterilized by autoclaving at 121°C for 15 minutes before use. Certified 

sterile plastic disposable coliform sample vessels with 100-mL fill line, 

thiosulfate tablet, and tamper seals are available through a variety of 

commercial suppliers and CPI International. Sterile Coli-Test bags are 

available through Nasco or CPI International. 

7.0 Reagents and Standards 

7.1 Modified ColitagTM product for P/A or MPN testing is provided as ready-touse, pre-measured dehydrated media. One packet dissolves directly into a 

100-mL sample for presence/absence testing or is dissolved with 100-mL 

sterile reagent water to make 100-mL of single strength medium. The 

medium is stable when stored at 4 to 30 degrees Centigrade away from 

light. The expiration date and lot number are indicated on each unit. 

Modified ColitagTM dehydrated medium has a shelf life of at least 18 

months from the date of manufacture under proper storage conditions. 

7.2 Sodium thiosulfate (Na2S2O3) is used to dechlorinate drinking water 

samples. 

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7.3 A reagent blank (one Modified ColitagTM test packet mixed with 100-mL of 

sterile distilled water) may be incubated along with the test samples as a 

negative control standard. A Modified ColitagTM color comparator may be 

used as a positive control standard. If the sample has yellow color equal or 

greater than the comparator, coliforms are present. If the fluorescence of 

the sample is equal or greater than the fluorescence of the comparator then 

E. coli are present in the sample. 

8.0	 Sample Collection, Dechlorination, Preservation, Shipment 

and Storage 

8.1 Water Sample Collection: Sampling procedures are described in the 

USEPA Manual for the Certification of Laboratories Analyzing Drinking 

Water and the Standard Methods for the Examination of Water and 

Wastewater, part 90005

. 

8.1.1 Water samples should be collected in sterile, plastic or glass leakproof 

containers and sufficient sodium thiosulfate to neutralize chlorine at the 

time of collection. For drinking water, 0.1-mL of a 3% solution of Na2S2O3 

in a 120-mL bottle will neutralize up to 5 mg/L of residual chlorine. 

8.1.2 Storage temperature and handling conditions: Samples should be tested as 

soon as possible after collection and should be placed on ice or refrigerator 

at a temperature of 1-10oC during transit to the laboratory. Use insulated 

containers to transport sample vessels to maintain proper storage 

temperatures. Sample vessels should be packed so they do not become 

immersed should the ice melt during transit or storage. 

8.1.3 Drinking water samples should be analyzed within 30 h of collection. 

9.0 Quality Control (QC): 

Each lot of Modified ColitagTM should be tested for quality. Four presence/absence 

packs or single strength MPN tubes will be needed; the first as a blank 

(uninoculated), and the others to be inoculated with a representative positive and 

negative cultures. For additional guidance or clarity regarding QC, refer to 

Standard Methods, Sections 9223 B and 9020 A. The QC results for a particular lot 

are valid until the expiration date is realized. 

9.1 Incubate the individual tubes or bottles containing Modified ColitagTM

medium with a loopful of growth from an 18-24 hour broth culture of the 

following: E. coli (ATCC 25922 or a characterized MUG positive strain), 

Klebsiella pneumoniae (ATCC 13883 or another characterized non6 Rev 08/28/2009 
 

ATP D05-0035 

fluorescing coliform species), Salmonella subsp. enterica serovar 

Typhimurium (ATCC 14028 or another characterized non-coliform). 

Include an uninoculated tube or bottle of Modified ColitagTM as an 

additional control. 

9.2 Incubate QC samples at 35±0.5°C for 16-48 hours. If results are intended 

to be read before 22 hours incubation, samples must be pre-warmed for 7­

10 minutes in a 44.5 ±0.2°C water bath. Observe for the following 

reactions: 

Modified ColitagTM Control Organism Reactions — 24 hours 

Organism Color Fluorescence 

E. coli Yellow Positive 

Klebsiella species Yellow Negative 

Salmonella subsp. enterica serovar 

Typhimurium 

Colorless Negative 

None Clear Negative 

9.3 For additional quality control testing, positive Modified ColitagTM may be 

inoculated into Brilliant Green Lactose Bile, EC-MUG or other media used 

for confirmation. 

10.0 Calibration and Standardization 

10.1 Check incubation temperatures twice daily (morning and afternoon) to 

ensure proper temperature regulation. 

10.2 Temperature measurement devices should be calibrated annually against a 

NIST-certified thermometer or one traceable to NIST. 

11.0 Procedure 

11.1 Test Procedure for the Presence/Absence method 

11.1.1 Aseptically add Modified ColitagTM medium from one packet to a 

100-mL water sample in a sterile nonfluorescing, leakproof, 

container. 

11.1.2 Shake to begin dissolution. 

11.1.3 Incubate bottles at 35±0.5°C for 16-48 hours. If results are 

intended to be read before 22 hours incubation, samples must be 

pre-warmed for 7-10 minutes in a 44.5 ±0.2°C water bath. 

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11.1.4 Read results (refer to section 11.3, below). 

11.2 Test procedure for the MPN method: Refer to Section 9221C of the 

Standard Methods for the Examination of Water and Wastewater: 

11.2.1 Method A: 

11.2.1.1 Select the appropriate number of tubes per sample for the MPN 

test (10 tubes x 10-mL or 5 tubes x 20-mL). 

11.2.1.2 Mix 100-mL of a water sample with one packet of Modified 

ColitagTM medium in a sterile vessel. Shake to dissolve medium. 

11.2.1.3 Aseptically dispense the sample into the tubes. 

11.2.1.4	 Incubate tubes at 35± 0.5 oC for 16-48 hours. 

11.2.1.5	 Read results (refer to section 11.3, below) and report as the most 

probable number (MPN)/100-mL using values from Table 

9221:III, Section 9221 C in the Standard Methods for the 

Examination of Water and Wastewater. 

11.2.2 Method B: 

11.2.2.1 Dissolve Modified ColitagTM in sterile distilled water to make up 

desired quantity of medium. One packet contains sufficient 

contents to make 100-mL of single strength medium, or 50-mL 

of double strength medium. 

11.2.2.2 Perform serial dilutions and inoculations according to the MPN 

method described in The Standard Methods for the 

Examination of Water and Wastewater, 21st Ed. Section 9221C, 

Estimation of Bacterial Density. 

11.3 Sample Interpretation 

11.3.1 Visually check each bottle, bag or tube for a yellow color. If the 

sample (P/A bottles or bags or MPN tubes) are yellow, coliform 

bacteria are present. 

11.3.2 The absence of yellow color in the sample after 16-48 hours 

incubation indicates the sample is negative for coliform bacteria. 

11.3.3 If samples are yellow, examine for fluorescence using a long 

wavelength 365 nm UV lamp in a darkened environment. If a 

bright blue fluorescence is present, the sample is confirmed for the 

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presence of E. coli. As with the ONPG test, known positive and 

negative cultures may be run parallel to the unknown sample, or 

the sample may be compared to a ColitagTM comparator 

(description included in this section). 

11.3.4 The absence of fluorescence in the sample under longwave UV 

light after 16-48 hours incubation indicates the sample is negative 

for E. coli bacteria. 

11.3.5 For quality control, the test sample may be compared to any of the 

following: 

•	 Negative control – either sterile water with CPI Modified 

ColitagTM and or a negative control culture with Modified 

ColitagTM, incubated at 35±0.5°C for 16-48 hours (see 

Table 9.2 in the quality control section). 

•	 Positive control – Coliform-positive, MUG-positive 

organism with Modified ColitagTM, incubated at 35±0.5°C 

for 16-48 hours (see Table 9.2 in the quality control 

section). 

•	 Color Comparator -- ColitagTM comparator transferred to a 

similar test vessel- if the sample is equal or darker yellow 

than the ColitagTM comparator, the sample is confirmed for 

the presence of total coliforms. If the sample fluoresces 

with equal or greater intensity than the comparator, the 

sample is confirmed for the presence of E. coli. The color 

comparator should be stored protected from light when not 

in use. 

11.3.6	 Use Modified ColitagTM medium on or before the printed expire 

date. For optimal performance, it is recommended that the 

powdered medium be stored in cool (preferably 4-7 degrees C), 

dry conditions, protected from light and moisture. 

12.0 Data Analysis, Calculation, Interpretation and Reporting 

Results 

12.1 Follow the same interpretation directions from 11.3 above. 

12.1.1 Presence/Absence: Report results as presence/absence of total 

coliforms/E. coli per 100-mL. No further data analysis or 

calculation is required. 

12.1.2 MPN Format: Report results as the most probable number of total 

coliforms/E. coli (MPN) per 100-mL. MPN values and further 

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information are available in Section 9221C of the Standard 

Methods for Examination of Water and Wastewater, refer to tables 

9221: II, III, and IV. 

13.0 Method Performance Characteristics of Modified ColitagTM 

13.1	 Specificity: 93.8% overall (16-48 hours incubation) when compared to 

SM9221B (LTB, BGLB) and 96.1% overall compared to SM9221F 

(EC+MUG). 

13.2	 Comparability: Modified Colitag was found to have an overall agreement 

of 94.5% with LTB, BGLB for total coliforms and 95.9% with EC-MUG 

for E.coli detection. 

13.3 Sensitivity: 95.2% overall (16-48 hours incubation) when compared to 

SM9221B (LTB, BGLB) and 92.8% overall compared to SM9221F 

(EC+MUG). 

14.0 Pollution Prevention 

14.1 Wherever possible, it is recommended that laboratory personnel use 

pollution control techniques to minimize waste generation. When waste 

cannot be reduced at the source, recycling is recommended. 

15.0 Waste Management 

15.1 It is the responsibility of each laboratory to comply with all federal, state 

and local regulations governing waste management, particularly to 

hazardous waste identification rules and land disposal restrictions. In 

addition, it is the responsibility of each laboratory to protect the air, water 

and land by minimizing and controlling all release from fume hoods and 

bench operations. Compliance is also required with any National Pollutant 

Discharge Elimination System (NPDES) Permits and regulations.10 For 

further information, Federal, State or local agencies should be contacted. 

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16.0 References 

16.1	 ColitagTM product insert, prepared by CPI International. 

16.2	 "Colitag: An Indole- and MUG-based Medium for Detecting and Recognizing Fecal 

Coliforms and Escherichia coli." Abstract with poster presentation, 1991 ASM 

General Meeting, Dallas TX. 

16.3	 "A Comparison of the Colitag Method and the Standard Methods for the Detection of 

Fecal Coliforms and Escherichia coli in Urban Creeks "WEF/CWEA Collection 

Systems 2002 Conference. San Francisco, CA. 

16.4	 Chang, G. W. and Lum, R. A., 1992. Improved Recovery of Chlorine-Injured E. coli 

on Acidified Media. ColitagTM and mCT3. Abstract with poster presentation. 

American Society for Microbiology General Meeting, New Orleans, LA. 

16.5	 United States Environmental Protection Agency. National Primary Drinking Water 

Regulations, Total Coliforms (Including Fecal Coliforms and E. coli); Final Rule. 

Federal Register 54 (124): 27547-27568. Washington, D. C., Office of Federal 

Register. June 29, 1989. 

16.6	 American Public Health Association, American Water Works Association, Water 

Environment Federation. Microbiological Examination, Part 9000. IN: Standard 

Methods for the Examination of Water and Wastewater, 21st edition. 

16.7	 Krieg, N. R. and J. G. Holt., eds. Bergey's Manual of Systematic Bacteriology, vol. 1. 

Baltimore, Williams and Wilkins, 1989. 

16.8	 Unpublished data, CPI International Inc., 5580 Skylane Blvd, Santa Rosa, Ca, 95403. 

16.9	 American Public Health Association, American Water Works Association, Water 

Environment Federation. Microbiological Examination, Standard Methods for the 

Examination of Water and Wastewater, 21st edition, Sections 1090 H and 1090 J. 

16.10	 American Society of Testing Materials. Specifications for Reagent Water, (Type III 

Grade), D1193-91. In: Annual Book of ASTM Standards, 1991, vol. 11.01. p. 45. 

Philadelphia, PA. 

16.11	 Code of Federal Regulations Title 40 Protection of the Environment. Chapter I 

Environmental Protection Agency. Part 122 EPA Administered Permit Programs: 

The National Pollutant Discharge Elimination System. 

http://ecfr.gpoaccess.gov/cgi/t/text/textidx?c=ecfr&sid=344382d789a386c880645c300a514061&rgn=div5&view=text&nod 

e=40:21.0.1.1.12&idno=40. 

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