1 

Readycult® Coliforms 100 Presence/Absence Test for Detection and Identification 

of Coliform Bacteria and Escherichia coli in Finished Waters 

January 2007 

Version 1.1 

EPA-approved alternative version for 

November 2000 

Version 1.0 

MERCK KGaA 

Frankfurter Strasse 250 

64271 DARMSTADT 

GERMANY 
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Readycult® Coliforms 100 Presence/Absence Test for Detection and Identification 

of Coliform Bacteria and Escherichia coli in Finished Waters 

1.0 Scope and Application 

1.1 This method is for use in the Environmental Protection Agency’s (EPA’s) data 

gathering and monitoring programs under the Safe Drinking Water Act. 

1.2 Readycult® Coliforms is a selective and differential medium for determining the 

presence or absence of total coliforms and E. coli in finished waters. It is a onestep, ready-to-use, dehydrated, and granulated culture medium supplied in a 

gamma-irradiated snap pack. Each unit is added to a 100 ml water sample. The 

composition of Readycult® is identical to the composition of Merck ́s dehydrated 

culture medium named Fluorocult® LMX Broth (Manafi and Ossmer, Merck Cat. No. 

1.10620).

1.3 This method tests for coliform bacteria and/or E. coli in 24 ± 1 h. Confirmation or 

verification steps are not required. Optional confirmation or verification with the use 

of KOVAC ́s indole reagent (for the confirmation of E. coli) is approved but not 

required. 

1.4 The detection limit of Readycult® Coliforms is 1 colony forming unit (CFU) of coliform 

bacteria or E. coli per 100 ml of medium. 

2.0 Summary of Method 

2.1 This medium determines the presence or absence of coliform bacteria and E. coli in 

finished water. The content of one blister pack is added to a 100 ml sample of water 

followed by incubation at 36 ± 1°C or 35 ± 0.5°C for 24 ± 1 h. If coliform bacteria 

are present, the medium changes color from slightly yellow to blue-green. In 

addition, if E. coli is present, the medium will emit a bright blue fluorescence when 

subjected to a long wave (366 nm) ultraviolet (UV) light. 

2.2 Readycult® Coliforms is based on the detection of two enzymes (ß-galactosidase 

and ß-glucuronidase) that are characteristic of the total coliform group and E. coli, 

respectively (1, 2). Identification of these enzymes is accomplished by resuscitation 

of the target organisms with a combination of tryptose, salts, phosphate buffers, and 

carbohydrates, followed by hydrolysis of the chromogenic and/or fluorogenic 

enzyme substrates. At the same time, lauryl sulfate sodium salt selectively inhibits 

the growth of accompanying bacterial flora. 

2.3 Readycult® Coliforms contains the chromogenic enzyme substrate 5-bromo-4-

chloro-3-indolyl-ß-D-galactopyranoside (X-GAL) for the detection of ß-galactosidase 

(an enzyme indicative of the coliform group). Upon hydrolysis by ß-D-galactosidase, 

X-GAL releases a chromogenic compound (indigo-blue) that turns the medium from 

slightly yellow to a blue-green color. The endpoint of this reaction in the medium is 

distinct and not subject to interference in water samples discolored by humic acids, 

turbidity or other materials. 

2.4 Readycult® Coliforms contains the fluorogenic enzyme substrate 4-methylumbelliferyl-ß-D-glucuronide (MUG) for the detection of ß-glucuronidase (an enzyme 
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specific to E. coli). Upon hydrolysis by ß-glucuronidase, MUG releases 4-

methylumbelliferone that fluoresces when exposed to ultraviolet light. The 

fluorescence differentiates the presence of E. coli from the rest of the coliform group 

as required by the Total Coliform Rule (3) in the analysis of drinking water. 

2.5 Readycult® Coliforms contains the enzyme substrate tryptophane for the detection 

of tryptophanase (another enzyme specific to E. coli). Upon cleavage by 

tryptophanase, tryptophane releases indole that immediately forms a red ring when 

KOVAC ́s indole reagent is added directly to the broth. This reaction may be used 

as an additional confirmation for the presence of E. coli in vessels with positive 

fluorescence. 

3.0 Definitions

3.1 Defined Substrate - An enzyme substrate that has a specific known chemical 

structure. 

3.2 Chromogenic Enzyme Substrate - A substrate that releases a chromogenic 

compound upon hydrolysis by an enzyme. 

3.3 Fluorogenic Enzyme Substrate - A substrate that releases a fluorogenic compound 

upon hydrolysis by an enzyme. 

3.4 Specific Enzymes - Enzymes which react with only one particular substrate or very 

closely related compounds, e.g., ß-D-galactosidase is a specific enzyme that reacts 

with lactose or lactose analogs such as X-GAL. In addition, specific enzymes can 

also refer to those associated with a certain species or group of microorganisms. 

3.5 Hydrolysis - Enzyme catalyzed decomposition of a substrate by addition of a water 

molecule (H20). 

3.6 5-bromo-4-chloro-3-indolyl-ß-D-galactopyranoside (X-GAL). A defined substrate 

that changes color from colorless to blue-green after hydrolysis by ß-Dgalactosidase. 

3.7 4-Methylumbelliferyl-ß-D-glucuronide (MUG) - A defined enzyme substrate that 

fluoresces under long wave UV light after hydrolysis by ß-D-glucuronidase. 

3.8 Tryptophane - A defined enzyme substrate that releases indole upon cleavage by 

tryptophanase. A red ring forms in the presence of indole when KOVAC ́s indole 

reagent is added directly to the broth. 

3.9 Coliform Bacteria (Total Coliforms) - Bacteria that possess the enzyme ß-Dgalactosidase. In Readycult® Coliforms, this enzyme is capable of producing a bluegreen color. 

3.10 Escherichia coli - Bacteria that possess the enzymes ß-D-galactosidase, ßglucuronidase, and tryptophanase. In Readycult® Coliforms, these enzymes are 

capable of producing a blue-green color, fluorescence under UV light, and a positive 

indole reaction (red ring) upon addition of KOVAC’s indole reagent (if desired). 
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3.11 Positive Control - Addition of a known organism to medium that produces a positive 

reaction. 

3.12 Negative Control - Addition of a known organism to medium that produces a 

negative (no) reaction. 

4.0 Interferences

4.1 Chemical / Physical - There are no known interferences contained in drinking or 

potential source water that interfere with the distinct color change or development of 

fluorescence in Readycult® Coliforms 

5.0 Safety

5.1 The analyst must know and practice normal safety procedures for working in a 

microbiology laboratory where handling potentially biohazardous cultures is 

possible. 

5.2 Solid and liquid waste containing viable microorganisms should be decontaminated 

by autoclaving or using an appropriate disinfectant before discarding. 

5.3 Readycult® Coliforms is not a listed or suspected carcinogen. Contact with the skin 

or eyes may cause irritation or redness. 

6.0 Equipment and Supplies

6.1 Equipment 

6.1.1 Incubator or Waterbath – 36 ± 1°C or 35 ± 0.5°C. 

6.1.2 Long wave UV Light - 366 nm, 6W minimum 

6.2 Glassware and Supplies - All glassware and plastic vessels used for determining the 

presence of coliform bacteria or E. coli should be handled according to 

recommended guidelines(1). All glassware and containers should be washed with a 

suitable laboratory detergent, rinsed thoroughly with tap water followed by rinsing 

with reagent water(4) and sterilized by autoclaving at 121°C for 15 minutes prior to 

use. 

6.2.1 Sterile Graduated Glass or Plastic Sample Collection Vessels – minimum 

120 ml capacity for 100 ml water sample 

6.2.2 Biohazardous Waste Container 

6.3 Sterile Inoculating Loop or Needle 

7.0 Reagents and Standards

7.1 Readycult® Coliforms 100 is provided as ready-to-use, pre-measured, dehydrated, 

granulated culture medium in a snap pack for 100 ml water sample format. If 
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properly stored at room temperature (+15 to + 25 °C), the product has a maximum 

shelf life of 36 months from date of manufacture. 

7.2 Composition (gram/snap pack) of Readycult® Coliforms 100 

Tryptose 0.5 

Sodium chloride 0.5 

Sorbitol 0.1 

Tryptophan 0.1 

Di-potassium hydrogen phosphate 0.27 

Potassium dihydrogen phosphate 0.2 

Laurylsulfate sodium salt 0.01 

X-GAL 0.008 

MUG 0.005 

IPTG 0.01 

7.3 Readycult® Coliforms is provided in a ready-to-use, pre-measured, sterile snap pack 

that requires no preparation. This media should be purchased from a commercially 

available source and should not be prepared from basic ingredients. 

7.4 Sodium thiosulfate (Na2S203) - 3% solution (w/v) prepared with ACS grade Na2S203

(or equivalent) and reagent water. 

7.5 KOVAC ́s indole reagent is provided as a ready to use solution in 100 ml glass 

bottle or in a 30ml convenient dropper bottle (Bactident® Indole). 

8.0 Sample Collection, Dechlorination, Preservation, Shipment and Storage (1)

8.1 Water Sample Collection. Detailed guidance of current aseptic sample collection 

techniques may be found in: 1) Chapter V, 6.1-6.5 Manual for the Certification of 

Laboratories Analyzing Drinking Water: Criteria and Procedures Quality Assurance

– 5th Edition. U.S. EPA. Office of Ground Water and Drinking Water, EPA 815-R-05-

004. and/or 2) Section 9060 A-B. Standard Methods for the Examination of Water 

and Wastewater – 21st Edition – 2005. American Public Health Association, 

American Water Works Association, and Water Environment Federation. 

8.1.1 Collection Bottle or Bag – Sample containers should be clean, wide-mouth 

plastic or borosilicate glass bottles with non-leaking stoppers or caps that 

can withstand repeated sterilization. Presterilized plastic bottles or bags may 

also be used. The capacity of sample containers should be at least 120 ml. 

Containers should be non-fluorescing under long wave UV light. 

8.1.2 Sampling Procedure – Remove faucet attachments such as screen or splash 

guard. Open tap fully and let water run to waste for 2-3 minutes. Reduce 

water flow to permit filling bottle without splashing. 

8.2 Dechlorination - Chlorinated drinking water should be collected into a sterile 

collection vessel containing sufficient sodium thiosulfate (Na2S203) to neutralize the 

chlorine present. For a 100 ml water sample, 0.1 ml 3% Na2S203 should be used. 

Alternately, an equivalent amount of Na2S203 in tablet form may be substituted 

provided the tablet contains no binding agent that inhibits growth. 
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8.3 Preservation - Samples should be tested as soon as possible after collection. Water 

samples should be shipped or held at <10°C prior to testing and processed within 30 

hours of collection. See current regulations in Chapter V, 6.3-6.4 Manual for the 

Certification of Laboratories Analyzing Drinking Water: Criteria and Procedures 

Quality Assurance – 5th Edition. U.S. EPA. Office of Ground Water and Drinking 

Water, EPA 815-R-05-004. 

9.0 Quality Control

9.1 Each new lot of Readycult® Coliforms should be tested with positive and negative 

controls according to Chapter V, 5.1.6.4 Manual for the Certification of Laboratories 

Analyzing Drinking Water: Criteria and Procedures Quality Assurance – 5th Edition. 

U.S. EPA. Office of Ground Water and Drinking Water, EPA 815-R-05-004. 

9.2 For each quality control sample, aseptically transfer the content of one snap pack to 

100 ml sterile reagent water. Shake gently to completely dissolve. 

9.3 Inoculate the individual bottles of rehydrated Readycult® Coliforms with a loopful of 

growth from a 21 ± 3 h broth culture of the following: Citrobacter freundii (ATCC 

8090 or a characterized ß-D-galactosidase positive environmental strain) or 

Klebsiella pneumoniae (ATCC 31488) as a positive control for total coliform, E. coli

(ATCC 11775, ATCC 25922 or a characteristic MUG positive environmental strain) 

as a positive control for E. coli, and Enterococcus faecalis (ATTC 19433) or 

Pseudomonas Aeruginosa (ATCC 10145) as a negative control for both target 

organisms. Include an uninoculated bottle of rehydrated media as an additional 

negative control. 

9.4 Incubate at 36 ± 1°C or 35 ± 0.5°C for 24 ± 1 h and observe for the following results. 

Ensure that the specified incubation period is followed in air-type incubators 

according to Chapter V, 5.6.8 Manual for the Certification of Laboratories Analyzing 

Drinking Water: Criteria and Procedures Quality Assurance – 5th Edition. U.S. EPA. 

Office of Ground Water and Drinking Water, EPA 815-R-05-004. 

Readycult ® Coliforms Control Organism Reactions

Organism Color Fluorescence Indole 

Klebsiella pneumoniae Blue-Green Negative Negative 

Citrobacter freundii Blue-Green Negative Negative 

E. coli Blue-Green Positive Positive 

Pseudomonas aeruginosa Yellowish Negative Negative 

Enterococcus faecalis Yellowish Negative Negative 

 None Yellowish Negative Negative 
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9.5 For additional quality control testing, positive Readycult® Coliforms may be 

inoculated into brilliant green lactose bile, EC plus MUG or other media used for 

confirmation. 

9.6 Routine laboratory QA/QC procedures for applicable glassware, incubators, and 

other equipment should conform to : 1) Chapter V, Section 3, Manual for the 

Certification of Laboratories Analyzing Drinking Water: Criteria and Procedures 

Quality Assurance – 5th Edition. U.S. EPA. Office of Ground Water and Drinking 

Water, EPA 815-R-05-004. and/or 2) Section 9020. Standard Methods for the 

Examination of Water and Wastewater – 21st Edition – 2005. American Public 

Health Association, American Water Works Association, and Water Environment 

Federation. 

10.0 Calibration and Standardization

10.1 Readycult® Coliforms - there is no required calibration or standardization of the 

method. Acceptable performance of the Readycult® Coliforms by the user is 

determined by routine quality control using known cultures and is described in 

Section 9.0. 

10.2 Equipment: All equipment described in Section 6.0 should be maintained and 

calibrated according to the manufacturers' recommendations and the general QC 

guidelines in . 1) Chapter V, Section 3, Manual for the Certification of Laboratories 

Analyzing Drinking Water: Criteria and Procedures Quality Assurance – 5th Edition. 

U.S. EPA. Office of Ground Water and Drinking Water, EPA 815-R-05-004. and/or 

2) Section 9020. Standard Methods for the Examination of Water and Wastewater – 

21st Edition – 2005. American Public Health Association, American Water Works 

Association, and Water Environment Federation. 

11.0 Procedure

11.1 Test Procedure 

11.1.1 Add 100 ml water sample into a sterile, transparent vessel with at least a 120 

ml capacity. (refer to section 8.0) 

11.1.2 Take out one Readycult® Coliforms snap pack, tap the pack to ensure the 

granulated media does not stick to the bottom, and bend the upper part of 

the pack until it breaks open. Do not touch the opening. 

11.1.3 Add the contents to the water sample. Seal the vessel and shake to 

completely dissolve the granules. 

11.1.4 Incubation: 24 ± 1 h at 36 ± 1°C or 35 ± 0.5°C. Ensure that the specified 

incubation period is followed in air-type incubators according to Chapter V, 

5.3 Manual for the Certification of Laboratories Analyzing Drinking Water: 

Criteria and Procedures Quality Assurance – 5th Edition. U.S. EPA. Office of 

Ground Water and Drinking Water, EPA 815-R-05-004 
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11.2 Sample Interpretation 

11.2.1 Visually check each vessel for a blue-green color. If the sample is bluegreen, coliform bacteria are present in the test sample. 

11.2.2 If the solution is blue-green, examine for fluorescence using a long wave UV 

lamp. If fluorescence is present, the solution will glow a uniform, bright, lightblue throughout. The presence of fluorescence indicates that E. coli is 

present in the sample. 

11.2.3 Optional: If a secondary confirmation of E. coli is desired in the vessel with 

positive fluorescence, add approximately 2.5 ml KOVAC ́s indole reagent 

directly to the broth. A red ring will immediately form (indole reaction) to 

confirm the presence of E. coli. 

11.2.4 If no blue-green color is observed, the sample is negative for coliform 

bacteria. 

12.0 Data Analysis, Calculation, Interpretation and Reporting Results

12.1 Total Coliforms 

12.1.1 A change of color from slight yellow to blue-green indicates the presence of 

at least 1 CFU of coliform bacteria. Report as a positive Presence/Absence 

Test result. An absence of a change of color should be reported as a 

negative Presence/ Absence Test result. No further data analysis or 

calculation is required. 

12.2 E. coli

12.2.1 A fluorescence observed under long wave UV light indicates a positive result 

and the presence of at least 1 CFU of E. coli. Report as a positive 

Presence/Absence Test result. 

13.0 Method Performance Characteristics

13.1 Specificity - the specificity of Readycult® Coliforms was assessed for recovery of 

total coliforms and E. coli. Both positive and negative results were evaluated 

according to the EPA’s ATP(5) format for the presence or absence of total coliforms 

and E. coli as compared to the reference methods. Discrepancies between 

Readycult® Coliforms and reference method results in this study were resolved with 

an identification to species by a clinically approved commercial kit (#4345000 BBL 

Crystal Enteric/Nonfermenter). Both unadjusted (identification-to-species data not 

considered) and adjusted (identification-to-species data considered) results of that 

study appear below: 

13.1.1 Total Coliforms (unadjusted) - False positive error was 7.00%. False 

negative error was 5.10%. 

13.1.2 E. coli (unadjusted) - False positive error was 5.00%. False negative error 

was 6.86%. 
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13.1.3 Total Coliforms (adjusted) - False positive error was 3.0%. False negative 

error was 3.96%. 

13.1.4 E. coli (adjusted) - False positive error was 0.00%. False negative error was 

6.86%. 

13.2 Comparability - the comparability of Readycult® Coliforms was assessed for 

recovery of total coliforms and E. coli. Both positive and negative results were 

evaluated according to the EPA’s ATP(5) format for the presence or absence of total 

coliforms and E. coli as compared to the reference methods. Chi-square, CochranMantel-Haenszel, and Breslow-Day statistics were calculated on 16 sets of data for 

total coliforms and 15 sets of data for E. coli. The results are too extensive to 

summarize here. See report from Montana Environmental Laboratory. 

13.3 Additional note: The use of 120ml test vessel has been validated according to the 

EPA Microbiological Alternate Test Procedure (ATP) protocol for Drinking Water, 

Ambient Water, and Waste Water Methods. Based on a review of the study data, 

the EPA’s Office of Ground Water and Drinking Water (OGWDW) and Statistics and 

Analytical Support Branch (SASB) concluded that there is no significant difference 

between the performance of the method with 150ml bottles at 36°C versus when run 

in 120ml bottles at 35°C. These findings were ratified by the USEPA with a formal 

letter of approval dated 6th May 2005. 

13.4 Precision and Bias - This method is a qualitative test, and as such, precision and 

bias statements cannot be provided. 

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 and 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 sewage discharge permits and 

regulations (6). For further information, Federal, State or local agencies should be 

contacted. 
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16.0 References

1. 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, 18th ed. 

Greenberg, A.E., L.S. Clesceri, and A.D. Eaton, eds. Washington, D.C., American 

Public Health Association. 1993, pp. 9-1 9-69. 

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

Baltimore, Williams and Wilkins, 1989. 

3. U.S. 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. 

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

Grade), D1193-91. IN: Annual Book of ASTM Standards, vol. 11.01. Philadelphia, 

American Society for Testing Materials. 1991, p. 45. 

5. U.S. Environmental Protection Agency. Protocol for Alternative Test Procedures for 

Coliform Bacteria in Compliance with Drinking Water Regulations – Presence / 

Absence Liquid Culture Methods for Finished Waters – Version 1.2 December , 1995 

6. U.S. Environmental Protection Agency. National Primary and Secondary Drinking 

water Regulations: Analytical Methods for Regulated Drinking Water Contaminants; 

Proposed Rule, F6. Federal Register 58(129): 65626. Washington, D. C, Office of the 

Federal Register. December 15, 1993 

7. Manafi, M. and Rosmann, H., Evaluation of Readycult Presence-Absence Test for 

Detection of Total Coliforms and E. coli in Water. 98th. American Society for 

Microbiology, Atlanta, 17-21 May 1998 

8. Lee, J.V., Lightfoot, N.F. and Tillett, H.E. An evaluation of presence / absence tests for 

coliform organisms and Escherichia coli. International Conference on Coliforms and E. 

coli : Problem or solution? . 24-27 September 1995 , University of Leeds , UK 
UNITED STATES ENVIRONMENTAL PROTECTION AGENCY CINCINNATI, OHIO 45268 

Jennifer Singh 

EMD Chemicals 

Market Segment Manager 

Environmental & Molecular Microbiology 

480 S. Democrat Rd. 

Gibbstown, NJ 08027 

1/5/2007 

Dear Ms. Singh 

EPA’s Office of Ground Water and Drinking Water has reviewed EMD’s method 

“Readycult® Coliforms 100 Presence/Absence Test for Detection and Identification of 

Coliform Bacteria and Escherichia coli in Finished Waters, Version 1.1, January 2007” 

and has determined that it is acceptable for monitoring total coliforms and E. coli under 

the Total Coliform Rule (40 CFR 141.21). This current version incorporates changes that 

were approved in May 2005 (ATP Case No. BD03-001). 

We appreciate EMD Chemicals’ continued interest in the development of 

environmental compliance monitoring methods. If you have any questions regarding our 

determination, please contact Steven C. Wendelken at 513-569-7491. 

Sincerely, 

Steven C. Wendelken, Ph.D. 

ATP Coordinator 

Technical Support Center (MS-140) 

Office of Ground Water and Drinking Water