TECTATM EC/TC Method for the Simultaneous Detection

of Total Coliforms and E. coli in Drinking Water

1

TECTATM EC/TC medium and the TECTATM

Instrument: a Presence/Absence Method for 

the Simultaneous Detection of Total 

Coliforms and Escherichia coli (E. coli) in 

Drinking Water

May 22, 2014

Version 1.0
TECTATM EC/TC Method for the Simultaneous Detection

of Total Coliforms and E. coli in Drinking Water

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TECTATM EC/TC medium and the TECTATM Instrument, a 

Presence/Absence Method for the Simultaneous 

Detection of Total Coliforms and Escherichia coli (E. 

coli) in Drinking Water

1.0 Scope and Application 

1.1 The TECTATM EC/TC method allows for the detection of total coliforms (TC) and 

Escherichia coli (EC) within 18 hours of incubation at 35 ± 0.5°C without additional 

confirmation or verification steps. 

1.2 When using this method, the results are read automatically within the secure, simple to 

use incubator/reader instrument (the TECTATM Instrument). The TECTATM Instrument 

continuously monitors the test sample throughout the incubation period, and positive 

results may be obtained as early as after 2 hours of incubation, with final results being 

obtained after 18 hours of incubation. The TECTA system also allows for optional local 

and remote alerting (by email or SMS text message) upon detection of a positive result. 

No visual interpretation of the test results is required by the analyst, and personnel are 

not required to return to the laboratory to interpret test results at a specific time. 

1.3 The limit of detection of the TECTATM EC/TC method is 1 culturable cell of total coliform 

or E. coli bacteria in a 100 mL sample volume. Detection of the target organisms by a 

positive result is associated with inadequate water treatment, biofilm growth in the 

distribution system and/or other sources of contamination that may indicate a public 

health risk. 

2.0 Summary of the Method

2.1 The TECTATM EC/TC method contains two specific enzyme substrates that are used to

detect the activity of β-D-galactosidase and β-D-glucuronidase enzymes. The presence 

of β-D-galactosidase is indicative of the presence of total coliforms, while the presence 

of both enzymes indicates the presence of E. coli. These indicator enzymes are the 

basis of other commercially available and US EPA approved test methods. Nonselective nutritive components support the growth of organisms, and antimicrobial 

agents in the test medium suppress the growth of non-target bacteria.

2.2 The TECTATM EC/TC method detects the activity of the enzymes β-D-galactosidase and 

β-D-glucuronidase as they hydrolyze the specific substrates present in the medium. The 

cleavage products of the substrates are hydrophobic and fluorescent at different 

wavelengths, and diffuse into a polymer structure embedded in the base of the TECTATM

EC/TC cartridge where their presence is detected by the instrument. When the 

concentration of the products from the enzymatic reactions reaches a certain threshold 

of signal intensity, the TECTATM Instrument determines if total coliforms and E. coli are 

present. 
TECTATM EC/TC Method for the Simultaneous Detection

of Total Coliforms and E. coli in Drinking Water

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2.3 The method is performed by adding 100 mL of the water sample to be tested to a 

TECTATM EC/TC cartridge containing the dehydrated culture medium. After dissolution 

of the medium, the cartridge is incubated at 35 ± 0.5°C in the TECTATM Instrument, 

which serves as both an incubator and a fluorescence reader (“Standard Mode”). The 

incubation system maintains water sample temperature in the required range of 35 ± 

0.5°C throughout the incubation period. A minimum of three (3) temperature readings 

are automatically recorded with each test result report, and any incubation temperature 

excursion is automatically detected by the instrument and recorded on the test result 

report. Alternatively, the test sample may be incubated for 18 hours in a 35 ± 0.5°C 

incubator and the TECTATM Instrument employed after incubation to read the 

fluorescence level (“Read Mode”). 

The presence of total coliforms and E. coli is automatically detected by the TECTATM 

Instrument as soon as the preset detection threshold is reached in the instrument. No 

visual estimation of fluorescence levels is performed.

3.0 Method Definitions 

3.1 Total Coliform Bacteria – A group of bacteria belonging to the Enterobacteriaceae

family and defined by the presence of the enzyme β-D-galactosidase. When using the 

TECTA medium, total coliforms produce β-D-galactosidase, which hydrolyzes 

anthracene-galactoside, producing a hydrophobic, fluorescent product that is detected 

by the TECTATM Instrument and signals the presence of the target organisms. 

3.2 E. coli – A bacterial species belonging to the total coliform group present in the feces of 

warm blooded animals. Most E. coli strains produce the enzyme β-D-glucuronidase. 

When using the TECTATM medium, the E. coli produce β-D-glucuronidase that 

hydrolyzes the pyrene-glucuronide present in the medium, producing a hydrophobic, 

fluorescent product that is detected by the instrument. Products from the hydrolysis of 

both anthracene-galactoside and pyrene-glucuronide must be present and detected by 

the TECTATM Instrument to signal the presence of E. coli in the TECTATM method. 

3.3 Pyrene-glucuronide – a substrate which can be hydrolyzed by β-D-glucuronidase 

enzyme releasing hydroxy-pyrene, a hydrophobic, fluorescent compound. 

3.4 Anthracene-galactoside – a substrate which can be hydrolyzed by β-D-galactosidase 

releasing hydroxy-anthracene, a hydrophobic, fluorescent compound. 

3.5 Hydrolysis – biochemical reaction catalyzed by an enzyme resulting in the cleavage of 

a substrate by the addition of a water molecule. 

4.0 Interferences –Enterococcus faecalis, if present, may occasionally cause a false 

positive result for total coliforms. Levels over 10,000 cfu of the organism were not found 

to interfere with the E. coli result. 
TECTATM EC/TC Method for the Simultaneous Detection

of Total Coliforms and E. coli in Drinking Water

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5.0 Safety 

5.1 The laboratory must understand and practice normal safety procedures for working with 

microbes and for handling potentially biohazardous materials. 

5.2 Solid and liquid waste containing viable microorganisms should be decontaminated and 

disposed of by specific State and/or Federal guidelines. Decontamination of cultures or 

contaminated materials should be done by autoclaving or biohazard incineration. 

5.3 The TECTATM EC/TC medium is not listed as a suspected carcinogen. Refer to the 

Material Safety Data Sheet (MSDS) provided with the product for specific information 

relating to the potential hazards of this product by accidental ingestion or exposure. 

6.0 Equipment and Supplies 

6.1 Equipment: TECTATM Instrument or equivalent instrument certified by Endetec to 

properly read and incubate the TECTATM EC/TC cartridge.

A list of approved instruments is provided in Appendix A.

6.2 Supplies:

6.2.1 TECTATM EC/TC cartridges, which are pre-measured, sealed cartridges 

containing the TECTATM EC/TC medium. The cartridges (fill line marked at 100 

mL, capacity of 130 mL) are provided ready to use containing dehydrated media.

TECTATM EC/TC cartridges should be stored in the dark, at room temperature 

(18oC - 30

oC), prior to use. Cartridges must be used before the expiration date 

marked on the manufacturer’s certificate included in the package.

A list of approved consumables is provided in Appendix A.

6.2.2 Glassware or plastic containers suitable for sample collection. The head space 

in the sample collection bottles must be at least 1” to allow for adequate mixing of 

the sample. All glassware and plastic containers used must be appropriately 

washed and sterilized prior to being used for sample collection or analyses. 

Alternately pre-sterilized disposable vessels may be used. Samples should be 

premeasured using a sterile graduated cylinder and then transferred into the 

TECTATM EC/TC cartridge. 

6.2.3 Sodium thiosulfate in sample collection bottles sufficient to neutralize up to 5 ppm 

(mg/L) of residual chlorine.

6.2.4 Disposable sterile pipettes.
TECTATM EC/TC Method for the Simultaneous Detection

of Total Coliforms and E. coli in Drinking Water

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6.2.5 Calibrated pipette and sterile disposable tips.

7.0 Reagents and Standards

7.1 Purity of water: Reagent type purified water conforming to ASTM Type II water or better. 

7.2 Purity of reagents: Chemicals should conform to ACS or equivalent grade.

7.3 Medium: TECTATM EC/TC medium is provided ready to use in an incubation cartridge.

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TECTATM EC/TC Method for the Simultaneous Detection

of Total Coliforms and E. coli in Drinking Water

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8.0 Sample Collection, Preservation, and Storage

8.1 Sample collection - procedures are described in detail in Standard Methods for the 

Examination of Water and Wastewater 9060 (21st Edition). Briefly, sample collection 

bottles should be screw top borosilicate or other non-reactive glass or plastic of sufficient 

size to complete all testing. The sample collection vessels must be washed and rinsed 

thoroughly followed by a purified water rinse then sterilized. Alternately disposable 

bottles may be used. Sample volumes for each total coliform analysis in support of the 

Total Coliform Rule (40 CFR §141.852) must be 100 mL. Sample collection vessels 

must have at least a 1” headspace so that adequate mixing can occur before the sample 

is measured. 

8.2 Dechlorination – for samples that may contain residual chlorine or other halogens a 

reducing agent such as sodium thiosulfate should be added to the bottles. For 100 mL 

of a typical drinking water sample, 0.1 mL of a 3% sodium thiosulfate solution should be 

sufficient to neutralize up to 5 ppm (mg/L) residual chlorine.

8.3 Preservation and storage – Samples potentially containing chlorine should be 

neutralized at time of collection. Samples should be analyzed as soon as possible after 

collection. If they cannot be processed or refrigerated within one hour of collection, they 

should be held on wet ice or in a cooler containing gel-packs (1

oC - 10oC) for a 

maximum of 30 hours from collection to analysis. Sample hold times and transport 

conditions must adhere to appropriate State and Federal regulations.

9.0 Quality Control 

9.1 Quality control procedures should be consistent with guidance in Standard Methods for 

the Examination of Water and Wastewater 9020 (21st Edition) and the Manual for the 

Certification of Laboratories analyzing drinking water (2005, EPA 815-R-004). 

9.2 Each lot of TECTATM EC/TC Cartridges should be tested using known positive and 

negative organisms. The recommended inoculum level for positive organisms is 50-100 

colony forming units (cfu) per 100 mL. Negative strains should be tested at ≥1000 

cfu/100 mL. The control organisms should be added to 100 mL of sterile, purified water 

in a TECTATM EC/TC cartridge.

9.3 Each lot of medium should be tested for sterility and autofluorescence or as needed to 

meet regulations. Prepare a sterile medium blank by adding 100 mL of sterile, purified 

water to a TECTATM EC/TC cartridge and incubate in the TECTA instrument. The sterile 

water blank must produce a negative sample result at the end of the run to be valid. This 

also demonstrates the absence of autofluorescence in the medium lot.
TECTATM EC/TC Method for the Simultaneous Detection

of Total Coliforms and E. coli in Drinking Water

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9.4 Control Cultures – the following are recommended control cultures that can be used to 

evaluate the TECTATM EC/TC method and medium. Strains to be used for Quality 

Control purposes should be obtained either from a culture collection (American Type 

Culture Collection, ATCC, or equivalent) or from properly characterized and identified 

environmental isolates. Other relevant strains may be used. At least one positive control 

and one negative control culture should be run for each lot or at an interval consistent 

with State or Federal Regulations.

9.4.1 Total coliform Control Strains

9.4.1.1 Positive: Enterobacter aerogenes ATCC 13048

9.5.1.2 Negative: Pseudomonas aeruginosa ATCC 10145 

9.4.2 E. coli Control Strains

9.4.2.1 Positive: E. coli ATCC 25922 

9.4.2.2 Negative: E. aerogenes ATCC 13048

10.0 Calibration and Standardization

10.1 Each TECTATM Instrument is provided with a calibration cartridge that determines that 

the instrument is working properly and recording at both designated wavelengths. 

Follow manufacturer’s instructions for calibration and periodic verification.

10.2 The TECTATM Instrument provides a built-in system for continuous management, 

reading and recording of the sample incubation temperature. The incubation system 

must have been calibrated within the past year against a National Institute of Standards 

and Technology (NIST) calibrated thermometer. Note that this calibration may only be 

performed by a factory-authorized service technician. The incubation system maintains 

water sample temperature in the required range of 35 ± 0.5°C throughout the incubation 

period. A minimum of three (3) temperature readings are automatically recorded with 

each test result report, and any incubation temperature excursion is automatically 

detected by the instrument and recorded on the test result report for possible invalidation 

of the result by the analyst. 

For additional external temperature validation, the instrument chamber and media 

cartridges can be accessed with a National Institute of Standards and Technology 

(NIST) calibrated thermometer or temperature probe. Note that a temperature correction

of -1.1 degree C compared to the average of the maximum and minimum temperature 

measured over a 5-minute period must be applied to external temperature 

measurements to reflect sample incubation temperature. Refer to detailed 

manufacturer’s instructions.
TECTATM EC/TC Method for the Simultaneous Detection

of Total Coliforms and E. coli in Drinking Water

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11.0 Procedure

11.1 Cartridge Preparation Procedure

11.1.1 Mix the dechlorinated water sample thoroughly, add 100 mL to TECTATM EC/TC 

cartridge and securely close cap.

11.1.2 Swirl cartridge with sample by hand for approximately 2 minutes until medium is 

dissolved. A slight amount of medium may remain undissolved; this will not 

affect the test. Shaking the cartridge is not required or recommended but will not 

affect the test.

11.1.3 If the built-in incubation function of the TECTATM Instrument is to be used, follow 

the Instrument Procedure (Standard Mode) described in Section 11.2. 

Alternatively, cartridges may be incubated in an external air incubator and 

subsequently read by the TECTATM Instrument. Follow Read Mode Procedure 

described in Section 11.4.

11.2 Instrument Procedure (Standard Mode)

11.2.1 Switch power on and allow the TECTATM Instrument to reach temperature 

equilibrium at 35 ± 0.5oC, and the automated start-up routine is completed

(approximately 3 minutes).

11.2.2 Select “User ID” from the menu on Log-in screen, enter user ID (if required). 

11.2.3 Select “Test” on the touch screen and enter sample identification information for 

each sample to be run and record the specific incubation/detection chamber 

using the touch screen.

11.2.4 Select the “Standard Mode Operation” for the TECTATM EC/TC test. In this 

mode, the TECTA EC/TC detection system will continuously incubate and read 

the samples throughout the 18 hour incubation period.

11.2.5 Unlock the lid using the lid icon on the touch screen and open.

11.2.6 Load the TECTATM EC/TC test cartridges containing the sample(s) to the 

specified chambers.

11.2.7 Close the lid. Incubation, detection and timing of results will start automatically 

upon closure of the lid. The lid will lock automatically. Additional samples may 

be added/withdrawn by pressing the appropriate on-screen control. The lid may 

not be kept open for more than 180 seconds. An on-screen warning will appear.
TECTATM EC/TC Method for the Simultaneous Detection

of Total Coliforms and E. coli in Drinking Water

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11.3 Completion of Run, Test Results and Data Recording (Standard Mode)

11.3.1 During the test, the progress of each chamber is indicated by a sample progress 

indicator on the chamber screen indicating elapsed time of the test. This 

displays the progress until the end of the incubation/detection cycle at 18 hours

(or earlier if sample is positive for both EC and TC). 

11.3.2 When a test sample is detected as a positive anytime during the incubation, the 

positive indicator is displayed. At the end of the cycle, all negative tests are 

indicated by the negative test indicator. For each target organism detected, the 

time at which a positive result was detected is also recorded. 

11.3.3 At the completion of a test run, the incubator chamber will turn off and the results 

at 18 hours (target organism present or absent) will continue to be displayed until 

the user clears the Overview screen and removes the completed samples. 

11.3.4 Select the individual chamber on the Overview screen for a completed test 

summary and temperature recording. Repeat for each test sample. The data can 

also be retrieved and downloaded for storage using the “Reports” icon and 

accessing via the USB port(s). Data from each run are stored in the non-volatile

internal memory of the instrument. Onboard memory storage capacity is 

sufficient to store several years of test data.

11.3.5 When complete, unlock the lid using the Lid icon and remove the completed test 

samples from the chamber.

11.3.6 Discard TECTATM EC/TC test cartridges as potential biohazards according to 

State and Federal regulations. 

11.4 Read Mode (Alternate Procedure) 

11.4.1 As an alternate procedure, TECTATM EC/TC tests can be incubated for 18 hours

in a separate, calibrated, laboratory-grade air incubator and read in the TECTATM

Instrument using the “Read mode”. Incubation temperature must be 35 ± 0.5°C

and cartridges must be placed in the incubator with a minimum 0.5 inch (1 cm) 

air gap between adjacent cartridges to allow for incubator air circulation. 

Sample results must be read immediately (within 30 minutes) of removal from the 

incubator.

11.4.2 Select “New Tests” on the touch screen and enter the Sample ID. Select “Read 

Mode” as test type. Select “Add”, the next available chamber will be allocated.
TECTATM EC/TC Method for the Simultaneous Detection

of Total Coliforms and E. coli in Drinking Water

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11.4.3 Select “Overview”, and unlock the lid by touching the Lid icon. Open the lid and 

add the pre-incubated TECTATM test cartridge(s) containing the sample or 

controls to the specified chamber. Repeat for each pre-incubated sample.

11.4.4 The sample results are read automatically upon the closing of the lid. The 

Read Mode requires approximately 1 minute per sample to complete.

11.4.5 Remove samples on completion of reading and discard TECTATM EC/TC test 

cartridges as potential biohazards according to State and Federal regulations. 

11.4.6 Presence/absence test results are displayed on-screen and are also recorded in 

the non-volatile memory of the instrument.
TECTATM EC/TC Method for the Simultaneous Detection

of Total Coliforms and E. coli in Drinking Water

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12.0 Data Analysis and Calculations

12.1 The instrument continually reads and records change in fluorescence at two different 

wavelengths for each of the enzyme products specific to each of the target organisms. 

12.2 As soon as the amount of fluorescence reaches a designated threshold for each

wavelength, the instrument records a positive result for Total Coliforms and E. coli and 

an indication is displayed on-screen. 

12.3 A positive result is designated by the on-screen display for the specific water sample 

cartridge and a time to detection (TTD) in hours and minutes is also shown. In general, 

positive samples with shorter TTD results contain higher initial concentrations of the 

target organism.

12.4 A negative result is produced when the instrument has detected no significant 

development of fluorescence after 18 hours of incubation. 
TECTATM EC/TC Method for the Simultaneous Detection

of Total Coliforms and E. coli in Drinking Water

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13.0 Method Performance

The TECTATM EC/TC Method was compared to defined reference methods according to the 

2010 EPA Microbiological Alternate Test Procedure (ATP) Protocol for Drinking Water, Ambient 

Water, Wastewater and Sewage Sludge Monitoring Methods. Primary wastewater effluent 

samples from 10 different geographical sources were used to test total coliforms and E. coli at 

an independent laboratory. Each sample was chlorinated to obtain a 2 - 4 log reduction, and 

then neutralized with sodium thiosulfate. The samples were diluted in oxidant free drinking 

water to obtain low numbers of injured target organisms and tested with the TECTATM EC/TC 

method and lauryl tryptose broth (LTB) in a presence-absence format using replicates of 20 for 

each sample. Both the TECTATM EC/TC and LTB results were confirmed using LTB/brilliant 

green lactose bile broth for total coliforms and LTB and Escherichia coli broth with MUG 

(EC+MUG) for E. coli. 

The overall results for the total coliform comparison are shown in Table 1. 

Table 1. Summary of total coliform data from the ATP Study

The sensitivity of the TECTATM EC/TC method for total coliforms is 

(191/(191+18))*100 = 91.4%

 

The specificity of the TECTATM EC/TC method for total coliforms is 

(88/(88+3))*100 = 96.7%

Based on all samples run during this study for the detection of total 

coliforms, by calculation the False Positive Rate is 3.3% and the False 

Negative Rate is 8.6%

The overall results for E. coli from the same study are presented in Table 2.

Table 2. Summary of E. coli data from the ATP Study

True Positive False positive True Negative False Negative

191 12 86 31

The sensitivity of the TECTATM EC/TC method for E. coli is 

(191/(191+31))*100 = 86.0%

 

The specificity of the TECTATM EC/TC method for E. coli is 

(86/(86+12))*100 = 87.8%

True Positive False positive True Negative False Negative

191 3 88 18
TECTATM EC/TC Method for the Simultaneous Detection

of Total Coliforms and E. coli in Drinking Water

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Using all samples run in the study for the detection of E. coli, by 

calculation the False Positive Rate is 12.2% and the False Negative Rate 

is 14.0% 

The TECTATM EC/TC Method was shown to have acceptable performance at an 18 hour 

incubation time and equivalent or greater sensitivity for the defined target organisms in 

comparison to the standard reference procedures during the ATP study.

14.0 Pollution Prevention

14.1 Every effort should be made to reduce or eliminate the quantity and/or toxicity of waste 

at the site of generation. 

14.2 Each lab, wherever possible, should review the process along with materials and 

reagents used and strive to use these as efficiently as possible so as to generate the 

least waste and hazardous material. 

14.3 Recycling should be considered whenever possible. 

15.0 Waste Management

15.1 Waste management should be consistent with all local and Federal rules and 

regulations. Any lab using the TECTATM EC/TC Method should be cognizant of and 

must comply with these regulations. 

15.2 Information given in the Package insert and MSDS should be used as a basis for waste 

management in addition to determining the scope of all applicable rules and regulations.

15.3 Any samples, glassware, disposable materials that may have bacterial contamination 

must be sterilized or decontaminated via autoclaving or incineration prior to disposal. 

 
TECTATM EC/TC Method for the Simultaneous Detection

of Total Coliforms and E. coli in Drinking Water

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16.0 References

16.1 USEPA. 2010. EPA Microbiological Alternate Test Procedure (ATP) Protocol for Drinking 

Water, Ambient Water, Wastewater and Sewage Sludge Monitoring Methods. EPA-821-

B-10-001. 

16.2 APHA. 2005. Standard Methods for Examination of Water and Wastewater. 21

th Edition. 

American Public Health Association. 1015 15th St, NW, Washington, DC 20005

16.3 American Society for Testing and Materials (ASTM). Standard Specification of Reagent 

Water, Designation D1193-06 (2011). In: Book of Standards Volume 11.01

16.3 American Chemistry Society. 2005. Reagent Chemicals. In American Chemical Society 

Specifications, 10th Edition. American Chemical Society, Washington D.C.

16.4 U.S. Environmental Protection Agency. Manual for the Certification of Laboratories 

analyzing drinking water, 2005, EPA 815-R-004, USEPA, Cincinnati OH

16.5 USEPA. 1978. EPA Microbiological Methods for Monitoring the Environment, EPA600/8-78-017

17.0 Tables, Diagrams, Flowcharts, and Validation Data

Refer to Section 13.0 for details on the Method Performance. 
TECTATM EC/TC Method for the Simultaneous Detection

of Total Coliforms and E. coli in Drinking Water

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Appendix A: Approved Instrument and Consumable List

The following instruments and consumables have been approved for use with this method:

Instrument:

 TECTATM B16: Instrument Rev. 1.0; Software Rev. 1.0.3;

 TECTATM B16: Instrument Rev. 1.0; Software Rev. 2.0.4.

Consumable:

 TECTA TM EC/TC cartridge Rev 1.0.