TECTA TM EC/TC Method for the Simultaneous Detection

of Total Coliforms and E. coli in Drinking Water

1 

# TECTA TM 

# EC/TC medium and the TECTA TM



# Instrument: a Presence/Absence Method for

the Simultaneous Detection of Total

Coliforms and Escherichia coli ( E. coli ) in Drinking Water

May 22, 2014

Version 1.0TECTA TM EC/TC Method for the Simultaneous Detection

of Total Coliforms and E. coli in Drinking Water

2 

## TECTA TM 

## EC/TC medium and the TECTA TM 

## 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 TECTA TM EC/TC method allows for the detection of total coliforms (TC) and

E scherichia coli (EC) within 18 hours of incubation at 35 ± 0.5°C without additional confirmation or verification steps.

1. 2 When using t his 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 b y 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 TECTA TM 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 o f the Metho d

2.1 The TECTA TM EC/TC method c ontains 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. Non

selective nutritive components support the growth of organisms, and antimicro bial agents in the test medium suppress the growth of non

- target bacteria.

2.2 The TECTA TM 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 TECTA TM

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

concentrati on of the products from the enzym atic reactions reaches a certain threshold of signal intensity, the TECTATM I nstrument determines if total coliforms and E. coli are present.TECTA TM EC/TC Method for the Simultaneous Detection

of Total Coliforms and E. coli in Drinking Water

3 2.3 T he method is performed by adding 100 mL of the water sample to be tested to a TECTA TM EC/TC cartridge containing the dehydrated culture medium

. A f ter 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

incu bation 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

exc ursion 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 TECTA TM 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, t otal coliforms produce β

- D

- galactosidase, which hydrolyze s
anthracene

- galactoside

, producing a hydrophobic, fluorescent product that is detected by the TECTATM I nstrument 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 hydrolyze s the pyrene

- glucuronide present in the me dium, 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. co li 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 wate r 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.TECTA TM EC/TC Method for the Simultaneous Detection

of Total Coliforms and E. coli in Drinking Water

4 

## 5.0 Safety

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

microbes and for handling potentially bio hazardous materials

.

5.2 Solid and liquid waste containing viable microorganisms should be decontaminated and disposed of by specif ic State and/or Federal guidelines. Decontamination of cultures or contaminated materials should be done by autoclaving or biohazard incineration.

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

M aterial Safety Data S heet ( M SDS) 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: TECTA TM I nstrument or equivalent instrument certified by Endetec to properly read and incubate the TECTA TM EC/TC cartridge.

A list of approved instruments is provided in Appendix A.

6 .2 Supplies:

6.2.1 TECTA TM EC/TC c a rtridges, which are pre

- measured, sealed cartridges containing the TECTATM EC/TC m edi um . The ca rtridges (fill line marked at 100

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

TECTA TM EC/TC cartridges should be stored in the dark, at room temperature ( 18 o C

- 3 0 o C) , 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

TECTA TM 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

.TECTA TM EC/TC Method for the Simultaneous Detection

of Total Coliforms and E. coli in Drinking Water

5 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 : TECTA TM EC/TC medium is provided ready to use in an incubation cartridge

.



### THE REMAINDER OF THIS PAGE IS INTENTIONALLY LEFT BLANKTECTA TM EC/TC Method for the Simultaneous Detection

of Total Coliforms and E. coli in Drinking Water

6 

## 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 (21 st 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 s o 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 d rinking 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. S amples should be analyzed as soon as possible after collection. If they cannot be process ed or refrigerated within one hour of collection , they

should be held on wet ice or in a cooler containing gel

- packs ( 1 o C

- 10 o C) for a

maximum of 30 h ours from col lection 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 (21 st Edition) and the Manual for the Certification of Laboratories analyzing drinking water ( 2005, EPA 815

- R

- 004 ).

9. 2 Each lot of TECTA TM EC/TC Cartridges should be tested using known positive and negative organisms. The recommended inocul um 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, puri fied water

in a TECTA TM 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 TECTA TM EC/TC cartridge a nd incubat e 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.TECTA TM EC/TC Method for the Simultaneous Detection

of Total Coliforms and E. coli in Drinking Water

7 9.4 Control Cultures

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

evaluate the TECTA TM 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 f rom 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 N egative: E

. aerogenes ATCC 13048



## 10.0 Calibration and Standardization

10.1 Each TECTA TM I nstrument 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 TECTA TM I nstrument provides a built

- in system for continuous management, reading and recording of the sample incubation temperature. The incubation system

must have be en calibrated within the past year against a National Institute of Standards and Technology (NIST) calibra ted thermometer. Note that this calibration m ay 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 re port 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 te mperature. Refer to detailed

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

of Total Coliforms and E. coli in Drinking Water

8 

## 11.0 Procedure

11.1 Cartridge Preparation Procedure

11.1.1 Mix the dechlorinated water sample thoroughly, add 100 mL to TECTA TM 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 th e built

- in incubation function of the TECTA TM 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 TECTA TM 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 TECTA TM Instrument to reach temperature equilibrium at 35 ± 0.5 o C, and the automated start

- up routine is completed (approximately 3 minutes).

11.2.2 Select “U ser 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 M ode O peration ” for the TECTA TM EC/TC test. In this

mode, t he TECTA EC/TC d etection s ystem 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 t ouch screen and open.

11.2.6 Load the TECTA TM EC/TC t est c artridges containing the sample (s) to the

specified chambers.

11.2.7 Close the lid . I ncubation, detection and timing of resul ts will start automatically

upon closure of the lid . T he 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 18 0 seconds. An on

- screen warning will appear.TECTA TM EC/TC Method for the Simultaneous Detection

of Total Coliforms and E. coli in Drinking Water

9 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 c hamber 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 display ed. 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 c hamber 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 O verview 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

int ernal 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 TECTA TM 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, TECTA TM EC/TC tests can be incubated for 18 h ou r s in a separate , calibrated, laboratory

- grade ai r incubator and read in the TECTA TM
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 “ A dd” , the next available chamber will be allocated.TECTA TM EC/TC Method for the Simultaneous Detection

of Total Coliforms and E. coli in Drinking Water

10 11.4. 3 Select “ Overview ”

, and unlock the lid by touching the Lid icon. Open the lid and

add the pre

- incubated TECTA TM 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 min ute per sample to complete.

11.4. 5 Remove samples on completion of reading and d iscard TECTA TM 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.TECTA TM EC/TC Method for the Simultaneous Detection

of Total Coliforms and E. coli in Drinking Water

11 

## 12.0 Data Analysis and C alculations

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 T otal C oliforms 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 o f fluorescence after 18 hours of incubation.TECTA TM EC/TC Method for the Simultaneous Detection

of Total Coliforms and E. coli in Drinking Water

12 

## 13.0 Method Performance

The TECTA TM 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 an d 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 TECTA TM EC/TC

method a nd lauryl tryptose broth (LTB) in a presence

- absence format using replicates of 20 for each sample. Both the TECTA TM EC/TC and LTB results were confirmed using LTB/brilliant green lactose bile broth for total coliforms and LTB and Escherichia coli broth w ith 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

T he sensitivity of the TECTA TM EC/TC method for total coliforms is (19 1 /(19 1 +18))*100 = 91.4%

The specificity of the TECTA TM EC/TC method for total coliforms is (88/(88+3 ))*100 = 9 6.7 %

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

. S ummary of E. coli data from the ATP Study

True Positive False positive True Negative False Negative

19 1 1 2 8 6 31

T he sensitivity of the TECTA TM EC/TC method for E. coli is (191 /(19 1+31))*100 = 86.0 %

T he specificity of the TECTA TM EC/TC method for E. coli is (86/(86+12))*100 = 87.8 % True Positive False positive True Negative False Negative

19 1 3 88 18TECTA TM EC/TC Method for the Simultaneous Detection

of Total Coliforms and E. coli in Drinking Water

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

calculation the False Positive Rate is 1 2.2 % and the False Negative Rate

is 14. 0 %

The TECTA TM EC/TC M ethod was shown to have acceptable performance at an 18 h our
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 materia l.

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 TECTA TM EC/TC Method should be cognizant of and must comply with these regulations.

15.2 Information given in the Package insert and M SDS should be used as a basis for waste
management in addition t o determining the scope of all applica ble rules and regulations.

15.3 Any samples, glassware, disp osable materials that may have bacterial contamination must be sterilized or decontaminated via autoclaving or incineration prior to disposal.TECTA TM EC/TC Method for the Simultaneous Detection

of Total Coliforms and E. coli in Drinking Water

14 

## 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 . 2 1 th Edition.

American Public Health Association. 1015 15 th 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 Ame rican Chemistry Society. 2005. Reagent Chemicals. In American Chemical Society Specifications, 10 th Edition. American Chemical Society, Washington D.C.

16.4 U.S. Environmental Protection Agency. Manual for the Certification of Laboratories analyzing drin king water, 2005, EPA 815

- R

- 004, USEPA, Cincinnati OH

16.5 USEPA . 1978. EPA Microbiological Methods for Monitoring the Environment, EPA

600/8

- 78

- 017



## 17.0 Tables, Diagrams, Flowcharts, and Validation Data

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

of Total Coliforms and E. coli in Drinking Water

15 

## Appendix A: Approved Instrument and Consumable List

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

Instrument :

 TECTA TM B16: Instrument Rev. 1.0; Software Rev. 1.0.3;

 TECTA TM B16: Instrument Rev. 1.0; Software Rev. 2.0.4.

Consumable :

 TECTA TM EC/TC cartridge Rev 1.0.