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GreenLight® Model 910
Software Use and Water
Sample Analysis Manual
September 23, 2013
Page 2 of 45
Table of Contents
Introduction ..................................................................................................................................... 6
Instrument Set-Up ........................................................................................................................... 6
Software Installation ....................................................................................................................... 6
Main Start-Up Screen ..................................................................................................................... 7
Administrator ................................................................................................................................ 12
Manage Languages ..................................................................................................................... 13
Administrator Options ................................................................................................................ 13
Manage Sample Templates ......................................................................................................... 14
Manage Preparation Lists ........................................................................................................... 15
Manage Calibration ....................................................................................................................... 16
View calibrations .......................................................................................................................... 17
Reference Check ........................................................................................................................... 18
Multipoint Calibrations ................................................................................................................. 19
Quick Calibration .......................................................................................................................... 20
Manual Calibration ....................................................................................................................... 21
Table 1: Manual Calibration Calculation ............................................................................ 24
Table 2: Decimal Hour Calculator ...................................................................................... 24
Preparing a Method ....................................................................................................................... 26
Running an Analyses .................................................................................................................... 30
Water Analyses ............................................................................................................................. 31
Equipment ..................................................................................................................................... 31
Chemicals ...................................................................................................................................... 31
Safety ............................................................................................................................................ 32
Aerated Dilution Water ................................................................................................................. 32
Hydration of PolySeed® Seed ...................................................................................................... 32
Wastewater Secondary Treatment Biomass Seed ......................................................................... 33
Experimental Procedure ................................................................................................................ 34
Water Sample .............................................................................................................................. 34
Page 3 of 45
Sample Collection, Storage, and Holding Time ......................................................................... 34
Analysis Pretreatment ................................................................................................................. 34
Prior to analyses ...................................................................................................................... 34
GreenLight® Bacterial Activity Test for Water ......................................................................... 35
Water Test Sample ...................................................................................................................... 35
GreenLight® 910 Software Procedure ......................................................................................... 38
Table 4: Examples for Continuous Test.............................................................................. 38
Continuous Test .......................................................................................................................... 39
Multi Point Test .......................................................................................................................... 39
Quality Control ............................................................................................................................. 43
Duplicate Relative Percent Difference ........................................................................................ 43
Control Blank .............................................................................................................................. 44
References ..................................................................................................................................... 45
Page 4 of 45
Table of Figures
Figure 1: GreenLight® 910 Start-Up Screen .................................................................................. 7
Figure 2: File Tab Screen ................................................................................................................ 8
Figure 3: Page Setup Screen ........................................................................................................... 8
Figure 4: Global Reports Screen ..................................................................................................... 9
Figure 5: Tools Tab Screen ............................................................................................................. 9
Figure 6: Options Function Screen. .............................................................................................. 10
Figure 7: Events Log Screen ......................................................................................................... 10
Figure 8: Help Tab Screen. ........................................................................................................... 11
Figure 9: Help Screen. .................................................................................................................. 11
Figure 10: About Screen. .............................................................................................................. 12
Figure 11: Administrators Screen ................................................................................................. 12
Figure 12: Manage Languages Screen .......................................................................................... 13
Figure 13: Administrator Options Screen ..................................................................................... 13
Figure 14: Manage Templates Screen. .......................................................................................... 14
Figure 15: Manage Preparations Screen. ...................................................................................... 15
Figure 16: Information added to a Preparation. ............................................................................ 15
Figure 17: Manage Calibrations Screen ........................................................................................ 16
Figure 18: View Calibrations Screen ............................................................................................ 17
Figure 19: Reference Check Screen. ............................................................................................. 18
Figure 20: Reference Check vials. ................................................................................................ 18
Figure 21: Multipoint Calibration Screen with 3 Calibration Points Selected. ............................ 19
Figure 22: Multipoint Calibration Linear Fit with Formula and R2 Screen. ................................. 20
Figure 23: Quick Calibration Screen with Single Calibration Points Selected. ........................... 21
Figure 24: Quick Calibration Linear Fit with Formula and R2 Screen. ........................................ 21
Figure 25: Manual Calibration Screen. ......................................................................................... 22
Figure 26: Excel Manual Calibration Plot .................................................................................... 25
Figure 27: Manage Methods Screen ............................................................................................. 27
Figure 28: New Method Screen .................................................................................................... 28
Page 5 of 45
Figure 29: Edit Method Screen ..................................................................................................... 29
Figure 30: PolySeed® Aeration .................................................................................................... 33
Figure 31: Sample Being Aerated ................................................................................................. 35
Figure 32: Mixed Sample with Media A and Media B ................................................................. 36
Figure 33: Sample in a 15 mL Vial Being Transferred into a GreenLight® 910 ......................... 36
Figure 34: 15 mL GreenLight® Vials in the Hot Block Incubator ............................................... 37
Figure 35: New Test Screen .......................................................................................................... 38
Figure 36: Continuous Analysis Screen ........................................................................................ 39
Figure 37: Vial Read Screen for Multipoint test ........................................................................... 40
Figure 38: Vial Description Screen for Multipoint test ................................................................ 41
Figure 39: Multipoint Test Incubating Main Screen..................................................................... 41
Figure 40: Multipoint Incubating Analyses Screen ...................................................................... 42
Figure 41: Example of RPD Chart for Water Analyses ................................................................ 43
Page 6 of 45
Introduction
This SOP will cover the software set-up, use and basic sample analyses for the GreenLight®
Model 910 instrument. The use of the instrument requires familiarization with current computer
operating software Microsoft® Windows (XP, 7 or 8) and for the manual calibration
calculations, Microsoft® Office Excel (XP, 7, 10, or 13).
Instrument Set-Up
Refer to the “GreenLight® Series Model 910 Operators Manual” for the Model 910 instrument:
Unpacking the Instrument
Setting Up the System
Parts and Controls
Software Installation
1. A computer program is required to operate the GreenLight™ model 910 system. This
program must be installed on a computer before the instrument can be used. The software is
provided on CD.
2. The GreenLight™ application comes with one master “Install” CD. This CD ROM contains
licensed or copyrighted programs.
NOTE: Installation of this software requires “Administrator” level access to the computer.
Check the Windows reference material for assistance in logging in at the correct access level.
3. To install the GreenLight™ application, insert the MOCON GreenLight™ model 910
Software CD into your CD-ROM drive; if “Auto-Play” is enabled, the installation program
will start automatically. If “Auto-Play” is disabled, select “Run” from the Windows start
menu and type in “D:\Setup.exe” where “D” is the CD-ROM drive letter, and then select
“OK”.
NOTE: The “Disable Login” check box is used to disable all “Application Level” security
functions. If you do not need users to “Log In” to the GreenLight application we suggest you
check this box to disable the security functions in the application. The application must be
reinstalled to enable the security functions.
4. When installing the software you will be required to enter a “Serial Number”. The “Serial
Number” will have been provided (on the back of the CD case) with the software when it
was purchased. You will also be asked to accept MOCON’s license agreement.
5. Follow the instructions to complete the installation. If you have any questions or problems
with the installation call MOCON Technical Services in the USA at (763) 493-7250.
NOTE: If the “Disable Login” box was not checked, you must set up the appropriate groups
and group assignments before the application can be used. For more information on setting
Page 7 of 45
up groups and user permissions see the “Read me” file on the software CD and the
“Application Security” topics in the software help system.
Main Start-Up Screen
1. Turn on the 910 instrument and the computer.
2. Select the GreenLight® icon and open the Start-Up screen.
Figure 1: GreenLight® 910 Start-Up Screen
3. Select File tab
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Figure 2: File Tab Screen
4. Select the page setup function.
5. Set up the margins, page orientation, and paper size.
Figure 3: Page Setup Screen
6. Select the Global Reports function.
Page 9 of 45
Figure 4: Global Reports Screen
7. Set the Global Reports format.
8. Do not use the Print Barcodes function as this is not used with the instrument software.
9. Select the Tools tab.
Figure 5: Tools Tab Screen
10. Select the Options function.
Page 10 of 45
Figure 6: Options Function Screen.
11. Set the functions or sounds required for each software driven function. Such as, sound for
incubation time expired or passing reading.
12. Select the Events Log function.
13. Review the Events Log as required for advanced troubleshooting.
Figure 7: Events Log Screen
14. Select the Help tab.
15. Utilize the Help functions as needed or contact Baseline for advanced troubleshooting.
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Figure 8: Help Tab Screen.
Figure 9: Help Screen.
16. The about screen will provide information needed for advanced help. Such as, instrument
serial number or software version number.
Page 12 of 45
Figure 10: About Screen.
Administrator
1. Select the Administrator function.
Figure 11: Administrators Screen
2. Select the Manage Languages function.
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Manage Languages
Figure 12: Manage Languages Screen
3. Note that currently in the software there is no additional language that you can select.
Administrator Options
4. Select the Administrator Options function.
Figure 13: Administrator Options Screen
5. Set the following functions:
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a. Default Signal Threshold, which is where you want the test to stop.
b. Default Pass/Fail time in minutes.
c. Default Continuous Incubation time in minutes.
d. Default Multipoint Incubation period in 15 min. increments.
e. Default Multipoint Read period in 15 min. increments.
f. Default Incubation Temperature in degrees centigrade.
g. Food Safety Analysis Name.
h. Hide Inactive Records: On/Off
i. Delta Threshold: On/Off.
Note: All functions selected under this Administrator Option can be changed in individual
methods or calibrations.
Manage Sample Templates
6. Select the Manage Templates function.
Figure 14: Manage Templates Screen.
7. The Manage Templates function performs no useful function in this software, but a
template must be defined for use in a multipoint sample analysis test. You may create a
new template, assign and name to it, and utilize it with no additional information
required.
Page 15 of 45
Manage Preparation Lists
8. The Manage Preparations function allows the analyst to set up a check off sheet that is
required prior to the beginning of a selected sample analysis. These preparation
instructions remind the analysts to perform a certain functions such as:
a. Record sample information.
b. Add media.
9. This information is illustrated in Figure 16.
Figure 15: Manage Preparations Screen.
Figure 16: Information added to a Preparation.
Page 16 of 45
Manage Calibration
1. Select the Manage Calibrations function to go to the Calibration screen
Figure 17: Manage Calibrations Screen
2. This screen is divided into five functions that can be utilized by the analyst.
3. The five functions are:
3.1. View Calibrations: This provides a screen which lists all calibrations of record for either
the Delta function being on or the Delta function being off.
3.2. Reference Check: This is a reference check file system to determine if the instrument is
operating within parameters set at the factory.
3.3. New Multipoint Calibration: This is the function that the analyst will use to create a
calibration file utilizing data collected by the instrument.
3.4. Quick Calibration: This is a single point calibration utilizing one calibration file
collected by the analyst.
3.5. New Manual Calibration: This is a function that allows the analyst to enter data
determined from instrument analysis and the Excel calibration function file.
Page 17 of 45
View calibrations
Figure 18: View Calibrations Screen
4. The information you will see on the View calibrations Screen is:
4.1. Selection box to export to a file.
4.2. Calibration name.
4.3. Calibration active checkbox.
4.4. Calibration gain.
4.5. Calibration offset.
4.6. Calibration units.
4.7. Vial size, either 2 mL or 15 mL.
4.8. Threshold for analysis completion.
4.9. Temperature set point for the analysis.
4.10. User, default is admin.
4.11. Date calibration file was created.
4.12. Type of calibration file, either a standard file created with the instrument software or
a manual calibration created from the Excel calibration utility.
4.13. You will also be able to edit, copy, or delete any of these calibration files. Also,
import, or export these files to or from another computer utilizing the GreenLight®
Model 910 software.
Page 18 of 45
Reference Check
Figure 19: Reference Check Screen.
5. To perform a reference check, select the Scan button and insert the reference check files as
directed by the program.
6. Check the acceptance range for either the Low Range or the High Range vials utilizing the
values on the inside of the Check Vials container.
7. If the reference values are outside the vial range, contact MOCON technical support.
Figure 20: Reference Check vials.
Page 19 of 45
Multipoint Calibrations
8. To generate a multiple point calibration utilizing calibration data points stored in the
software, select the Multipoint Calibration function.
9. The Multipoint Calibrations screen will provide a scrolling menu containing each calibration
file stored in the software.
10. Select the files that will be utilized to generate the calibration line. For these files, you must
know the actual bacteria concentration either from a plate, membrane filter, or most probable
number bacteria test. This additional information is needed to assign a value to the time to
threshold that was determined.
11. Figures 20 and 21 below illustrate the process in which three data points were selected, the
actual bacteria concentration assigned and a calibration line generated with the linear formula
and R2 determined.
Figure 21: Multipoint Calibration Screen with 3 Calibration Points Selected.
Page 20 of 45
Figure 22: Multipoint Calibration Linear Fit with Formula and R2 Screen.
Quick Calibration
12. To generate a quick calibration utilizing a single calibration data point stored in the software,
select the Quick Calibration function.
13. The Quick Calibrations screen will provide a scrolling menu containing each calibration file
stored in the software.
14. Select the single file that will be utilized to generate the single point calibration line. For this
file, you must know the actual bacteria concentration either from a plate, membrane filter, or
most probable number bacteria test. This additional information is needed to assign a value to
the time over threshold that the was determined.
15. Figures 20 and 21 below illustrate the process in which one data point was selected, the
actual bacteria concentration assigned and a calibration line generated with the linear formula
and R2 determined.
Page 21 of 45
Figure 23: Quick Calibration Screen with Single Calibration Points Selected.
Figure 24: Quick Calibration Linear Fit with Formula and R2 Screen.
Manual Calibration
16. To generate a manual calibration utilizing calibration data points stored in the software,
select the Manual Calibration function.
17. The manual calibration screen will provide a table in which values must be entered.
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18. The values obtained for the Gain and the Offset can be obtained from the View Calibrations
window or can be determined from experimental data entered into the ancillary Excel
worksheet provided.
19. The values entered into the Manual Calibration window are:
19.1. Calibration Name: This name will be assigned by the analyst.
19.2. Gain: this is the slope of the linear function defined by an X:Y plot of the Time to
Signal Threshold in decimal hours versus the log(10) of the bacteria concentration.
Typically, the bacteria concentration is reported as CFU per 100 mL.
20. Figure 25 illustrates the Manual Calibration screen.
Figure 25: Manual Calibration Screen.
21. To utilize the Excel worksheet, open the Excel file in either Excel 2007, 2010, or 2013..
22. The file will have a tab called Chart Data. That worksheet will contain the two tables
illustrated below.
23. Enter into the column for each specific sample and dilution, the amount of bacteria
determined by a traditional plate, membrane filter, or most probable number bacteria test and
the time to threshold in decimal hours.
24. The software provides the time to threshold in hours and seconds and that must be converted
to decimal hours so as to calculate a gain and offset that can be used for the manual
calibration.
Page 23 of 45
25. The Excel worksheet, also has a logic function applied to the final gain and offset value
calculated and if that value is outside the range excepted by the software, the word “Error”
will be displayed instead of a value that can be utilized by the software.
26. Figure 26, below, illustrates the calibration chart that is automatically generated in the
calibration chart worksheet.
Page 24 of 45
Table 1: Manual Calibration Calculation
Sample
Dilution #
CFU per 100 mL Calibration Chart Values
Sample 1
MPN or
CFU per 100
mL
Time
(Decimal
Hours)
Sample 2
MPN or
CFU per
100 mL
Time
(Decimal
Hours)
Sample 3
MPN or
CFU per
100 mL
Time
(Decimal
Hours)
Sample 4
MPN or
CFU per
100 mL
Time
(Decimal
Hours)
Sample 5
MPN or
CFU per
100 mL
Time
(Decimal
Hours)
Average
Time
(Decimal
Hours)
Log 10 of the
Average
CFU per 100
mL
1 10,000.00 4.03 4.03 4
2 115,000.00 2.47 2.47 5.06069784
3 120,000.00 2.22 2.22 5.079181246
Gain -1.58
Offset 10.37
R Squared 0.99
Table 2: Decimal Hour Calculator
Decimal Hour Calculator
Hours Seconds Decimal Hours
4 10 4.17
Page 25 of 45
Figure 26: Excel Manual Calibration Plot
y = -1.5838x + 10.37 R² = 0.9874
0.00
0.50
1.00
1.50
2.00
2.50
3.00
3.50
4.00
4.50
0 1 2 3 4 5 6
Tim
e (
De
cim
al H
rs.)
Log 10 of the Average CFU per 100 mL
GreenLight® Model 910 Manual Calibration Chart
Log 10 of the Average CFU per 100 mL
Linear (Log 10 of the Average CFU per 100 mL)
Page 26 of 45
Preparing a Method
1. Select the Manage Methods function.
2. From the manage methods screen you will see a scroll down menu containing all currently
stored methods in the software.
Note: you will only see the methods that either have a Delta function On or Off.
3. The Manage Methods screen will provide you with current method parameters that include:
3.1. Method name
3.2. Method type: either a test method or a calibration method.
3.3. Test type: a multipoint, pass/fail, or continuous test.
3.4. Preparation name: name of the preparation assigned for that method.
3.5. Active: if this box is checked, then when you set up a new test, this method will be
available for the test type being run.
3.6. Incubation temperature in degrees Celsius.
3.7. Initial incubation time in minutes.
3.8. Incubation time in minutes
3.9. Read time in minutes: which is the time it will attempt to read the vial. Default is 5
min.
3.10. Maximum time in hours: minutes.
3.11. Calibration file name used.
3.12. Pass/fail criteria used in scientific notation.
3.13. Units of bacteria concentration.
3.14. Signal threshold for completion of the test.
3.15. Vial size: either 15 mL or 2 mL.
Page 27 of 45
Figure 27: Manage Methods Screen
4. If you select the New function a window will appear in which you must enter the following
information:
4.1. Method name
4.2. Method type: either a test method or a calibration method.
4.3. Test type: a multipoint, pass/fail, or continuous test.
Note: some information required in this section may be different depending on the Test Type
selected.
4.4. Preparation name: name of the preparation assigned for that method.
4.5. Calibration Name: if needed.
4.6. Incubation temperature in degrees Celsius.
4.7. Initial incubation time in minutes.
4.8. Incubation time in minutes
4.9. Read time in minutes: which is the time it will attempt to read the vial. Default is 5
min.
4.10. Maximum time in hours: minutes.
4.11. Calibration file name used.
4.12. Pass/fail criteria used in scientific notation.
4.13. Units of bacteria concentration.
Page 28 of 45
4.14. Signal threshold for completion of the test.
4.15. Vial size: either 15 mL or 2 mL.
5. Once all of that method information has been entered, save the method, and it will show up
on the manage methods scroll down window as an active method.
Figure 28: New Method Screen
6. If you select the edit function, you'll be allowed to edit the method that is highlighted and
modify the parameters currently assigned to the method. This is illustrated in figure 29 below
Page 29 of 45
Figure 29: Edit Method Screen
7. Parameters you can edit are:
7.1. Method name
7.2. Test type: a multipoint, pass/fail, or continuous test.
7.3. Preparation name: name of the preparation assigned for that method.
7.4. Incubation temperature in degrees Celsius.
7.5. Incubation time in minutes
7.6. Signal threshold for completion of the test.
7.7. Vial size: either 15 mL or 2 mL.
8. Once all of that method information has been entered, save the method, and it will show up
on the manage methods scroll down window as an active method.
9. If you select the copy function, you'll be allowed to copy the method that is highlighted and
assign it a new name.
10. If you select the delete function, you'll be allowed to delete the method that is highlighted.
Page 30 of 45
Running an Analyses
Page 31 of 45
Water Analyses
Equipment
1. GreenLight® Model 910
2. GreenLight® 2 mL or 15 mL vials
3. GreenLight® 2 mL or 15 mL Hot Block Incubator (if required for 910 Multipoint Analysis)
4. Computer with GreenLight® operational software loaded and all required cables and power
converters.
5. Magnetic Stir-bars
6. Magnetic Stir-plates
7. 2000, 1000, 250 mL Glass Beakers
8. 10 mL and 100 mL Glass Graduated Cylinders
9. 1-10 mL adjustable pipette with disposable tips.
10. Aquarium Air Pump (if needed to aerate sample)
11. Aquarium Stainless Steel Diffuser Stone (if needed to aerate sample)
12. Plastic Tubing for Aquarium Air Pump and Diffuser Stone (if needed to aerate sample).
Chemicals
1. GreenLight® Media A (BD BBL™ Trypticase™ Soy Broth powder or suitable substitute)
2. GreenLight® Media B (Ethanol, absolute-200 Proof, Sigma-Aldrich #459844 or suitable
substitute)
3. Sterile Water (if needed to dilute sample or run control blank)
4. De-chlorination reagent (Ascorbic acid or Sodium thiosulfate) if needed.
5. PolySeed® bacteria seed or Wastewater Secondary Treatment Biomass (settled).
5.1. PolySeed® Buffering and Mineral Dilution Water Media
5.1.1. Buffer Solution pH 7.2 ± 0.2 at 20 ˚C, APHA for BOD (Hach® #431-49 or
suitable substitute)
5.1.2. Ferric Chloride Solution, APHA for BOD (Hach® #429-43 or suitable substitute)
5.1.3. Magnesium Sulfate Solution, APHA for BOD (Hach® #430-49 or suitable
substitute)
5.1.4. Calcium Chloride Solution, APHA for BOD (Hach® #428-49 or suitable
substitute)
5.1.5. Distilled or Reverse Osmosis (RO) water.
Page 32 of 45
Safety
1. PolySeed® Microbial Formulation: The product formulation consists of a range of naturally-
occurring microorganisms which are known to be non-pathogenic to humans, livestock, and
agricultural crops. Keep dry and at room temperature. Ensure containers remain sealed.
Avoid inhalation, ingestion and exposure to skin. Wash hand thoroughly after use.
2. Wastewater Secondary Treatment Biomass: This material consists of a range of naturally-
occurring microorganisms which are known to be pathogenic to humans, livestock, and
agricultural crops. Avoid inhalation, ingestion and exposure to skin. Wash hand thoroughly
after use.
3. Media A: Prevent dispersion of material. Wipe up with damp sponge or mop.
4. Media B: Avoid contact with skin and eyes. Avoid inhalation of vapor or mist. Keep away
from sources of ignition.
5. Buffer Solution pH 7.2 ± 0.2: Refer to Hach® MSDS or selected chemical supplier.
6. Ferric Chloride Solution: Refer to Hach® MSDS or selected chemical supplier.
7. Magnesium Sulfate Solution: Refer to Hach® MSDS or selected chemical supplier.
8. Calcium Chloride Solution: Refer to Hach® MSDS or selected chemical supplier.
Aerated Dilution Water
1. Prepare dilution water as needed.
2. Transfer the required amount of distilled or RO water into a suitable size inert container
containing a magnetic stir bar.
3. Aerate the water with an aquarium pump and stir. (~ 1 hour).
4. Transfer 1 mL of the following buffering and mineral chemical per liter of dilution water
while it is stirring:
1.1.1. Buffer Solution pH 7.2 ± 0.2 at 20 ˚C
1.1.2. Ferric Chloride Solution
1.1.3. Magnesium Sulfate Solution
1.1.4. Calcium Chloride Solution
For best results, the aerated dilution water should be used within three (3) hours of aeration.
Hydration of PolySeed® Seed
1. Place the entire contents of one PolySeed® capsule (discard the gelatin capsule) into 500 mL
of dilution water in a 2000 mL beaker with a stir bar.
Bran, which acts as the carrier for the microorganisms, will neither dissolve nor inhibit
microbial activity, but must be settled out of the PolySeed® solution prior to use.
Page 33 of 45
2. Next, aerate the PolySeed® solution with an aquarium pump with a diffuser stone and stir the
PolySeed® solution for at least sixty (60) minutes.
Figure 30: PolySeed® Aeration
3. Allow the PolySeed® bran suspension to settle and decant the supernatant carefully into a
1000 mL beaker with as stir bar so as not to allow any bran to carry over.
4. Stir and aerate the PolySeed® suspension. Remove the aliquots of bacteria suspension as
needed from the aerated mixed suspension.
For best results, the PolySeed® solution should be used within six (6) hours of rehydration of
the capsule.
Wastewater Secondary Treatment Biomass Seed
1. Collect sufficient Final Effluent from the Secondary Treatment system of the wastewater
plant after the final clarifier and before the disinfection process in a HDPE or brown glass
bottle.
2. Keep out of direct sunlight.
3. Cool to 1 ˚C to 6 ˚C until analyses.
For best results use within 6 hours.
4. Next, aerate the Final Effluent solution with an aquarium pump with a diffuser stone and stir
for at least thirty (30) minutes.
Page 34 of 45
5. Allow any suspended solids to settle and decant the supernatant carefully into a 1000 mL
beaker with as stir bar so as not to allow any suspended solids to carry over.
6. Stir and aerate the Final Effluent suspension. Remove the aliquots of bacteria suspension as
needed from the aerated mixed suspension.
For best results, the Final Effluent solution should be used within six (6) hours of collection.
Experimental Procedure
Water Sample
Sample Collection, Storage, and Holding Time
1. Collect a minimum 100 mL of water from a sample point in a sterile plastic or glass sample
bottle.
2. Add a de-chlorination reagent to remove any chlorine based disinfection chemicals if needed.
3. Keep out of direct sunlight.
4. Cool to 1 ˚C to 6 ˚C until analyses.
5. Maximum storage time: 6 hours.
Analysis Pretreatment
Prior to analyses:
1. Bring water up to room temperature.
2. If water sample needs to be aerated
2.1. Transfer up to 1000 mL of water sample to a 2000 mL glass beaker with a stir bar.
2.2. Aerate the water samples with stirring for 15 minutes at room temperature.
Page 35 of 45
Figure 31: Sample Being Aerated
GreenLight® Bacterial Activity Test for Water
Water Test Sample
1. Transfer 100 mL of aerated sample water into a 250 mL glass beaker that has a magnetic stir
bar.
2. Stir to mix
3. Add ½ teaspoon of Nutrient A to the stirring water sample.
4. Once Nutrient A has dissolved, add 1 mL of Nutrient B with an adjustable pipet while
stirring.
Page 36 of 45
Figure 32: Mixed Sample with Media A and Media B
5. Transfer 2 ml or 15 mL of sample to a GreenLight® 2 mL or 15 mL vial and run the Beach
Water GreenLight® test on the 910.
Figure 33: Sample in a 15 mL Vial Being Transferred into a GreenLight® 910
6. If a Multipoint test on the 910 is run, then the 2 mL or 15 mL vials must be kept incubated in
the Hot Block Incubator. Set the incubator temperature to 40 ˚C and allow it to come to
equilibrium before starting the GreenLight® tests.
Page 37 of 45
Figure 34: 15 mL GreenLight® Vials in the Hot Block Incubator
Page 38 of 45
GreenLight® 910 Software Procedure
1. Open the GreenLight® Model 910 software program on the computer.
2. The program can be opened before you start sample analyses so as to allow the instrument to
warm up to its operational temperature (40 ºC)
3. Select the New Test tab in either the GreenLight® 910.
Figure 35: New Test Screen
4. Enter the following data in the GreenLight® 910 or 930 fields
Table 4: Examples for Continuous Test
Field Name 910
Instrument Not Required.
Test ID Enter your test ID number.
Test Type Enter “Continuous” or “Multipoint”
Method Select method.
Rack ID Only Required for Multipoint
Sample Template Select Sample template “
Vial Description Check box for Multipoint. Vial descriptions entered after initial reading
for Multi Point test.
Page 39 of 45
5. Select the Next Button
Continuous Test
1. Place the 2 mL or 15 mL tube in the instrument and the internal bar code reader will start the
analysis.
2. Once the GreenLight® signal for a sample vial has crossed the threshold in the method, the
time to threshold is reported in Hours and Minutes.
3. Values for bacteria concentration will be reported.
Note: Use the threshold crossover time for the calculations
Figure 36: Continuous Analysis Screen
Multi Point Test
4. Select “Assign Vial Description” check box in the new test window.
5. Place each 2 mL or 15 mL tube in the multipoint sample set into the GreenLight® 910
instrument and the internal bar code reader will beep, read the bar code and initial signal for
each vial.
6. Remove each of the 2 mL or 15 mL tubes after they have been read and place it in the Hot
Block Incubator.
NOTE: If the vial is not read by the bar code reader
6.1. Select “Read” block in the test window.
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Figure 37: Vial Read Screen for Multipoint test
6.2. Enter the vial number from the screen in the AP Check vial window. Include the “0s” in
front of each vial number.
6.3. Select “Read” in this window to enter the vial # and the initial signal value.
7. A “Vial Description” window will appear after the vials have been read. Additional
descriptive information for each vial can be entered.
8. Select “Save” in this window to save your vial description information.
9. Select the “Begin Incubation” block to start the tests
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Figure 38: Vial Description Screen for Multipoint test
10. Observe the incubating time countdown window on the GreenLight® 910 screen. Once the
indicator bar has reached “Read”, reinsert each 2 mL or 15 mL tube in the multipoint sample
set into the GreenLight® 910 instrument and the internal bar code reader will read the
barcode and the oxygen signal in the sample tube.
Figure 39: Multipoint Test Incubating Main Screen
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Figure 40: Multipoint Incubating Analyses Screen
11. Repeat steps 2 and 3 until the oxygen signal has passed the threshold for all of the samples in
the multipoint sample set.
12. Once the GreenLight® signal for a sample vial has crossed the threshold in the method,
bacteria concentration will be reported.
13. The test will continue until all vials have crossed the threshold.
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Quality Control
Duplicate Relative Percent Difference
1. It is suggested that a duplicate of one sample per batch be run. Acceptable control limits
are developed per laboratory QC SOPs.
2. Calculate the Relative Percent Difference (RPD).
Sample1 - Sample1Duplicate*100 = RPD
Sample1 + Sample1Duplicate
2
Sample1 = Water Sample 1
Sample 1 Duplicate = Water Sample 1 Duplicate
Figure 41: Example of RPD Chart for Water Analyses
3. Run control charts of the RPD as per Standard Methods 1020 (Eaton 2012)
0
5
10
15
20
25
1
3
5
7
9
11
13
15
17
19
21
23
25
27
29
31
Rel
ati
ve
Per
cen
t D
iffe
ren
ce
Test Number
Water Sample Bacteria Concentrations RPDs
UAL UWL RPD
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Control Blank
1. Run 100 mL of dilution water as a sample when bacteria cross contamination is suspected in
the dilution water or media.
2. If the blank sample crosses the threshold within 24 hours, then there is a bacteria cross
contamination.
3. Run a Root Cause Analyses to determine source of bacterial contamination.
4. Replace all suspected sources of bacteria cross contamination.
5. Correct bacteria contamination problem as per laboratory QC policy. Further information
can be obtained in Standard Methods for the Examination of Water and Wastewater Part
9000 (Eaton 2012).
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References
Eaton, A., D., Baird R.B.., Rice, E., W. (2012). Standard Methods for the Examination of Water
and Wastewater 22nd Edition. Washington D.C., APHA, AWWA, WEF.
