GreenLight Model 910 Software Use and Water Sample Analysis Manual. September 23, 2013

Transcription

1 GreenLight Model 910 Software Use and Water Sample Analysis Manual September 23, 2013

2 Table of Contents Introduction... 6 Instrument Set-Up... 6 Software Installation... 6 Main Start-Up Screen... 7 Administrator Manage Languages Administrator Options Manage Sample Templates Manage Preparation Lists Manage Calibration View calibrations Reference Check Multipoint Calibrations Quick Calibration Manual Calibration Table 1: Manual Calibration Calculation Table 2: Decimal Hour Calculator Preparing a Method Running an Analyses Water Analyses Equipment Chemicals Safety Aerated Dilution Water Hydration of PolySeed Seed Wastewater Secondary Treatment Biomass Seed Experimental Procedure Water Sample Page 2 of 45

3 Sample Collection, Storage, and Holding Time Analysis Pretreatment Prior to analyses GreenLight Bacterial Activity Test for Water Water Test Sample GreenLight 910 Software Procedure Table 4: Examples for Continuous Test Continuous Test Multi Point Test Quality Control Duplicate Relative Percent Difference Control Blank References Page 3 of 45

4 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 Figure 7: Events Log Screen Figure 8: Help Tab Screen Figure 9: Help Screen Figure 10: About Screen Figure 11: Administrators Screen Figure 12: Manage Languages Screen Figure 13: Administrator Options Screen Figure 14: Manage Templates Screen Figure 15: Manage Preparations Screen Figure 16: Information added to a Preparation Figure 17: Manage Calibrations Screen Figure 18: View Calibrations Screen Figure 19: Reference Check Screen Figure 20: Reference Check vials Figure 21: Multipoint Calibration Screen with 3 Calibration Points Selected Figure 22: Multipoint Calibration Linear Fit with Formula and R 2 Screen Figure 23: Quick Calibration Screen with Single Calibration Points Selected Figure 24: Quick Calibration Linear Fit with Formula and R 2 Screen Figure 25: Manual Calibration Screen Figure 26: Excel Manual Calibration Plot Figure 27: Manage Methods Screen Figure 28: New Method Screen Page 4 of 45

5 Figure 29: Edit Method Screen Figure 30: PolySeed Aeration Figure 31: Sample Being Aerated Figure 32: Mixed Sample with Media A and Media B Figure 33: Sample in a 15 ml Vial Being Transferred into a GreenLight Figure 34: 15 ml GreenLight Vials in the Hot Block Incubator Figure 35: New Test Screen Figure 36: Continuous Analysis Screen Figure 37: Vial Read Screen for Multipoint test Figure 38: Vial Description Screen for Multipoint test Figure 39: Multipoint Test Incubating Main Screen Figure 40: Multipoint Incubating Analyses Screen Figure 41: Example of RPD Chart for Water Analyses Page 5 of 45

6 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) 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 6 of 45

7 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. 3. Select File tab Figure 1: GreenLight 910 Start-Up Screen Page 7 of 45

8 4. Select the page setup function. Figure 2: File Tab Screen 5. Set up the margins, page orientation, and paper size. 6. Select the Global Reports function. Figure 3: Page Setup Screen Page 8 of 45

9 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. 10. Select the Options function. Figure 5: Tools Tab Screen Page 9 of 45

10 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. Page 10 of 45

11 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 11 of 45

12 Administrator 1. Select the Administrator function. Figure 10: About Screen. 2. Select the Manage Languages function. Figure 11: Administrators Screen Page 12 of 45

13 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. 5. Set the following functions: Figure 13: Administrator Options Screen Page 13 of 45

14 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 14 of 45

15 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 15 of 45

16 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 Reference Check: This is a reference check file system to determine if the instrument is operating within parameters set at the factory New Multipoint Calibration: This is the function that the analyst will use to create a calibration file utilizing data collected by the instrument Quick Calibration: This is a single point calibration utilizing one calibration file collected by the analyst 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 16 of 45

17 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 Calibration name Calibration active checkbox Calibration gain Calibration offset Calibration units Vial size, either 2 ml or 15 ml Threshold for analysis completion Temperature set point for the analysis User, default is admin Date calibration file was created Type of calibration file, either a standard file created with the instrument software or a manual calibration created from the Excel calibration utility 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 17 of 45

18 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 18 of 45

19 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 R 2 determined. Figure 21: Multipoint Calibration Screen with 3 Calibration Points Selected. Page 19 of 45

20 Quick Calibration Figure 22: Multipoint Calibration Linear Fit with Formula and R 2 Screen. 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 R 2 determined. Page 20 of 45

21 Figure 23: Quick Calibration Screen with Single Calibration Points Selected. Manual Calibration Figure 24: Quick Calibration Linear Fit with Formula and R 2 Screen. 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. Page 21 of 45

22 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: Calibration Name: This name will be assigned by the analyst 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 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 22 of 45

23 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 23 of 45

24 Table 1: Manual Calibration Calculation CFU per 100 ml Calibration Chart Values Sample Dilution # 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, , , Gain Offset R Squared 0.99 Table 2: Decimal Hour Calculator Decimal Hour Calculator Hours Seconds Decimal Hours Page 24 of 45

25 Time (Decimal Hrs.) GreenLight Model 910 Manual Calibration Chart y = x R² = Log 10 of the Average CFU per 100 ml Linear (Log 10 of the Average CFU per 100 ml) Log 10 of the Average CFU per 100 ml Figure 26: Excel Manual Calibration Plot Page 25 of 45

26 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 Test type: a multipoint, pass/fail, or continuous test Preparation name: name of the preparation assigned for that method 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 Incubation temperature in degrees Celsius Initial incubation time in minutes 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 Maximum time in hours: minutes Calibration file name used Pass/fail criteria used in scientific notation Units of bacteria concentration Signal threshold for completion of the test Vial size: either 15 ml or 2 ml. Page 26 of 45

27 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 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 Preparation name: name of the preparation assigned for that method Calibration Name: if needed Incubation temperature in degrees Celsius Initial incubation time in minutes 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 Maximum time in hours: minutes Calibration file name used Pass/fail criteria used in scientific notation Units of bacteria concentration. Page 27 of 45

28 4.14. Signal threshold for completion of the test 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 28 of 45

29 7. Parameters you can edit are: 7.1. Method name Figure 29: Edit Method Screen 7.2. Test type: a multipoint, pass/fail, or continuous test Preparation name: name of the preparation assigned for that method Incubation temperature in degrees Celsius Incubation time in minutes 7.6. Signal threshold for completion of the test 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 29 of 45

30 Running an Analyses Page 30 of 45

31 Water Analyses Equipment 1. GreenLight Model 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 , 1000, 250 ml Glass Beakers ml and 100 ml Glass Graduated Cylinders 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 # 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) PolySeed Buffering and Mineral Dilution Water Media Buffer Solution ph 7.2 ± 0.2 at 20 C, APHA for BOD (Hach # or suitable substitute) Ferric Chloride Solution, APHA for BOD (Hach # or suitable substitute) Magnesium Sulfate Solution, APHA for BOD (Hach # or suitable substitute) Calcium Chloride Solution, APHA for BOD (Hach # or suitable substitute) Distilled or Reverse Osmosis (RO) water. Page 31 of 45

32 Safety 1. PolySeed Microbial Formulation: The product formulation consists of a range of naturallyoccurring 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 naturallyoccurring 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: Buffer Solution ph 7.2 ± 0.2 at 20 C Ferric Chloride Solution Magnesium Sulfate Solution 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 32 of 45

33 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 33 of 45

34 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 Aerate the water samples with stirring for 15 minutes at room temperature. Page 34 of 45

35 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 35 of 45

36 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 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 36 of 45

37 Figure 34: 15 ml GreenLight Vials in the Hot Block Incubator Page 37 of 45

38 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 Test ID Test Type Method Rack ID Not Required. Enter your test ID number. Enter Continuous or Multipoint Select method. 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 38 of 45

39 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 Multi Point Test Figure 36: Continuous Analysis Screen 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. Page 39 of 45

40 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 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 Page 40 of 45

41 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 Page 41 of 45

42 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. Page 42 of 45

43 Relative Percent Difference 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 25 Water Sample Bacteria Concentrations RPDs 20 UAL UWL RPD Test Number Figure 41: Example of RPD Chart for Water Analyses 3. Run control charts of the RPD as per Standard Methods 1020 (Eaton 2012) Page 43 of 45

44 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). Page 44 of 45

45 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. Page 45 of 45