BIOSCAN* ATP method for biocide (toxicant) evaluations

Transcription

1 Water Technologies & Solutions microbiological procedure MB012 BIOSCAN* ATP method for biocide (toxicant) evaluations summary of method This method can identify the most effective biocides to treat the microbial flora in the cooling system. Microbes in the cooling water samples are exposed to a variety of biocides at various concentrations. After the contact time, biocide efficacy is determined by measuring microbial ATP via BIOSCAN ATP detection technology. ATP is a high-energy compound in living cells, and the amount found in water samples is proportional to cell numbers and activity. A decrease of 90% in microbial ATP as compared to initial microbial ATP control levels may be used to identify the biocides most efficacious in the actual cooling system. Results can be used to identify the most effective biocide for the cooling system. apparatus required code Bottle, 1 gallon HDPE L106 Cylinder, graduated, plastic, 50 ml L486 Kit, BIOSCAN 2 ATP Meter L6570 with accessories Pipetter, µl Adjustable L1587 Pipetter Tips, µl for L1587 L12025 Pipetter, Socorex Calibra , µl (EMEA only) L8034 Pipetter Tips, 1000 µl for L8034 (EMEA only) L8037 media or chemicals required 1 ATP Standard, 1.0 ng/l; 5 vials/package Biocide Samples Kits: code L6583 NAM Commodity Biocide Kit (L code may vary according to your country location) L4100 NAM Proprietary Biocide Kit (L code may vary according to your country location) L4101 BIOSCAN Total ATP Pens; 10/box (green cap) BIOSCAN Free ATP Pens, 10/box (red cap) L6586 L6587 MB Dilution Water, sterile, 99 ml, 72/box L Review the Safety Data Sheets (MSDS/SDS) for the chemicals that are used. Use the recommended personal protective equipment. Dispose of reacted solutions according to local, state and federal regulations. Refer to the Safety Data Sheets for disposal information for unused reagents. Refer to the environmental, health and safety staff for your facility and/or local regulatory agencies for further disposal information. procedure Preparation of Water Sample 1. It is best to collect samples before the nonoxidizing biocide treatment and/or after halogen feed has been stopped. Ideally the total halogen residual should be <0.1 ppm. Higher levels of halogen (>0.1ppm) will falsely increase efficacy on the other biocides. 2. Determine the number of aliquots required for the test by the formula below: (# of biocides to be tested) x (# ofconcentrations to be tested) + 2 aliquots for controls For example: if 4 biocides will be tested at 5 concentrations then 22 aliquots are needed for the test. 3. Empty enough 99 ml sterile water blanks to use for the aliquots of cooling sample. Find a contact near you by visiting and clicking on Contact Us. *Trademark of SUEZ; may be registered in one or more countries SUEZ. All rights reserved. Mar-17

2 4. Add 1 tablespoon of biofilm/slime deposit to 1/2 gal of system bulk water and shake well to mix. If clumps of deposit remain, decant the supernatant to a second 1- gal jug. BIOSCAN ATP procedure BIOSCAN pens and storage 5. Dispense 50 ml aliquots into the emptied, sterile, 99 ml water dilution bottles in Step 3. preparation of biocide stock solutions: 1. Make a 10,000 mg/l (10,000 ppm) working stock solution for each biocide by adding the contents of the biocide sample vial into a fresh 99 ml water dilution blank. To ensure the entire contents of the vial are added, drop the emptied vial into the dilution blank and shake. Use stock biocide solutions within 1 hour of preparation. preparation of test samples 1. Each biocide stock solution (10,000 mg/l or ppm). Add the stock solution to separate 50 ml samples of cooling water according to the chart below. Page 2 Concentration (ppm) Stock solution added (ml) Each bottle is shaken after addition of biocide. Other biocide concentrations may be prepared if desired. 3. The controls are cooling water samples without any added biocide. 4. Run BIOSCAN ATP to determine the microbial RLUs in the initial /0 hour controls. The procedure for BIOSCAN ATP is below. contact time 1. Incubate biocide treated samples and controls at a temperature similar to system temperature. 2. Treated samples are exposed to biocide for four hours and twenty-four hours after which the efficacy of biocide is determined via BIOSCAN ATP procedure below. The Total ATP pens have a green colored cap and the pen contains extractant on the sampling stick (Figure 2). The extractant releases ATP from the cells into the sample liquid which also contains free floating ATP. The Free ATP pens have a red cap and they have no extractant coating on the sampling stick (Figure 2). The Free pen will detect free floating ATP in the sample. Both pens contain buffer for dilution buffering and neutralization of the sample. Proper storage of pens is important. The box should be stored with the large arrows on the side pointing up (Figure 2). The pens should be stored at 2-8 C (35-46 F). Excessive heat can damage the reagents and will produce inaccurate results. Quick Steps: 1. Turn on the BIOSCAN luminometer by pressing the "On/Off" button on the front panel (Figure 1). 2. Remove cap from Free ATP pen and dip sampling stick into sample to cover the grooves for approximately one second (Figures 3 and 4). 3. Push in the grooved white part of the pen (area where the stick enters the pen body) until a click or snap is heard (Figures 5, 6 and 7), and then turn (screw) cap section until it contacts the lower part (Figures 8 and 9). 4. Immediately shake the pen vigorously 10 times mix the sample with the reagent (foam and yellow color may appear). 5. Immediately after shaking, insert the pen into the chamber with the colored cap down (no

3 pressure is needed) (Figure 1). Gently close the lid to start the measurement. 6. When the measurement is complete, the lid will open automatically with the result displayed on the screen. Results are reported as Relative Light Units (RLU), and as Log 10 RLU. 7. Steps 2-6 should be repeated with a Total ATP pen. 8. Subtract the Free ATP pen RLUs from the Total ATP pen RLUs to determine the microbial (m) ATP in RLUs. Total ATP Free ATP = Microbial ATP detailed BIOSCAN testing protocol for cooling water samples 1. Obtain a water sample from a cooling water system, using the procedure described in MB012 above (Preparation of Water Sample). Samples should be representative of the system. Testing should be performed immediately after sampling. For accurate results, it is important to run both Free and Total ATP pens. 2. Turn on the BIOSCAN luminometer by pressing the "On/Off" button on the front panel (Figure 1). The instrument will then perform a self-check, after which the lid/flap of the pen chamber will open automatically. The temperature compensator should always be left on. ON is the default state for temperature compensation; thermometer icon is visible in the lower left of the screen. 3. Remove cap from Free ATP pen and dip sampling stick into sample to cover the grooves for approximately one second (Figures 3 and 4). Note: If both Total and Free ATP determinations are to be performed from the same sample, perform the Free ATP test first. Note: Care must be taken to immerse only the grooved portion of the sampling stick. Do not flick or shake after removing the stick from the water sample. 4. On a solid surface, gently press the stick into the cuvette chamber using continuous pressure. Note: Do not slam the pen to drive in the stick. When pressed in correctly, the bottom of the sampling stick should be flush with the bottom of the pen (Figure 5, 6, 7, and 8). 5. Then turn the screw cap section of the pen. The pen s stick will contact the lower section and break the foil membrane (Figures 8 and 9). Note: This opens the foil of the reagent chamber by slitting it with the sharp end of the stick. 6. Immediately shake the pen vigorously 10 times mix the sample with the reagent (foam and a yellow color may appear). Immediately after shaking, insert the pen into the chamber (Figure 1) with the colored cap down (no pressure is needed). Gently close the lid to start the measurement. Note: Delays in inserting and reading the pen will negatively affect results. 7. When the measurement is complete, the lid will open automatically with the result displayed on the screen. Results are reported as Relative Light Units (RLU) and as Log 10 RLU. Record the result. Remove the pen from the luminometer. Note: Dispose of the pen in the trash unless local plant regulations prohibit this disposal. 8. Repeat steps 3 through 7 with a Total ATP pen (green button pen). 9. To determine levels of Microbial ATP, subtract the Free ATP RLU value from the Total ATP RLU value: Total ATP Free ATP = Microbial ATP untreated test controls 1. Average microbial ATP (Total ATP minus Free ATP) is determined at zero hour in the control samples. After the contact times (four and twenty-four hours), microbial ATP in the treated samples as well as untreated controls are determined. Control matp = control1 (Total ATP - Free ATP) + Control 2 (Total ATP - Free ATP)/2 2. Microbial ATP (matp) values in the controls should stay the same or increase slightly from time zero (initial control count) to the other contact times (4 and 24 hour control (matp) counts). If control microbial ATP (matp) values decrease significantly, results should be viewed Page 3

4 with caution, as decreases in ATP in treated samples may be a result of factors other than biocide activity. biocide treatment calculations 1. To determine levels of Microbial ATP (matp), subtract the Free ATP RLU value from the Total ATP RLU value for the treated samples: matp (treated) = Total ATP (treated) - Free ATP (treated) 2. Determine the % Microbial ATP (matp) Reduction by the following formula: % matp Reduction = [(matpcontrol - matp treated sample)/ matp Control] X A decrease of 90% in microbial ATP as compared to initial microbial ATP control levels is used to identify the biocides most efficacious in the actual cooling system. quality control The ATP standard provides a quality control method for evaluating sampling pen performance. 1. Select a representative pen and run an ATP test using the ATP standard. Test the standard as a water sample. Acceptable results should be between 300 and 600 RLU for both Free and Total pens. 2. If the pen s result is out of range, run a second pen from the box. If both pens are out of range, discard the box of pens. 3. The same standard vial can be used for testing both Free ATP and Total ATP pens quality. Do not sample from the vial more than two times. Note: ATP standard should be stored at 2-8 C Page 4

5 Insert pen Directional Arrows for Storage Total ATP Free ATP Pens Total ATP Pens On/Off button Free ATP Pen Figure 1: BIOSCAN and Accessories Figure 2: Pen boxes and pens Grooves Gap Screw Cap Grooves Figure 3: Free ATP pen without cap Figure 4: Sampling with pen Page 5

6 Gap Grooves Figure 5: Pen before inserting sample into pen Figure 6: Firmly push sampling stick into pen Gap Screw Cap Figure 7: Sampling stick inserted in pen Figure 8: Sampling stick inserted after click Page 6

7 No gap Sampling stick inside pen Figure 9: Screw cap tightened pen ready to shake 10 X Page 7