Water Technologies & Solutions microbiological procedure MB013 BIOSCAN* ATP method for monitoring of biofilm/sessile microorganisms in cooling systems summary of method This method can be used to determine the biofilm/sessile organisms in the cooling systems. The BIOSCAN* method provides a rapid, easy procedure that measures ATP levels of the biofilm/sessile organisms. ATP is a high-energy compound found in all living cells. Results can be used to: 1. Monitor the development of biofilm/sessile organisms in cooling systems by utilizing the corrosion bypass rack coupons as the biofilm forming surface. 2. Determine the effectiveness of the microbial treatment. media or chemicals required 1 code ATP Standard, 1 ng/ml, 5 vials/package BIOSCAN Total ATP Pens 10/Package (Green Cap) BIOSCAN Free ATP Pens, 10/Package (Red Cap) MB Dilution Water, Sterile, 9 ml, 70/box apparatus required code Set BIOSCAN 2 ATP Meter with Accessories L6583 L6586 L6587 L1045 L6570 Coupon Bypass Rack, PVC, 1 2013971 Coupons, Stainless Type 304, 10/package L9002 Coupons, Stainless Type 316, 10/package 14 ml Sterile Polystyrene Round Bottom Tubes, 25/package L9003 L796 1 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. coupon rack installation 1. Install the Coupon Bypass Rack as stated in the instructions with the rack. The number of coupon ports will depend upon sample frequency. 2. Stainless steel coupons are recommended for microbiological (MB) testing as they provide a uniform, consistent surface. 3. Wear gloves when handling coupons to avoid coating the coupon with natural oil from the skin or with joint compound, as this will affect biofilm formation. 4. Allow at least one week for microbes to colonize the coupon surface and form a biofilm. 5. Use the same coupon exposure time for each coupon sampling. removal of microbes from coupons ( Shake Method ) 1. The Shake Method is used to remove and disperse the biofilm/sessile organisms into sterile water to determine the biofilm s ATP. The amount Find a contact near you by visiting www.suezwatertechnologies.com and clicking on Contact Us. *Trademark of SUEZ; may be registered in one or more countries. 2017 SUEZ. All rights reserved. Dec-16
of microbial ATP corresponds to the number of biofilm organisms on the coupon surface. 2. Add contents of a 9 ml sterile dilution water blank to the 14 ml plastic tube. 3. Using gloves carefully remove coupons without disturbing the biofilm layer, and place in the 14 ml plastic tube described in Step 2. Note: Biofilm may not be visible to the naked eye Note: Avoid handling the coupons without gloves, because hands contain ATP that can contaminate the coupon and therefore your ATP determination would not be accurate. 4. Cap the 14 ml plastic tube and vigorously shake up and down for 30 seconds to dislodge biofilm from the coupon This sample is ready for processing using the ATP Bioscan Method. 5. Process the samples within 1 hour using the BIOSCAN Method below. BIOSCAN pens and storage BIOSCAN quick step procedure to determine biofilm/sessile organisms 1. Turn on the BIOSCAN luminometer by pressing the "On/Off" button on the front panel (Figure 1). 2. Remove cap from the Free ATP pen (red capped pen) and for 1 second dip sampling stick into sample (prepared by Shake Method ) to cover the grooves (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 to 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 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. 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 cells into the sample liquid which also contains the 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. Touching the ATP pen s sampling stick with bare hands can lead to false results. Recommend wearing gloves. 7. Steps 2-6 should be repeated with a Total ATP pen (green capped pen). 8. Subtract the Free ATP pen RLUs from the Total ATP pen RLUs to determine the microbial ATP in RLUs. Multiply the microbial RLUs by 9 and divide that RLU number by the surface area of the coupon (19.35 cm 2 for Stainless Steel coupons) to determine the number of biofilm organisms per unit area. [(Total ATP-Free ATP) X 9]/ Coupon Surface Area 19.35cm 2 = Biofilm RLUs/Surface Area (cm 2 ). detailed BIOSCAN testing protocol for biofilm determination in cooling systems 1. Prepare the sample via the Shake Method described above in the Removal of Microbes from Coupons section. Page 2
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 the Free ATP pen (red capped pen) and for 1 second dip sampling stick into sample (prepared by Shake Method ) to cover the grooves (Figures 3 and 4). Note: 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 to 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 the method using the Total ATP pen (green capped pen) steps 3-7. biofilm ATP calculation If the same coupon type and size is always used, data can be reported as RLU/coupon or simply as RLU. The RLU result (also displayed as Log 10 RLU) is visible on the BIOSCAN screen. To determine the RLU/cm 2 (surface area) result, multiply the RLU value by 9 (for the 9 ml of sterile water) and then divide by the surface area of the coupon. For 304 or 316 stainless steel the surface area is 19.35 cm 2. [(Total ATP-Free ATP) X 9]/ Coupon Surface Area 19.35cm 2 = Biofilm RLUs/Surface Area (cm 2 ). Quality Control The ATP standard provides a quality control method for evaluating sampling pen performance. 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. 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. 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 3
Insert pen here 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 Screw Cap Gap Gap Screw Cap Coupon Grooves Grooves Figure 3: Free ATP pen without cap Figure 4: Sampling pen in tube with coupon Page 4
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 5
No gap Sampling stick inside Figure 9: Screw cap tightened pen ready to shake 10 X Page 6