Performing Tests - QuenchGone21 Wastewater

Using a new wide-bore pipette tip, add 1mL of well-mixed sample to a QG21W Extraction Tube. Cap and invert twice to mix. Allow at least 1 minute for incubation.

NOTE: At this point, the contents of the QG21W Extraction Tube can be capped and stored refrigerated between 2-8oC for up to 1 week prior to 2.2.
TIP: Wide-bore pipette tips are those that have sufficiently large openings to prevent tip plugging by sample particles. In general, standard 1-5mL and wide-mouth 100-1000µL pipette tips are sufficient for most samples. If required, increase the bore size of a tip using a clean pair of scissors.

Pour the QG21W Extraction Tube contents into a QG21W Dilution Tube. Transfer the mixture back and forth between the two tubes several times for best mixing accuracy. Cap and invert twice to mix. Allow beads to settle.

NOTE: At this point, the contents of the QG21W Dilution Tube are stable at room temperature for up to 4 hours.
TIP: If beads do not settle or settle slowly, tap the QG21W Dilution Tube gently to assist settling.
TIP: If the QG21W Extraction Tube cannot be poured into the QG21W Dilution Tube, simply pour the QG21W Dilution Tube contents into the QG21W Extraction Tube to liquefy its contents.

Using a new pipette tip, add 100µL of the QG21W Dilution Tube contents and 6 drops (300µL) of LuminaseW to a new 12x55mm test tube (the Assay Tube), swirl gently five times, and insert into the Luminometer. Read and record RLUtATP for use in Step 5.

NOTE: If RLUtATP < 10 on a Kikkoman Lumitester C-100 or C-110, you are below the low–detection limit. Report tATP (ng ATP/mL) = 0 in Step 5.
TIP: If “Scale Over” is returned, repeat the tATP analysis using 100µL of sample in 2.1 (EXTRACTION) and modify the dilution factor in Step 5 as noted.

Using a new pipette tip, add 100µL of well-mixed sample to a QG21W LumiSolve Tube. Cap and invert twice to mix. Allow at least 1 minute for incubation.

NOTE: At this point, the QG21W Dilution Tube can be capped and stored between 2 to 8oC for up to 1 week prior to Part 3.2.

Using a new pipette tip, add 100µL of the QG21W LumiSolve Tube contents and 6 drops (300µL) of LuminaseW to a new 12x55mm test tube (the Assay Tube), swirl gently five times, and insert into the Luminometer. Read and record RLUdATP for use in Step 5

NOTE: If RLUdATP < 10 on a Kikkoman Lumitester C-100 or C-110, you are below the low–detection limit. Report dATP (ng ATP/mL) = 0 in Step 5.

Place an unused piece of 2” x 2” 250µm mesh over the mouth of an unused specimen container. Using a new wide-bore pipette tip, slowly and carefully pipette 2.5mL of well-mixed sample onto the top of the mesh and allow the filtrate to be collected in the specimen container. Allow 1 minute for filtration to occur.

TIP: Because your hands and surroundings can contain ATP, use care to touch only the corners of the mesh piece with your hands. For maximum care, use laboratory gloves or clean foreceps to transfer the mesh piece.
TIP: The filtrate and used specimen container may be disposed following this step.
TIP: Wide-bore pipette tips are those that have sufficiently large openings to prevent tip plugging by sample particles. In general, standard 1-5mL and wide-mouth 100-1000µL pipette tips are sufficient for most samples. If required, increase the bore size of a tip using a clean pair of scissors.

Carefully transfer the 2” x 2” 250µm mesh used in 4.1 into a second new, unused specimen container. Using a new pipette tip, add 5mL of UltraLyse 3021 to the container. Cap and mix the container thoroughly to ensure complete contact between UltraLyse 3021 and the mesh piece. Allow at least 5 minutes of incubation.

NOTE: fbATP Extracts can be capped and stored refrigerated between 2 to 8^oC for up to 1 week prior to dilution.
TIP: If the mesh piece sticks to the side walls of the specimen container, simply place the container on its side during the incubation period with the mesh immersed in UltraLyse 3021.

Using a new pipette tip, transfer 3mL of the contents from the specimen container into a QG21W Dilution Tube. Cap and invert twice to mix. Allow beads to settle.

NOTE: At this point, the contents of the QG21W Dilution Tube are stable at room temperature for up to 4 hours.
TIP: If beads do not settle or settle slowly, tap the QG21W Dilution Tube gently to assist settling.

Using a new pipette tip, add 100µL of the QG21W Dilution Tube contents and 6 drops (300µL) of LuminaseW to a new 12x55mm test tube (the Assay Tube), swirl gently five times, and insert into the Luminometer. Read and record RLUfbATP for use in Step 5.

NOTE: If RLUfbATP < 10 on a Kikkoman Lumitester C-100 or C-110, you are below the low–detection limit. Report fbATP (ng ATP/mL) = 0 in Step 5.
TIP: If “Scale Over” is returned, repeat the fbATP analysis using 250µL of sample in 4.1 (FILTRATION) and modify the dilution factor in Step 5 as described.