Troubleshooting Guide:
Below you will find solutions to a number of possible troubleshooting scenarios. Simply click the heading to expand the information.
| Possible Scenario |
Explanation |
Solution |
| 1. User is reporting RLU instead of ATP concentration. |
dATP RLUs can be and often are greater than tATP RLUs. This is due to the dilution factor being significantly less for dATP tests. However, once ATP concentrations have been calculated taking into account these dilution factors and the UltraCheck1 calibration value, it is impossible for the concentration of dATP to be greater than that of tATP. |
Ask the user if they are referring to RLUs or actual ATP concentrations. The solution is often as simple as carrying out the remaining calculations to achieve the correct result. |
| 2. User not following procedure correctly. |
Dilution factors are built into ATP calculations, so using incorrect sample and/or reagent volumes can totally change the end result. |
Consult the Quick-Reference Guide and/or User’s Guide and confirm that all steps are being performed correctly. |
| 3. Detection limit of kit is surpassed |
Kit used to test a sample may not be appropriate for the level of biomass present. For example, the TCB kit should not be used in low-biomass samples (i.e. cooling water) because the low-end detection limit is not sensitive enough. |
See the Kit Selection Guide to determine the appropriate kit. |
| 4. Inhibition may be effecting result |
For certain samples, factors such as turbidity or heavy metals may inhibit the light assay, resulting in inaccurate results. |
Perform tATP and dATP tests using the standard protocol, then add 1mL of diluted extract (from tATP) to 1mL of UltraBuffTM, and assay. If there is no inhibition, the resulting RLU should be ½ the value obtained from the standard protocol. |
| 5. Reagents may be contaminated |
If the user is not careful, it is quite easy to cross-contaminate LuminUltra reagents. This can be done using the same pipet tip multiple times touching plasticware with your hands or other objects, etc. |
Always use a new tip when pipetting new reagents or samples. Also, never touch plasticware in areas that will contact reagents. |
| Possible Scenario |
Explanation |
Solution |
| 1. Incorrect dilution |
Samples and extracts must be diluted exactly as described in the procedures. If a sample has a high biomass content and is not diluted enough, the light output will be more than the luminometer can read. |
Consult the Quick-Reference Guide and/or User’s Guide and confirm that all steps are being performed correctly. |
| 2. Detection limit of kit is surpassed |
Kit used to test a sample may not be appropriate for the level of biomass present. For example, the TCM kit should not be used in high-biomass samples (i.e. anaerobic sludge) because the high-end detection limit is too low. |
See the Kit Selection Guide to determine the appropriate kit. |
| Possible Scenario |
Explanation |
Solution |
| 1. Sampling Error |
A poor location or perhaps poor sampling technique may be used. |
Take multiple samples from the same location and analyze tATP and dATP individually on each sample. If the sampling location is well mixed and the samples are of good quality, repeatability should be good. If the variability is high, this could be indicative of insufficient reactor mixing, some other biological problem, or a poor sampling location. |
| 2. Analytical Error |
The user may have made an error when analyzing the sample. |
Perform three tATP and three dATP tests on the same sample. Compare only the measured results (i.e. tATP and dATP) for repeatability. Less than 10% variability should be achieved. If not, take the 3 tATP extracts, pool them, then dilute and assay the pooled extract. If the problem was poor-sub-sampling, the results should come very close to the average of the 3 previous measurements. |
| 3. Detection limit of kit is surpassed |
Kit used to test a sample may not be appropriate for the level of biomass present. This will result in erratic and inaccurate results. For example, the TCB kit should not be used in low-biomass samples (i.e. cooling water) because the low-end detection limit is not sensitive enough. |
See the Kit Selection Guide to determine the appropriate kit. |
| 4. Luminase not at room temperature |
If not allowed to reach room temperature prior to use, the light output from the assay will not be representative of what it should be. |
Always allow 1 hour for Luminase to reach room temperature before use. If the Luminase is pipetted into the 12x55mm culture tubes, this time can be decreased. |
| 5. New technician performing tests |
When new staff begins performing ATP tests, it is normal for there to be some anomalies in the data until the new user becomes accustomed to the protocols. |
Ask the client if this is the case. Also, ask if they have been adequately trained in sampling, pipetting, and analysis techniques. |
| 6. Inadequate or inconsistent sample mixing |
Samples must be mixed to ensure homogeneity before analyses. This is especially important with high-solids samples since they tend to settle after mixing. |
Always mix samples thoroughly (but not too much - 3 to 4 inversions is usually adequate) immediately before testing. When different operators perform tests from day to day, encourage consistent mixing technique to minimize error. |
| Possible Scenario |
Explanation |
Solution |
| 1. Changes are occurring in the process |
ATP monitoring is extremely sensitive, and as such, will be affected by process changes. TSS measurements have several interferences and therefore do not have the same variability as ATP measurements. |
Compare results of ATP tests to major process variables (i.e. BOD, oxygen and nutrient feeds, pH, etc.). Changes in any of these quantities WILL affect the biomass. |
| 2. Inconsistent sampling |
Factors such as sampling time, sampling location, and the vessel used to collect the sample are all possible sources of error when collecting samples. |
As with all other aspects of ATP test protocols, consistency is key. Try to sample from the same location at the same time each day with a clean vessel. |
| 3. Pipettors are out of calibration |
Pipettors that are not properly calibrated will produce poor results. The accuracy of measurement devices is paramount to achieving proper results. |
Perform the calibration procedure as outlined in the “Pipetting Guide” |
| 4. Inconsistent test methods |
Factors such as incubation times and pipetting technique can adversely affect the results if not done consistently. |
Follow the test protocols carefully and try to use consistent techniques on a day to day basis. |
| Possible Scenario |
Explanation |
Solution |
| 1. Pipettor is not calibrated properly |
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| 2. Incorrect pipette tip size is being used |
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| 3. Pipettor needs cleaning and/or maintenance |
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| Possible Scenario |
Explanation |
Solution |
| 1. Solids content of sample is too high. |
The sample may contain too much solids to filter. Therefore, the QGA kit may not be appropriate for the level of biomass present. For example, the QGA kit should not be used with samples containing high-solids content (i.e. pulp & paper process water). |
See the Kit Selection Guide to determine the appropriate kit. Water samples with high solids usually requires the TCM kit. |
| 2. Wrong filter is being used. |
0.7μm GMF Syringe Filters (25mm diameter) are used with Quench-Gone kits in all cases except for Ultrapure water, where 0.2 μm filters are used. |
Check the filter porosity and confirm it is the correct size. |
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