Because microorganisms do not distribute evenly throughout samples, the variability associated with microbiological tests is higher than with chemical analyses. Typical variation when conducting microbiological testing by conventional plate count ranges from 15% < RSD < 35% compared to chemical assays which range from 1% < RSD < 3%). Reference: USP 1223 - Validation of Alternative Microbiological methods.
Sampling consistency is key
Sampling error, inconsistent sampling location, poor technique, collection container not being clean, inconsistent timing, and inconsistent flushing time (when collecting samples from taps), can all introduce variability in the results. Be sure to mix the sample well before each analysis. To determine if sampling is the cause of poor results, collect and test multiple samples from the same location. If the sample is from the same location and is well mixed, repeatability should be good. If the variability is high, it could be indicative of insufficient mixing within the process.
There are also key steps in the protocol where variability may be introduced. To test the repeatability for each user and reproducibility between users, have each tester perform three ATP analyses on the same sample. Compare the results for reproducibility and repeatability (CV < 15% is good). If there is significant variability between testers - walk through the protocol and take care to notice areas of inconsistency. One of the key steps is the timing of the assay - after the Luminase and extracted ATP (from the final dilution tube) are mixed the reading should be taken in the Luminometer within 5-15 seconds. Pipetting errors may also cause variability so ensure that you are using clean and accurate pipettes.
If the luminase enzyme is not allowed to reach ambient temperature prior to use, Luminase activity will change, impacting your ATP results. Always allow 1 hour for Luminase to reach room temperature before use.
Process and operational changes
Process and operational changes will most certainly be seen in ATP results, whether the change(s) benefited or harmed the biomass. If you notice significant changes in the ATP levels, investigate potential equipment breakdown or maintenance, changes in biocide or disinfectant levels, changes in source water, or other process changes that may have impacted the biomass.
Reagents or plastic consumables
Reagents or plastic consumables may be contaminated. This can be done by using the same pipette tip multiple times, touching plastic consumables with hands or other objects, allowing dust or debris to collect in open vessels or bags, and so on. Never reuse pipette tips! Ensure that all materials are stored in sealed containers or bags when not in use. Backgrounds can be done to check the assay tubes for contamination - simply add 100 uL of Luminase to a new assay tube and take the reading in the luminometer - backgrounds should be less than 10 RLU. If the result is significantly higher, discard and use a new assay tube.
If you are testing highly pigmented samples or high-solids samples where you are concerned about interference or inhibition contact support.