Sampling Consistency
Microorganisms do not distribute evenly throughout samples, leading to higher variability in microbiological tests compared to chemical analyses. To improve repeatability, ensure sampling consistency by avoiding errors like inconsistent sampling locations, poor technique, unclean collection containers, inconsistent timing, and inconsistent flushing times (when collecting samples from taps). Mix samples well before each analysis and collect and test multiple samples from the same location to assess variability. If the sample is from the same location and is well mixed, repeatability should be good.
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.
Protocol and Testing
There are also key steps in the protocol where variability may be introduced. To test repeatability and reproducibility, have each tester perform three ATP analyses on the same sample. Compare the results for reproducibility and repeatability (CV < 15% is good). Pay attention to protocol steps, especially the timing of the assay, and ensure clean and accurate pipetting to minimize variability.
Luminase Handling
Allow the Luminase enzyme to reach ambient temperature before use to prevent changes in activity that can impact ATP results. Always allow 1 hour for Luminase to reach room temperature before use.
What is Luminase and How Should I Handle and Store it for ATP Testing?
Process and Operational changes
Changes in process or operations can affect ATP results. 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 if you notice significant changes in ATP levels.
Reagents and Plastic Consumables
Ensure reagents and plastic consumables are not contaminated. Avoid reusing pipette tips and store all materials in sealed containers or bags when not in use. Perform background checks on assay tubes for contamination by adding 100 uL of Luminase to a new assay tube and taking a reading in the luminometer - backgrounds should be less than 10 RLU. If the result is significantly higher, discard and use a new assay tube.
Special Samples
For highly pigmented samples or high-solids samples where interference or inhibition is a concern, contact support for guidance.