ATP testing and bacterial culture on solid agar plates are two completely different techniques. While culture method only measures culturable bacteria, i.e., the ones able to grow on a given media, ATP testing measures the quantity of ATP in a sample. As this molecule is produced and found in all living bacteria, ATP testing measures all the bacteria, culturable or not. This major difference makes them difficult to compare.
However, when validating a new technique, it is natural to compare it with the conventional method. To avoid bias in result interpretation, here are some general tips.
General tips, not limited to these two techniques
- Be aware of what each technique measures: ATP test measures the ATP and thus indirectly the total bacteria, while culture only measures culturable bacteria.
- Each technology has its own limits. ATP test uses a defined convention to estimate the number of bacteria in the sample (1 pgATP ≈ 1 000 bacteria). As for the culture method, it does not count the VBNC (Viable But Nonculturable) bacteria. Yet, according to literature, only 0.01 to 1% of bacteria grow on HPC media. Bacterial culture is limited by the choice of culture media, of incubation time and incubation temperature.
- It is necessary to work on a large concentration range, i.e., on several LOG.
- Perform each measurement at least in triplicate to obtain a significative value for each method.
- All the samples must be treated in the same way, whatever the analysis method. One of the most frequent mistakes is to collect the sample in a bottle with sodium thiosulfate for culture analysis and without sodium thiosulfate for ATP analysis. In the first case, the biocide action will be stopped, while in the second case, the biocide will keep its action, eliminating the biomass. The comparison would then be distorted. Therefore, it is essential to perform the analyses on the same sampling bottle. Likewise, if a dilution is necessary, it must be diluted in sterile water or physiological serum for both methods.
- Last but not least: it is primordial to look at the results with a critical eye. You should be able to identify outlying results.
Specific tips on ATP tests vs Culture comparison
On top of the previous tips, here are some advice specific to these two technologies:
- Liquid culture media bias ATP results. Indeed, there is a large amount of free ATP and inhibitors in culture media. To avoid misleading results, dilute the samples in water or rinse the filter membrane.
- Bacteria cultures are not representative of the actual sample. As those bacteria are prepared for growth on culture media, a large part of them will grow and form colonies, whereas we know that only 0.01 to 1% of environmental bacteria are able to grow on culture media. Thus, it is important to compare the methods on real samples with complex ecosystems.
- Even if ATP test has a very high sensitivity, it cannot demonstrate sterility.
ARTICLES AVAILABLE ONLINE
Several quantitative ATP tests vs culture comparisons have been published over the last few years:
- Cross-validation of the ATP thresholds – CNR-IRSA and SMAT (2018)
- ATP tests in WSPs – Régie des eaux de Montpellier (2018)
- Monitoring of the microbial quality of drinking water – SUEZ and Odace (2014)