Automated thresholding method (ATM) for biomass fraction determination using FISH and confocal microscopy

Research output: Contribution to journalArticleResearchpeer-review

45 Citations (Scopus)


Background: Fluorescence in situ hybridization (FISH) together with confocal laser scanning microscopy (CLSM) is widely used for the analysis and quantification of biomass fractions of activated sludge or biofilm systems in wastewater treatment research. Unlike the FISH technique, the CLSM image analysis and quantification is not generally unique and thus, several manual and automated methods exist that can lead to very different results. Results: Amethod based on the analysis of negative control images is reported in this paper. Histograms of negative control images were used in the segmentation of positive images (hybridized with FISH probes). The threshold (Ithr) was defined as the minimum intensity value that satisfies the following condition: the proportion of pixels with intensity I ≤ Ithr in the negative control images is greater than or equal to a value a. It was found that the optimal α-value was 99.90% for the specific case studied (nitrifying activated sludge samples) by comparing the automatic thresholding with manual thresholding by three expert operators. Conclusion: The method developed was useful for following systematically the evolution of ammonia oxidizing bacteria (AOB) and nitrite oxidizing bacteria (NOB) fractions in a nitrifying system under changing operating conditions. © 2009 Society of Chemical Industry.
Original languageEnglish
Pages (from-to)1140-1145
JournalJournal of Chemical Technology and Biotechnology
Issue number8
Publication statusPublished - 27 Jul 2009


  • Automated thresholding method
  • CLSM
  • FISH
  • Nitrification
  • Quantification


Dive into the research topics of 'Automated thresholding method (ATM) for biomass fraction determination using FISH and confocal microscopy'. Together they form a unique fingerprint.

Cite this