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Here, plate count, quantitative real-time polymerase chain reaction (q-PCR) and peptide nucleic acid probe-fluorescence <jats:italic>in situ<\/jats:italic> hybridization (PNA-FISH) were employed to quantitate cystic fibrosis multispecies biofilms. Growth of <jats:italic>Pseudomonas aeruginosa<\/jats:italic>, <jats:italic>Inquilinus limosus<\/jats:italic> and <jats:italic>Dolosigranulum pigrum<\/jats:italic> was assessed in dual- and triple-species consortia under oxygen and antibiotic stress. Quantification methods, that were previously optimized and validated in planktonic consortia, were not always in agreement when applied in multispecies biofilms. Discrepancies in culture and molecular outcomes were observed, particularly for triple-species consortia and antibiotic-stressed biofilms. Some differences were observed, such as the higher bacterial counts obtained by q-PCR and\/or PNA-FISH (\u22644 log<jats:sub>10<\/jats:sub> cells\/cm<jats:sup>2<\/jats:sup>) compared to culture. But the discrepancies between PNA-FISH and q-PCR data (eg <jats:italic>D<\/jats:italic>. <jats:italic>pigrum<\/jats:italic> limited assessment by q-PCR) demonstrate the effect of biofilm heterogeneity in method\u2019s reliability. As the heterogeneity in biofilms is a reflection of a myriad of variables, tailoring an accurate picture of communities\u00b4 changes is crucial. This work demonstrates that at least two, but preferentially three, quantification techniques are required to obtain reliable measures and take comprehensive analysis of polymicrobial biofilm-associated infections.<\/jats:p>","DOI":"10.1038\/s41598-018-27497-9","type":"journal-article","created":{"date-parts":[[2018,6,18]],"date-time":"2018-06-18T09:05:32Z","timestamp":1529312732000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":34,"title":["Quantitative assessment of individual populations within polymicrobial biofilms"],"prefix":"10.1038","volume":"8","author":[{"given":"Susana Patr\u00edcia","family":"Lopes","sequence":"first","affiliation":[]},{"given":"Nuno Filipe","family":"Azevedo","sequence":"additional","affiliation":[]},{"given":"Maria Ol\u00edvia","family":"Pereira","sequence":"additional","affiliation":[]}],"member":"297","published-online":{"date-parts":[[2018,6,22]]},"reference":[{"key":"27497_CR1","doi-asserted-by":"publisher","first-page":"114","DOI":"10.1038\/nrd1008","volume":"2","author":"D Davies","year":"2003","unstructured":"Davies, D. 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