Validation of fluorescent in situ hybridization combined with flow cytometry for assessing interindividual variation in the composition of human fecal microflora during long-term storage of samples

Journal of Microbiological Methods
Violaine RochetJoel Doré

Abstract

This work was conducted to assess the accuracy of in situ hybridization to show differences in human microflora composition between volunteers and to optimize the storage of fecal samples to allow delayed analysis of gut microflora composition in humans. Fecal samples from 25 healthy subjects (14 women, 11 men aged 24-51) were collected. The samples were fixed in 4% Paraformaldehyde (PFA) solution at 4 degrees C overnight and stored at -70 degrees C. Twenty samples were analysed to quantify the variation due to interindividual differences in the composition of fecal microflora. The five remaining samples were stored either after PFA fixation or directly frozen at -70 degrees C and were monitored on a 12-month period. The fecal microflora was analysed by in situ hybridization combined with flow cytometry detection. Ribosomal RNA-targeted probes were used to assess the relative proportions of four phylogenetic groups: Clostridium coccoides-Eubacterium rectale (Erec 482), Bacteroides (Bac 303), Faecalibacterium prausnitzii (Fprau 645) and Bifidobacterium (Bif 164). Our results demonstrated that the method used is adapted to detect significant differences in fecal microflora composition in humans. Moreover, samples stored in PFA so...Continue Reading

References

Sep 19, 2000·Journal of Microbiological Methods·A Moter, U B Göbel
Jun 1, 2002·Applied and Environmental Microbiology·Hermie J M HarmsenGjalt W Welling

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Citations

Aug 11, 2011·PloS One·Francesc Peris-BondiaGiuseppe D'Auria
Jul 1, 2008·Journal of Microbiological Methods·S GüntherS Müller
Aug 27, 2014·The Proceedings of the Nutrition Society·Johanna Maukonen, Maria Saarela
Dec 17, 2009·FEMS Microbiology Ecology·Jie JiaAnne L McCartney
Sep 26, 2016·Systematic and Applied Microbiology·Lena M BruderWolfgang Liebl
May 22, 2008·Journal of Pediatric Gastroenterology and Nutrition·Fabien MagnePhilippe Pochart
Jan 16, 2007·The British Journal of Nutrition·Elise AlvaroJoël Doré
Mar 28, 2012·Nature Reviews. Gastroenterology & Hepatology·Marianne H FraherEamonn M M Quigley
May 26, 2017·Applied Microbiology and Biotechnology·Joanna Maria Radziwill-BienkowskaMagdalena Kowalczyk

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