title: Preparation of a standardised faecal slurry for ex-vivo microbiota studies which reduces inter-individual donor bias.
abstract: In-vitro gut fermentation systems provide suitable models for studying gut microbiota composition and functionality. However, such methods depend on the availability of donors and the assumption of reproducibility between microbial communities before experimental treatments commence. The aim of this study was to develop a frozen standardised inoculum (FSI) which minimizes inter-individual variation and to determine its stability over time using culture-dependent and culture-independent techniques.
  
  
  
  A method for the preparation difference of a FSI is described which involves pooling the faecal samples, centrifugation and pelleting of the cell biomass and finally homogenising the cell pellets with phosphate buffer and glycerol. Using this approach, no significant difference in total anaerobe cell viability was observed between the fresh standardised inoculum (before freezing) and the 12days post freezing FSI. Moreover, Quantitative PCR revealed no significant alterations in the estimated bacterial numbers in the FSI preparations for any of the phyla. MiSeq sequencing revealed minute differences in the relative abundance at phylum, family and genus levels between the FSI preparations. Differences in the microbiota denoted as significant were limited between preparations in the majority of cases to changes in percentage relative abundance of ±0.5%. The independently prepared FSIs revealed a high degree of reproducibility in terms of microbial composition between the three preparations.
  
  
  
  This study provides a method to produce a standardised human faecal inoculum suitable for freezing. Based on culture-dependent and independent analysis, the method ensures a degree of reproducibility between preparations by lessening the effect of inter-individual variation among the donors, thereby making the system more suitable for the accurate interpretation of the effects of experimental treatments.
authors: Teagasc Food Research Centre, Moorepark, Fermoy, Co. Cork, Ireland; APC Microbiome Institute, University College Cork, Ireland.
authors: Michelle M
authors: MM
authors: O'Donnell
authors: Teagasc Food Research Centre, Moorepark, Fermoy, Co. Cork, Ireland; APC Microbiome Institute, University College Cork, Ireland.
authors: Mary C
authors: MC
authors: Rea
authors: Teagasc Food Research Centre, Moorepark, Fermoy, Co. Cork, Ireland; APC Microbiome Institute, University College Cork, Ireland.
authors: Órla
authors: Ó
authors: O'Sullivan
authors: Kerry Ingredients, Tralee Road, Listowel, Co. Kerry, Ireland.
authors: Cal
authors: C
authors: Flynn
authors: Kerry Ingredients & Flavours, Americas Regional Headquarters, 3330 Millington Road, Beloit, WI 53511, United States.
authors: Beth
authors: B
authors: Jones
authors: Kerry Ingredients, Tralee Road, Listowel, Co. Kerry, Ireland.
authors: Albert
authors: A
authors: McQuaid
authors: APC Microbiome Institute, University College Cork, Ireland.
authors: Fergus
authors: F
authors: Shanahan
authors: Teagasc Food Research Centre, Moorepark, Fermoy, Co. Cork, Ireland; APC Microbiome Institute, University College Cork, Ireland; School of Microbiology, University College Cork, Cork, Ireland. Electronic address: p.ross@ucc.ie.
authors: R Paul
authors: RP
authors: Ross
mesh_terms: D044822:Biodiversity; D004269:DNA, Bacterial; D005243:Feces / Q000382:microbiology*; D005285:Fermentation; D005615:Freezing; D006801:Humans; D064307:Microbiota; D011309:Preservation, Biological / Q000379:methods*; D060888:Real-Time Polymerase Chain Reaction; D012015:Reference Standards; D015203:Reproducibility of Results; D013048:Specimen Handling / Q000379:methods*
chemical_list: D004269:DNA, Bacterial
keywords: Cryo-preservation; Fermentation; MiSeq; Microbiota; Standardisation