Role of Intestinal Microbiota in Ulcerative Colitis

Methodology part
6. Methodology, considerations and choices
to the system, if the amount of testing compound was sufficient. The in vitro fermentation
procedure is further described in the following manuscripts; Paper 3 ‐ 6.
6.3. Continues systems
Dynamic in vitro digestion methods are suited models to study the microbial ecology of the
gastrointestinal tract. One of the most known physio‐chemical models is the validated Simulator of
the Human Intestinal Microbial Ecosystem (SHIME). This model is multi‐compartmental and
consists of a series of 5 vessels, simulating the stomach, small intestine and the three colon
regions; ascending, transverse and descending (Molly et al., 1993). Hence, measurements of
fermentation activity and composition of the colon microbial community in the different colonic
regions can be performed (Yoo and Chen, 2006). The SHIME is unable to simulate selective
absorption of metabolites and fluids, and can only simulate the growth of the luminal bacteria,
whereas the adhesion of bacteria to mucus is omitted. However, Abbeele et al. (2011a) have
recently developed a dynamic in vitro gut model incorporating mucin‐covered microcosms into the
SHIME. This model named M‐SHIME simulates both the lumen and mucus environment, and
allows studying the mucosal microbiota and interaction between luminal and mucosal microbial
communities. Other in vitro models have previously been used to screen the adhering potency of
intestinal microbes. They include adhesion assays to various components of the intestinal surface:
e.g. intestinal mucus (Ouwehand et al., 2002b), mucins (Van den Abbeele et al., 2009), colonic
tissue (Ouwehand et al., 2002a) or cell lines (Laparra and Sanz, 2009). However, they often only
provide short‐term information regarding axenic cultures and tend to ignore the microbial
interaction between and within the luminal and mucosal microbiota.
The M‐SHIME set up used in the experimental part of this thesis consisted of two vessels
simulating the stomach and the small intestine, and six ascending colon vessels, which were run in
parallel without the transverse and descending colon. All ascending colon vessels were modified
by incorporating mucin‐covered microcosms into the luminal suspension. The procedure for the
M‐SHIME is described in details in Paper 2.
6.4. DNA extraction
DNA extraction is a key factor affecting any approach for analyzing microbial diversity. Several
methods have been reported for the isolation of microbial DNA from human stool samples
(Machiels et al., 2000;Clement and Kitts, 2000;Zhang et al., 2006). However, problems can arise in
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