full issue - Association of Biotechnology and Pharmacy

Current Trends in Biotechnology and Pharmacy
Vol. 5 (3) 1338 -1345 July 2011, ISSN 0973-8916 (Print), 2230-7303 (Online)
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the mesentery manually (11). Next, the entire
intestine is transferred to a Petri dish containing
Krebs-Ringer saline, and quickly extended to its
full length. At this point it may be cut into
segments of convenient length i.e. 5cm of
jejunum segment was taken and washed out with
normal saline solution (0.9% w/v NaCl) using a
syringe equipped with blunt end.
Preparation of Everted intestinal sac: Intestinal
segments (5cm) were everted according to the
method described earlier (12). A narrow glass
rod was inserted into one end of the intestine. A
ligature was tied over the thickened part of the
glass rod and the sac was everted by gently
pushing the rod through the whole length of the
intestine (Fig. 1). Krebs-Ringer saline with the
pH 7.3 was used as the incubation medium. The
loose ligature over the needle was tightened and
2 ml of the Krebs Ringer Saline (in mM: 118
NaCl, 4.7 KCl, 25 NaHCO 3
, 1.2 CaCl 2
, 1.2
MgSO 4
, 11.1 glucose, pH 7.3) was injected into
the sac (Fig. 2). All the ligatures have to be firm
enough to prevent leaks but not too tight so as
damage the tissue.
The compartment containing the buffer
in the sac was named serosal fluid compartment
and the buffer in the petridish was named as
mucosal fluid compartment respectively. The
distended sacs was placed in petri dish (13) with
10 ml of the same medium and incubated for a
suitable period of 45 minutes at 37°C, with
oxygen entering through a plastic tube. The loss
of substrate from the medium was taken as uptake
by the sac while gain in substrate was taken as
the release of the metabolite by the sac (Fig. 3).
Sample collection: After incubation, the sacs
were removed from the petridish and the serosal
fluid from the sac was collected through a small
incision and transferred into a test tube. The
emptied sac was shaken gently to remove the
adhered fluid whereas the mucosal fluid was
Fig. 3. Everted intestinal sac
In vitro Evaluation of Gluconeogenesis