Ultrastructure of E. coli: An in vitro study of cells cultured in the presence of four gastrointestinal (GI) hormones

Abstract

Hormones are found in exocrine secretions entering the gut. They alter the morphology of many eukaryotic cells; whether they affect the morphology of enteric flora is unknown. In this study, we examined the ultrastructure of E. coli, a common bacterium in the mammalian gut, for morphological changes resulting from exposure to GI hormones.E. coli (#11775 from American Type Culture Collection) were grown in protease-free trypticase soy broth (TSB) at 37°C for 18 hr to a concentration of 2 x 107 cells/ml. Pure synthetic hormones were used: sulfated C-terminal cholecystokinin octapeptide (CCK), pentagastrin (PG), cyclic somatostatin tetradecapeptide (SS), or the porcine form of secretin (SEC). These were individually added to. bacterial cultures in TSB to make 1 x 107 organisms/ml and 0.0, 0.5, 2.5, or 5.0 μg of hormone/ml, then incubated for 30 min at 37°C. The cultures were rapidly chilled and added to equal volumes of cold 6% glutaraldehyde in 0.2 M cacodylate buffer. After 30 min, the bacteria were concentrated by centrifugation (15 min at 4000 RPM) and the pellets suspended in cold 3% glutaraldehyde for an additional 15 min, followed by centrifugation. The pellets were resuspended in cold cacodylate buffer and stored at 2°C for 1-7 d. The cells were again centrifuged and the pellets were blotted with a strip of filter paper to remove excess fluid, then mixed with a drop of warm 2% agar. The agar suspensions were pipetted into cold saline. The resulting solidified extrusions were cut by hand into 2 mm segments for further processing in 1% OsO4 (with or without en bloc staining in 2% uranyl acetate (UA) in ethanol). Following dehydration in ethanol, rinsing in propylene oxide, and encapsulation in Epon-Araldite, thin sections were examined and photographed with a JEOL-100C microscope.