Double-staining method for differentiation of morphological changes and membrane integrity of Campylobacter coli cells

Abstract

We developed a double-staining procedure involving NanoOrange dye (Molecular Probes, Eugene, Oreg.) and membrane integrity stains (LIVE/DEAD BacLight kit; Molecular Probes) to show the morphological and membrane integrity changes of Campylobacter coli cells during growth. The conversion from a spiral to a coccoid morphology via intermediary forms and the membrane integrity changes of the C. coli cells can be detected with the double-staining procedure. Our data indicate that young or actively growing cells are mainly spiral shaped (green-stained cells), but older cells undergo a degenerative change to coccoid forms (red-stained cells). Club-shaped transition cell forms were observed with NanoOrange stain. Chlorinated drinking water affected the viability but not the morphology of C. coli cells.

FIG. 1.

Double staining of C. coli cells demonstrates the morphology and membrane integrity changes during growth in broth and plate media. (A) Viable (green) spiral form cells of a 20-h culture on NB2, stained with Syto 9, visualized with an XF-53 filter; (B) transition and coccoid form cells of a 48-h culture on NA2, stained with NanoOrange, visualized with a B2-A filter; (C) club-shaped cells of a 24-h culture on NA2, stained with NanoOrange, visualized with a B2-A filter; (D) viable (green) and dead (red) club-shaped cells of a 24-h culture on NA2, stained with the BL stain mixture, visualized with an XF-53 filter.