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.Campylobacter cells are mostly slender, spirally curved rods (16). When Campylobacter jejuni is exposed to suboptimal conditions, its characteristic curved, spiral morphology undergoes a transition via numerous intermediary forms until the cells finally adopt a coccoid morphology (3,12). Cells in old cultures may form coccoid bodies, which are considered degenerative forms rather than a dormant stage of the organism (6). From the results of hundreds of studies published since 1982, it is now more clearly understood that a lack of nutrients, low temperatures, high pressure, sharp changes in pH or salinity, and other environmental factors can initiate a complex series, or cascade, of cellular events that include changes in cellular morphology and in concentration and/or structure of major biopolymers (proteins, membrane lipids, and nucleic acids) and cessation of ability to grow on solid or liquid laboratory media that would otherwise support growth of the bacterial strain employed in the studies (2).Recently, new nucleic acid-specific dyes with different quantum yields on DNA and RNA have been developed (10). The LIVE/DEAD BacLight kit (catalog no. L-7012; Molecular Probes, Eugene, Oreg.) stain mixture (BL) distinguishes viable bacterial cells from dead ones on the basis of membrane integrity. The kit contains two nucleic acid stains. The green fluorochrome (Syto 9) is a small molecule that can penetrate intact plasma membranes, while the larger red fluorochrome (propidium iodide) penetrates only compromised membranes. Bacterial suspensions incubated in the presence of both stains simultaneously will fluoresce either green (i.e., viable) or red (i.e., dead), depending on their viability. The excitation and emission maxima for these dyes are about 480 and 500 nm for Syto 9 and 490 and 635 nm for propidium iodide, respectively (Handbook of fluorescent probes and research chemicals, Molecular Probes).The fluorescent protein stain NanoOrange (catalog no. N-6666; Molecular Probes) has been used recently for visualizing bacterial flagella (4). The NanoOrange reagent is virtually nonfluorescent in aqueous solutions, but upon interaction with hydrophobic regions of proteins, it undergoes a dramatic fluorescence enhancement, with ex...

Chitosan/TPP and Chitosan/TPP-hyaluronic Acid Nanoparticles: Systematic Optimisation of the Preparative Process and Preliminary Biological Evaluation

“…The LIVE/DEAD double staining kit (Sigma, St Louis, MO, USA), which allows the simultaneous fluorescence staining of viable (Calcein-AM, excitation 495 nm, emission 515 nm) and dead (Propidium iodide, PI, excitation: 535 nm, emission: 617 nm) cells (41,42), was used. Macrophages J774.2 were seeded in a 96-well plate at a count 8,000 cells/ well and incubated for 24 h with 0.1 mg/ml of coated or uncoated CSNPs.…”

Use of Flow Cytometry To Monitor Legionella Viability

“…The existence of viable but nonculturable (VBNC) bacteria (13,21,24), which reduce the sensitivity of culture-based assays, has been demonstrated. Analysis of membrane integrity in order to distinguish between viable and dead cells in various bacterial species has been proposed (2,14,17,19); some of these assays are based on double staining combining Syto 9 and propidium iodide (PI) (6,9,26). To date, only three studies dealing with the detection of Legionella cells by flow cytometry have been reported (15,27,29), and none of them analyzed the presence of VBNC cells, which was undertaken in this study.…”

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