Double-Staining Method for Differentiation of Morphological Changes and Membrane Integrity of Campylobacter coli Cells

We developed a double-staining procedure involving NanoOrangedye (Molecular Probes, Eugene, Oreg.) and membrane integritystains (LIVE/DEAD BacLight kit; Molecular Probes) to show themorphological and membrane integrity changes of Campylobactercoli cells during growth. The conversion from a spiral to acoccoid morphology via intermediary forms and the membrane integritychanges of the C. coli cells can be detected with the double-stainingprocedure. Our data indicate that young or actively growingcells are mainly spiral shaped (green-stained cells), but oldercells undergo a degenerative change to coccoid forms (red-stainedcells). Club-shaped transition cell forms were observed withNanoOrange stain. Chlorinated drinking water affected the viabilitybut not the morphology of C. coli cells.

INTRODUCTION

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Introduction

Campylobacter cells are mostly slender, spirally curved rods(16). When Campylobacter jejuni is exposed to suboptimal conditions,its characteristic curved, spiral morphology undergoes a transitionvia numerous intermediary forms until the cells finally adopta coccoid morphology (3, 12). Cells in old cultures may formcoccoid bodies, which are considered degenerative forms ratherthan a dormant stage of the organism (6). From the results ofhundreds of studies published since 1982, it is now more clearlyunderstood that a lack of nutrients, low temperatures, highpressure, sharp changes in pH or salinity, and other environmentalfactors can initiate a complex series, or cascade, of cellularevents that include changes in cellular morphology and in concentrationand/or structure of major biopolymers (proteins, membrane lipids,and nucleic acids) and cessation of ability to grow on solidor liquid laboratory media that would otherwise support growthof the bacterial strain employed in the studies (2).

Recently, new nucleic acid-specific dyes with different quantumyields on DNA and RNA have been developed (10). The LIVE/DEADBacLight kit (catalog no. L-7012; Molecular Probes, Eugene,Oreg.) stain mixture (BL) distinguishes viable bacterial cellsfrom dead ones on the basis of membrane integrity. The kit containstwo nucleic acid stains. The green fluorochrome (Syto 9) isa small molecule that can penetrate intact plasma membranes,while the larger red fluorochrome (propidium iodide) penetratesonly compromised membranes. Bacterial suspensions incubatedin the presence of both stains simultaneously will fluoresceeither green (i.e., viable) or red (i.e., dead), depending ontheir viability. The excitation and emission maxima for thesedyes are about 480 and 500 nm for Syto 9 and 490 and 635 nmfor propidium iodide, respectively (Handbook of fluorescentprobes and research chemicals, Molecular Probes).

The fluorescent protein stain NanoOrange (catalog no. N-6666;Molecular Probes) has been used recently for visualizing bacterialflagella (4). The NanoOrange reagent is virtually nonfluorescentin aqueous solutions, but upon interaction with hydrophobicregions of proteins, it undergoes a dramatic fluorescence enhancement,with excitation and emission peaks averaging about 485 and 590nm. Nucleic acids do not interfere with protein quantitationwhen NanoOrange reagent is used (Handbook of fluorescent probesand research chemicals, Molecular Probes).

In this paper we describe a simple and rapid method for facilitatingthe characterization of changes in viability and morphologyduring the growth of Campylobacter coli cells. This method enablesvisualization of cell shapes and membrane integrity changesof C. coli cells with the fluorescent membrane integrity dyesSyto 9 and propidium iodide and the fluorescent protein stainNanoOrange.

Culture conditions.

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Culture conditions.

C. coli strain NCTC 11366 was employed during these studies.To monitor morphological and membrane integrity changes, theorganism was grown under a microaerobic atmosphere (10% CO₂,5% O₂, 85% N₂) for 24 h at 37°C on a Campylobacter agarbase (Oxoid) supplemented with 5% lysed sheep blood (Oxoid)(NA2). Two culture media were inoculated to produce exponential-or stationary-phase cells. A 24-h plate culture was suspendedin a 100-ml Erlenmeyer flask of nutrient broth no. 2 (Oxoid)supplemented with 5% sheep blood (NB2) and was incubated at37°C under a microaerobic atmosphere. Additionally, a 24-hplate culture was plated onto a Campylobacter agar base supplementedwith 5% lysed sheep blood and incubated at 37°C under amicroaerobic atmosphere. Cultures of broth and plate media weresampled at 6, 12, 20, 24, 48, and 72 h postinoculation. Themorphological and membrane integrity changes were studied bythe BL-NanoOrange staining method described below. All the experimentswere carried out in duplicate.

Chlorine inactivation.

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Chlorine inactivation.

An inactivation experiment was conducted as follows: samplestaken at different interval times (20, 24, 48, and 72 h) fromthe broth and plate media described above were washed twicewith phosphate-buffered saline and suspended in 250 µlof filter-sterilized chlorinated drinking water (0.81 mg oftotal chlorine/liter and 0.75 mg of free chlorine/liter). Chlorinelevels were measured with an amperometric titrator (model 716DMS Titrino; Methrom, Herisau, Switzerland), and the physicochemicalparameters of drinking water (Table 1) were determined accordingto American Public Health Association standards (1). Drinkingwater-inoculated samples were incubated for 15 min at room temperatureand examined by the BL-NanoOrange staining method.

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TABLE 1. Physicochemical characteristics of tap water^a

BL-NanoOrange staining method.

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Bl-nanoorange staining method.

Samples (1 ml) were mixed with 3 µl of a mixture of Syto9 and propidium iodide (1:1), nucleic acid stains from a LIVE/DEADBacLight viability kit, and were incubated in the dark for 15min at room temperature. C. coli cells stained with the BL mixturewere immobilized on glass surfaces. BL-stained bacteria (5 µl)were applied to the poly-L-lysine area at the center of a cleanglass microscope slide. A 0.25-µl portion of undilutedNanoOrange stock solution was added to the 5 µl of immobilizedBL-stained bacteria on the microscope slide. An 18-mm² coverslipwas placed over the suspension and sealed with petrolatum. Grossartet al. (4) reported that a 1:20 dilution also worked well forstaining bacterial flagella. These authors (4) observed thatstaining times of 10 to 15 min in the dark ensured adequatestaining of bacterial flagella, whereas shorter staining timesresulted in images that were qualitatively less bright and prolongedstaining times (up to 6 h) did not change the quality of theimage. We examined the slide after a 10-min staining periodat a magnification of x1,250 with an epifluorescence microscope(Labophot; Nikon, Tokyo, Japan) equipped with a mercury lampand a Nikon B2-A filter set for NanoOrange stains and an Omega(Brattleboro, Vt.) XF-53 filter set for BL stains. Ideally,healthy (live) bacteria with intact plasma membranes fluorescegreen and the dead or injured cells with compromised membranesfluoresce red. In accordance with the manufacturer's instructions(Handbook of fluorescent probes and research chemicals, MolecularProbes), all green cells were considered viable and red cellswere considered dead. Images were recorded with a digital camera(model DP-10; Olympus, Hamburg, Germany).

All cells observed in samples examined after 6 and 12 h werespiral shaped. Changes in cell morphology from spiral to coccoidforms and in membrane integrity were detected after BL and NanoOrangestaining, as shown in Fig. 1. After 20 h, C. coli cells grownon NA2 were in spiral form and had maintained membrane integrity(Fig. 1A). Similar results were observed with C. coli cellsgrown on NB2. After 24 h, C. coli cells grown on NA2 showeda transition form (Fig. 1B), but the C. coli cells grown onNB2 still presented the spiral form. Ng et al. (12) observedthat more than 99% of C. coli cells grown in Bacto BrucellaBroth and incubated at 37°C in a modified atmosphere (7%CO₂) for 24 h were spiral shaped. All the transition forms detectedwere club shaped. These transition forms of Campylobacter specieshave been demonstrated previously by using transmission electronmicroscopy (9, 12) and scanning electron microscopy (12, 15).The transition forms (Fig. 1C) were sometimes viable (with intactmembranes) (Fig. 1D) and sometimes dead (with injured membranes)(Fig. 1D). Lázaro et al. (9) indicated that two formsof nonculturable C. jejuni cells may exist—viable andnonviable—which may not correspond with spiral and coccoidforms, respectively. Several authors (7, 13) have reported atransition to the viable but nonculturable (VBNC) stage andhave found a conversion to coccoid forms, some with a slightlyenlarged periplasmic space or with membrane budding. Lázaroet al. (9) observed bleb-like membrane vesicles around C. jejunicells incubated at 4°C. These vesicles could be due to aprocess of cell volume adjustment by bleb formation, part ofa survival strategy for minimizing cell maintenance requirementsand enhancing substrate uptake with a high surface-to-volumeratio (9). Thomas et al. (15) found that the typical spiralform of C. jejuni, evident during logarithmic phase, undergoeselongation during stationary phase before becoming coccoid viathe formation of membrane blebs and budded forms in declinephase. Cellular elongation and coccoid formation occurred despitethe inhibition of protein synthesis and without a detectablechange in the protein components of the inner and outer cellmembranes (15). Jacob et al. (7) found no changes in whole-cellprotein or lipooligosaccharide patterns as the cells becamenonculturable. These authors (7) concluded that coccoid formsmay be considered dormant forms that would not be detected inwater by conventional microbiological methods. Tholozan et al.(14) recorded an increase in cell volume of VBNC C. jejuni cells,which had significantly lower potassium contents and membranepotentials than cells in the culturable stage. All these traitswere suggested to be involved in strategies for minimizing cellmaintenance requirements. The involvement of membrane blebsand budding in the process of coccoid formation in the absenceof de novo protein synthesis and without changes in membraneprotein composition indicates that the process is passive andpotentially degenerative (15). There is evidence that the formationof coccoid C. jejuni cells is not an active process but representsa degeneration resulting from oxidative damage (5). After 72h, C. coli cells showed coccoid forms. It has been proposedthat even though these coccoid forms are not culturable, theymay still be viable (13). However, the evidence that the coccoidforms can revert back to the spiral forms is generally weak,and there is considerable controversy surrounding the wholephenomenon of VBNC cells (8).

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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.

All Campylobacter cells (spiral, transition, and coccoid forms)taken at different time intervals (20, 24, 48, and 72 h) frombroth and plate media and suspended in 250 µl of filter-sterilizedchlorinated drinking water for 15 min showed injured membranes(they stained red). Chlorinated drinking water affected theviability but not the morphology of C. coli cells. The samecell shape observed on NA2 or NB2 was detected after cell suspensionin chlorinated drinking water. Only membrane integrity was compromised.Exposure to chlorine has been shown to damage cell membranes(11).

In summary, we describe a novel staining method for rapid visualizationof morphological and membrane integrity changes of C. coli cells.Like the NanoOrange method for demonstrating bacterial flagella(4), the BL-NanoOrange method does not require fixation of thecells and involves very little sample manipulation; thus, artifactsdue to fixation, dehydration, or excessive sample manipulationcan be avoided.

ACKNOWLEDGMENTS

This work was supported by grant no. ALI99-0539 from CICYT.S.M. has a graduate student fellowship from the UniversidadPolitécnica de Valencia.

We thank Mariano Ferris for physicochemical characterizationof drinking water and Richard Connon for correcting the Englishin the manuscript.

FOOTNOTES

* Corresponding author. Mailing address: Instituto de Hidrología y Medio Natural, Universidad Politécnica, Camino de Vera 14, 46022 Valencia, Spain. Phone: 96-3877090. Fax: 96-3877090. E-mail: jalonso{at}ihdr.upv.es.

REFERENCES

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