JOURNAL OF BACTERIOLOGY, OCt. 1985, p. 338-3430021-9193/85/100338-06$02.00/0Copyright © 1985, American Society for Microbiology

Vol. 164, No. 1

Morphological Forms and Viability of Campylobacter SpeciesStudied by Electron Microscopy

LAI-KING NG,' RICHARD SHERBURNE,2 DIANE E. TAYLOR,'2* AND MICHAEL E. STILES1'3Departments of Microbiology,1 Medical Microbiology,2 and Food Science,3 University ofAlberta, Edmonton, Alberta,

Canada T6G 2H7

Received 18 March 1985/Accepted 12 June 1985

Electron microscopic studies of Campylobacter revealed that different morphological forms predominate atdifferent parts of a colony. At the periphery, cells were almost all spirals, while in the center of the colony cellswere mainly coccus shaped. Unusual ring-shaped cells, "donuts", were observed in the raised, peripheralregion of the colony. Donut or ring forms have not previously been reported for Campylobacter organisms. Ourdata indicate that young or actively growing cells are mainly spiral shaped. Older ceUs undergo a degenerativechange to coccoid forms. The donut shape appears to be an intermediate stage between spirals and cocci.Comparisons of plate counts of actively growing and inactive cells confirmed that coccoid cells are probablynonviable.

Campylobacter spp. are gram-negative, non-spore-forming, microaerophilic organisms. Campylobacter jejuni,C. coli, and "C. laridis" (5) have all been implicated inoutbreaks of gastroenteritis. Direct examination of fecalspecimens by dark-field or phase contrast microscopy issometimes used as part of the routine diagnostic procedurefor Campylobacter spp. (4). The spiral shape and dartingmotility may be used as characteristics to differentiate themfrom oth'er enteric organisms (5). Several morphologicalforms of Cainpylobacter organisms have been reported,including spirals, S shapes, gull shapes, commas, dimpledshapes, and coccoid shapes (5, 10, 15). The spiral formspredominate in young cultures, while coccoid forms arefound mainly in old cultures (2, 11, 12, 15, 16). The coccoidforms of C. fetus and C. jejuni are believed to be degenera-tive (2, 15) and to resemble similar forms in some of thechemoheterotrophic spirilla (15).

In this study, single colonies of C. coli, C. jejuni, and "C.laridis" were studied by scanning electron microscopy(SEM) to demonstrate the transition from spiral to coccoidforms by examining various areas of a colony. Thin sectionsof C. jejuni and "C. laridis" were studied by transmissionelectron microscopy (TEM). The viability of the coccoidforms of C. jejuni and C. coli was determined by compara-tive plating of the cultures.

MATERIALS AND METHODSElectron microscopy. C. jejuni, C. coli, and "C. laridis"

were grown on Columbia blood agar containing 10%defibrinated sheep blood. Plates were incubated at 37°C in anincubator containing 7% CO2. The cells were harvested after48 to 72 h of' incubation.For SEM, agar blocks containing a single colony were cut

from the plates, placed in stall petri dishes containing 1%(wt/vol) osmium tetroxide in Veronal buffer (pH 6.1), andheld overnight at room temperature to fix the cells in thecolony. After fixation, colonies were floated off the agarblocks. The colonies were then dehydrated in a gradedalcohol series (25, 50, 75, 90, and 100% ethanol). Aftercritical-point drying, the colonies were mounted onto SEM

* Corresponding author.

specimen stubs and sputter coated with gold. Samples wereexamined with a Cambridge 250 scanning electron micro-scope.For TEM, colonies of C. jejuni and "C. laridis" were fixed

and dehydrated as described for SEM. After dehydration inethanol, the specimens were placed in two changes ofpropylene oxide for 15 min each, followed by 30 mim in 12%Araldite in propylene'oxide and 1 h in 25% Araldite inpropylene oxide. The specimens were then transferred to50% Araldite in propylene oxide, left in an uncapped vial atroom temperature fQr 24 h, and then transferred to pUreAraldite and left for 1 h. The Araldite was replaced, and thespecimens were cured for 24 h at 60°C. Ultrathin sections (60nm thick) were prepared with an ultramicrotome (Ultracut;Reichert-Jung). The sections were placed on 400-mesh cop-per grids and stained with 5% uranyl acetate in methanol for10 min and in lead citrate for 4 min.Suspensions from C. jejuni colonies were also studied by

TEM by negative staining with 1% sodium phosphotungstateat pH 7.0. These preparations were made on Formvar andcarbon-coated 200-mesh grids. Both the thin sections and thenegatively stained specimens were examined in a PhilipsEM300 electron microscope.

Viability study. C. coli NCTC 11353 and C. jejuni NCTC11168 were grown in Bacto Brucella Broth (Difco Laborato-ries) at 37TC under a 7% CO2 atmosphere for 24 h. Appro-priate dilutions of 24-h cultures were plated onto Mueller-Hinton (MH) agar (Oxoid Ltd.), so that each plate containedabout 300 colonies after incubation. After 5, 7, and 10 daysof incubation, one of the plates was removed to prepare acell suspension by transferring sufficient colonies to 0.85%saline to give a turbidity equivalent to the McFarland num-ber 2 standard. Appropriate dilutions of the saline suspen-sions were plated in triplicate onto MH agar to measure thenumber of CFU per milliliter of the suspensions. Simulta-neously, the saline suspensions were diluted 1:1 or 1:4 with0.5% phenol. After treatment with phenol for 5 min, the cellswere nonmotile, and the microscopic counts of the differentmorphological forms were determined with a Petroff-Hausser bacteria counter under phase contrast. The exper-iment was duplicated for each culture, and the microscopiccounts were compared with the number of CFU per milliliteron MH agar plates.

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FORMS AND VIABILITY OF CAMPYLOBACTER SPP. 339

FIG. 1. Scanning electron micrographs of C. jejuni cells at different magnifications: (a) whole colony, bar represents 200 ,um; (b)enlargement of a portion of the edge of the colony (indicated in panel a), bar represents 10 ,um; (c) magnification of cells at periphery of colony,bar represents 1 ,um; (d) magnification of cells at ridge of colony. shown in panel b, bar represents 1 p.m; (e) and (f) enlargements ofdonut-shaped cells, bar represents 400 nm; (g) magnification of cells at center of colony showing coccus-shaped cells and amorphous material,bar represents 2 pum.

RESULTS

SEM. Colonies of Campylobacter spp. grown for 72 h onColumbia blood agar had raised centers and narrow, flatedges. The average diameter of the colonies after 72 h ofincubation at 37°C was 2 mm. Figure la shows an entirecolony of C. jejuni that had been removed from the agarsurface and mounted on an SEM specimen stub. Figure lb

shows a magnified area of the edge of the colony in Fig. la.In the flat region at the edge of the colony, where activelygrowing cells would be expected, the cells were mainly spiralshaped (Fig. ic). In contrast, cells farther from the periph-ery, at the raised portion (ridge) of the colony, were mainlyring or donut shaped (Fig. ld). The ring- or donut-shapedcells are shown at higher magnification in Fig. le and f,illustrating the hollow center of this form. Cells toward the

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FIG. 2. Scanning electron micrographs of Campylobacter cells, showing various morphological shapes observed when the surface layersof cells were removed by an agar overlay technique: (a) C. coli, toward the center of the colony; and (b) C. jejuni, at the periphery of thecolony. Bar represents 1 ,um.

center of the colony (Fig. lg) showed a greater predomi-nance of coccoid forms, with an amorphous material on thesurface of the colony. Colonies of C. coli and "C. laridis"were similar in appearance to C. jejuni, except that donut-shaped cells were not observed.The cells shown in Fig. 2a and b were obtained by pouring

a layer of purified Agar Noble (Difco) on top of a colonysimilar to that shown in Fig. la and then removing the agarlayer so that the upper mass of cells was removed. Figure 2a,which shows a mixture of spiral and coccoid cells from a C.coli colony, represents cells observed at the center of the

colony. In fact, the cells are pleomorphic, including S-shaped, gull-shaped, and ribbon-shaped spirals, as well asdimpled and round coccus forms. In contrast, Fig. 2b, whichshows primarily spiral-shaped cells from a colony of C.jejuni, represents cells at the periphery of the colony. In thiselectron micrograph, the end view of a spiral cell may beseen (see arrow in Fig. 2b). The spirals do not have a ringshape when viewed from one end, owing to the smallamplitude of the helix.TEM. Negative staining of cell suspensions prepared from

C. jejuni cells grown on Columbia blood agar showed both

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FORMS AND VIABILITY OF CAMPYLOBACTER SPP. 341

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FIG. 3. Transmission electron micrograph of negative-stained cellscells. Bar represents 0.5 urm.

spiral and coccoid forms (Fig. 3). Although most coccus-shaped cells appeared to have lost their flagella, some stillhad flagella attached. Thin sections of colonies of C. jejuni(Fig. 4a) and "C. laridis" (Fig. 4b through e), viewed byTEM, showed a variety of cell forms. The results for both C.jejuni and "C. laridis" were similar. In Fig. 4a, the colonythat had been floated off the agar was sectioned and showedthat the cells adjacent to the agar surface were most proba-bly spirals and their cytoplasm was close to the cell wall. Thecells farther from the agar surface were more likely to becoccus shaped, and the cytoplasm of these cells was gener-ally separated from the cell wall. Sometimes more than onecytoplasmic mass was observed within a cell envelope (Fig.4b). The cytoplasm of these cells was not as dense as that ofthe spirals, and their cell wall seemed to be stretched,indicating that these cells might be in a degenerative state. InFig. 4b and c, thick bands could be seen, as indicated by thearrows. Serial sections showed that the band was close tothe end of the cells. A similar band was observed in allCampylobacter spp. studied. Some cell types had a largediameter, and the cytoplasm was separated from the cell wall(Fig. 4c). Most of these cells were circular, indicating thatthey might be coccoid. Some were club-shaped (Fig. 4d),indicating that they might be intermediate forms. Serialsections illustrated that the cytoplasm in the coccoid cells

jejuni, showing flagellation of both spiral- and coccus-shaped

was of variable shape, even though their outline in thinsections was generally circular (Fig. 4e).

Viability study. More than 99% of the cells of C. coli andC. jejuni grown in Bacto Brucella Broth and incubated at37°C in a modified atmosphere (7% C02) for 24 h were spiralshaped. No coccus-shaped cells were observed under phasecontrast. When the 24-h broth culture was diluted and platedonto MH agar, almost 100% of the cells formed colonies (seeTable 1). Similarly, when C. coli and C. jejuni were grown onMH agar for 24 h, the predominating cell shape was spiral.After 5 days of incubation, the predominant cell shape hadshifted to the coccoid form. When appropriate dilutions of asuspension prepared from these colonies, which contained106 to 107 spiral- and 109 coccus-shaped cells per ml, wereplated, only 106 cells formed colonies on MH agar. Both theplate count and the spiral cell count were 1,000-fold less thanthe total microscopic count.

After 7 and 10 days of incubation, colonies were sampledto include cells from the periphery and the center of thecolony. Spiral cells were still detected after 10 days ofincubation. The microscopic counts for samples taken after7 and 10 days ranged from 1.0 x 109 to 2.2 x 109 coccoidcells and 1.9 x 106 to 5.6 x 106 spiral-shaped cells. The platecount ranged from 6.7 x 105 to 5.1 x 106 CFU/ml.The log1o counts were compared statistically by paired t

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c d e

FIG. 4. Transmission electron micrographs of thin sections of (a) C. jejuni and (b) to (e) "C. laridis", showing different morphologicalforms of the cells. The lower surface of cells in panel a was adjacent to the agar surface. Bar represents 0.5 ,um.

test analysis. The logl0 mean counts of cell suspensions,containing spiral and coccoid forms of C. coli or C jejunigrown on MH agar are shown in Table 1. The total micro-scopic counts for both C. coli and C. jejuni were significantlyhigher than the plate counts. The number of spiral-shaped

TABLE 1. Microscopic counts of coccoid and spiral forms of C.coli and C. jejuni compared with plate counts on MH agar

Log1o mean micro- Log1oStraiadcNo. of scopic count: mean plate

replicates . . countscocci/mi spirals/mI (CFU/ml)

C. coli NCTC 1135324-h Bacto Brucella 1 NDb 8.50 8.41

Broth cultureGrowth on MH 6a 9.14 6.59 6.46

C. jejuni NCTC 1116824-h Bacto Brucella 1 ND 8.60 8.49

Broth cultureGrowth on MH 6a 9.28 6.90 6.10a The number of replicates is based on duplicate trials with three samplings

(5, 7, and 10 days) from MH agar plates.bND, None detected.

cells of C. coli enumerated microscopically was the same asthe plate count. However, the number of spiral-shaped cellsof C. jejuni enumerated microscopically was slightly higherthan the plate count (P = 0.0025), indicating that not allspiral cells formed colonies. However, the difference is lessthan one log cycle, which may not be of practical impor-tance.

DISCUSSION

The cells in a single colony are heterogeneous in age andphysiological state. It is assumed that at the periphery of thecolony the cells are actively growing, while at the center andon the upper surface of the colony nutrients are less avail-able and cells are more likely to be old and inactive. SEM ofa single colony of C. jejuni showed that cells with charac-teristic morphological forms predominate at different loca-tions within the colony. Spiral forms predominate at theedge, while coccoid forms predominate in the center, sug-gesting that these forms represent actively growing andinactive cells, respectively. The donut forms were observedin a small region between the area where the spiral andcoccoid forms were observed. This suggests the possibilitythat they are an intermediate form between spiral andcoccus-shaped cells. The mechanism of donut formation isnot clear; however, other microorganisms such as Micro

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FORMS AND VIABILITY OF CAMPYLOBACTER SPP. 343

cyclus and Spirosoma spp. are known to form ringlikestructures (13).Coccoid forms have been reported for other spiral-shaped

bacteria, including Spirillum, Vibrio, Oceanospirillum, andDesulfovibrio spp. (7). The coccoid forms are believed to beresting stages in Spirillum spp. (8). Reversion of coccoid tospiral forms in C. fetus has been reported previously (11).However, this reversion was observed when broth or agarcultures which had less than three spirals per 10 fields undermicroscopic examination (considered to be 100% coccoidforms) were transferred to fresh broth or agar. After 24 h to48 h of incubation, only spiral forms were detected. Incontrast, Baker and Park (1), using a slide cultivation tech-nique, demonstrated that coccoid forms in Vibrio spp. werenonviable and that survival of the culture depended on thefew rod forms that were present.

In our study, enumeration of viable cells by plating on MHagar indicated that coccoid cells were probably unable toform colonies. This was supported by our observations fromthin sections of C. jejuni and "C. laridis" cells. The thinsections of "C. laridis" (Fig. 4) showed a high incidence ofbleb formation. This has been reported by others (2, 12) toindicate loss of cell wall integrity, which represents a degen-erative change. The anatomical features that we observed bySEM and TEM were similar to those reported for C. fetus,including the presence of thick bands at the flagellated poles(6, 14) and the absence of a flagellar sheath (3, 9, 14). Thissuggests that our strains were morphologically similar toother Campylobacter strains that have been studied.

It is well documented that all Campylobacter spp. exist indifferent morphological forms. Our studies showed thatspiral forms are probably actively growing cells, whereascoccoid forms are old, inactive, and possibly degenerativecells. The mechanism of coccoid cell formation remainsunknown, but our observation of ring- or donut-shaped cellssuggests a progressive change associated with degenerationof the cell wall. The lack of viability of the coccoid forms ofC. coli and C. jejuni was indicated in our study by theinability of these cells to form colonies on MH agar. Propa-gation of cultures therefore requires the presence of sprialforms. Without repair of the cell wall, solid media areunlikely to support the growth of coccoid cells. Hence, platecount methods cannot be used for quantitative study of thecoccoid cells.

ACKNOWLEDGMENTSWe thank G. D. Braybrook, Department of Entomology,

University of Alberta, Edmponton, Alberta, Canada, for technicalassistance with the SEM, San Vinh for printing the electronmicrographs, and R. Whitehouse for his interest in the project.

L.-K.N. is supported by an Alberta Heritage Foundation forMedical Research (AHFMR) Studentship, and D.E.T. is supportedby an AHFMR Scholarship. The Reichert-Jung Ultracut microtomewas purchased with funds from AHFMR.

LITERATURE CITED1. Baker, D. A., and R. W. A. Park. 1975. Changes in morphology

and cell wall structure that occur during growth of Vibrio sp.NCTC4716 in batch culture. J. Gen. Microbiol. 86:12-28.

2. Buck, G. E., K. A. Parshall, and C. P. Davis. 1983. Electronmicroscopy of the coccoid form of Campylobacter jejuni. J.Clin. Microbiol. 18:420-421.

3. Ferris, F. G., T. J. Beveridge, M. L. Marceau-Day, and A. D.Larson. 1984. Structure and cell envelope associations offlagellar basal complexes of Vibrio cholerae and Campylobacterfetus. Can. J. Microbiol. 30:322-333.

4. Goossens, H., M. DeBoeck, H. Van Landuyt, and J. P. Butzler.1984. Isolation of Campylobacter jejuni from human feces, p.39-50. In J. P. Butzler (ed.), Campylobacter infection in manand animals. CRC Press Inc., Boca Raton, Fla.

5. Karmali, M. A., and M. B. Skirrow. 1984. Taxonomiy of thegenus Campylobacter, p. 1-20. In J. P. Butzler (ed.), Campylo-bacter infection in man and animals. CRC Press Inc., BocaRaton, Fla.

6. Keeler, R. F., A. E. Ritc4ie, J. H. Bryner, and J. Elmore. 1966.The preparation and characterization of cell walls and thepreparation of flagella of Vibrio fetus. J. Gen. Microbiol. 43:439-454.

7. Krieg, N. R. 1976. Biology of the chemoheterotrophic spirilla.Bacteriol. Rev. 40:55-115.

8. Krieg, N. R., and P. B. Hylemon. 1976. The taxonomy of thechemoheterotrophic spirilla. Annu. Rev. Microbiol. 30:303-325.

9. McCoy, E. C., D. Doyle, H. Wiltberger, K. Burda, and A. J.Winter. 1975. Flagellar ultrastructure and flagella-associatedantigens of Campylobacterfetus. J. Bacteriol. 122:307-315.

10. Merrell, B. R., R. I. Walker, and J. C. Coolbaugh. 1981.Campylobacter fetus ss. jejuni, a newly recognized entericpathogen: morphology and intestinal colonization. ScanningElectron Microsc. 4:125-131.

11. Ogg, J. E. 1962. Studies on the coccoid form of ovine Vibriofetus. 1. Cultural and serologic investigations. Am. J. Vet. Res.23:354-358.

12. Pead, P. J. 1979. Electron microscopy of Campylobacterjejuni.J. Med. Microbiol. 12:383-385.

13. Raj, H. D. 1977. Microcyclus and related ring-forming bacteria.Crit. Rev. Microbiol. 5:243-269.

14. Ritchie, A. E., R. F. Keeler, and J. H. Bryner. 1966. Anatomicalfeatures of Vibrio fetus: electron microscopic survey. J. Gen.Microbiol. 43:427-438.

15. Smibert, R. M. 1978. The genus Campylobacter. Annu. Rev.Microbiol. 32:673-709.

16. Tritz, G. J., and J. E. Ogg. 1967. Physical and chemicalconditions inducing the coccoid form of ovine Vibrio fetus. Am.J. Vet. Res. 28:123-129.

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