INFECrION AND IMMUNITY, Sept. 1975, p. 630-637Copyright 0 1975 American Society for Microbiology

Vol. 12, No. 3Printed in U.S.A.

Scanning Electron Microscopic Study of Virulent and AvirulentColonies of Neisseria gonorrhoeael

THEODOR ELMROS, PER HORSTEDT, AND BENGT WINBLADDepartments of Bacteriology and Pathology, University of Umed, S-901 87 Umea, Sweden

Received for publication 17 March 1975

A preparation procedure for scanning electron microscopy was developed bywhich gonococcal colonies can be studied directly on the agar surface. Afterglutaraldehyde fixation directly in petri dishes, small agar pieces were cut outand dehydrated stepwise in increasing concentrations of ethanol. The blocks werethereafter transferred to a critical-point drying apparatus, via steps of increasedgradients up to 100% amyl acetate. By this method five different gonococcalcolony types could be distinguished analogous to light microscopic observationsmade by others. At higher magnifications an abundance of intercellular strandswas found between the cells in virulent type 1 and 2 colonies, but not in theavirulent types 3 through 5. These strands seemed to anchor the cells to eachother and to the agar surface. The presence of such structures probably explainsthe highly convex surface of virulent colonies and explains why colonies ofavirulent strains exhibit a radial extension and a flat upper surface. The natureof these filamentous intercellular strands is discussed.

Morphological properties of bacterial coloniesare of both practical and theoretical interest inmany areas of microbiology. Despite this, rela-tively little is known about factors determiningthe shape of colonies.

In studies of Neisseria gonorrhoeae, lightmicroscopy has been extensively used both forroutine and scientific purposes. Five differentgonococcal colony types have been distin-guished by this method. Types 1 and 2 havebeen isolated from patients, whereas types 3, 4,and 5 can be isolated during subcultivation inthe laboratory (7, 11, 19). Colony types 1 and 2can, even after 700 passages, produce manifestinfections in man (10). Moreover, gonococcivirulent for man are also pathogenic for chim-panzee and chicken embryos (5, 13). The othercolony types appear to contain avirulent cells.Transmission electron microscopy has re-

vealed a gonococcal cell envelope similar to thatof other gram-negative bacteria (8, 21). Recentinvestigations indicate that there also is achemical similarity (9). By transmission elec-tron microscopy, cells belonging to colony types1 and 2 have been shown to possess pili struc-tures often occurring as large aggregates. It hasbeen suggested that virulence is related to thepresence of such appendages (8, 21).Scanning electron microscopy (SEM) has

been used relatively little in investigations on' Address reprint requests to: Dr. Theodor Elmros, Depart-

ment of Clinical Bacteriology, University of Umea, S-901 87UmeA, Sweden.

bacterial colonies. This method has, in compar-ison with light microscopy, up to 200 timeslarger depth of focus. It seemed likely to us thatSEM could thus add an extra dimension to themorphological knowledge of the gonorrhoeaeand especially the colony types.The aim of this investigation was to develop a

preparation method for SEM by which the fivecolony types could be analyzed in situ withminimal distortion of morphology.

MATERIALS AND METHODS

Microorganisms. Colony types 1 through 4 fromone strain of N. gonorrhoeae (82409/55) were obtainedfrom A. Reyn, Copenhagen. Colony type 5 was derivedfrom a type 3 colony. The strains were stored at - 60 Cin GC Medium Base without agar but with 20%glycerol.

Cultivation methods. The gonococci were cul-tivated on GC Medium Base plus supplement B(Difco) (11). After drying at 37 C for 30 min, the plateswere inoculated and incubated at 36 C for 3, 22, or 30h in an incubator with 6% CO2 and in 80% humidity.When studying microcolonies, 1 million gonococcalcells were spread on the agar surface with a glass rod.Incubation was performed for 3 h as above. Afterpreparation (see below), the microcolonies weresearched for in the scanning electron microscope.Macrocolonies were obtained by restreaking individ-ual colonies. For colony typing, the gonococci wereexamined after 22 h of incubation in a plate micro-scope with oblique transmitted light.

Materials. The following enzymes were obtainedfrom Sigma Chemical Co., St. Louis, Mo.: trypsin(from bovine pancreas: activity, 12,500 N-benzoyl-L-

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SEM AND N. GONORRHOEAE 631

arginine ethyl ester units/mg). Pronase (from Strep-tomyces griseus; activity; 0.7 to 1.0 units/mg), deoxy-ribonuclease (DNase) I (from beef pancreas; activity;780 Kunitz units/mg), hyaluronidase (from bovinetestes; activity, 360 national formulary units/mg),,-glucoronidase (from Escherichia coli; activity, 95units/mg), and a-amylase (from Bacillus subtilis;activity, 99 units/mg).Enzyme test procedure. Virulent gonococci (type

2) were cultivated for 22 h as described above. Thefollowing enzymes were used at a concentration of 200isg/ml:trypsin, Pronase, DNase, a-amylase, ,B-glucuronidase, and hyaluronidase. A 10-ml amount ofthe enzyme solution was gently poured over the agarsurface. All incubations were performed in tris (hy-droxymethyl)aminomethane-hydrochloride, 0.05 M,for 20 min at room temperature. pH 7.2 was used forall incubations except trypsin (pH 8.2) and Pronase(pH 7.4). DNase was incubated together with 0.01 MMgCl2. Before fixation (see below) the solution waspoured away.

Fixation. Glutaraldehyde, 3% in phosphate buffer,pH 7.2 (18), was very gently poured over the agarsurface. Fixation was carried out at 4 C for 18 h.

Cutting procedure. Areas with individual intactcolonies were selected after examination in a platemicroscope. Agar pieces, about 5 by 5 mm in size and2 mm thick, were cut out.

Drying. Dehydration of the agar blocks was per-formed by the critical-point drying procedure (2).Agar blocks were placed in baskets and dehydratedthrough an increasing gradient of ethanol, followed bytransfer through solutions of 75% ethanol-25% amylacetate, 50% ethanol-50% amyl acetate, 25%ethanol-75% amyl acetate, and 100% amyl acetate(10 min in each solution) to a Polaron E 3000critical-point drying apparatus. The agar blocks werefinally dried in liquid carbon dioxide (17).

Metal evaporation. The specimens were coatedwith gold in an Edwards vacuum-coating unit M4 (6).The evaporation of gold on the surfaces on thespecimens was carried out in a vacuum of 10-5 torr,under continuous rotation and tilting of the speci-mens.

Microscopy. The specimens were studied in aCambridge Stereoscan S 4 scanning electron micro-scope operated at 20- and 30-kV accelerating voltage,beam current 190 MA, and final aperture size 150 um.The results were recorded on Agfapan Professional 100film with a Linhof Super Rollex camera.

RESULTS

Preparation procedure. For the optimumconservation of N. gonorrhoeae colonies, thefollowing points should be stressed. Fixationshould be carried out directly in the petri dish toavoid loss and deformation of colonies. Beforecutting the agar pieces, the fixative should begently poured away. The time from removal ofthe glutaraldehyde solution until the agarpieces are put into ethanol should not exceed 5min. Agar slices thicker than 2 mm should beavoided since thicker pieces gave heavy shrink-age of the agar, thereby changing the colony

morphology. Dehydration in ethanol andethanol-amyl acetate should be carried outstepwise in room temperature by incubation forat least 10 min in each concentration beforecritical-point drying. If this procedure was notfollowed, cracking and deformation of colonieswas observed. Dehydration followed by air-dry-ing or freeze-drying resulted in severe shrinkageand cracks in the agar and in the colonies.Colony morphology after 22 h of incubation

as revealed by low-magnification SEM. Type1 colonies were smaller and higher than types3-5. The edge was markedly rounded. Thecolony exhibited a high convex upper surface(Fig. 1A and 2A).Type 2 colonies were of about the same size

and height as those of type 1. However, the edgewas more vertical and the colonies showed atriangular upper surface (Fig. 1B and 2B). Itwas also noted that colony types 1 and 2 had amore compact appearance than types 3-5.Type 3 colonies appeared to be less adherent

to the agar surface than types 1 and 2. Theywere larger than both types 1 and 2, and showeda sharp edge and a flat convex upper surface(Fig. 1C and 2C).Type 4 colonies could not be distinguished

from type 3 colonies by SEM. However, duringthe preparation procedure the type 4 colonieswere more adherent to the agar surface thanwere the type 3 colonies.Type 5 colonies were similar to type 3 colonies

except for the irregular margin, somewhat flat-ter upper surface, and slightly steeper edge (Fig.1E and 2E).Colony morphology after 30 h of incubation

as revealed by SEM. During prolonged incuba-tion, most colonies of type 1 or 2 will segregateavirulent offsprings, resulting in colonies con-taining cells of other colony types. Such coloniesgrown for 30 h had at one edge segregated outtype 3 cells (Fig. 3). The great difference indensity was evident, the type 3 surface cellsappearing considerably more granular than thesmooth surface of type 2 (Fig. 3A and C). Thesharp edge of the type 3 part of the colony was incontrast to the vertical margin of the rest of thecolony (Fig. 3B). At higher magnifications,considerable differences between the two celltypes could be illustrated. In the virulent part ofthe colony intercellular filamentous strandswere abundant, whereas such strands were rarein the type 3 part of the colony (Fig. 3D and E).Colony morphology after 3 h of incubation

as revealed by SEM. The intercellular strandswere common in all virulent colonies observed.In contrast, avirulent derivatives showed noneor only few of these structures. To see whetherthese intercellular bridges also characterizedmicrocolonies of virulent specimens, the same

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INFECT. IMMUN.632 ELMROS, HORSTEDT, AND WINBLAD

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FIG. 1. Low-magnification scanning electron micrograph of the five gonococcal types on the agar surfaceincubated for 22 h, viewed from above. 0° angle and x240 magnification. (A-E) Colony types 1 through 5,respectively.

preparation procedure was applied to gonococciincubated for only 3 h. In Fig. 4 an avirulenttype 4 and a virulent type 2 microcolony isdepicted. The former appeared already at thisearly stage to extend radially. In contrast, thevirulent cells exhibited a greater tendency togrow in a vertical direction. Individual cellswere all held together by strands appearingidentical to those observed in virulent mac-rocolonies. These strands could also be seenextending from the cells to the agar surface.Colony morphology after enzyme treatment

as revealed by SEM. In the experimental

conditions used, no effect of the enzymes couldbe noted on the strands or on the cells.

DISCUSSIONBy using scanning electron microscopy, sev-

eral workers have attempted to observe bacter-ial colonies located on the agar surface. The re-sult has often been discouraging, mainly becauseboth colonies and agar are heavily distortedduring the preparation procedure (1, 23). Toavoid such problems some investigators haveinoculated bacteria on membrane filters (Mil-lipore Corp.) or dialysis membrane strips that

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SEM AND N. GONORRHOEAE 633

FIG. 2. Medium-magnification scanning electron micrograph of the five gonococcal types on the agar surfaceincubated for 22 h, viewed from the side. 850 angle and x 1,200 magnification. (A-E) Colony types 1 through 5,respectively.

were in direct contact with the agar surface (1,3). However, in our hands N. gonorrhoeae type 4did not form visible colonies when incubated for22 h on Millipore or Nucleopore filters. Eventhough visible colonies could be observed ondialysis strips, they were only of about half thesize of colonies grown directly on the agarsurface. Moreover, type 2 colonies during theseconditions showed an irregular instead ofsmooth colony margin. Kraus and Glassman(12) tried to shake the gonococcal colony awayfrom the agar surface and float the colony on aglass slide. Even with this method there wereproblems in preparing colonies free from ar-tifacts.Dehydration of the agar has been one critical

problem. In the present investigation, increasedagar concentrations as suggested by Bibel andLawson (4) were tried. However, this had amarked influenrce on the gonococcal colonymorphology. The different colony types couldconsequently not be distinguished by light mi-croscopy. Moreover, higher agar concentrationscaused a prolonged and insufficient penetrationof alcohol and amyl acetate during critical-point drying, resulting in considerable distor-tions of the agar surface and of the colonymorphology. By using critical-point drying, lessthan 10% shrinkage of the agar blocks wasregistered. It is likely that colonies are moresusceptible to surface tension due to theirmacrostructure as compared with cells grown in

FIG. 3. Low-, medium-, and high-magnification scanning electron micrographs of a type 2 colony incubatedfor 30 h. The type 2 colony has segregated out cells with type 3 colony morphology. Notice the apparently moresmooth and compact surface and the more vertical edge of the large type 2 colony (A and B, x400; C, x 1,200).At high magnification many intercellular strands are seen in the type 2 part of the colony but not in the type 3part (D, x6,000). Spherical bodies are seen on the individual cells and on the intercellular strands (E, x24,000).

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636 ELMROS, HORSTEDT, AND WINBLAD

_ ~~~~~~~~~~~~~~~~~~~~~~4FIG. 4. High-magnification scanning electron micrograph of a type 4 microcolony (A, x24,000) and a type 2

microcolony (B, x24,000) incubated for 3 h. Cell aggregation with a vertical growth tendency can be seen in thetype 2 colony. A striking difference between the two colony types is the presence of strands connecting theindividual virulent type 2 cells to each other and to the agar surface.

solution (4). Therefore, critical-point dryingwhose principle involves elimination of all sur-face tensions of ambient liquids should be theproper method for preparing gonococcal colo-nies for SEM.Several workers have described differences in

colony morphology between virulent and aviru-lent strains of N. gonorrhoeae (7, 11, 19). It istherefore possible that the factor(s) determiningthe virulent colony morphology also is causingthe virulence of the cell itself. Transmissionelectron microscopy has revealed pili in virulentcolonies, which were lacking in avirulent deriva-tives. The possibility that these structures maybe correlated with virulence in gonococci hasbeen suggested (8, 21).SEM at high magnification showed an abun-

dance of intercellular strands in all type 1 and 2colonies observed. In rare cases analogous struc-tures were also found in type 4 colonies. Theirthickness varied somewhat, 50 to 100 nm, andtheir length was between 500 and 1,000 nm. Nodifferences with regard to number and dimen-sions were found between type 1 and type 2colonies. The exact nature of these intercellularstrands is not known. They are thicker than asingle pilus. However, an aggregation tendencyfor gonococcal pili has been reported (8, 21). It istherefore possible that the structures observedhere represent bundles of pili. On the otherhand, elongated protrusions of cell wall mate-rial have been described by using freeze-frac-ture and freeze-etch techniques (20). Pili arenormally proteinous in nature and would there-fore be sensitive to trypsin digestion. The find-

ing that neither trypsin, Pronase, DNase, #-glucuronidase, a-amylase, nor hyaluronidaseaffected the strands in a morphological sensesuggests a more complex biochemical structure.A model for colony formation in bacteria in

general has been presented (15). This modeldistinguishes between an initial sphericalgrowth phase when the supply of nutrients issufficient for all cells, and a radial growth phasewhen sufficient nutrition is available only forthe peripheral cells of the colony. Virulent typesof gonococci have almost spherical colonies(Fig. 2A, 2B, and 3B), in contrast to avirulentderivatives (Fig. 2C, to E and 3B), whichapparently have a radial growth tendency.The presence of intercellular strands is proba-

bly the explanation for the virulent colonymorphology. They act as a "glue" between cellsand between cells and agar surface, explainingin part the marked tendency for virulent colo-nies to stick to the agar surface during prepara-tion for SEM. The same tendency to stick to theagar surface in the type 2 colony has been noted(12, 16). This "glue" also explains the markedlyrounded margins and highly convex upper sur-face observed in virulent colonies. Absence ofintercellular strands results in a radial exten-sion, with a sharp margin and low convex uppersurface. The surface of virulent colonies ap-peared considerably smoother than that of avir-ulent derivatives because of the strands. How-ever, within the colony avirulent cells were moredensely packed.The difference in growth patterns of virulent

and avirulent cells were observed already after a

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SEM AND N. GONORRHOEAE 637

few cellular divisions. In microcolonies virulentcells were already anchored to the agar surfaceand to each other, whereas no such connectionswere found in the radially extending avirulentmicrocolony.

It is likely that the SEM observations donehere may be extrapolated to in vivo situations,thus suggesting that gonococcal virulence isrelated to the ability of a cell to attach to theepithelial surface (22).

Spherical bodies of varying size located bothon cells and on strands were seen in all colonytypes (Fig. 3E) as also noted by Kraus andGlassman (12). We could not demonstrate anyclear difference in the number of sphericalbodies present in various colony types after 22 hof incubation. However, after 3 h of incubationthe type 2 microcolonies displayed a largernumber of spherical bodies in comparison withtype 4 microcolonies. The few large ones werelocated close to a cell and might be the result ofan anomalous cell division. The smaller spheri-cal bodies were situated on cells and on strands.These can be parts of the outer membrane of thegonococci (Fig. 3E). Similar structures havebeen observed surrounding gonococci in ure-thral secretions (14). Zollinger et al. (24) haveshown that N. meningitidis, when grown inliquid medium, releases parts of the outermembrane. It is possible that N. gonorrhoeaereleases part of its "endotoxin" in this wayduring normal growth. In contrast, in Npharyn-gitis we found very few spherical bodies asrevealed by SEM.The use of SEM might be a promising ap-

proach in the study of bacterial cell and colonymorphology and its relation to virulence.

ACKNOWLEDGMENTSWe wish to thank S. E. Holm, S. Normark, and L. G.

Burman for encouraging criticism and inspiring suggestionsand Gunnar Sandstrom for skillful technical assistance.

This work was supported by grants from the SwedishNational Association against Heart and Chest Diseases,The Swedish Medical Science Research Council (no.04769-01. grants to Staffan Normark), and the EdvardWelander Foundation.

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3. Bibel, D. J., and J. W. Lawson. 1971. A simple method forscanning electron microscopy of L-form colonies grownon agar. Can. J. Microbiol. 17:822-823.

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9. Johnston, K. H., and E. C. Gotschlich. 1974. Isolationand characterization of the outer membrane of Neis-seria gonorrhoeae. J. Bacteriol. 119:250-257.

10. Kellogg, D. S., Jr., I. R. Cohen, L. C. Norins, A. L.Schroeter, and G. Reising. 1968. Neisseria gonorrhoeae.II. Colonial variation and pathogenicity during 35months in vitro. J. Bacteriol. 96:596-605.

11. Kellogg, D. S., Jr., W. L. Peacock, Jr., W. E. Deacon, L.Brown, and C. I. Pirkle. 1963. Neisseria gonorrhoeae. I.Virulence genetically linked to clonal variation. J.Bacteriol. 85:1274-1279.

12. Kraus, S. J., and L. H. Glassman. 1974. Scanningelectron microscope study of Neisseria gonorrhoeae.Appl. Microbiol. 27:584-592.

13. Lucas, C. T., F. Chandler, Jr., E. Martin, Jr., and J. D.Schmale. 1971. Transfer of gonococcal urethritis fromman to chimpanzee. J. Am. Med. Assoc. 216:1612-1614.

14. Ovcinnikov, N. M., and V. V. Delektorskij. 1971. Electronmicroscope studies of gonococci in urethral secretionsof patients with gonorrhoea. Br. J. Vener. Dis.47:419-439.

15. Pirt, S. J. 1967. A kinetic study of the mode of growth ofsurface colonies of bacteria and fungi. J. Gen. Micro-biol. 47:181-197.

16. Reyn, A., E. Jephcott, and H. Ravn. 1971. Brief report.Neisseria gonorrhoeae. Colony variation II. Acta Pa-thol. Microbiol. Scand. Sect. B 79:435-436.

17. Rohrschneider, I., I. Schinko, and R. Wetzstein. 1973.Vergleichende rasterelektronenmikroskopische Unter-suchungen an der Froschlunge nach Lufttrocknung undnach Anwendung der "critical-point"-Methode. Mik-roskopie Band 29:116-121.

18. Sabatini, D. D., K. Bensch, and R. J. Barrnett. 1963.Cytochemistry and electron microscopy. The preserva-tion of cellular ultrastructure and enzymatic activityby aldehyde fixation. J. Cell. Biol. 17:19-58.

19. Sparling, P. F., and A. R. Yobs. 1967. Colonial morphol-ogy of Neisseria gonorrhoeae isolated from males andfemales. J. Bacteriol. 93:513.

20. Swanson, J. 1972. Studies on gonococcus infection. II.Freeze-fracture, freeze-etch studies on gonococci. J.Exp. Med. 136:1258-1271.

21. Swanson, J., S. J. Kraus, and E. C. Gotschlich. 1971.Studies on gonococcus infection. I. Pili and zones ofadhesion: their relation to gonococcal growth patterns.J. Exp. Med. 134:886-906.

22. Ward, M. E., and P. J. Watt. 1972. Adherence ofNeisseria gonorrhoeae to urethral mucosal cells. Anelectron-microscopic study of human gonorrhea. J.Infect. Dis. 126:601-605.

23. Whittaker, D. K., and D. B. Drucker. 1970. Scanningelectron microscopy of intact colonies of microorga-nisms. J. Bacteriol. 104:902-909.

24. Zollinger, W. D., D. L. Kasper, B. J. Veltri, and M. S.Artenstein. 1972. Isolation and characterization of anative cell wall complex from Neisseria meningitidis.Infect. Immun. 6:835-851.

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