JOURNAL OF CLINICAL MICROBIOLOGY, Nov. 1977, p. 511-517Copyright (© 1977 American Society for Microbiology

Vol. 6, No. 5Printed in U.S.A.

Simple Method for Distinguishing Gonococcal Colony TypesELLIOT JUNI* AND GLORIA A. HEYM

Department ofMicrobiology, University ofMichigan, Ann Arbor, Michigan 48109

Received for publication 3 March 1977

Gonococcal colony types can be distinguished by a new procedure that makesuse of a dissecting microscope with a concave mirror and a fluorescent lamp.Critical adjustment of the mirror angle results in illumination similar to thatobtained in the dark-field microscope. When the concave mirror is set at acertain angle, colonies of the lenticular types 1 and 2 refract the light comingthrough them in such a way that an edge of the microscope stage is focused ineach colony. By contrast, colonies of types 3 and 4, which are relatively flat, failto refract incident light. Although distinguishable from each other by differencesin color, type 3 and 4 colonies do not display the focusing effect typical for type1 and 2 colonies and appear uniformly illuminated. This new technique permitsthe rapid identification and isolation of even a single type 1 or 2 colony in a fieldof type 3 or 4 colonies, making it possible to obtain and maintain competentcolonies (type 1 or 2) for the genetic transformation assay for Neisseria gonor-rhoeae strain identification as well as for other purposes.

Neisseria gonorrhoeae has been shown toundergo a relatively rapid variation of unknownorigin that results in the appearance of severaldifferent colony types when cultures are platedon suitable transparent media and observed witha dissecting microscope under special conditionsof illumination (4, 8). Strains of N. gonorrhoeaefreshly isolated from infected individuals giverise predominantly to colony types 1 and 2,whereas cells producing only colony types 3 and4 are most frequently found in cultures thathave been maintained in the laboratory by re-peated subculture (5, 9, 13). It is possible, how-ever, to maintain colony types 1 and 2 by usingspecial media (9). This colony type variation isof considerable significance since only cells fromcolony types 1 and 2 are pathogenic; cells derivedfrom colony types 3 and 4 are unable to initiateinfection in human volunteers (7, 8). Unlike cellsfrom colony types 3 and 4, cells from colonytypes 1 and 2 are piliated (5, 10, 14) and arealso highly competent for genetic transformation(3, 12).

It is usually not possible to distinguish N.gonorrhoeae colony types by examining colonieswith the naked eye. Colony types have beendifferentiated with low-power dissecting micro-scopes with either diffuse and angled transmit-ted light (1, 2, 7, 8) or with a double system ofangled substage lighting (4). During recent stud-ies in our laboratory leading to the developmentof a genetic transformation assay for identifica-tion of N. gonorrhoeae strains (3), it was neces-sary to be able to distinguish colonies of com-

petent cells (types 1 and 2) from colonies ofnoncompetent cells (types 3 and 4). By using astereoscopic dissecting microscope, we discov-ered a simple and highly effective method forobservation and differentiation of the N. gon-orrhoeae colony types. The present communi-cation describes this new procedure and illus-trates the appearance ofthe various colony typesof N. gonorrhoeae by this method.

MATERIALS AND METHODSOrganisms and media. Most of the N. gonor-

rhoeae strains examined in the -present study werederived from clinical specimens obtained from thediagnostic laboratory of St. Joseph Mercy Hospital,Ann Arbor, Mich. A few strains were obtained fromthe American Type Culture Collection, Rockville, Md.The photographs (Fig. 2 and 4-7) show the variouscolony types of stain 488, a uracil auxotroph that isused as a tester strain in the genetic transformationassay for N. gonorrhoeae strain identification (3). Allcultures were stored in 67% glycerol at -45°C aspreviously described (6). The transparent mediumused to grow and observe the various colony types inthe present study was medium Ml (3). Other mediasuitable for colony type observation include the GCB,GCBB, and GC8-2DS media of Kellogg et al. (8), andmedium M9 (3). Cultures were incubated at 35 to36°C in a candle jar, usually for 16 to 18 h. Photo-graphs were taken through a Bausch and LombStereoZoom 7 microscope at magnifications of x20 tox50 with a Bausch and Lomb photomicrographic cam-era and Polaroid type 107 film.

Identification of colony types. The various N.gonorrhoeae colony types were determined initiallyby the procedure of Jephcott and Reyn (4).

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512 JUNI AND HEYM

RESULTS AND DISCUSSIONBasic method for observing colony types.

The method to be described was originally de-veloped with a Bausch and Lomb StereoZoom7 microscope. A small modification must bemade when other microscopes are employed. Itis important that a concave mirror be used forbest results. However, it is possible to use a flatmirror. The basic method will be described foruse with the Bausch and Lomb microscope witha concave mirror. The modification required foruse with other microscopes and a proceduremaking it possible to use a flat mirror will thenbe given.The plate to be observed is illuminated with

a fluorescent lamp adjusted so that light reachesthe substage mirror both from above and frombelow the microscope stage (Fig. 1A). Focus themicroscope on colonies on a plate and removethe plate from the stage. Do not refocus themicroscope during subsequent steps of the pro-

cedure. Rotate the concave mirror so that it is

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FIG. 1. Illumination used with the Bausch andLomb StereoZoom 7 microscope. (A) Position of flu-orescent lamp for critical illumination. The arrow

indicates the opaque microscope stage edge discussedin the text. (B) Illumination with a vertical metalrod (arrow) in the light path.

horizontal and facing up. The field now observedthrough the unrefocused microscope should bedark. While looking through the microscope,slowly rotate the mirror toward the light source.As rotation is continued, a bright field of illu-mination will appear. This is followed by a darkfield that is then followed by a second brightfield. Upon observing the second bright field,stop rotating the mirror. Carefully reverse thedirection of rotation, turning back into the darkfield, until the approximate center of this darkfield is reached. At this point, the mirror angleis adjusted for critical illumination. The darkfield results from the blockage of light by thesolid edge of the microscope stage, indicated bythe arrow in Fig. 1A. At this angle, all the lightreaching the concave surface of the mirror isreflected from it to the stage at various angles,none being directed vertically into the micro-scope. Although the empty field appears darkat critical illumination, objects that reflect orrefract the angled light will appear brightly il-luminated. Any scratches on the glass stage, forexample, will appear as bright lines.

Figure 2A shows the appearance of a mixtureof type 2 and 3 colonies of N. gonorrhoeae 488on a plate that was placed on the microscopestage after critical illumination was achieved asdescribed above. The lenticular (double-convex-shaped) type 2 colonies (7, 11) act as small lensesand focus the edge of the microscope stage (ar-row in Fig. 1A) in each colony so that it appearsas a dark horizonal bar in the center of a brightlyilluminated colony. Minute adjustment of themirror angle will serve to center this dark barin each colony if the original adjustment didnot result in perfect centering. At this point, itis possible to check the lamp angle for optimumpositioning. The bright areas above and belowthe dark bar in each type 2 colony should beequally right. If this is not the case, the angleof the lamp should be changed to obtain equalcolony illumination. It will be noted that adjust-ment of the lamp angle is not nearly as criticalas adjustment of the mirror angle. By contrastwith type 2 colonies, type 3 colonies will berelatively dimly but fairly uniformly illuminatedand will not have central horizontal dark bars(Fig. 2A).To verify further that the horizontal dark bar

observed in each type 2 colony (Fig. 2A) wasindeed due to focusing of the stage edge (arrowin Fig. 1A) in the colony, the colonies shown inFig. 2A were viewed with a vertical metal rodplaced in the path of the light directed towardthe mirror (Fig. 1B). The results of this changein illumination are shown in Fig. 2B, in whicheach type 2 colony now has focused in it thevertical rod as well as the horizontal microscope

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DIFFERENTIATING GONOCOCCAL COLONY TYPES

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FIG. 2. Appearance of a mixture of type 2 and 3 colonies of strain 488 with critical illumination. (A) Inthis typical view, all type 2 colonies have dark central bars in contrast to the type 3 colonies, which areuniformly illuminated. (B) The same colony mixture illuminated with a vertical bar in the light path. (C)The same colony mixture illuminated with the vertical bar extending down only to the edge of the microscopestage. (D) The same colony mixture photographed with random illumination.

stage edge. When the vertical rod was raised sothat it extended down to the edge of the micro-scope stage, and not below, each type 2 colonyonly focused the vertical rod in its top half (Fig.2C). When no attempt was made to obtain thecritical mirror angle and essentially random il-lumination was used, the type 2 and 3 coloniesappeared as shown in Fig. 2D.

Critical illumination with microscopesother than the Bausch and Lomb micro-scope. The critical illumination just describedwas obtained readily with Bausch and LombStereoZoom 7 or Series B microscopes, so itcame as a surprise to find that critical illumina-

tion could not be achieved with an AmericanOptical Cycloptic microscope equipped with aconcave mirror. Further investigation of thissituation revealed that differences in the dimen-sions of the stage edge (arrow in Fig. 3A) andrelative placement of the mirror mounting inthe American Optical microscope resulted ininability to obtain complete dark-field illumina-tion with this unit. This situation could be com-pensated for, however, by a small increase insize of the effective light-blocking edge of themicroscope stage. This was accomplished byplacing an opaque object, such as the rulershown in Fig. 3B, at the edge of the glass stage

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514 JUNI AND HEYM

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FIG. 3. Illumination used with the American Op-tical Cycloptic microscope. (A) The arrow indicatesthe opaque microscope stage edge discussed in thetext. (B) Position of fluorescent lamp for critical il-lumination with a microscope stage edge extender(ruler) as discussed in the text.

nearest the lamp. In the absence of this stageedge extender, and at the mirror angle corre-sponding to critical illumination, it could be seenthat the microscope field was not entirely dark,bright areas being visible at the top and bottomof a central dark area. With the stage edgeextender in place (ruler in Fig. 3B), the completefield was dark at critical illumination.With the ruler in place and the mirror now

adjusted for critical illumination, a mixture oftype 2 and 3 colonies appeared exactly as theydo in Fig. 2A. Once the best position for theruler was determined, the ruler could be re-

placed by a piece of opaque tape, such as blackplastic electrical tape. This strip of tape is notvisible through the microscope and may be leftin place permanently, since it will not interferewith other applications of the microscope.One of the dissecting microscopes encoun-

tered during this study has a U-shaped metalframe supporting a rectangular sheet of glasswith no opaque edge. Placement of an opaque

strip, such as a ruler or a piece of opaque tape,at the edge of the stage nearest the lamp servedto make it possible to achieve critical illumina-tion. In this case, as well as the one previouslydescribed, the opaque tape can remain in placepermanently without interfering with other usesof this microscope.Modification required for use of a flat

mirror. When a flat mirror was used in placeof a concave mirror, it was found to be impossi-ble to obtain critical illumination, since only anarrow dark band was visible even with theBausch and Lomb microscopes. Upon makingthe field as dark as possible by adjustment ofthe mirror angle, a mixture of type 2 and 3colonies appeared as shown in Fig. 4A. Althoughthe two types of colonies are different in severalways, both of them have horizontal dark barsthrough their centers (Fig. 4A). It was possibleto darken the entire field by extending the stageedge of the Bausch and Lomb microscope witha ruler in a manner similar to that illustrated inFig. 3B. With this modification, the more ex-treme extension of the opaque microscope edgeresulted in relatively little light being able toreach the flat mirror with the lamp in the posi-tion shown in Fig. 1A. By simply rotating thelamp 90° so that more light could reach themirror from both above and below the extendedstage edge, it was possible to remedy this situa-tion. The picture in Fig. 4B shows the samegroup of colonies seen in Fig. 2A and 4A, withthe stage edge extended and the lamp rotated900 as indicated above. Under these conditionsof illumination, type 2 colonies have character-istic dark bars through their centers, whereastype 3 colonies do not (Fig. 4B).Some microscopes are routinely supplied with

only a flat mirror, the opposite side usually beinga flat diffuse surface. It is possible to modifysuch microscopes by taping a concave mirrorfrom an old unused, conventional high-magnifi-cation microscope to the flat mirror or the flatdiffuse surface. Although the flat mirror can beused successfully to distinguish gonococcal col-ony types, as illustrated above, the concave mir-ror is distinctly superior and warrants either themodification just described or purchase of aconcave mirror when none is available.Observation of type 3 and 4 colonies.

Type 3 and 4 colonies of N. gonorrhoeae arewell known to be interconvertible in manystrains (4, 7, 8). Figure 5A shows an artificialmixture of type 3 and 4 colonies of N. gonor-rhoeae 488. Critical illumination of the kinddescribed above is, therefore, also useful in en-abling differentiation of colony types 3 and 4.Although both colony types are essentially thesame size, type 3 colonies are distinctly whiter

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DIFFERENTIATING GONOCOCCAL COLONY TYPES

than type 4 colonies, the latter being relativelymore transparent. It may be somewhat difficultto be certain about the identification of sometype 4 colonies when viewed alone, since coloniesof this type from a particular strain may besomewhat whiter than type 4 colonies from an-other strain. When the colony type 3 to 4 inter-conversion takes place, however, it is alwayspossible to recognize the type 3 colonies as therelatively whiter ones. When N. gonorrhoeae isplated on the medium Ml used in these studies,colony types 3 and 4 are somewhat larger thancolony types 1 and 2 (see Fig. 2A). Figure 5B

shows the same group of type 3 and 4 coloniesas in Fig. 5A, photographed by the techniquedescribed above that employs a flat rather thana concave mirror. Although it is possible todistinguish colonies of type 3 from colonies oftype 4 in Fig. 5B, the use of the concave mirror(Fig. 5A) is clearly superior.

Colonies of type 4 of certain N. gonorrhoeaestrains have been shown to revert only rarelyto colonies of type 3, whereas type 3 colonies ofthese strains revert very rapidly to type 4. Insuch cases, it is almost impossible to maintaina fairly uniform population of type 3 colonies.

FIG. 4. Appearance of the same mixture of type 2 and 3 colonies of Fig. 2, seen through the Bausch andLomb StereoZoom microscope with a flat mirror. (A) Critical illumination without a microscope stage edgeextender. (B) Critical illumination with a microscope stage edge extender.

FIG. 5. Critical illumination of a mixture of type 3 and 4 colonies of strain 488. (A) Illumination with a

concave mirror. (B) Illumination with a flat mirror and microscope stage extender as described in the text.

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516 JUNI AND HEYM

In other strains, however, it is possible to main-tain populations of either type 3 or 4 coloniesbecause of the relatively low rate of interconver-sion of colony types in either direction.Differentiation of colony types 1 and 2.

Colony types 1 and 2 are both characterized inthe present system of illumination by a horizon-tal dark bar in the center of each colony(Fig. 6). Colonies of type 2 (Fig. 6B), which arebrighter than colonies of type 1, have sharp-edged horizontal bars, whereas the horizontalbars of colonies of type 1 (Fig. 6A) have edgesthat are considerably less sharply defined. Col-onies of type 2 are significantly more translucentthan colonies of type 1, the type 1 colonies beingmore transparent. Furthermore, the horizontalbars in colonies of type 1 tend to be darker thanthose of type 2. It is easily possible to distinguishcolonies of type 1 from colonies of type 2 whenthese factors are kept in mind.Appearance of areas of confluent colo-

nial growth. Previous studies of gonococcalcolony types with other methods of illuminationhave emphasized that colony type differentia-tion is best made for colonies that are isolatedand not confluent (2, 4, 8). Figures 6A and 6Bshow the appearance of confluent growth areasas well as isolated colonies of colony types 1and 2, as viewed with the critical illuminationof the present procedure. As the colonies becomemore crowded, the horizontal bars in coloniesof types 1 and 2 become thicker and are occa-sionally curved. Confluent growth areas havethe same shade of darkness as the bars in theindividual colonies. The typical horizontal barsare still evident in colonies that are very close

to areas of confluent growth. Isolated coloniesof types 3 and 4 are uniform in appearance (Fig.5A) under the present system of illumination,and the color of the colonies does not changewhen growth becomes confluent.Relationship of colony types 1 and 2 to

colony types 3 and 4. As a result of carefulstudies of colony type interconversion of strain488, we have been able to confirm the observa-tion of Jephcott and Reyn (4) that colony type2 mutates chiefly to colony type 3 and colonytype 1 mutates chiefly to colony type 4. Al-though the reverse mutations to colony types 1and 2 occur less frequently, we have found thattype 3 colonies give rise to type 2 colonies andtype 4 colonies give rise to type 1 colonies.Colonies of type 1 do occur rarely in a populationof type 2 colonies, and these type 1 colonieshave been isolated and shown to breed true.When starting from a population of type 2 col-onies, perhaps the simplest way to obtain apopulation of type 1 colonies is to select andstreak out one of the type 3 colonies that appearspontaneously at a fairly frequent rate. Subcul-ture of type 3 colonies will eventually revealtype 4 colonies that also arise by spontaneousmutation. A type 4 colony is then subculturedand will in time give rise to colonies showinghorizontal bars when viewed with critical illu-mination, such colonies invariably being of type1.

It should be emphasized that although colo-nies of types 1 and 2 mutate to colonies of types4 and 3, respectively, the ability to show thereverse mutation is strain specific, some strainsbeing able to revert readily whereas other strains

FIG. 6. Confluent growth of colony types 1 and 2 of strain 488, viewed with critical illumination. (A)Colony type 1. (B) Colony type 2.

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DIFFERENTIATING GONOCOCCAL COLONY TYPES 517

FIG. 7. Critical illumination ofa mixture ofcolonytypes 1, 2, 3, and 4 of strain 488.

revert very poorly or not at all during extensivesubculture. Strains able to revert colony type 3to colony type 2 are also able to revert colonytype 4 to colony type 1.

Figure 7 shows the appearance of an artificialmixture of colony types 1, 2, 3, and 4 as viewedwith critical illumination. Although all the pho-tographs shown are of colonies of strain 488,many other clinical isolates of N. gonorrhoeaehave also been studied by the present technique.Except for minor differences, colony types ofother strains were similar in appearance to thecolony types of strain 488.

ACKNOWLEDGhMENT'S

We thank E. M. Britt of St. Joseph Mercy Hospital, AnnArbor, Mich., for supplying us with Thayer-Martin platesstreaked with clinical specimens. We are grateful for thehelpful suggestions made by M. I. Dolin and J. E. Juni duringthe course of this study. J. E. Juni took the photographs ofthe microscopes used in this report.

This investigation was supported by Public Health Service

grant AI-10107 from the National Institute of Allergy andInfectious Diseases.

LITERATURE CITED

1. Braun, W. 1965. Bacterial genetics, p. 153-163. W. B.Saunders Co., Philadelphia.

2. Brown, W. J., and S. J. Kraus. 1974. Gonoccocal colonytypes. J. Am. Med. Assoc. 228:862-863.

3. Janik, A., E. Juni, and G. A. Heym. 1976. Genetictransformation as a tool for detection of Neisseria gon-orrhoeae. J. Clin. Microbiol. 4:71-81.

4. Jephcott, A. E., and A. Reyn. 1971. Neisseria gonor-rhoeae. Colony variation I. Acta Pathol. Microbiol.Scand. Sect. B 79:609-614.

5. Jephcott, A. E., A. Reyn, and A. Birch-Anderson.1971. Neisseria gonorrhoeae. III. Demonstration of pre-sumed appendages to cells from different colony types.Acta Pathol. Microbiol. Scand. Sect. B 79:437-439.

6. Juni, E. 1977. Genetic transformation assays for identifi-cation of strains of Moraxella urethralis. J. Clin. Mi-crobiol. 5:227-235.

7. Kellogg, D. S., Jr., I. R. Cohen, L. C. Norins, A. L.Schroeter, and G. Reising. 1968. Neisseria gonor-rhoeae. II. Clonal variation and pathogenicity during35 months in vitro. J. Bacteriol. 96:596-605.

8. Kellogg, D. S., Jr., W. L. Peacock, Jr., W. E. Deacon,L. Brown, and C. I. Pirkle. 1963. Neisseria gonor-rhoeae. I. Virulence genetically linked to clonal varia-tion. J. Bacteriol. 85:1274-1279.

9. La Scolea, L., J., Jr., M. J. Dul, and F. E. Young.1975. Stability of pathogenic colony types of Neisseriagonorrhoeae in liquid culture by using the parametersof colonial morphology and deoxyribonucleic acid trans-formation. J. Clin. Microbiol. 1:165-170.

10. Punsalang, A. P., Jr., and W. D. Sawyer. 1973. Roleof pili in the virulence of Neisseria gonorrhoeae. Infect.Immun. 8:255-263.

11. Reyn, A., A. E. Jephcott, and H. Ravn. 1971. Neisseriagonorrhoeae. Colony variation II. Acta Pathol. Micro-biol. Scand. Sect. B 79:435-436.

12. Sparling, P. F. 1966. Genetic transformation of Neisseriagonorrhoeae to streptomycin resistance. J. Bacteriol.92:1364-1371.

13. Sparling, P. F., and A. R. Yobs. 1967. Colonial mor-phology of Neisseria gonorrhoeae isolated from malesand females. J. Bacteriol. 93:513.

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

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