JOURNAL OF BACTERIOLOGY, Oct. 1972, p. 602-610Copyright ( 1972 American Society for Microbiology

Vol. 112, No. 1Printed in U.S.A.

Development of Streptococcal L-Form ColoniesDAVID J. BIBEL AND JOHN W. LAWSON

Division of Medical Microbiology and Immunology, School of Public Health, University of California,Berkeley, California 94720

Received for publication 13 June 1972

The development and architecture of L-form agar colonies produced fromprotoplasts and L-phase bodies were studied by both light and scanning elec-tron microscopy. Agar blocks containing L-phase microcolonies of group AStreptococcus strains ADA and GL8 and group D Streptococcus strain F24 as

well as longitudinal sections of mature colonies were used as samples. Initially,granules of about 0.5 um in diameter were produced by multiple condensationand fragmentation of protoplasts and large bodies. Surface growth by granulesensued and infiltration into agar occurred only after 10 to 11 hr of incubationat 37 C. Club-shaped granules were noted and division seemed to take place bysimple fission. The configuration of large bodies and granules in mature colo-nies suggested budding as another means of' replication. Acellular spaces insidethe colonies appeared to have been formed by lysis of large bodies or by theenvelopment of space by the extending growth of minute granules. Whereas no

significant strain variation was noted in colonies of less than 24 hr of incuba-tion, fully mature colonies were differentiated on uniform media.

The development of the characteristic L-phase colony on agar has been the subject ofintense interest, and varying observations havebeen described (7, 8, 11, 12). Investigation ofearly growth has been hampered by the rela-tively low magnification of light microscopy.Transmission electron microscopy is severelylimited by the lack of perspective and difficul-ties in sample preparation. In a previous studywe examined the growth in broth of the L-phase variants of' streptococci and demon-strated the scanning electron microscope(SEM) to be an effective tool in studying mor-phological changes during growth (3). In thisreport we present and compare our findings bylight and scanning electron microscopy of thedevelopment and structure of' L-phase agarcolonies derived from both stable broth cul-tures and newly induced protoplasts.

MATERIALS AND METHODS

Microorganisms. Stable L-phase cultures ofgroup A Streptococcus strains ADA and GL8, origi-nally produced by L. Dienes and J. T. Sharp, weresupplied by R. M. Cole. Group D streptococci andtheir L-phase strains F24 and F24-L (ATCC 19634,19635) were received from H. Gooder. Protoplasts ofstrain F24 were induced by lysozyme according tothe method of Bibb and Straughn (1).

Media. Group D L-phase streptococci were cul-tured in the media described by King and Gooder (5).Group A L-phase streptococci were grown in amedium consisting of Trypticase soy broth (BBL)and 3% (w/v) NaCl. For colony formation 1.2% agar(Difco) was added.

Colony formation. Diluted samples from brothcultures at stationary phase or from a protoplastsuspension were inoculated upon replicate plates.Samples containing 104 to 106 colony-forming units(CFU)/ml were used for studies of young colonies

FIG. 1-7. Development of L-form colony from L-phase of' group A Streptococcus strain ADA. Unless oth-erwise stated, magnification is x540. Fig. 1, 5 hr. Bar = 10 gm. Fig. 2, 8 hr. Fig. 3, 9 hr. Colonies haverounded out and are increasing in height. Fig. 4, 11 hr. Granules have penetrated the agar. A, Focal plane atsurface of agar. B, Focal plane below surface of agar. Fig. 5, 15 hr. Arrou points to acellular space. Other suchzones are in early formation. Fig. 6, 18 hr. Further development of acellular areas and vacuolated largebodies. Fig. 7, 18 hr. Colony viewed at x 120 with oblique lighting. Bar = 50 um.

FIG. 8-16. Development of L-form colony from protoplasts of group D Streptococcus strain F24. Unlessotherwise stated, magnification is x540. Fig. 8, 3 hr. Protoplasts uith irregular edge. Bar = 10 gim. Fig. 9, 5hr. Fig. 10, 6 hr. Fig. 11, 7 hr. Fig. 12, 8 hr. The faster rate of growth is apparent when compared to Fig. 2.Fig. 13, 9 hr. Arrow indicates the envelopment of a space by growing granules. Fig. 14, 11 hr. Infiltration ofgranules into agar. Fig. 15, 21 hr. Fig. 16, 24 hr. The "fried egg" form of the colony is clearly developed.Oblique lighting. x 120. Bar = 50 gm.

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VOL. 112, 1972 DEVELOPMENT OF STREPTOCOCCAL L-FORM COLONIES

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BIBEL AND LAWSON

whereas suspensions of 102 to 103 CFU/ml were em-ployed for photography of more mature colonies.Before selecting representative colonies for photogra-phy, all plates were inspected for colony size andstructure to guard against effects of crowding.Measurement of growth. The diameter of the

colony was used as a parameter of growth followingPirt (10) and Palumbo et al. (9). Averages of 15 to 20representative colonies were used to determinegrowth curve.

Light microscopy. Photographs were obtained byemploying a Polaroid ED-10 camera (Cambridge,Mass.) and type 107 pack film. Satisfactory calibra-tion was achieved by photography of the ruled gridof a certified hemacytometer.SEM. Preparative techniques, including glutaral-

dehyde fixation and critical-point drying, have beendescribed in a previous report (2). Whenever possi-ble, samples used for light microscopy were also se-lected for SEM. Polaroid film type 55 P/N was uti-lized for photomicrographs. The Cambridge Ster-eoscan electron microscope was operated at 20 kv.

Cross-sections. L-colonies were longitudinallycut by two methods. Fixed and dried agar blockswith individual colonies were embedded in paraffinand sliced by a microtome. Sections (10-.um thick)were placed on 15-mm diameter cover glasses,treated with xylene to remove the wax, and coatedwith gold/palladium. In the second procedure, fixedblocks were quick-frozen in liquid nitrogen andimmediately split with a very thin razor blade.Pieces were then dried by the critical-point methodand prepared for the SEM.

RESULTSLight microscopy of growth. Agar plates

were inoculated with stable cultures of strepto-coccal strains F24-L, ADA-L, and GL8-L andincubated at 37 C for 48 hr. During the first 2hr, single units, mainly large bodies, and occa-sional small clusters of 2 to 4 units were found.By 5 hr, growth was detected and consisted ofshort chains of 2 to 3 units extending from thecentral group in various directions (Fig. 1).Growth was in the form of granules 0.5 to 1 gmin diameter which seemed to undergo fission.Between 8 and 10 hr of incubation, the micro-colony developed a circular appearance andbegan to increase in height (Fig. 2 and 3). Be-ginning at 10 to 11 hr, granules penetrated theagar and formed the characteristic "yolk" ofthe L-colony (Fig. 4 and 14). Yolk productionwas delayed or even inhibited in excessively

moist plates. This infiltration generally startedfrom a small focus within an area close to thecenter of the colony. Upon further incubation,the subsurface zone increased in size but re-mained distinctly granular. Surface units,however, lost their defined edges and becamenondistinct and confluent. Between 12 and 15hr, acellular areas apparently formed by thecircular envelopment of granules around aspace (Fig. 5, 13) were first observed. By 18 hr(Fig. 6) of incubation the colony was of typical"fried egg" form consisting of a deep, granularcenter and a lacey surface periphery (Fig. 7,16). For the first 24 hr there were no signifi-cant differences among the various strains.Afterwards, strain variances with respect tosize and subtle surface features were noted.

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FIG. 17. Comparison of the growth rates of par-ent, stable L-phase, and newly induced L-phase col-onies of Streptococcus faecium strain F24. *ATCC19635. **Our collection. L-phase growth penetratesagar at 1Oto 11 hr.

FIG. 18-25. Scanning electron micrographs of the development of L-form colony from the L-phase ofStreptococcus strain ADA. Unless otherwise stated, magnification bar = 1 Aim. Fig. 18, Cluster of large bodyand adjacent granule after 1 hr of incubation. Note pit on surface of granule. Fig. 19, Condensation of largebody with loss of smooth surface. I hr. Fig. 20, Two large bodies after 1.5 hr. One has collapsed with in-creased condensation; the other is still spherical but possesses several pits. Fig. 21, Granules formed by ap-parent fragmentation. 1.5 hr. Fig. 22, 4 hr. Granules are small and often club-shaped. Fig. 23, 7 hr. Fig. 24, 10hr. Bar = 5 gim. Fig. 25, Detail of the surface of a 12-hr colony.

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The formation of the L-form colony fromprotoplasts of group D streptococci followedthe same sequence of development as its L-phase (Fig. 8-16). The f'irst indications ofgrowth were detected at 3 hr by the scallopedappearance of the edge of protoplasts (Fig. 8).Smaller bodies were also seen along the out-side of the membrane which suggested frag-mentation or budding as the initial mode ofreplication. Penetration into agar by the newlyinduced L-phase also began between 10 and 11hr of incubation.The early growth of the newly induced L-

phase, prior to infiltration into agar, appearedfaster than that of the stable culture (ATCC19635) (Fig. 17). Whether the comparativelyrapid rate of growth is a function of initial L-phase growth from the protoplastic stage orwhether the slower growth of' the stable culturewas due to mutation and selection during theprocess of stabilization was not determined.The growth curve of another stable L-phaseculture, also derived from strain F24 strepto-cocci, was compared to those depicted in Fig.17. This variant, which has been subculturedin our laboratory for 3 years, produced an ex-ponential growth curve whose slope appearedslightly less than that of the newly induced L-form, but greater than that of strain F24-L,ATCC 19635.Scanning electron microscopy of growth.

After absorption of the suspending fluid by theagar, pits were observed on the surface of thestable L-phase bodies (Fig. 18, 20). Pits did notincrease in number with exposure to the elec-tron beam, nor were they encountered in oldercultures. During the f'ollowing hour largebodies seemed to have condensed, collapsed,and lost their smooth surf'ace (Fig. 19, 20). Theflattened, irregularly edged body continued tofragment and formed multiple granules whichthereafter multiplied (Fig. 21). These granulesoften measured under 0.5 um in diameter, andoccasionally club-shaped f'orms were noted(Fig. 22 and 23).

Previous studies had shown that protoplastsof Streptococcus faecium possess a somewhatsmooth surface (2). When protoplasts wereplaced upon the agar medium, the membranesoon developed wrinkles and, in a manner sim-ilar to that of L-phase large bodies, condensedand fragmented (Fig. 26-28). Granules tendedto remain close together, but when found suffi-ciently separate, apparent fission was indi-cated (Fig. 29 and 30). Whereas the colony ofgroup A strain ADA-L was distinctly convexby 10 hr (Fig. 24), the newly induced L-form

colony of' strain F24 remained low to the sur-face (Fig. 31 and 32).Examination of mature L-colonies showed a

wide variation in the size of bodies, measuringfrom 0.3 to over 5 pm. The range in diameterof large bodies also varied with the region ofthe colony. Groups of smaller bodies were seenat the top and center of the colony whereas thelargest bodies were found at the bottom edge(Fig. 34). Besides possible multiplication byfission, granules seemed to undergo budding aswell (Fig. 35). Throughout all our observationsoccasional dimpling of the surface of largebodies was noted (Fig. 25, 32, 36, 37).

Inspection of split colonies showed the usualvariation in size of large bodies. Growth wasnot confluent, however, and small acellularspaces were observed. The growing edge of thecentral yolk shown in Fig. 37 was of interest.Occasional large bodies of up to 1.5 pm werenoted, but the vast majority of bodies weregranules of' 0.3 to 0.8 pm in diameter.

DISCUSSIONA search of' the literature produced only four

reports describing in extensive detail the se-quential development of the colony of L-phaseor cell wall-defective organisms. The study bySchuhmann and Taubeneck (12) of the growthof an L-phase colony of Escherichia coli in-volved time-lapse photography. In theirsystem the L-phase bodies were extremelypleomorphic; amorphous masses underwentspreading and irregular fragmentation. Colo-nies did not resemble fried eggs but were sur-face growth of' brain-like appearance. Marraroet al. (7) investigated cell wall-defective var-iants of S. faecalis over a 36-day period. Littleinformation regarding earliest growth and agarpenetration was presented. They described theformation of' the L-colony as stemming fromthe massive granulation of a vacuolated, "cart-wheel' microcolony. This initial structure wasalso described by Young and Armstrong in S.Iiquefaciens (13). Marston studied the induc-tion of the L-form colony of staphylococci anddescribed its development in terms of the ag-gregation and multiplication of large bodies (8).Liebermeister (6) investigated the L-formgrowth of' Proteus and showed "sproutinggrowth via pseudopodia-like protrusions."

Although differing considerably from pre-vious findings, our results are generally con-sistent with the observations and postulationsof Dienes (4), who also studied streptococcal

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FIG. 26-32. Scanning electron micrographs of the development of the L-form colony from protoplasts ofStreptococcus strain F24. Unless otherwise stated, magnification bar = 1 ,m. Fig. 26, Protoplast after 1 hrincubation. Fig. 27, Condensation and collapse of protoplast membrane. I hr. Fig. 28, 2 hr. Fragmentationinto several granules. Fig. 29, Granules showing apparent fission, 2.5 hr. Fig. 30, 3 hr. Fig. 31, 6 hr. Fig. 32, 10hr. Wide surface growth of granules and occasional large bodies.

strains ADA-L and GL8-L. If media, species, shares a common pattern of development withand strain differences are responsible for the group A strains. Dienes also observed con-varying sequences of maturation, then it ap- densations along the periphery of isolated largepears that group D Streptococcus strain F24-L bodies as the first sign of colony formation.

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bodies. Both features may actually be differentstages of the same process. Perhaps these de-pressions are a result of membrane invagina-tion leading to the production of vacuoles.

FIG. 33. Scanning electron micrographs of thedevelopment of the L-form colony from protoplastsof Streptococcus strain F24. Convex colony at 15 hr.Bar = 10 gm.

FIG. 35. Detail of L-colony of strain GL8. Arrowspoint to configurations suggestive of budding.

FIG. 34. Mature 48-hr colony o! Streptococcusstrain GL8-L. Note variation in size of large bodies.Bar = 10 inm.

Granules formed in the areas of condensation,multiplied, and penetrated the agar.Photomicrographs taken with the SEM

clearly showed the process of condensation andmultiple production of granules from proto-plasts as well as from large bodies. The pres-

ence of deep pits in L-phase bodies during thefirst hour of incubation on agar was a curiousand consistently observed phenomenon. Wehave often noted dimples or small depressionson the surface of a small percentage of L-phase

FIG. 36. Dimpling of surface of large bodies.Colony of ADA-L.

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FIG. 37. Cross-section of a 48-hr L-colony ofstrain ADA showing the edge of growth penetratinginto agar.

Once granules were created, they seemed toundergo fission. Dienes observed a more com-

plex process by which granules elongate, fol-lowed by condensation at the ends and thebreakdown of the central material. We havealso seen elongated granules, but there was no

evidence of a collapsed central area betweendouble granules. Furthermore, configurationsof large bodies and granules in photomicro-graphs of mature L-form colonies suggest bud-ding (Fig. 35). Another possibility would be thegrowth of only one daughter granule, since theformation of viable granules is inefficient.The foamy appearance of the L-form colony

as seen by light microscopy is for the mostpart due to vacuolated large bodies, but inaddition may also be due to the absence ofgrowth or to lysis. Spaces were also noted byMarraro et al. (7). They believed, however,that the zones were a result of variances ininternal colony pressure rather than the mere

absence of growth. Figures 5 and 13, neverthe-less, indicate the latter possibility as the more

probable explanation.Razin and Oliver (11) and Liebermeister (6)

described L-phase growth to occur initially in-

side agar rather than on the surface. Lieber-meister further showed that in his system allyoung growth occurred at the deep center ofthe colony and forced older elements to theperiphery. Our photographs, however, are inagreement with Dienes (4) and Marraro et al.(7), who described infiltration into agar fromsurface growth.

609

The penetrating edge of the yolk was viewedby Marraro et al. (7) to be composed of"ghost" cells lacking cytoplasmic material.One would expect that this zone should becomposed of active, viable cells. Of course it ispossible that the leading cells are used as bat-tering rams forced into the agar by the pres-

sure of the growing colony. Our own photomi-crographs, showing infiltrating bodies to besmall, separate, and spherical granules, suggesta more active role.The development of L-form colonies of var-

ious strains is essentially similar during earlygrowth; however, mature colonies differ. Wehave compared the L-phase colonies of severalstrains of group A streptococci, group D strep-tococci, and Neisseria gonorrhoeae grown upon

uniform medium, and noted subtle variationby both light microscopy and SEM (Bibel,Ph.D. dissertation, Univ. of California). Strainvariation was enhanced when grown upon Mil-lipore membrane filters (3).

ACKNOWLEDGMENTS

This work was supported by grant no. 5-SO1-FR5441-09from The Division of Research Facilities and Resources. Thescanning electron microscope was operated under grantGM17523 from the National Institute of General MedicalSciences. D. Bibel was supported by training grant 2 TO1-A100332-03 from the National Institute of Allergy and Infec-tious Disease. All three grants were from the Public HealthService.We thank Harold Sampson for his excellent technical as-

sistance.

LITERATURE CITED

1. Bibb, W. R., and W. R. Straughn. 1962. Formation ofprotoplasts from Streptococcus faecalis by lysozyme.

J. Bacteriol. 84:1094-1098.2. Bibel, D. J., and J. W. Lawson. 1972. Scanning electron

microscopy of L-phase streptococci. I. Development oftechniques. J. Microscopy 95:453-458.

3. Bibel, D. J., and J. W. Lawson. 1972. Scanning electronmicroscopy of L-phase streptococci. II. Growth inbroth and upon millipore filters. Can. J. Microbiol.18:1179-1184.

4. Dienes, L. 1967. Morphology and reproductive processesof the L forms of bacteria. I. Streptococci and staphy-lococci. J. Bacteriol. 93:693-702.

5. King, J. R., and H. Gooder. 1970. Induction of entero-coccal L-forms by the action of lysozyme. J. Bacteriol.103:686-691.

6. Liebermeister, K. 1960. Morphology of the PPLO and Lforms of Proteus. Ann. N.Y. Acad. Sci. 79:326-343.

7. Marraro, R. V., R. M. Pfister, M. S. Rheins, and I. Ka-petonavic. 1971. The morphology of induced wall-defective variants of Streptococcus faecalis as studiedby light and electron microscopy. Can. J. Microbiol.17:365-371.

8. Marston, J. 1968. Production and cultivation of staphy-lococcal L-forms, p. 212-220. In L. B. Guze (ed.), Mi-crobial protoplasts, spheroplasts, and L-forms. TheWilliams and Wilkins Co., Baltimore.

9. Palumbo, S. A., M. G. Johnson, V. T. Rieck, and L. D.Witter. 1971. Growth measurements on surface colo-

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nies of bacteria. J. Gen. Microbiol. 66:137-143.10. Pirt, S. J. 1967. A kinetic study of the mode of growth of

surface colonies of bacteria and fungi. J. Gen. Micro-biol. 47:181-197.

11. Razin, S., and 0. Oliver. 1961. Morphogenesis of myco-plasma and bacterial L-form colonies. J. Gen. Micro-biol. 24:225-237.

12. Schuhmann, E., and U. Taubeneck. 1971. L-formen vonEscherichia coli K 12 (X). I. Unduktion und vermeh-rungsweise. Z. Allg. Mikrobiol. 11:205-219.

13. Young, L. S., and D. Armstrong. 1969. Induction, colonymorphology, and growth characteristics of the L formof Streptococcus liquefaciens. J. Infect. Dis. 120:281-291.

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