JOURNAL OF BACTERIOLOGY, May 1970, p. 561-572Copyright a 1970 American Society for Microbiology

Vol. 102, No. 2Printed in U.S.A.

Ultrastructure of Mycoplasma laidlawiiDuring Culture Development

JACK MANILOFF

Department of Microbiology and Department of Radiation Biology and Biophysics, School of Medicine andDentistry, University ofRochester, Rochester, New York 14620

Received for publication 4 December 1969

Mycoplasma laidlawii B has been studied by phase-contrast and electron micros-copy (thin-section and negative staining). Exponential-phase cells are filaments withthe following structures: surface unit membrane, nuclear material, ribosomes, andintracellular granular region. These cells appear to reproduce by binary fission. Instationary phase, the filamentous cells have numerous constrictions, giving them a

beaded appearance. Nonviable death-phase cultures contain cellular debris: swollencells, granular bodies, and other aberrant forms.

The morphological features and life cycles ofthe Mycoplasma are currently unresolved. Theseproblems are interdependent, since (except forM. gallisepticum; 13, 16) it has been necessary topostulate reproductive mechanisms based oninterpretations of morphological studies. Thesedata on Mycoplasma ultrastructure, morphology,and reproduction have recently been reviewed byFreundt (5) and Anderson (1).Growth studies have shown differences in

Mycoplasma viability and turbidity curves, witha sharp nonlinear absorbance increase at high celldensities, suggesting that changes in cellularmorphology and size could be occurring duringliquid-culture development (12). This possibility,which would explain some of the variation re-reported for Mycoplasma structure, was investi-gated in the studies described here and presentedbriefly elsewhere (Maniloff, Bacteriol. Proc.,p. 78, 1968).

In this study, M. laidlawii B has been examinedby thin-section electron microscopy during cul-ture development. Marked morphologicalchanges are noted in the stationary and deathphases of the culture. The morphology of cellswas also compared by phase-contrast microscopyand electron microscopy of negatively stainedpreparations. These data show the filamentousmorphology of exponentially growing M. laid-lawii and the appearance of granular bodies andother aberrant forms during cellular degenerationand death.

MATERIALS AND METHODSMycoplasma. The organism used in this study,

obtained from M. E. Tourtellotte (University ofConnecticut, Storrs), was M. laidlawii B.

Media and assay. The tryptose broth medium(containing 1% glucose and 1% serum fraction) hasbeen described previously (12). Assaying the numberof clone-forming units per milliliter (CFU/ml) wasdone by using blood agar plates, a method which hasbeen shown to provide an accurate measure of theviability of M. laidlawii liquid cultures (12).

Phase-contrast microscopy. A portion of culturewas mixed with an equal volume of 12.5% glutaralde-hyde in 0.1 M sodium cacodylate (final fixative con-centration, 6.25%) and fixed at 4 C for 2 hr. A dropof the cell suspension was then put on a carbon-Formvar-coated London Finder electron microscopegrid (E. F. Fullam Inc., Schenectady, N.Y.). Thedrop was drained off with filter paper, and the gridwas placed on a glass slide in a drop of 2% phos-photungstic acid (PTA), pH 7.0, for examination.

Phase-contrast microscopy was done by using aReichert Zetopan microscope with a 10X oil-immersion fluorite objective. Pictures were taken,by using a green filter, on Kodak Tri-X Pan profes-sional film at a magnification of 730X. These gridswere subsequently examined by electron microscopy.

For the observation of unfixed cells, a drop of theliquid culture was examined directly with the phase-contrast microscopic apparatus described above.

Electron microscopy. For negative-staining studies,the cells, placed on London Finder grids in a dropof 2% PTA, were used after the phase-contrastmicroscopic examination. The PTA was drained offwith filter paper, and the grid was dried and examined.

For thin-section studies, 500 ml of culture wasmixed with an equal volume of 12.5% cold glutaralde-hyde (in 0.1 M cacodylate), fixed in the cold for 2 hr,harvested by centrifugation at 14,000 X g for 10 min(at 4 C), and stored overnight in cold 0.1 M cacodylate.Some pellets were postfixed by using 1% osmiumtetroxide by the procedure of Kellenberger, Ryter,and Sechaud (8). The samples were then dehydrated,embedded in Epon, sectioned, and stained with

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either uranyl or uranyl and lead, as previously de-scribed (14).

Grids were examined with a Siemens Elmiskop I(modified to IA) electron microscope, with a doublecondenser, liquid nitrogen decontamination device,50-nm objective aperature, and 80 kv of operatingvoltage. Micrographs were recorded on Kodakelectron image plates.

RESULTSGrowth curve. The M. laidlawii growth curve

(Fig. 1) is similar to those previously reported (12,17); it shows no measurable lag phase, an expo-nential increase in CFU/ml, a short stationaryphase (about 12 hr), and a rapid death phase. Insamples at 75, 96, 108, and 120 hr, no CFU wereobserved. Considering the dilutions plated, at a95% confidence limit, the maximum titers whichcould have been present at these late times wereabout 102 CFU/ml.

Ultrastructure during culture development. Fromthe culture used for growth curve studies, 500-ml

109

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TIME, hrsFIG. 1. Semilogarithmic plot of M. laidlawii B

growth (CFU/ml) as afunction oftime after inoculation.Samples for electron microscopy were taken at thetimes indicated by the arrows.

portions were removed at 8, 36, 75, and 108 hr (asindicated by the arrows in Fig. 1) and preparedfor thin-section electron microscopy. Each un-fixed culture sample was also examined by phase-contrast microscopy and showed morphologicalforms similar to those observed by electron mi-croscopy; i.e., dense filamentous cells at 8 and 36hr and large refractile bodies at 75 and 108 hr.At 8 hr, the culture was growing exponentially

-about 108 CFU/ml. The M. laidlawii cells ap-peared as filaments (Fig. 2 and 3) about 0.4 to0.5 Am wide. Only small lengths of most filamentswere in the sections. However, filaments up to 2,um in length were frequently seen (Fig. 2b and c),and filaments as long as 5 Am in the plane ofsection have been observed (Fig. 2a). Branchedfilaments (Fig. 2d and e) have also been seen.These forms may be more numerous in the cul-ture, but were seen only infrequently as a conse-quence of the low probability of sectioningthrough a branched structure.Each cell (filament) was bounded by a unit

membrane about 7 nm thick, appearing as twodense lines, each about 2 nm thick, separated by aless dense area, about 3 nm wide. This membranestructure was seen best in preparations that hadbeen fixed in both glutaraldehyde and osmiumtetroxide (e.g., Fig. 3). The membrane outer denseline usually appeared fuzzy, probably because ofthe binding of media to the membrane duringfixation; the size measurements were made onmembrane areas that did not show this phenome-non.The intracytoplasmic area contained fibrillar

material, which is probably the nuclear materialof the cell and granules of various sizes and den-sities. The largest granules, approximately 20 nm,are probably ribosomes.The M. laidlawii cells also contain another in-

tracytoplasmic structure, appearing as a finelygranular region, partitioning the filament (Fig.2a-c, 3). This granular region, which is as wide asthe cell, is about 0.2 to 0.3 ,um long. No more thanone such granular region has ever been observedper filamentous cell.The 36-hr culture (about 109 CFU/ml) was in

late-stationary phase. The filamentous cells hadnumerous constrictions (Fig. 4 and 5). Branchedfilaments were seen (Fig. 4 and Sb), and a fewcells at this stage of culture development haddegenerated into swollen bodies (Fig. 5a). Somecells, in these preparations, contained granularregions (Fig. 4 and Sa), but none of these regionswere noted at the points of filament constriction.At 75 and 108 hr, no viable cells were found in

the cultures. Therefore, the morphological formsseen at these times represent dead cells. Since the

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FIG. 2. Exponential M. laidlawii B cells (8 hr) stainied with uranyl and lead. Fine structural elementsinclude: surface unit membrane, nuclear material, ribosomes, and granular region. Fixation: (a-c) glutaralde-hyde and (d, e) glutaraldehyde and osmium. Magn(ificationi: (a, d, and e) X 40,500, (b and c), X 48,600.Abbreviations: m, membrane; ni, nuclear material; r, ribosomes; and g, granular region.

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FIG. 3. Exponential M. laidlawii B cells (8 hr), fixed with glutaraldehyde and osmium, stained withuranyl, and showing cellularfine structure. X 120,000.

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ULTRASTRUCTURE OF M. LAIDLA WII

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FIG. 4. Stationary-phase M. laidlawii B cells (36 hr) fixed in glutaraldehyde and stained with uranyl. X 64,800.

micrographs at 75 and 108 hr are similar, these in diameter, with various degrees of loss of intra-data are presented together. cytoplasmic material. The poor intracytoplasmicThe cells at 75 (Fig. 6 and 7) and 108 hr (Fig. 8) preservation reflected the lack of viability of these

appear as round bodies, mostly from 0.5 to 1 ,um cells. A number of abnormal forms were seen in

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Large swollen cell (arrow), X 56,700; (b) branched cell, X 64,800.

the nonviable cultures, probably resulting fromcellular degeneration. These include dense mem-brane-bound intracytoplasmic inclusions (Fig. 7),intracellular membranes (Fig. 8), and emptyextracellular membraneous vesicles, sometimesconnected together (Fig. 6 and 8).Morphology. The use of London Finder grids

to examine exponentially growing cells allowedthe same cell, or cluster of cells, to be located and

compared by both phase-contrast and electronmicroscopy. Although this necessitated the use offixed preparations, it was noted that the fixed cellson grids appeared the same in the phase-contrastmicroscope as the unfixed-cell suspension growingin culture. Therefore, it was concluded that thefixed cells used for these studies give a good repre-sentation of the cellular morphology.

Figure 9 shows samples of the cells, taken from

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ULTRASTRUCTURE OF M. LAIDLAWII

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FiG. 6. Nonviable M. laidlawii cells (75 hr), fixed with glutaraldehyde and osmium, and containing empty

vesicles (arrows). X 40,500. (a) Uranyl stained; (b) uranyl and lead stained.

exponentially growing M. laidlawii cultures, ob-served by both phase-contrast and electron mi-croscopy. The chain of cells in the phase-contrastmicrograph (Fig. 9b) is about 5.5 ,um long and

in the electron micrograph (Fig. 9a) is about 5.3,um long. This agreement in size is typical of thedata and indicates that, in preparation for electronmicroscopy, the fixed cells do not undergo sub-

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FIG. 7. Nonviable M. laidlawii cells (75 hr), fixed with glutaraldehyde and osmium, stained with uranyland lead, and showing dense membrane-bound intracytoplasmic inclusions (arrow) which have formed inlarge swollen cell. X 81,000.

stantial shrinkage or other morphological alter-ations during drying.The phase-contrast pictures (Fig. 9b, d, and f)

showed filamentous structures with a beaded ap-pearance. The corresponding electron micro-graphs (Fig. 9a, c, and e) showed that each struc-ture was in fact made up of several filmentous andcoccoid cells. For example, the phase-contrastmicrograph of Fig. 9f appeared to be a largebranched filamentous cell, but the electron micro-graph (Fig. 9e) showed that the structure is threefilamentous cells. Similarly, the beaded structureof Fig. 9d was made up of many smaller cells(Fig. 9c).

DISCUSSIONThe difficulty in using phase-contrast micro-

scopic data for studies of Mycoplasma morphol-

ogy and growth is demonstrated in Fig. 9. This isprimarily because of the small size of the cells andthe closeness of the cells within a cluster. In termsof resolving power, Barer (2) has stated thatphase-contrast microscopic resolution should beabout that of an ordinary microscope with thesame numerical aperature (NA). Therefore, theresolving power should be given by the equation,minimum resolvable separation = 0.61 X/NA, inwhich X is the wavelength of the light (19). Forgreen light (X = 525 nm) and the objective(NA = 1.30) used in these studies, the resolutionis 0.25 gm. This value is probably typical for mostphase-contrast microscopy, and, hence, the closepacking of Mycoplasma cells cannot be resolved.That is, as shown in Fig. 9e and f, one cannotdistinguish between a filamentous cell and severalcells stuck together. This resolving power also

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FIG. 8. Nonviable M. laidlawii cells (108 hr) fixed with glutaraldehyde and osmium, stained with uranyland lead, and showing swollen cells, vesicles, and cell which appears to have an intracellular membrane(arrow). X 40,500.

makes it difficult to evaluate the morphologicaldetails of structures that are only about 0.5 ,umwide. For example, in their phase-contrast micro-graphs of M. gallisepticum strain A5969, Razinand Cosenza (17) observed chains and clusters ofcells; however, they were neither able to resolvethem into the individual viable tear-drop-shapecells nor able to observe the small bleb reproduc-tive structures of these cells, as identified in theelectron micrographs of Maniloff, Morowitz, andBarrnett (15) and Morowitz and Maniloff (16).

In addition, the "halo" effect, an optical arti-fact inherent in phase-contrast microscopy whichis caused by light diffraction at the phase-changingannulus (2), acts to emphasize a beaded image ofthe cells. The bright halos tend to mask adjacentstructures, in this case obscuring the continuity ofthe filamentous cells and giving them a beadedappearance (e.g., compare Fig. 9a and b).

Freundt (4, 5) has proposed that most, if notall, Mycoplasma species replicate by endofilamen-

tous or endomycelial formation of elementarybodies, which are released by the fragmentationof the filament or mycelium, each elementarybody then forming a new cell and repeating thecycle. This reproductive scheme is based on theinterpretation of whole-cell preparations, exam-ined primarily by phase-contrast microscopy andelectron microscopy of unfixed dried and shad-owed cells. The limitations of phase-contrastmicroscopy for such studies are discussed above,and Anderson (1) has reviewed the problems ofpreparing the extremely plastic Mycoplasmaspecies for electron microscopy. Aside from suchinferential considerations, there is, as yet, nodirect evidence for the existence of viable elemen-tary bodies, let alone their playing a role in Myco-plasma reproduction.

Razin and Cosenza (17) have studied M. laid-lawii B by phase-contrast microscopy, using thesame growth medium as that used in the studiesreported here. During exponential growth of the

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FIG. 9. Three clusters of exponentially growing glutaraldehyde-fixed M. laidlawii B cells examined byphase-contrast and electron microscopy. (a, c, and e) Electron micrographs, X 20,520; (b, d, and f), phase-contrast micrographs, X 2,190.

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ULTRASTRUCTURE OF M.LAIDLAWI5

culture, they observed short branched filaments.At 36 hr, the filaments changed and appeared aschains of coccoid elements. The late-stationary-and death-phase cultures showed large cells andsmall vesicles. These data are consistent with theelectron microscopic data reported here. If thechain of coccoid elements is to be identified withelementary bodies, then, since the entire culturereaches this stage at about 36 hr, there should be asharp rise in viability at this time. However, thegrowth curve shows an exponential increase fromthe time of inoculation (Fig. 1; also 12, 17) andthe 36-hr culture is actually in stationary phase.This inconsistency has been noted by Anderson(1), who also mentioned it with respect to similarobservations on other Mycoplasma species.

Exponentially growing M. laidlawii B culturescontain filamentous cells (Fig. 2 and 3). Somecells appear to be branched. The cells are boundedby a unit membrane of 7-nm thickness, which is inagreement with the 8-nm value reported by Razin,Morowitz, and Terry (18). This surface membranewas the only membraneous structure found in thecell. The intracytoplasmic structures of the cellinclude nuclear material, ribosomes, and a granu-lar region. The identification of the nuclear mate-rial and ribosomes is based on the similarity oftheir stained appearance with the nuclear materialand ribosomes of M. gallisepticum, which havebeen chemically analyzed (15).

Although only one granular region per cell hasbeen observed thus far, it may be that, because ofthe filamentous cellular structure, if there aremore than one per cell, the probability of gettingthem in a section is very small. These regionscould possibly be either a form of septum or somesort of intracellular storage granule. It does notseem very probable that they are division septa,since the granular regions are rarely seen in areasof cellular constriction (e.g., Fig. 2c). However,although no structural basis can be seen for thewavy appearance of the filaments, it can be notedthat the granular regions of the filaments areusually thinner than the adjacent areas (Fig.2 and 3).Although it is difficult to make detailed deduc-

tions regarding the life cycle of a cell from a setof micrographs, a number of conclusions can bedrawn. The pictures of exponentially growingcells must contain organisms distributed over allstages of the life cycle. Since these micrographs ofM. laidlawii B contain no evidence of elementarybodies or filament fragmentation at multiplepoints, these replicative modes are improbable.The data are consistent with what would be ex-pected to be seen in a population of filamentouscells reproducing by binary fission, with eachfilament dividing to form two daughter cells.

Hence, it is probable that exponentially growingM. laidlawii B cells reproduce by binary fission,but it is not obvious how this process would occurin the branched cells or whether the branched cellsare, in fact, viable. The growing filamentous cellsare found, in liquid culture, in clusters (Fig. 9).The binary fission mode of replication, describedhere for M. laidlawii B, has also been reported forM. orale (6), M. pneumoniae (7), M. gallisepticum(13, 16), M. hominis (11), and several otherMycoplasma (9).The filamentous cellular structure and the

binary replicative mode refer to morphologicalobservations. From these data, no conclusionscan yet be made regarding nuclear events; i.e.,binary cellular reproduction does not exclude thepossibility of coenocytic growth of the filamentouscells.

Stationary-phase cultures contain cells withconstrictions along the filaments (Fig. 4 and 5).Since these cultures have a constant titer, the con-stricted filaments must not fragment, with eachfilament yielding many viable units. In fact, it maybe that these cells do not fragment at all, but in-stead swell when going into death phase.The death-phase cultures contain rounded cells

and a variety of cellular debris (Fig. 6-8). If thefilamentous cells (2 ,um long and 0.5 Am wide)swell to give spheres of equivalent volumes, thenO.9-,um round cells should be observed. Most ofthe spherical forms are about this size, suggestingthat many of the constricted filaments remainintact and swell to give these bodies, rather thanfragmenting to smaller forms. The larger bodiesfound in these cultures may have arisen by cellfusion. A variety of vesicles (Fig. 6 and 8) andgranular bodies (Fig. 7) are found in these deadcultures and must represent cellular debris. Thesemicrographs are similar to those of Domermuthet al. (3); hence, it seems probable that they exam-ined a nonviable culture.

Different Mycoplasma species have their owncharacteristic morphology. M. laidlawii B cellsare filaments, as reported here; M. gallisepticumA5969 cells are tear-drop-shaped (13, 15, 16);Caprine PPLO, strain 14, cells are streptococcal-like chains (10); M. hominis species H39 cells areellipsoids (11); and other examples are reviewedby Anderson (1). M. laidlawii and M. gallisepti-cum were found to have subcellular organeles;hence, the Mycoplasma are not simply membrane-bound cytoplasmic material. It would appear,then, that the Mycoplasma species represent amorphologically and ultrastructurally diversegroup of organisms.

ACKNOWLEDGMENTS

I wish to thank Madalyn Smith for her excellent technicalassistance.

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This investigation was supported by Public Health Servicegrant Al 07939 from the National Institute of Allergy and Infec-tious Diseases and by University of Rochester Atomic EnergyProject (report no. UR-49-1141).

LITERATURE CITED

1. Anderson, D. R. 1969. Ultrastructural studies of Mycoplasmasand the L-phase of bacteria, p. 365-402. In L. Hayflick (ed.),The mycoplasmatales and the L-phase of bacteria. Apple-ton-Century-Crofts, N.Y.

2. Barer, R. 1959. Phase, interference, and polarizing microscopy,p. 169-272. In R. C. Mellors (ed.), Analytical cytology.McGraw-Hill Book Co., N.Y.

3. Domermuth, C. H., M. H. Nielsen, E. A. Freundt, and A.Birch-Andersen. 1964. Ultrastructure of Mycoplasmaspecies. J. Bacteriol. 88:727-744.

4. Freundt, E. A. 1958. The Mycoplasmataceae. Munksgaard,Copenhagen, Denmark.

5. Freundt, E. A. 1969. Cellular morphology and mode of repli-cation of the mycoplasmas, p. 281-315. In L. Hayflick (ed.),The mycoplasmatales and the L-phase of bacteria. Apple-ton-Century-Crofts, N.Y.

6. Furness, G. 1968. Analysis of the growth cycle of Mycoplasmaorale by synchronized division and by ultraviolet irradiation.J. Infect. Dis. 118:436-442.

7. Furness, G., F. J. Pipes, and M. J. McMurtrey. 1968. Analysisof the life cycle of Mycoplasma pneumoniae by synchronizeddivision and by ultraviolet and X irradiations. J. Infect. Dis.118:7-13.

8. Kellenberger, E., A. Ryter, and J. Sechaud. 1958. Electronmicroscope study of DNA-containing plasms. II. Vegetativeand mature phage DNA as compared with normal bacterial

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nucleoids in different physiological states. J. Biophys. Bio-chem. Cytol. 4:671-678.

9. Kelton, W. H. 1960. Growth-curve studies of pleuropneu-monia-like organisms. Ann. N.Y. Acad. Sci. 79:422-429.

10. Maniloff, J. 1967. Organization of small cells: the pleuro-pneumonia-like organisms. J. Cell Biol. 35:87A.

11. Maniloff, J. 1969. Electron microscopy of small cells: Myco-plasma hominis. J. Bacteriol. 100:1402-1408.

12. Maniloff, J. 1969. Use of blood agar plates to study Myco-plasma physiology. Microbios 1:125-135.

13. Maniloff, J., and H. J. Morowitz. 1967. Ultrastructure and lifecycle of Mycoplasma gallisepticum A5969. Ann. N.Y. Acad.Sci. 143:59-65.

14. Maniloff, J., H. J. Morowitz, and R. J. Barrnett. 1965. Studiesof the ultrastructure and ribosomal arrangements of thepleuropneumonia-like organism A5969. J. Cell Biol. 25:139-150.

15. Maniloff, J., H. J. Morowitz, and R. J. Barrnett. 1965. Ultra-structure and ribosomes of Mycoplasma gallisepticum. J.Bacteriol. 90:193-204.

16. Morowitz, H. J. and J. Maniloff, 1966. Analysis of the lifecycle of Mycoplasma gallisepticum. J. Bacteriol. 91:1638-1644.

17. Razin, S. and B. J. Cosenza. 1966. Growth phases of Myco-plasma in liquid media observed with phase-contrast micro-scope. J. Bacteriol. 91:858-869.

18. Razin, S., H. J. Morowitz, and T. M. Terry. 1965. Membranesubunits of Mycoplasma laidlawii and their assembly tomembrane-like structures. Proc. Nat. Acad. Sci. U.S.A. 54:219-225.

19. Setlow, R. B., and E. C. Pollard. 1962. Molecular biophysics.Addison-Wesley Publishing Co., Reading.

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