Growth Phases of Mycoplasma in Liquid MediaObserved with Phase-Contrast Microscope
SHMUEL RAZIN' AND BENJAMIN J. COSENZA
Department ofBacteriology, University of Connecticut, Storrs, ConnecticutReceived for publication 9 September 1965
ABSTRACT
RAZIN, SHMUEL (University of Connecticut, Storrs), AND BENJAMIN J. COSENZA.Growth phases of Mycoplasma in liquid media observed with phase-contrast micro-scope. J. Bacteriol. 91:858-869. 1966-Growth of 11 Mycoplasma strains in liquidmedia was followed by phase-contrast microscopy. A similar pattern of developmentwas common to all strains. Branching filaments, 0.3 to 0.4 ,u thick, characterized theearly logarithmic phase of growth. The length of the filaments varied according to thestrain tested and the growth medium. Addition of oleic acid to the medium inducedthe formation of very long filaments by M. laidlawii strain B. Upon aging, the fila-ments were found to break up into chains of coccoid elements. These chains furtherfragmented to yield shorter chains and single coccoid elements, which characterizedthe stationary and decline phases of growth. The size of the coccoid elements in-creased from 0.3 to 0.4 A,u when formed in the filaments, to 0.6 to 0.8 , after beingreleased from the chains. Further increase in the size of the cells took place at thedecline phase of growth, leading to the formation of very large cells reaching adiameter of 10 to 20 ,. However, these large cells had the appearance of emptyvesicles and were apparently nonviable as indicated by viable-count experiments.
The high plasticity of Mycoplasma cells, result-ing from the absence of cell walls, and the minutedimensions of the organisms have hampered mor-phological studies of this group. In spite of nu-merous morphological investigations carried outover the past 60 years, no agreement has yet beenreached concerning the mode of reproduction ofMycoplasma. According to Freundt (9, 10), My-coplasma cells go through a cycle of morphologi-cal changes during growth, commencing with asmall spherical particle, referred to as an elemen-tary body. From this elementary body, one ormore short thin filaments emerge. The filamentscontinue to grow in length, and frequently branch.Thus, during the first 12 to 18 hr of growth, amycelial structure develops. Then, within the fila-ments, regularly spaced spheres of uniform sizeand shape are formed. Subsequently, constrictionsdevelop between the spheres, dividing the filamentinto a chain of coccoid elements. The sphericalelements are liberated by disintegration of thechain and are considered to be new elementarybodies. Klieneberger-Nobel (13) and Dienes (4)
'Present address: Department of Clinical Micro-biology, The Hebrew University-Hadassah MedicalSchool, Jerusalem, Israel.
claimed that the elementary body of Mycoplasmagrows to a large disclike or spherical cell, referredto as a "large body." The cytoplasm of the "largebody" undergoes segmentation to give manyelementary bodies, released by rupture of the"large body." Reproduction of Mycoplasma bybudding was suggested by several authors (7, 8,14), and simple binary fission was claimed tooccur in M. gallisepticum (12). Anderson andBarile (1) suggested that Mycoplasma cells haveseveral alternate modes of reproduction, depend-ing on cultural conditions.The mycelial theory of growth suggested by
Freundt was rejected by several investigators, whoclaimed that the filaments were artifacts of themicroscopic procedure, resulting from the ex-treme plasticity of the cells (4, 14). Though theseauthors conceded that filaments are found in M.mycoides cultures, they did not accept filamentousgrowth as a feature common to all Mycoplasmaspecies. The present investigation, based on exam-ination of unfixed specimens with a phase-con-trast microscope, under conditions minimizingartifact formation, demonstrates the presence of afilamentous phase of growth in a representativeseries of Mycoplasma species.
MATERIALS AND METHODSOrganisms. M. laidawii strain A (PG 8), M.
laidawii strainB (PG 9), M. neurolyticum (PG 28), andM. bovigenitalium (PG 11) were obtained from D. G.ff. Edward (Wellcome Research Laboratories, Becken-ham, Kent, England). M. gallisepticum strain A5969,M. gallinarum, and M. agalactiae var. bovis (11) wereobtained from M. E. Tourtellotte (Department ofAnimal Diseases, University of Connecticut, Storrs).M. hominis ATCC 14152 and M. arthritidis ATCC14124 were kindly provided by R. G. Wittler (WalterReed Army Institute of Research, Washington,D.C.). M. hominis strain H39 was obtained from P. F.Smith (Department of Microbiology, School ofMedicine, University of South Dakota, Vermillion).Mycoplasma sp. strain 14 (goat strain), was obtainedfrom H. E. Adler (School of Veterinary Medicine,University of California, Davis).
Media and growth conditions. M. laidlawii strains Aand B, M. gallisepticum, and Mycoplasma sp. strain 14were grown in tryptose broth of the following com-position (per liter): tryptose (Difco), 20 g; NaCl, 5 g;tris(hydroxymethyl)aminomethane (Tris), 5 g; glu-cose, 7 g; penicillin G (crystalline), 100,000 units; andPPLO Serum Fraction (Difco), 10 ml. The pH of themedium was 8.2 to 8.4 without adjustment. All otherMycoplasma strains were grown in a modified liquidEdward medium (17). The effect of oleic acid ongrowth and morphology of M. laidlawii strain B wastested in the tryptose broth devoid of PPLO SerumFraction, and supplemented with 0.4% of bovinealbumin fraction V (Calbiochem), extracted withacetone as described by Razin and Rottem (18). Thefatty acid was added to the medium in ethyl alcoholicsolution.
Growth was carried out in 50-ml quantities ofmedium, dispensed in 200-ml bottles. Each bottlereceived 1 ml of a 1:100 dilution of a 24-hr culture asthe inoculum. The bottles were incubated statically at37 C, and samples were withdrawn at various timeintervals for viable counts and microscopic examina-tion. The number of viable particles in cultures wasestimated by the technique of Butler and Knight (3),with the use of tryptose broth for dilutions and thesame medium solidified with 1% agar (Difco, certified)for plating.
Microscopy and photomicrography. Small drops ofeach culture of the Mycoplasma strains were put onglass slides, covered with cover slips, and examinedwith a Wild M20 microscope. The Wild Fluotar phaseobjective HI 50/1.00 and a X10 or X20 eyepiecewere routinely used. The illumination source was aWild low-voltage lamp. The organisms were photo-graphed, as soon as they settled on the slide, with aLeitz Wetzler camera, by use of Kodak ContrastProcess panchromatic film. Exposure time was usually2 to 3 sec. The total microscopic magnification was625 times. Prints were made on Kodabromide (F-3)paper to a final magnification of 1,250, 2,700, or 3,850times.
RESULTSGrowth curves of M. laidlawii strain B, M.
gallisepticum, and Mycoplasma sp. strain 14 in
F MYCOPLASMA 859
tryptose broth are presented in Fig. 1 and 2. M.laidlawii strain B could grow in tryptose brothdevoid of PPLO Serum Fraction, whereas M.laidlawii strain A grew very poorly in this medium.M. gailisepticum and Mycoplasma sp. strain 14could not grow in tryptose broth without PPLOSerum Fraction.Growth of M. laidlawii strain B in tryptose
broth without PPLO Serum Fraction was charac-terized by a steep decline in the number of viablecells after the logarithmic phase of growth (Fig.1). Addition of 1 % PPLO Serum Fraction to themedium reduced this decline in viable counts. Amore pronounced effect on viability was shown byreplacing the PPLO Serum Fraction with a mix-ture of oleic acid and acetone-extracted bovinealbumin (Fig. 1). M. Iaidlawii strain B grew inthis medium in very long and branching filaments,many of which were composed entirely, or in part,of uniform coccoid elements (Fig. 3 and 4). Longchains of coccoid elements could be seen even atthe decline phase of growth; however, many ofthe coccoid elements became swollen and ghost-like (Fig. 6). Very thin threads could be seenconnecting the cocci (Fig. 5). When M. laidlawiistrain B was grown in tryptose broth supple-mented with 1 % PPLO Serum Fraction, shortbranching filaments were observed only at theearly logarithmic phase of growth. Shortly after,
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FIG. 1. Growth of Mycoplasma laidlawii strain B intryptose broth containing: 1% PPLO Serum Fraction(0); 0.4% acetone-extracted bovine albumin plus 50,Ag/ml of oleic acid (A); or no PPLO Serum Fraction(0).
FIG. 2. Growth of Mycoplasma sp. strain 14 (0)and M. gallisepticum (A) in tryptose broth containing1% PPLO Serum Fraction.
the filaments were fragmented, giving rise tochains of coccoid elements of different diameters(Fig. 7). At the stationary and decline phases ofgrowth, some large cells surrounded by numerousbuds or vesicles could frequently be seen (Fig. 8).Growth of M. laidlawii strain B in tryptose brothwith no PPLO Serum Fraction or added oleicacid was distinguished by a very short filamentousphase, followed by the appearance of short chainsof cocci (Fig. 9). The decline phase of growth inthis medium was characterized by the formationof very large ghosts (Fig. 10), some of which werestill arranged in chains (Fig. 11).Growth of M. laidlawii strain A in tryptose
broth containing 1% PPLO Serum Fraction fol-lowed basically the same pattern showed by M.laidlawi strain B. Long branching filaments, manyof which were beaded, characterized the earlylogarithmic phase of growth (Fig. 12 and 13).Microcolonies composed of entangled filamentscould be seen in the liquid medium (Fig. 12). Asthe culture aged, the filaments fragmented to givevery short chains of cocci (Fig. 14 and 15). At thedecline phase of growth, the cocci, now appearingmostly singly, became bigger and swollen, havinga ghostlike appearance.
Branching filaments and chains of small coccicharacterized the early logarithmic phase of M.gallisepticum (Fig. 16 and 17). The chains became
shorter as the culture aged (Fig. 18-20). Cocco-bacillary bodies (13), assembled in small aggre-gates, formed the typical picture at the end of thelogarithmic phase (Fig. 21). Cells with attachedbuds or "budlike" structures are seen in Fig. 22.Mycoplasma sp. strain 14 grew as branching
filaments in the tryptose broth enriched with 1%PPLO Serum Fraction (Fig. 23). Cells of thisstrain demonstrated a much higher tendency tocollapse on contact with the glass surface of theslide than did other Mycoplasma cells (Fig. 24).Good photomicrographs of filaments could there-fore be obtained only within a short time intervalafter the filaments had settled on the slide. Aswith the other Mycoplasma, the filaments weretransformed to chains of coccoid elements andbecame much shorter at the late logarithmic phaseof growth (Fig. 25 and 26). The chains disinte-grated finally to yield single cells (Fig. 27). Duringthe decline phase of growth, most of these cellslost their contents and appeared as empty ghosts.A sticky precipitate usually appeared at the bot-tom of the growth medium during the late declinephase, and consisted essentially of agglutinatedghosts and cell debris (Fig. 28). Agglutination wasapparently caused by deoxyribonucleic acidreleased from cells (Razin, unpublished data).Some of the forms appearing at the decline phaseare presented in Fig. 29-32. These forms includethe remnants of the chains characterizing earlierphases of growth (Fig. 29 and 32), and someforms described frequently in the literature, suchas the "large body" containing dark granules(Fig. 30) and an "asteroid" (Fig. 31).In all other Mycoplasma strains examined, es-
sentially the same morphological changes oc-cuffed during the various growth phases. Branch-ing filaments or chains of cocci, which appearedearly in the logarithmic phase, changed into shortchains of cocci, or single cells, at later growthphases. The length of the filaments formed de-pended on the strain. For example, M. neurolyt-icum produced extremely long filaments (Fig.38-40). The filament in Fig. 38 is more than 160 ,uin length. M. hominis and M. arthritidis formedfilaments of moderate length (Fig. 33-36),whereas M. agalactiae var. bovis (Fig. 42 and 43),M. bovigenitalium (Fig. 37), and M. gallinarumproduced very short filaments or chains of cocci.
Size of organisms. An estimation of the size ofthe elements appearing in Mycoplasma culturescould be obtained by measuring the dimensions oftheir images on the photomicrographic prints.The pronounced variety in size of Mycoplasmaorganisms is well known, and could be also estab-lished in the present investigation. This propertywas especially striking on analysis of cultures at
FIG. 3-6. Mycoplasma laidlawii strain B. Growth medium: tryptose broth devoid ofPPLO Serum Fraction ancdsupplemented with 0.4% acetone-extracted bovine albumini plus 50 lg/ml of oleic acid; (3) 13-hr culture; (4) 36-hrculture; (5 and 6) 60-hr culture. The gradual disinitegration of the filamelnts and the appearance of ghosts are no-
FIG. 12-15. Mycoplasma laidlawii strain A. Growth medium: tryptose broth conitaining 1% PPLO Serum Frac-tion; (12) 15-hr culture, a microcolony composed of entangled filaments, X 1,250; (13) 36-hr culture, filamentsand long chains ofcocci are still seen, X 1,250; (14 and 15) 45-hr culture, most cells appear singly, in pairs, or invery short chains, X 2,700.
the decline phase of growth, where many of thecells swelled enormously and formed "largebodies" typical of aged Mycoplasma cultures.However, the size of cells was more uniform at theearlier stages of growth. Thus, the caliber of thefilaments appearing in all Mycoplasma strainstested was quite uniform, varying between 0.3 and0.4 ,u. The diameter of the coccoid elements lib-erated by fragmentation of the filaments wasabout 0.3 to 0.4 IA just after fragmentation, butusually increased at later growth phases to 0.6 to0.8 ,u, without significant decrease in the density
of the cytoplasm. However, the diameter of theindividual cells of the more exacting Mycoplasma,especially those of M. hominis and M. bovigeni-talium, rarely exceeded 0.4 A.
DIscussIoN
The demonstration of a filamentous phase ofgrowth in a representative series of Mycoplasmaspecies confirms the observations of Freundt (9,10), and supports his theory on the mode of re-
production of Mycoplasma. In agreement withFreundt, the "large bodies" observed in the pres-
FiG. 16-22. Mycoplasma gailisepticum. Growth medium: tryptose broth containing 1% PPLO Serum Fraction;(16) 16-hr culture, a small microcolony composed of entangled filaments, X 1,250; (17) 16-hr culture, short andbranching filaments, X 1,250; (18-20) 24-hr culture, various forms of short, sometimes branched, chains of cocci,X 2,700; (21) 48-hr culture, aggregates of coccobacillary organisms, X 2,700; (22) 48-hr culture, "budlike" pro-trusions on cells, X 3,850.
FIG. 23-28. Mycoplasma sp. strain 14 (goat strain). Growth medium: tryptose broth containting 1% PPLO SerumFraction; (23) 21-hr culture, higlhly brancthed filaments, X 1,250; (24) 21-hr culture, collapsing cells on the glassslide, X 1,250; (25) 27-hr culture, branching filamentts are still seen, X 2,700; (26) 39-hr culture, the filamentsfragmenited to yield short chains of cocci, X 2,700. (27) 64-hr culture, cells appear mostly sinigly, many have a di-ameter greater than I I-, X 2,700; (28) 64-hr culture, aggregates of swollen distorted cells and ghosts, X 2,700.
ent study seem to be involution forms of low vi-ability, and do not represent a reproductive stageas suggested by others (4, 13). However, not beingable to follow the growth of a single Mycoplasmacell in a liquid medium, all our assumptions re-garding the mode of reproduction must be basedon the sequence of microscopic pictures, and can-not, therefore, constitute a definite proof.A problem still to be answered is whether bud-
ding plays any role in multiplication of Myco-plasma. Cells containing one or more "budlike"structures are most commonly seen at the latelogarithmic phase of growth. These "budlike"structures may be interpreted as remnants of theplasma thread that connected the cells in thechain. The growing or swelling of one cell withina short chain of Mycoplasma may also produce apicture reminiscent of budding.The high sensitivity of Mycoplasma cells to
collapse on contact with a solid surface, such asthe glass slide, was noticed in all microscopicpreparations, and was especially pronouncedwith Mycoplasma sp. strain 14. The collapse ofspherical Mycoplasma cells to form flat discikebodies is known to take place on an agar surface(13) or on grids prepared for electron microscopy.The term "platycytes" was coined by Shifrine,Pangborn, and Adler (19) to describe these flatcells. The collapse of Mycoplasma cells on a solidsurface is apparently responsible for the formationof some other morphological entities describedfrequently in the literature, such as the "asteroid"form. To avoid as much as possible the morpho-logical changes that follow the contact of Myco-plasma cells with the glass surface, the photomi-crographs in the present study were taken justafter the organisms had settled on the glass sur-face. At that time the organisms which had settledstill had retained the shape of those moving freelyin the liquid phase. Weibull and Lundin (20, 21,22) photographed cells of several Mycoplasmastrains moving freely in a liquid medium, with anelectronic flash as the light source. Although theirphotomicrographs show little detail, they demon-strate the presence of filaments in several Myco-
plasma, and the fragmentation of the filamentsduring growth. When the filaments were trans-ferred onto a solid medium, they were convertedto spherical bodies, apparently by collapsing (21).
Although the filaments produced by the differ-ent Mycoplasma strains were approximately ofthe same caliber (0.3 to 0.4 A), the cells resultingfrom fragmentation of these filaments reacheddifferent sizes in the various strains. Thus, cellsof M. laidlawii reached a diameter of 0.6 to 0.8 ,without any decrease in density of the cytoplasm,indicating their viability, whereas cells of M.hominis did not exceed 0.4 ,u in diameter. Thesedifferences in size of cells can perhaps be corre-lated with recent findings showing marked differ-ences in the size of the genome of the variousstrains. The genome of M. hominis strain H39 wasestimated to have a molecular weight of 380 x106 daltons (2), whereas that of M. laidlawii seemsto have a molecular weight of about 800 x 106daltons (H. J. Morowitz, personal communica-tion). Determination of the size of Mycoplasmaorganisms by the method used in the present in-vestigation appears to be preferable to methodsinvolving measurements of the diameter of sec-tioned cells (1, 5) or filtration through Milliporefilters (16). The values obtained by measuringsectioned cells vary with the plane of the sectionthrough the cell, and filtration experiments sufferfrom an unknown degree of squeezing of theplastic cells when they are forced through thepores of the filter under pressure.Of special significance is the finding of filamen-
tous growth in M. gallisepticum. The identity ofthis organism as a "true" Mycoplasma has beenquestioned by Klieneberger-Nobel (13), and in-deed thin sections of this organism have shownsome unique features not found in any otherMycoplasma (6, 15). The formation of filamentsby this organism favors its inclusion in the Myco-plasma group.The effects of oleic acid and other long-chain
fatty acids on growth, morphology, and osmoticfragility of M. laidlawii will be described in detailelsewhere.
FIG. 29-32. Mycoplasma sp. strain 14 (goat strain). Growth medium: tryptose broth containing 1% PPLO SerumFraction; (29 and 32) 64-hr culture, damaged and swollen cells still showing the original chain arrange-ment, X 2,700; (30) 64-hr culture, a "large body" containing dense granules, X 2,700; (31) 64-hr culture, an "as-teroid" form, X 3,850.
FIG. 33-34. Mycoplasma hominis ATCC 14152; 24-hr culture in Edward broth showing filamentous growth.X 2,700.
FIG. 35. Mycoplasma hominis strain H39; 24-hr culture in Edward broth showing beadedfilaments. X 2,700.FIG. 36. Mycoplasma arthritidis; 24-hr culture in Edward broth showing a longfilament. The point ofattachment
of the filament to the glass slide is marked by the bleb that appeared in the filament at that point. X 2,700.FIG. 37. Mycoplasma bovigenital:um; 24-hr culture in Edward broth showing short chains of cocci. X 2,700.
FIG. 38-41. Mycoplasma neurolyticum. Growth medium: Edward broth; (38) 24-hr culture, an extremely longfilament, X 1,250; (39) 24-hr culture, entangled beadedfilaments, X 1,250; (40) 24-hr culture, a beadedfilament,at the point ofattachment to the glass, an asteroidform was produced, X 2,700; (41) 48-hr culture, the filamentsdisintegrated to form very short chains ofcocci, X 1,250.
FiG. 42-43. Mycoplasma agalactiae var. bovis; 24-hr culture in Edward broth showing thepresence ofshort fila-ments; (42) X 1,250; (43) X 2,700.
Shmuel Razin held a Senior Foreign ScientistFellowship from the National Science Foundationduring this work.
Contribution no. 128 of the Institute of CellularBiology, University of Connecticut.
LITERATURE CITED
1. ANDERSON, D. R., AND M. F. BARILE. 1965.Ultrastructure of Mycoplasma lhominiis. J.Bacteriol. 90:180-192.
2. BODE, H. R., AND H. J. MOROWITZ. 1965. DNAcontent of the PPLO (Strain H-39) genomeusing the Kleinschmidt procedure. Proc. Bio-physical Soc., p. 109.
3. BUTLER, M., AND B. C. J. G. KNIGHT. 1960. Thesurvival of washed suspensions of Mycoplasma.J. Gen. Microbiol. 22:470-477.
4. DIENES, L. 1960. Controversial aspects of themorphology of PPLO. Ann. N.Y. Acad. Sci.79:356-368.
5. DOMERMUTH, C. H., M. H. NIELSEN, E. A.FREUNDT, AND A. BIRCH-ANDERSEN. 1964.Ultrastructure of Mycoplasma species. J.Bacteriol. 88:727-744.
6. DOMERMUTH, C. H., M. H. NIELSEN, E. A.FREUNDT, AND A. BIRCH-ANDERSEN. 1964.Gross morphology and ultrastructure of Myco-plasma gallisepticumn. J. Bacteriol. 88:1428-1432.
7. DUTrA, S. K., R. E. DIERKS, AND B. S. POMEROY.1965. Electron microscopic studies of themorphology and the stages of development ofMycoplasma gallisepticaim. Avian Diseases9:241-251.
8. EDWARDS, G. A., AND J. FOGH. 1960. Fine struc-ture of pleuropneumonia-like organisms inpure culture and in infected tissue culture cells.J. Bacteriol. 79:267-276.
9. FREUNDT, E. A. 1958. The Mycoplasmataceae.Munksgard, Copenhagen, Denmark.
10. FREUNDT, E. A. 1960. Morphology and classifica-
tion of the PPLO. Ann. N.Y. Acad. Sci. 79:312-325.
11. HALE, H. H., C. F. HELMBOLDT, W. N.PLASTRIDGE, AND E. F. STULA. 1962. Bovinemastitis caused by a Mycoplasma species.Cornell Vet. 52:582-591.
12. KELTON, W. H. 1962. Synchronized division ofavian pleuropneumonia-like organisms. J.Bacteriol. 83:948-955.
13. KLIENEBERGER-NOBEL, E. 1962. Pleuropneumonia-like organisms (PPLO): Mycoplasmataceae.Academic Press, Inc., New York.
14. LIEBERMEISTER, K. 1960. Morphology of thePPLO and L forms of Pr-oteits. Ann. N.Y.Acad. Sci. 79:326-343.
15. MANILOFF, J., H. J. MOROWITZ, AND R. J.BARRNETT. 1965. Ultrastructure and ribosomesof Mycoplasma gallisepticmni. J. Bacteriol. 90:193-204.
16. MOROWITZ, H. J., M. E. TOURTELLOTTE, AND M.E. POLLACK. 1963. Use of porous celluloseester membranes in the primary isolation andsize determination of pleuropneumonia-likeorganisms. J. Bacteriol. 85:134-136.
17. RAZIN, S. 1963. Osmotic lysis of Mycoplasma. J.Gen. Microbiol. 33:471-475.
18. RAZIN, S., AND S. ROTTEM. 1963. Fatty acidrequirements of Mycoplasma laidlawil. J. Gen.Microbiol. 33:459-470.
19. SHIFRINE, M., J. PANGBORN, AND H. E. ADLER.1962. Colonial growth of Mycoplasmna galli-septicum observed with the electron micro-scope. J. Bacteriol. 83:187-192.
20. WEIBULL, C., AND B. M. LUNDIN. 1962. Size andshape of pleuropneumonia-like organisms(PPLO) grown in liquid media. J. Bacteriol. 84:513-519.
21. WEIBULL, C., AND B. M. LUNDIN. 1963. Morphol-ogy of pleuropneumonia-like organisms andbacterial L forms grown in liquid media. J.Bacteriol. 85:440-445.
22. WEIBULL, C., AND B. M. LUNDIN. 1963. Directdemonstration of the fragmentation of Myco-plasma (PPLO) filaments grown in liquidmedium. Exptl. Cell Res. 32:414-415.
