THE JOURNAL OF INFECTIOUS DISEASES • VOL. 134, NO.3· © 1976 by the University of Chicago. All rights reserved.
SEPTEMBER 1976
Morphology, Ultrastructure, and Mode of Division of Mycoplasma fermentans, Mycoplasma hominis, Mycoplasma orale, and Mycoplasma salivarium Geoffrey Furness, Jack Whitescarver, * Marsha Trocola, and Maria DeMaggio
From the Department of Microbiology, College of Medicine and Dentistry of New Jersey, New Jersey Medical School, and the Graduate School of Biomedical Sciences, Newark, New Jersey
T-mycoplasmas in exponential-phase cultures (in T-broth) divide by budding, and the buds are produced by invagination of the plasma membrane [1]. We have reported that the classical human mycoplasmas, Mycoplasma fermentans, Mycoplasma hominis, Mycoplasma orale types 1 and 2, and Mycoplasma salivarium, in the exponential phase of growth (in Eaton agent broth cultures) also replicate by budding [2]. However, the ultrastructure of these mycoplasmas during the process of division was not studied. Therefore, we have compared the gross mor-
phology, ultrastructure, and mode of division of budding mycoplasmas by the techniques used previously for study of the T-mycoplasmas [1]. Materials and Methods
The source of M. fermentans, M. hominis, M. orale types 1 and 2, and M. salivarium have been reported [3]. The techniques for titrating viable mycoplasmas by colony counts [4] and for obtaining single-cell suspensions from cfu containing more than one organism have been described [3]. Screw-capped tubes of modified Eaton agent broth [3], which had been filtered through Nalgene membranes (pore diameter, 0.2 urn; Sybron Corp., Rochester, N.Y.) for removal of any artifacts resembling mycoplasmas [1], were inoculated with single-cell suspensions of the mycoplasmas and incubated in a waterbath at 37 C for 30-50 hr, at which time the organisms were in the log phase [2]. That the broth cultures from which aliquots were removed for microscopy were in the exponential phase of growth was confirmed by consecutive viable counts.
Received for publication October 30, 1975, and in revised form February 5, 1976. This research was supported by grant no. RO 1 A108282 from the National Institute of Allergy and Infectious Diseases. Please address requests for reprints to Dr. Geoffrey Furness, Department of Microbiology, College of Medicine and Dentistry of New Jersey, New Jersey Medical School, 100 Bergen Street, Newark, New Jersey 07103. * Present address: Department of Microbiology, Harvard School of Public Health, 665 Huntington Avenue, Boston, Massachusetts 02115.
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The morphology of viable Mycoplasma [ermentans, Mycoplasma hominis, Mycoplasma orale types 1 and 2, and Mycoplasma salivarium was studied in broth cultures by interference microscopy and in thin sections by electron microscopy. Only spherical cells were seen by interference microscopy. M. hominis had a capsule-like outer layer. Except for M. orale type I, mycoplasmas in thin sections were 0.3-1 urn in diameter, with a bounding trilaminar membrane 7.5-10 nm thick. The mycoplasmas contained DNA fibrils and randomly distributed ribosomes. No polyribosomes were seen. Dividing mycoplasmas elongated slightly; the membrane invaginated, forming one bud. Sometimes M. hominis and M. salivarium produced one bud by elongation, and the bud was attached by a tube. This method of division is not considered as characteristic but rather as due to centrifugal force separating unfixed cells during preparation for electron microscopy. Cross-septa were never observed. In thin sections M. orale type 1 was elongated and without buds, an observation which suggested that preparation for electron microscopy distorted the mycoplasmas.
Morphological Study of Human Mycoplasmas
copy to contain spherical or slightly ovoid mycoplasmas (figure 1). However, both single cells and clumps of M. hominis had a blurred outline, an observation which indicated that this mycoplasma produced a gelatinous capsule-like matrix (figure 2). Electron microscopy. With the exception of M. orale type 1, which in fixed preparations consisted of elongated forms without buds or evidence of replication (figure 3), individual exponentialphase mycoplasmas were seen in electron micrographs of thin sections as round or ovoid microorganisms, 0.3-1 urn in diameter. The majority of cells had a diameter of 0.4-0.6 um (figure 4). The mycoplasmas were bounded by a trilaminar membrane 7.5-10 run thick and contained DNA strands and ribosomes randomIy distributed throughout the cells (figure 4). No polyribosomes or geometric patterns of ribosomes were observed in any of these classical species.
Results
Viable exponential-phase broth cultures of the five species of human mycoplasmas, M. fermentans, M. hominis, M. orale types 1 and 2, and M. salivarium, were shown by interference micros-
Figure 1. Viable Mycoplasma salivarium from an exponential-phase broth culture showing that classical mycoplasmas are spheres (interference [Nomarski] microscopy, X 6,000; the bar represents 1 f-lm).
Figure 2. Viable Mycoplasma hominis from an exponential-phase broth culture showing clumps and single cells surrounded by a gelatinous matrix (interference [Nomarski] microscopy, X 4,680; the bar represents 1 urn).
Downloaded from http://jid.oxfordjournals.org/ at Columbia University Libraries on February 14, 2015
Light and electron microscopy. The gross morphology of viable mycoplasmas was observed directly in wet preparations. A drop of the broth culture was placed on a microscope slide, covered with a coverslip, sealed with a mixture of paraffin and vaseline to prevent evaporation, and examined (magnification, Xl,OOO) by interference (Normarski) illumination. For photography, the cells were fixed in situ by mixing of the broth culture with an equal volume of warm trypticase soy broth containing gelatin (20% wt/vol) [1]. For electron microscopy, pellets of mycoplasmas were obtained by centrifugation (14,500 g for 30 min) of 100-mI portions of exponentialphase broth cultures containing about 107 mycoplasmas/rnl. Thin sections of the pellets were prepared by the technique used previously for the T-mycoplasmas [1].
225
226
Figure 4. Thin section of Mycoplasma [ermentans illustrating round to ovoid cells containing DNA fibrils and randomly dispersed ribosomes (electron microscopy, X50,000; the bar represents 1 um) .
Division of the mycoplasmas most frequently commenced with slight elongation of the cell. At this time ribosomes tended to concentrate at each end of the cell, and invagination of the membrane began. The invagination deepened, and the mycoplasma divided unequally to form a single bud densely packed with ribosomes (figure 5) . Thereafter, on completion of invagination (figure 6) the cells could separate. Sometimes M. hominis and M. salivarium formed buds by elongation, and the single bud was attached to the parent cell by a tubular structure (figures 7 and 8). Budding by elongation was seen much less frequently than was division by invagination. A cross-septum was not formed; no mycoplasmas were observed producing two buds simultaneously. Moreover, the capsule-like outer layer of M. hominis was not detected in thin sections.
Discussion
Only spherical or ovoid mycoplasmas were seen by interference (Nomarski) microscopy in exponential-phase broth cultures of the human classical mycoplasmas, M. [ermentans, M. hominis, M. orale types 1 and 2, and M. salivarium. A capsular matrix also was visible around single cells and clumps of M. hominis. These observations of viable cells confirm our previous report of the morphology of the same strains seen by electron microscopy in pseudoreplicas [2]. None of these mycoplasmas had any distinguishing gross morphological features as do Mycoplasma gallisepticum, which has a terminal bleb [5], Mycoplasma pneumoniae, which may have a terminal knob-like structure [6], and the T-mycoplasmas, which have a dense outer layer of hair or pilus-like structures [1, 7, 8].
Downloaded from http://jid.oxfordjournals.org/ at Columbia University Libraries on February 14, 2015
Figure 3. Thin section of Mycoplasma orale type 1 from an exponential-phase broth culture showing elongated forms without buds (electron microscopy, X 32,000; the bar represents 1 um),
Furness et al.
Morphological Study of Human Mycoplasmas
227
Figure 6. Thin section of Mycoplasma hominis when invagination of the membrane was complete and a daughter cell was formed (electron microscopy, x60,OOO; the bar represents 1 urn).
Thin sections have the advantage of revealing not only the gross morphology of the mycoplasmas but also their ultrastructure, which cannot be observed either by interference microscopy or by the pseudoreplica technique. With the
exception of M. orale type 1, thin sections of these classical mycoplasmas showed that they contained DNA fibrils and randomly distributed ribosomes. This finding contrasts markedly with that in T-mycoplasmas, which form polyribo-
Figure 7. Thin section of Mycoplasma salivarium showing final stage of budding by elongation. The parent cell is elongated and is connected to the daughter cell by a membrane tubule (electron microscopy, x50,OOO; the bar represents 1 urn).
Figure 8. Thin section of Mycoplasma hominis showing two round cells connected by a membrane tubule (electron microscopy, x60,OOO; the bar represents 1 um).
Downloaded from http://jid.oxfordjournals.org/ at Columbia University Libraries on February 14, 2015
Figure S. Thin section of Mycoplasma hominis showing characteristic unequal division of budding cells with smaller daughter cell densely packed with ribosomes (electron microscopy, X50,OOO; the bar represents 1 urn).
Furness et al.
228
duced during the preparation of the mycoplasmas for electron microscopy. Although fixation prior to harvesting undoubtedly reduces the incidence of distortion of mycoplasmas during preparation for microscopy [13, 14], these observations confirm our previous findings, namely, that the effects of techniques for preparing mycoplasmas for electron microscopy are unpredictable, that pseudoreplication is the more reliable method for study of the gross morphology of mycoplasmas by electron microscopy, and that the gross morphology of viable cells should be ascertained by interference microscopy before preparation of mycoplasmas for electron microscopy [15].
References 1. Whitescarver, J., Furness, G. T-mycoplasmas: a study of the morphology, ultrastructure and mode of division of some human strains. J. Med. Microbiol. 8:349-355, 1975. 2. Furness, G. The growth and morphology of mycoplasmas replicating in synchrony. J. Infect. Dis. 122: 146-158, 1970. 3. Furness, G. Differential responses of single cells and aggregates of mycoplasmas to ultraviolet irradiation. Appl. Microbiol. 18: 360-364, 1969. 4. Furness, G., Pipes, F. J., McMurtrey, M. J. Susceptibility of human mycoplasmata to ultraviolet and X irradiations. J. Infect. Dis. 118: 1-6, 1968. 5. Allen, T. C., Stevens, J. 0., Florance, E. R., Hampton, R. O. Ultrastructure of Mycoplasma gallisepticum isolate 1056. J. Ultrastruct. Res. 33:318-331, 1970. 6. Biberfeld, G., Biberfeld, P. Ultrastructural features of Mycoplasma pneumoniae. J. Bacteriol. 102: 855-861, 1966. 7. Williams, M. H. Electron microscopy of T-strains. Ann. N.Y. Acad. Sci. 143:397-400, 1967. 8. Black, F. T., Birch-Andersen, A., Freundt, E. A. Morphology and ultrastructure of human T-mycoplasmas. J. Bacteriol. 111: 254-259, 1972. 9. Furness, G., DeMaggio, M. The growth cycle of Mycoplasma mycoides var. mycoides. J. Infect. Dis. 127:563-566, 1973. 10. Furness, G., DeMaggio, M. Binucleate classical mycoplasmas pathogenic for goats. Infec. Immun. 5:433-441, 1972. 11. Maniloff, J., Morowitz, H. J. Cell biology of the mycoplasmas. Bacteriol. Rev. 36:263-290, 1972. 12. Anderson, D. R., Barile, M. F. Ultrastructure of
Downloaded from http://jid.oxfordjournals.org/ at Columbia University Libraries on February 14, 2015
somes with a characteristic geometric arrangement of the constituent ribosomes [2, 7]. Both the classical and the T-mycoplasmas have a trilaminar plasma membrane and divide by budding, a form of replication that also is observed in pseudoreplicas [1, 2]. However, in the pseudoreplica technique, the classical mycoplasmas produce one bud [2], whereas the type species, Mycoplasma mycoides var. mycoides [9], Mycoplasma mycoides var. capri [10], and the T-mycoplasmas [1] can form two buds simultaneously. Elongated forms of M. hominis with a daughter cell connected by a tubule have been observed; this mode of division is considered typical of the species [11]. Mycoplasmas dividing by both invagination of the membrane and elongation of the cell (the latter mode of division resulting in a daughter cell attached to the parent mycoplasma by a tubule) were observed simultaneously in exponential-phase broth cultures of M. hominis and M. salivarium. Mycoplasmas were seen most often to be dividing by invagination; consequently, invagination is considered to be the characteristic mode of division of these mycoplasmas. These cultures were cloned from colonies grown from single-cell suspensions and thus are pure cultures [2]. Therefore, it is believed that the two methods of budding found in the same culture are similar in that they do not involve the formation of a septum. Cells joined by a tubule are not seen in pseudoreplicas [2]; such joined cells appear only in thin sections, and their appearance is probably related to the separation of the cells. The joined cells could be a consequence of centrifugal force on unfixed, dividing mycoplasmas during the preparation of the pellet for thin sectioning since the larger parent cell is unlikely to be deposited at the same rate as the smaller daughter cell. This factor could cause pulling apart of the two cells. In exponential-phase broth cultures, M. orale type 1 was seen by interference microscopy to consist of round to ovoid cells and by electron microscopy of pseudoreplicas as spheres producing a single bud [2]. Thin sections of similar cultures of the same strain contained elongated forms without buds or any evidence of replication. Therefore, even though these elongated forms have been observed previously [12, 13], we consider that they are distorted cells pro-
Morphological Study of Human Mycoplasmas
Mycoplasma orale isolated from patients with leukemia. J. Natl. Cancer Inst. 36: 161-167, 1966. 13. Lemcke, R. M. Osmolar concentration and fixation of mycoplasmas. J. Bacteriol. 110: 1154-1162, 1972. 14. Maniloff, J., Morowitz, H. J., Barrnett, R. J. Ultra-
229
structure and ribosomes of Mycoplasma gallisepticum. J. Bacteriol, 90: 193-204, 1965. 15. Furness, G., DeMaggio, M., Whitescarver, J. Morphological changes induced during fixation of Mycoplasmas mycoides for electron microscopy. Tex. Rep. BioI. Moo. 33:415-422, 1976.
Downloaded from http://jid.oxfordjournals.org/ at Columbia University Libraries on February 14, 2015
SEPTEMBER 1976
Morphology, Ultrastructure, and Mode of Division of Mycoplasma fermentans, Mycoplasma hominis, Mycoplasma orale, and Mycoplasma salivarium Geoffrey Furness, Jack Whitescarver, * Marsha Trocola, and Maria DeMaggio
From the Department of Microbiology, College of Medicine and Dentistry of New Jersey, New Jersey Medical School, and the Graduate School of Biomedical Sciences, Newark, New Jersey
T-mycoplasmas in exponential-phase cultures (in T-broth) divide by budding, and the buds are produced by invagination of the plasma membrane [1]. We have reported that the classical human mycoplasmas, Mycoplasma fermentans, Mycoplasma hominis, Mycoplasma orale types 1 and 2, and Mycoplasma salivarium, in the exponential phase of growth (in Eaton agent broth cultures) also replicate by budding [2]. However, the ultrastructure of these mycoplasmas during the process of division was not studied. Therefore, we have compared the gross mor-
phology, ultrastructure, and mode of division of budding mycoplasmas by the techniques used previously for study of the T-mycoplasmas [1]. Materials and Methods
The source of M. fermentans, M. hominis, M. orale types 1 and 2, and M. salivarium have been reported [3]. The techniques for titrating viable mycoplasmas by colony counts [4] and for obtaining single-cell suspensions from cfu containing more than one organism have been described [3]. Screw-capped tubes of modified Eaton agent broth [3], which had been filtered through Nalgene membranes (pore diameter, 0.2 urn; Sybron Corp., Rochester, N.Y.) for removal of any artifacts resembling mycoplasmas [1], were inoculated with single-cell suspensions of the mycoplasmas and incubated in a waterbath at 37 C for 30-50 hr, at which time the organisms were in the log phase [2]. That the broth cultures from which aliquots were removed for microscopy were in the exponential phase of growth was confirmed by consecutive viable counts.
Received for publication October 30, 1975, and in revised form February 5, 1976. This research was supported by grant no. RO 1 A108282 from the National Institute of Allergy and Infectious Diseases. Please address requests for reprints to Dr. Geoffrey Furness, Department of Microbiology, College of Medicine and Dentistry of New Jersey, New Jersey Medical School, 100 Bergen Street, Newark, New Jersey 07103. * Present address: Department of Microbiology, Harvard School of Public Health, 665 Huntington Avenue, Boston, Massachusetts 02115.
224
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The morphology of viable Mycoplasma [ermentans, Mycoplasma hominis, Mycoplasma orale types 1 and 2, and Mycoplasma salivarium was studied in broth cultures by interference microscopy and in thin sections by electron microscopy. Only spherical cells were seen by interference microscopy. M. hominis had a capsule-like outer layer. Except for M. orale type I, mycoplasmas in thin sections were 0.3-1 urn in diameter, with a bounding trilaminar membrane 7.5-10 nm thick. The mycoplasmas contained DNA fibrils and randomly distributed ribosomes. No polyribosomes were seen. Dividing mycoplasmas elongated slightly; the membrane invaginated, forming one bud. Sometimes M. hominis and M. salivarium produced one bud by elongation, and the bud was attached by a tube. This method of division is not considered as characteristic but rather as due to centrifugal force separating unfixed cells during preparation for electron microscopy. Cross-septa were never observed. In thin sections M. orale type 1 was elongated and without buds, an observation which suggested that preparation for electron microscopy distorted the mycoplasmas.
Morphological Study of Human Mycoplasmas
copy to contain spherical or slightly ovoid mycoplasmas (figure 1). However, both single cells and clumps of M. hominis had a blurred outline, an observation which indicated that this mycoplasma produced a gelatinous capsule-like matrix (figure 2). Electron microscopy. With the exception of M. orale type 1, which in fixed preparations consisted of elongated forms without buds or evidence of replication (figure 3), individual exponentialphase mycoplasmas were seen in electron micrographs of thin sections as round or ovoid microorganisms, 0.3-1 urn in diameter. The majority of cells had a diameter of 0.4-0.6 um (figure 4). The mycoplasmas were bounded by a trilaminar membrane 7.5-10 run thick and contained DNA strands and ribosomes randomIy distributed throughout the cells (figure 4). No polyribosomes or geometric patterns of ribosomes were observed in any of these classical species.
Results
Viable exponential-phase broth cultures of the five species of human mycoplasmas, M. fermentans, M. hominis, M. orale types 1 and 2, and M. salivarium, were shown by interference micros-
Figure 1. Viable Mycoplasma salivarium from an exponential-phase broth culture showing that classical mycoplasmas are spheres (interference [Nomarski] microscopy, X 6,000; the bar represents 1 f-lm).
Figure 2. Viable Mycoplasma hominis from an exponential-phase broth culture showing clumps and single cells surrounded by a gelatinous matrix (interference [Nomarski] microscopy, X 4,680; the bar represents 1 urn).
Downloaded from http://jid.oxfordjournals.org/ at Columbia University Libraries on February 14, 2015
Light and electron microscopy. The gross morphology of viable mycoplasmas was observed directly in wet preparations. A drop of the broth culture was placed on a microscope slide, covered with a coverslip, sealed with a mixture of paraffin and vaseline to prevent evaporation, and examined (magnification, Xl,OOO) by interference (Normarski) illumination. For photography, the cells were fixed in situ by mixing of the broth culture with an equal volume of warm trypticase soy broth containing gelatin (20% wt/vol) [1]. For electron microscopy, pellets of mycoplasmas were obtained by centrifugation (14,500 g for 30 min) of 100-mI portions of exponentialphase broth cultures containing about 107 mycoplasmas/rnl. Thin sections of the pellets were prepared by the technique used previously for the T-mycoplasmas [1].
225
226
Figure 4. Thin section of Mycoplasma [ermentans illustrating round to ovoid cells containing DNA fibrils and randomly dispersed ribosomes (electron microscopy, X50,000; the bar represents 1 um) .
Division of the mycoplasmas most frequently commenced with slight elongation of the cell. At this time ribosomes tended to concentrate at each end of the cell, and invagination of the membrane began. The invagination deepened, and the mycoplasma divided unequally to form a single bud densely packed with ribosomes (figure 5) . Thereafter, on completion of invagination (figure 6) the cells could separate. Sometimes M. hominis and M. salivarium formed buds by elongation, and the single bud was attached to the parent cell by a tubular structure (figures 7 and 8). Budding by elongation was seen much less frequently than was division by invagination. A cross-septum was not formed; no mycoplasmas were observed producing two buds simultaneously. Moreover, the capsule-like outer layer of M. hominis was not detected in thin sections.
Discussion
Only spherical or ovoid mycoplasmas were seen by interference (Nomarski) microscopy in exponential-phase broth cultures of the human classical mycoplasmas, M. [ermentans, M. hominis, M. orale types 1 and 2, and M. salivarium. A capsular matrix also was visible around single cells and clumps of M. hominis. These observations of viable cells confirm our previous report of the morphology of the same strains seen by electron microscopy in pseudoreplicas [2]. None of these mycoplasmas had any distinguishing gross morphological features as do Mycoplasma gallisepticum, which has a terminal bleb [5], Mycoplasma pneumoniae, which may have a terminal knob-like structure [6], and the T-mycoplasmas, which have a dense outer layer of hair or pilus-like structures [1, 7, 8].
Downloaded from http://jid.oxfordjournals.org/ at Columbia University Libraries on February 14, 2015
Figure 3. Thin section of Mycoplasma orale type 1 from an exponential-phase broth culture showing elongated forms without buds (electron microscopy, X 32,000; the bar represents 1 um),
Furness et al.
Morphological Study of Human Mycoplasmas
227
Figure 6. Thin section of Mycoplasma hominis when invagination of the membrane was complete and a daughter cell was formed (electron microscopy, x60,OOO; the bar represents 1 urn).
Thin sections have the advantage of revealing not only the gross morphology of the mycoplasmas but also their ultrastructure, which cannot be observed either by interference microscopy or by the pseudoreplica technique. With the
exception of M. orale type 1, thin sections of these classical mycoplasmas showed that they contained DNA fibrils and randomly distributed ribosomes. This finding contrasts markedly with that in T-mycoplasmas, which form polyribo-
Figure 7. Thin section of Mycoplasma salivarium showing final stage of budding by elongation. The parent cell is elongated and is connected to the daughter cell by a membrane tubule (electron microscopy, x50,OOO; the bar represents 1 urn).
Figure 8. Thin section of Mycoplasma hominis showing two round cells connected by a membrane tubule (electron microscopy, x60,OOO; the bar represents 1 um).
Downloaded from http://jid.oxfordjournals.org/ at Columbia University Libraries on February 14, 2015
Figure S. Thin section of Mycoplasma hominis showing characteristic unequal division of budding cells with smaller daughter cell densely packed with ribosomes (electron microscopy, X50,OOO; the bar represents 1 urn).
Furness et al.
228
duced during the preparation of the mycoplasmas for electron microscopy. Although fixation prior to harvesting undoubtedly reduces the incidence of distortion of mycoplasmas during preparation for microscopy [13, 14], these observations confirm our previous findings, namely, that the effects of techniques for preparing mycoplasmas for electron microscopy are unpredictable, that pseudoreplication is the more reliable method for study of the gross morphology of mycoplasmas by electron microscopy, and that the gross morphology of viable cells should be ascertained by interference microscopy before preparation of mycoplasmas for electron microscopy [15].
References 1. Whitescarver, J., Furness, G. T-mycoplasmas: a study of the morphology, ultrastructure and mode of division of some human strains. J. Med. Microbiol. 8:349-355, 1975. 2. Furness, G. The growth and morphology of mycoplasmas replicating in synchrony. J. Infect. Dis. 122: 146-158, 1970. 3. Furness, G. Differential responses of single cells and aggregates of mycoplasmas to ultraviolet irradiation. Appl. Microbiol. 18: 360-364, 1969. 4. Furness, G., Pipes, F. J., McMurtrey, M. J. Susceptibility of human mycoplasmata to ultraviolet and X irradiations. J. Infect. Dis. 118: 1-6, 1968. 5. Allen, T. C., Stevens, J. 0., Florance, E. R., Hampton, R. O. Ultrastructure of Mycoplasma gallisepticum isolate 1056. J. Ultrastruct. Res. 33:318-331, 1970. 6. Biberfeld, G., Biberfeld, P. Ultrastructural features of Mycoplasma pneumoniae. J. Bacteriol. 102: 855-861, 1966. 7. Williams, M. H. Electron microscopy of T-strains. Ann. N.Y. Acad. Sci. 143:397-400, 1967. 8. Black, F. T., Birch-Andersen, A., Freundt, E. A. Morphology and ultrastructure of human T-mycoplasmas. J. Bacteriol. 111: 254-259, 1972. 9. Furness, G., DeMaggio, M. The growth cycle of Mycoplasma mycoides var. mycoides. J. Infect. Dis. 127:563-566, 1973. 10. Furness, G., DeMaggio, M. Binucleate classical mycoplasmas pathogenic for goats. Infec. Immun. 5:433-441, 1972. 11. Maniloff, J., Morowitz, H. J. Cell biology of the mycoplasmas. Bacteriol. Rev. 36:263-290, 1972. 12. Anderson, D. R., Barile, M. F. Ultrastructure of
Downloaded from http://jid.oxfordjournals.org/ at Columbia University Libraries on February 14, 2015
somes with a characteristic geometric arrangement of the constituent ribosomes [2, 7]. Both the classical and the T-mycoplasmas have a trilaminar plasma membrane and divide by budding, a form of replication that also is observed in pseudoreplicas [1, 2]. However, in the pseudoreplica technique, the classical mycoplasmas produce one bud [2], whereas the type species, Mycoplasma mycoides var. mycoides [9], Mycoplasma mycoides var. capri [10], and the T-mycoplasmas [1] can form two buds simultaneously. Elongated forms of M. hominis with a daughter cell connected by a tubule have been observed; this mode of division is considered typical of the species [11]. Mycoplasmas dividing by both invagination of the membrane and elongation of the cell (the latter mode of division resulting in a daughter cell attached to the parent mycoplasma by a tubule) were observed simultaneously in exponential-phase broth cultures of M. hominis and M. salivarium. Mycoplasmas were seen most often to be dividing by invagination; consequently, invagination is considered to be the characteristic mode of division of these mycoplasmas. These cultures were cloned from colonies grown from single-cell suspensions and thus are pure cultures [2]. Therefore, it is believed that the two methods of budding found in the same culture are similar in that they do not involve the formation of a septum. Cells joined by a tubule are not seen in pseudoreplicas [2]; such joined cells appear only in thin sections, and their appearance is probably related to the separation of the cells. The joined cells could be a consequence of centrifugal force on unfixed, dividing mycoplasmas during the preparation of the pellet for thin sectioning since the larger parent cell is unlikely to be deposited at the same rate as the smaller daughter cell. This factor could cause pulling apart of the two cells. In exponential-phase broth cultures, M. orale type 1 was seen by interference microscopy to consist of round to ovoid cells and by electron microscopy of pseudoreplicas as spheres producing a single bud [2]. Thin sections of similar cultures of the same strain contained elongated forms without buds or any evidence of replication. Therefore, even though these elongated forms have been observed previously [12, 13], we consider that they are distorted cells pro-
Morphological Study of Human Mycoplasmas
Mycoplasma orale isolated from patients with leukemia. J. Natl. Cancer Inst. 36: 161-167, 1966. 13. Lemcke, R. M. Osmolar concentration and fixation of mycoplasmas. J. Bacteriol. 110: 1154-1162, 1972. 14. Maniloff, J., Morowitz, H. J., Barrnett, R. J. Ultra-
229
structure and ribosomes of Mycoplasma gallisepticum. J. Bacteriol, 90: 193-204, 1965. 15. Furness, G., DeMaggio, M., Whitescarver, J. Morphological changes induced during fixation of Mycoplasmas mycoides for electron microscopy. Tex. Rep. BioI. Moo. 33:415-422, 1976.
Downloaded from http://jid.oxfordjournals.org/ at Columbia University Libraries on February 14, 2015
