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An electron-microscopic study of methionine deficient Escherichia coli
J. ULTRASTRUCTURERESEARCH4, 213-221 (1960)
213
An Electron-microscopic Study of Methionine Deficient Escherichia colP KENNETH R. SMITH,Jr. with the technical assistance of GERALD R. SMITH
Department of Anatomy, Washington University School of Medicine, St. Louis, Missouri Received May 26, 1960 The morphology of E. coli K-12 (mutant 58/161 of Reubner) grown in a simple salt and glucose solution containing various concentrations of L-methionine and also in nutrient broth has been determined by examining electron micrographs of thin sections of osmium-fixed bacteria. The morphology of the organisms grown in high concentrations of methionine and in broth was similar. Bacteria grown in methionine deficient media contained numerous round, apparently hollow vesicles of 600 A diameter in their cytoplasm. The relationship of these vesicles to previously described cytoplasmic structures has been discussed. A m u t a n t of E. coli, strain K-12, isolated b y R e u b n e r (9), which required only L - m e t h i o n i n e for g r o w t h in a simple salt a n d glucose solution, can be used for the quantitative assay of methionine. A m o r p h o l o g i c a l study of this m u t a n t when g r o w n in v a r i o u s concentrations of m e t h i o n i n e is presented in this p a p e r .
MATERIALS AND METHODS A sample of E. coli K-12, mutant 58/161, was obtained from Reubner and maintained on agar slants at 4°C. Ten ml of nutrient broth was inoculated from a slant and incubated at 38°C for 12 hours. The tube was centrifuged, the pellet of bacteria washed three times with 0.9 % NaC1, the volume restored to 10 ml with saline, and 0.02 ml of this suspension inoculated into tubes each containing 5 ml basic medium plus 5 ml distilled water containing graded concentrations of L-methionine. The basic medium contained: Na~HPO4 14.0 g KH~PO4 3.0 g NH4C1 2.0 g Glucose 2.5 g MgSO4 0.02 g Distilled water to 1000.0 ml; p H adjusted to 7.2 with 0.1 N HC1 z This study was supported by Research Grants CRT 5009 and RG 3784 of the United States Public Health Service.
214
KENNETH R. SMITH AND GERALD R. SMITH ~o
3r~ g
M]CFIOGRAMS
L~METHIONINE PER ML,
FIG. 1. Growth of E. coli in 48 hours plotted against concentration of methionine in basic medium.
To determine a growth curve, inoculated tubes were selected from each culture medium and incubated at 38°C for 48 hours, after which their optical density to green light was determined with a Klett colorimeter (Fig. 1). Cultures from tubes containing 0.5, 5.0, 10.0, and 25.0 ~g/ml L-methionine were fixed after 10 and 24 hours' incubation. Cultures were also grown in Bacto-Nutrient broth (Difco) and fixed after 4 and 10 hours' incubation. The bacteria were fixed by centrifuging the culture tubes at 180 times gravity for 20 minutes, decanting the medium, pouring in 2 ml Dalton's chrome-osmium fixative at pH 7.6 (5), and dispersing the pellets. The tubes were allowed to stand at room temperature for 15 minutes, centrifuged as before, the fixative decanted, and the pellet covered with 2-3 drops of 2% agar at 40°C in which it was promptly dispersed. The agar was then allowed to gel. Dehydration was carried out by immersing the blocks in 20, 40, 60, 80, and 95% ethanol for 5 minutes in each concentration and for 1 hour in 100% ethanol. The blocks were then infiltrated with methacrylate (6:1 butyl-methyl mixture) for 1½ hours and cut into 2-mm cubes, which were embedded in gelatin capsules containing methacrylate and catalyst (0.2 % benzoyl peroxide). The methacrylate had been partially polymerized by heating before the embedding was done. Polymerization was completed by heating at 60°C for 18 hours. Silver sections (approximately 100-200 A in thickness) were cut with glass knives in a Porter-Blum microtome, placed on collodion-covered copper grids, and examined with an RCA EMU-2E electron microscope. Micrographs were taken at a magnification of 8500 and photographically enlarged.
FIG. 2. Electron micrograph of E. coli grown for 4 hours in nutrient broth (in logarithmic growth phase). The nuclear vacuoles (N) are clear areas scattered throughout the cytoplasm. The nuclear threads (7") wind through the vacuoles with focal accumulations of dense material on them. The cytoplasm is uniform throughout and is made up of many 100-200 A dense particles. The cell wall (C) is a dense membrane about 200 ~ thick, that is closely applied to the cytoplasmic membrane (CM). The cytoplasmic membrane is about one-half the thickness of the cell wall. The cell on the left is just completing division. × 60,000.
M E T H I O N I N E D E F I C I E N T E. COLI
215
216
KENNETH R. SMITH AND GERALD R. SMITH RESULTS
G r o w t h became visible in b r o t h after 2-3 hours, b u t i n the basic m e d i u m plus m e t h i o n i n e n o n e was seen until 8 hours after inoculation. G r o w t h apparently stopped after a b o u t 10 hours in broth, b u t bacteria were seen to be dividing after 24 hours i n basic m e d i u m with low concentrations of methionine. The various culture m e d i a used had no effect on the size of the bacteria. The organisms had distinct cell wails closely applied to thin, faint cytoplasmic m e m b r a n e s , a n d in several cells this true relationship was preserved (Figs. 2, 3, 4, a n d 6). I n m a n y instances, however, the processing disrupted the cell walls a n d separated t h e m from the cytoplasmic m e m b r a n e s (Figs. 5, 6, a n d 8). The electron-transparent nuclear areas (2) c o n t a i n e d tangled threads of n u c l e a r material with clumps of electron-dense material r a n d o m l y arranged along them. The nuclear threads were similar i n appearance in all cells regardless of b o t h their age a n d the media in which they were grown. I n cells from 24-hour cultures the n u c l e a r vacuoles were large a n d a p p r o x i m a t e d s m o o t h cylinders (Figs. 7 a n d 8), in contrast to those in 10-hour cultures which were small a n d irregular i n outline (Figs. 3 a n d 5).
FIG. 3. Electron micrograph of E. coli grown for 10 hours in basic medium containing 25/~g methionine/ml. The bacteria were in the rapid growth phase at the time of fixation. Their appearance is the same as that of E. coli grown for 4 hours in nutrient broth. The nuclear vacuole is markedly convoluted. The cytoplasm is structurally uniform and consists of dense 100-200 A particles. The cell wall is adherent to the cytoplasmic membrane. × 40,000. FIG. 4. Electron micrograph of E. coli grown for 10 hours in basic medium containing l0 #g methionine/ml. The nuclear vacuole is moderately convoluted. The cytoplasm consists of 100-200 A dense particles, but in addition contains 600/~ vesicles (V)having a rim of dense material surrounding an electron lucent center. This is one of the few bacteria that contained vesicles in this culture, x 40,000. FIG. 5. Electron micrograph of methionine deficient E. coli grown for 10 hours in basic medium containing only 0.5 #g methionine/ml. The nuclear vacuole is a narrow, greatly ramified structure containing the nuclear threads. The cytoplasm consists of densely packed 100-200/~ particles, and also contains several faintly outlined vesicles (arrows). The cell wall is separated from the cytoplasmic membrane at both poles, x 60,000. FI6. 6. Electron micrograph of a dividing E. coli grown for 24 hours in basic medium containing 5/~g methionine/ml. The nuclear vacuoles are highly convoluted and narrow, a characteristic feature of young cells. The three large dark dots are clumps of nuclear material on nuclear threads. The cytoplasm contains no vesicles. The cell wall and cytoplasmic membrane are indenting opposite sides of the parent cell. See Fig. 2 for end stage of cell division. The cell wall has been separated from the cytoplasmic membrane at each pole by fixation, x 65,000. FIG. 7. Electron micrograph of methionine deficient E. coli grown for 24 hours in basic medium containing 0.5 #g methionine/ml. The nuclear vacuole is a centrally located, pale, smooth cylinder containing tangled nuclear threads. The smooth cylindrical nuclear vacuole is characteristic of old (i.e. non-dividing) cells. The cytoplasm is made up of many 100-200/~ particles, but also contains several 600 ~ vesicles (V)with dense rims and less dense centers. × 60,000. FI6. 8. Electron micrograph of methionine deficient E. eoli grown for 24 hours in basic medium containing 0.5 /~g methionine/ml (from same culture as Fig. 7). The bacteria are cut in cross section. The round smooth nuclear vacuoles are seen centrally. The cytoplasm contains many vesicles (V) randomly distributed. The cell wall of the bacterium at the lower right is disrupted, while the cell at the upper left has an intact cell wall. x 60,000.
M E T H I O N I N E D E F I C I E N T E, C O L I
1 5 - - 60173312 J . Ultrastructure Research
217
218
K E N N E T H R. SMITH A N D G E R A L D R. SMITH
220
KENNETH R. SMITH AND GERALD R. SMITH
The cytoplasm of bacteria grown in broth or in 25 #g of methionine per ml was always of uniform density having a delicate, finely granular structure (Figs. 2 and 3). This was also the case when cells were grown in both 10 and 5 #g methionine/ml for 24 hours (Fig. 6). However, in almost all cells grown in 0.5 #g methionine/ml for 24 hours the cytoplasm contained numerous round vesicles with smooth dense rims and electron-transparent centers. The vesicles were of uniform size and about 600 A in diameter. Most were surrounded by cytoplasm (Figs. 7 and 8), but a few were separated from the cytoplasm by narrow clear zones. The vesicles were randomly distributed on cross sections, but were more numerous at the poles on longitudinal sections. A few cells grown in 10 #g methionine/ml for 10 hours contained such vesicles (Fig. 4), but none were present at the end of 24 hours in concentrations of 5 #g or more (Fig. 6). In cultures containing 0.5 #g/ml there was a suggestion of vesicle formation in some of the cells at 10 hours (Fig. 5).
DISCUSSION The morphology of broth-cultured E. coli of various strains as seen in high resolution micrographs of thin sections has been described by Ryter et al. (10). It was essentially similar to that just given for the E. coli grown in broth. However, they observed that the addition of CaC12 to their fixative caused the nuclear threads to appear fine and homogeneous, but that when versene was also added the threads clumped and became coarse as they do when fixed in Dalton's solution. They also noted dense cytoplasmic granules of about 500 • diameter, but only in E. eoli strain C stained with lanthanum and uranium. A number of structures lying in the cytoplasm of bacteria have been described. They include volutin granules (1), fat vacuoles (6), polysaccharide granules (3), parasporal bodies (7), peripheral bodies (4), sulphur granules (13), granules of ribonucleic acid measuring 100-400 ]~ (1, 12), and cytoplasmic granules 300-2000 in diameter (8) (these have been referred to as mitochondrial equivalents by some authors). None of these structures bears a close resemblance to the vesicles seen in the cytoplasm of methionine deficient E. coli, although very few of them have been pictured in high resolution micrographs of thin sections of well-fixed material. The volutin granules do have one thing in common with the vesicles of methionine deficient E. coli they were found in bacteria grown in deficient media for a long time (11). The chromatophores in some photosynthetic bacteria (13), the lanthanumstaining bodies in E. coli described by Caro et al. (2), and the dense cytoplasmic granules of E. coli strain C (stained with lanthanum and uranium) (10) bear perhaps
METHIONINEDEFICIENT E. COLI
221
the closest resemblance to the vesicles seen in methionine deficient E. coli, but they cannot be said to be morphologically or physiologically identical. It will not be possible to determine the relationship of the vesicles seen in the cytoplasm of methionine deficient E. coli to other cytoplasmic structures which have been described until high resolution electron micrographs of thin sections of bacteria containing these various inclusions become generally available.
REFERENCES 1. BRADHELD,J. R. G., in Bacterial Anatomy, Sixth Symposium of the Society for General Microbiology, London. Cambridge University Press, London, 1956. 2. CARo, L. G., VAN TtJBER6EN, R. P. and FORRO, F., J. Biophys. Biochem. Cytol. 4, 491 0958). 3. CEDERGREN,B. and HOLMS, T., 9". Ultrastructure Research 3, 70 (1959). 4. CHAPMAN,G. B. and HmLIER, J., J. Bacteriol. 66, 362 (1953). 5. DALTON, A. J., Anat. Record 121, 281 (1955). 6. GRULA, E. A. and HARTSELL,S. E., J. Bacteriol. 68, 498 (1954). 7. HANNAY, C. L., in Bacterial Anatomy, Sixth Symposium of the Society for General Microbiology, London. Cambridge University Press, London, 1956. 8. NIKLOWITZ,W., Zentr. Bakteriol. Parasitenk. 173, 12 (1958). 9. REUBNER, B., J. Lab. Clin. Med. 47, 140 (1956). 10. RYTER, A., KELLENRERGER,E., BmCrt-AND~RSON, A. and MAALO~, O., Z. Naturforsch. 13b, 597 (1958). 11. SMITH,I. W., WILKINSON,J. F. and DtJGUID, J. P., J. Bacteriol. 68, 450 (1954). 12. TISSIfZRES,A., Nature 174, 183 (1954). 13. VATTER, A. E. and WOLFE, R. S., or. Bacteriol. 75, 480 (1958).
213
An Electron-microscopic Study of Methionine Deficient Escherichia colP KENNETH R. SMITH,Jr. with the technical assistance of GERALD R. SMITH
Department of Anatomy, Washington University School of Medicine, St. Louis, Missouri Received May 26, 1960 The morphology of E. coli K-12 (mutant 58/161 of Reubner) grown in a simple salt and glucose solution containing various concentrations of L-methionine and also in nutrient broth has been determined by examining electron micrographs of thin sections of osmium-fixed bacteria. The morphology of the organisms grown in high concentrations of methionine and in broth was similar. Bacteria grown in methionine deficient media contained numerous round, apparently hollow vesicles of 600 A diameter in their cytoplasm. The relationship of these vesicles to previously described cytoplasmic structures has been discussed. A m u t a n t of E. coli, strain K-12, isolated b y R e u b n e r (9), which required only L - m e t h i o n i n e for g r o w t h in a simple salt a n d glucose solution, can be used for the quantitative assay of methionine. A m o r p h o l o g i c a l study of this m u t a n t when g r o w n in v a r i o u s concentrations of m e t h i o n i n e is presented in this p a p e r .
MATERIALS AND METHODS A sample of E. coli K-12, mutant 58/161, was obtained from Reubner and maintained on agar slants at 4°C. Ten ml of nutrient broth was inoculated from a slant and incubated at 38°C for 12 hours. The tube was centrifuged, the pellet of bacteria washed three times with 0.9 % NaC1, the volume restored to 10 ml with saline, and 0.02 ml of this suspension inoculated into tubes each containing 5 ml basic medium plus 5 ml distilled water containing graded concentrations of L-methionine. The basic medium contained: Na~HPO4 14.0 g KH~PO4 3.0 g NH4C1 2.0 g Glucose 2.5 g MgSO4 0.02 g Distilled water to 1000.0 ml; p H adjusted to 7.2 with 0.1 N HC1 z This study was supported by Research Grants CRT 5009 and RG 3784 of the United States Public Health Service.
214
KENNETH R. SMITH AND GERALD R. SMITH ~o
3r~ g
M]CFIOGRAMS
L~METHIONINE PER ML,
FIG. 1. Growth of E. coli in 48 hours plotted against concentration of methionine in basic medium.
To determine a growth curve, inoculated tubes were selected from each culture medium and incubated at 38°C for 48 hours, after which their optical density to green light was determined with a Klett colorimeter (Fig. 1). Cultures from tubes containing 0.5, 5.0, 10.0, and 25.0 ~g/ml L-methionine were fixed after 10 and 24 hours' incubation. Cultures were also grown in Bacto-Nutrient broth (Difco) and fixed after 4 and 10 hours' incubation. The bacteria were fixed by centrifuging the culture tubes at 180 times gravity for 20 minutes, decanting the medium, pouring in 2 ml Dalton's chrome-osmium fixative at pH 7.6 (5), and dispersing the pellets. The tubes were allowed to stand at room temperature for 15 minutes, centrifuged as before, the fixative decanted, and the pellet covered with 2-3 drops of 2% agar at 40°C in which it was promptly dispersed. The agar was then allowed to gel. Dehydration was carried out by immersing the blocks in 20, 40, 60, 80, and 95% ethanol for 5 minutes in each concentration and for 1 hour in 100% ethanol. The blocks were then infiltrated with methacrylate (6:1 butyl-methyl mixture) for 1½ hours and cut into 2-mm cubes, which were embedded in gelatin capsules containing methacrylate and catalyst (0.2 % benzoyl peroxide). The methacrylate had been partially polymerized by heating before the embedding was done. Polymerization was completed by heating at 60°C for 18 hours. Silver sections (approximately 100-200 A in thickness) were cut with glass knives in a Porter-Blum microtome, placed on collodion-covered copper grids, and examined with an RCA EMU-2E electron microscope. Micrographs were taken at a magnification of 8500 and photographically enlarged.
FIG. 2. Electron micrograph of E. coli grown for 4 hours in nutrient broth (in logarithmic growth phase). The nuclear vacuoles (N) are clear areas scattered throughout the cytoplasm. The nuclear threads (7") wind through the vacuoles with focal accumulations of dense material on them. The cytoplasm is uniform throughout and is made up of many 100-200 A dense particles. The cell wall (C) is a dense membrane about 200 ~ thick, that is closely applied to the cytoplasmic membrane (CM). The cytoplasmic membrane is about one-half the thickness of the cell wall. The cell on the left is just completing division. × 60,000.
M E T H I O N I N E D E F I C I E N T E. COLI
215
216
KENNETH R. SMITH AND GERALD R. SMITH RESULTS
G r o w t h became visible in b r o t h after 2-3 hours, b u t i n the basic m e d i u m plus m e t h i o n i n e n o n e was seen until 8 hours after inoculation. G r o w t h apparently stopped after a b o u t 10 hours in broth, b u t bacteria were seen to be dividing after 24 hours i n basic m e d i u m with low concentrations of methionine. The various culture m e d i a used had no effect on the size of the bacteria. The organisms had distinct cell wails closely applied to thin, faint cytoplasmic m e m b r a n e s , a n d in several cells this true relationship was preserved (Figs. 2, 3, 4, a n d 6). I n m a n y instances, however, the processing disrupted the cell walls a n d separated t h e m from the cytoplasmic m e m b r a n e s (Figs. 5, 6, a n d 8). The electron-transparent nuclear areas (2) c o n t a i n e d tangled threads of n u c l e a r material with clumps of electron-dense material r a n d o m l y arranged along them. The nuclear threads were similar i n appearance in all cells regardless of b o t h their age a n d the media in which they were grown. I n cells from 24-hour cultures the n u c l e a r vacuoles were large a n d a p p r o x i m a t e d s m o o t h cylinders (Figs. 7 a n d 8), in contrast to those in 10-hour cultures which were small a n d irregular i n outline (Figs. 3 a n d 5).
FIG. 3. Electron micrograph of E. coli grown for 10 hours in basic medium containing 25/~g methionine/ml. The bacteria were in the rapid growth phase at the time of fixation. Their appearance is the same as that of E. coli grown for 4 hours in nutrient broth. The nuclear vacuole is markedly convoluted. The cytoplasm is structurally uniform and consists of dense 100-200 A particles. The cell wall is adherent to the cytoplasmic membrane. × 40,000. FIG. 4. Electron micrograph of E. coli grown for 10 hours in basic medium containing l0 #g methionine/ml. The nuclear vacuole is moderately convoluted. The cytoplasm consists of 100-200 A dense particles, but in addition contains 600/~ vesicles (V)having a rim of dense material surrounding an electron lucent center. This is one of the few bacteria that contained vesicles in this culture, x 40,000. FIG. 5. Electron micrograph of methionine deficient E. coli grown for 10 hours in basic medium containing only 0.5 #g methionine/ml. The nuclear vacuole is a narrow, greatly ramified structure containing the nuclear threads. The cytoplasm consists of densely packed 100-200/~ particles, and also contains several faintly outlined vesicles (arrows). The cell wall is separated from the cytoplasmic membrane at both poles, x 60,000. FI6. 6. Electron micrograph of a dividing E. coli grown for 24 hours in basic medium containing 5/~g methionine/ml. The nuclear vacuoles are highly convoluted and narrow, a characteristic feature of young cells. The three large dark dots are clumps of nuclear material on nuclear threads. The cytoplasm contains no vesicles. The cell wall and cytoplasmic membrane are indenting opposite sides of the parent cell. See Fig. 2 for end stage of cell division. The cell wall has been separated from the cytoplasmic membrane at each pole by fixation, x 65,000. FIG. 7. Electron micrograph of methionine deficient E. coli grown for 24 hours in basic medium containing 0.5 #g methionine/ml. The nuclear vacuole is a centrally located, pale, smooth cylinder containing tangled nuclear threads. The smooth cylindrical nuclear vacuole is characteristic of old (i.e. non-dividing) cells. The cytoplasm is made up of many 100-200/~ particles, but also contains several 600 ~ vesicles (V)with dense rims and less dense centers. × 60,000. FI6. 8. Electron micrograph of methionine deficient E. eoli grown for 24 hours in basic medium containing 0.5 /~g methionine/ml (from same culture as Fig. 7). The bacteria are cut in cross section. The round smooth nuclear vacuoles are seen centrally. The cytoplasm contains many vesicles (V) randomly distributed. The cell wall of the bacterium at the lower right is disrupted, while the cell at the upper left has an intact cell wall. x 60,000.
M E T H I O N I N E D E F I C I E N T E, C O L I
1 5 - - 60173312 J . Ultrastructure Research
217
218
K E N N E T H R. SMITH A N D G E R A L D R. SMITH
220
KENNETH R. SMITH AND GERALD R. SMITH
The cytoplasm of bacteria grown in broth or in 25 #g of methionine per ml was always of uniform density having a delicate, finely granular structure (Figs. 2 and 3). This was also the case when cells were grown in both 10 and 5 #g methionine/ml for 24 hours (Fig. 6). However, in almost all cells grown in 0.5 #g methionine/ml for 24 hours the cytoplasm contained numerous round vesicles with smooth dense rims and electron-transparent centers. The vesicles were of uniform size and about 600 A in diameter. Most were surrounded by cytoplasm (Figs. 7 and 8), but a few were separated from the cytoplasm by narrow clear zones. The vesicles were randomly distributed on cross sections, but were more numerous at the poles on longitudinal sections. A few cells grown in 10 #g methionine/ml for 10 hours contained such vesicles (Fig. 4), but none were present at the end of 24 hours in concentrations of 5 #g or more (Fig. 6). In cultures containing 0.5 #g/ml there was a suggestion of vesicle formation in some of the cells at 10 hours (Fig. 5).
DISCUSSION The morphology of broth-cultured E. coli of various strains as seen in high resolution micrographs of thin sections has been described by Ryter et al. (10). It was essentially similar to that just given for the E. coli grown in broth. However, they observed that the addition of CaC12 to their fixative caused the nuclear threads to appear fine and homogeneous, but that when versene was also added the threads clumped and became coarse as they do when fixed in Dalton's solution. They also noted dense cytoplasmic granules of about 500 • diameter, but only in E. eoli strain C stained with lanthanum and uranium. A number of structures lying in the cytoplasm of bacteria have been described. They include volutin granules (1), fat vacuoles (6), polysaccharide granules (3), parasporal bodies (7), peripheral bodies (4), sulphur granules (13), granules of ribonucleic acid measuring 100-400 ]~ (1, 12), and cytoplasmic granules 300-2000 in diameter (8) (these have been referred to as mitochondrial equivalents by some authors). None of these structures bears a close resemblance to the vesicles seen in the cytoplasm of methionine deficient E. coli, although very few of them have been pictured in high resolution micrographs of thin sections of well-fixed material. The volutin granules do have one thing in common with the vesicles of methionine deficient E. coli they were found in bacteria grown in deficient media for a long time (11). The chromatophores in some photosynthetic bacteria (13), the lanthanumstaining bodies in E. coli described by Caro et al. (2), and the dense cytoplasmic granules of E. coli strain C (stained with lanthanum and uranium) (10) bear perhaps
METHIONINEDEFICIENT E. COLI
221
the closest resemblance to the vesicles seen in methionine deficient E. coli, but they cannot be said to be morphologically or physiologically identical. It will not be possible to determine the relationship of the vesicles seen in the cytoplasm of methionine deficient E. coli to other cytoplasmic structures which have been described until high resolution electron micrographs of thin sections of bacteria containing these various inclusions become generally available.
REFERENCES 1. BRADHELD,J. R. G., in Bacterial Anatomy, Sixth Symposium of the Society for General Microbiology, London. Cambridge University Press, London, 1956. 2. CARo, L. G., VAN TtJBER6EN, R. P. and FORRO, F., J. Biophys. Biochem. Cytol. 4, 491 0958). 3. CEDERGREN,B. and HOLMS, T., 9". Ultrastructure Research 3, 70 (1959). 4. CHAPMAN,G. B. and HmLIER, J., J. Bacteriol. 66, 362 (1953). 5. DALTON, A. J., Anat. Record 121, 281 (1955). 6. GRULA, E. A. and HARTSELL,S. E., J. Bacteriol. 68, 498 (1954). 7. HANNAY, C. L., in Bacterial Anatomy, Sixth Symposium of the Society for General Microbiology, London. Cambridge University Press, London, 1956. 8. NIKLOWITZ,W., Zentr. Bakteriol. Parasitenk. 173, 12 (1958). 9. REUBNER, B., J. Lab. Clin. Med. 47, 140 (1956). 10. RYTER, A., KELLENRERGER,E., BmCrt-AND~RSON, A. and MAALO~, O., Z. Naturforsch. 13b, 597 (1958). 11. SMITH,I. W., WILKINSON,J. F. and DtJGUID, J. P., J. Bacteriol. 68, 450 (1954). 12. TISSIfZRES,A., Nature 174, 183 (1954). 13. VATTER, A. E. and WOLFE, R. S., or. Bacteriol. 75, 480 (1958).