NOTES
Can. J. Microbiol. Downloaded from www.nrcresearchpress.com by University of Queensland on 11/19/14 For personal use only.
3. RAMSDALE,S. J., and R. E. WILKINSON. 1968. Identification of petroleum sources of beach pollution by gas-liquid chromatography. J. Inst. Petrol. 54: 326332. J. D., and R. R. COLWELL. 1974. Microbial 4. WALKER,
89 1
degradation of model petroleum at low temperatures. Microbial Ecol. 1: 63-95. 5. WESTLAKE,D. W. S., A. JOBSON. R. PHILLIPPE, and F. D. COOK.1974. Biodegradability and crude oil composition. Can. J. Microbial. 20: 9 15-928.
Electron microscopy of antibody-labelled cells of Streptococcus mutans R. G. EMYANITOFF Dc,pcrrtr?7ent q f Microbiology. Loyolti Uni\*c,rsity Stritck Scllool of M ~ d i c i n e Mtryn,ood, , l1linoi.s. U . S . A . 60153
T. E. RUCINSKY Department oJ'Microbiology, Penn.cyh~rrniaState University, University Prrrk, Penn.syIl~ania,U . S . A . 16802
AND
D. C. BIRDSELL Depcrrtment of Btrsic Dentrrl Sri~rrc.es,Urli~.rrsityc(fF1oridrr College. of Dentistry. Gninc,.svill(., Floridrr, U . S . A . 32610 Accepted March 1. 1976
EMYANITOFF, R. G . , T. E. RUCINSKY, and D. C. BIRDSELL. 1976. Electron microscopy of antibody-labelled cells of Streptococcwss mrrttrt1.s. Can. J. Microbiol. 22: 891-895. Examination of immune complexes between cells of Strcprococ~crr.~ r?lr~totr.sand homologous antiserum by the techniques of thin-sectioning and freeze-etching revealed that the cells were embedded within an extensive matrix 8C-90 nm thick with defined boundaries.
EMYANITOFF, R. G., T. E. RUCINSKYet D. C. BIRDSELL. 1976. Electron microscopy of antibody-labelled cells of Streptococclrs mrrtrrrls. Can. J . Microbiol. 22: 891-895. Les immuns complexes form& lors de la reaction entre S t r e p t o c o c c ~ trn~ttans .~ et un antiserum homologue ont ete examines en coupes minces et par decapage i froid. Cet examen montre des cellules enrobees dans une grosse gaine de 80 i 90 nm d'tpaisseur et dont les contours sont dkfinis. [Traduit par le journal]
T h e initial interaction between m a n a n d his coniplex oral flora is based upon specific adherence between surface components o f the organism a n d complimentary components o n the surface t o be colonized (tooth surface, epithelial cell, etc.) (4). Once this initial interaction has been established, most subsequent diversification of species is d u e t o bacterial cell -cell interaction with concomitant nutritional interaction (cross-feeding, etc.). T h e techniques recently developed by Listgarten et al. (9) t o examine dental plaque ill sit11 have underlined the complexity of the flora contained within the plaque matrix. Dental plaque plays m a j o r roles in t w o disease processes: (a) as a source of acids f o r caries production, a n d (6) a s a site f o r release of antigenic conlponents believed t o be involved in the i m m u n e etiology of gingivitis a n d perio'Received June 20, 1975.
dontal disease ( I I). A n understanding o f the specific etiology of either of these disease processes necessitates a n understanding of the interaction a n d possible synergism a m o n g a n d between the niembers of the oral flora. A n imp o r t a n t contribution t o o u r a p p r o a c h t o this understanding results from the work of Lai et al. (6, 7, 8). Using specific antiserum they have been able t o identify c o l u m n a r colonies of Sttvptoc o c c ~ t ssang~tiswithin plaque. Expansion o f this technique t o include other members o f the plaque microbiota would certainly p r o m o t e a better understanding of t h e complexities of dental plaque. T h e study described below was designed t o (a) demonstrate antiserum coating of S. tn~ttans, a n d (b) gain information a b o u t t h e three-dimensional organization o f the antiserum - bacterial cell complex b y using t h e techniques of freeze-etching. Streptococc~ts tnlttans strain O M Z 1 7 6 (sub-
Can. J. Microbiol. Downloaded from www.nrcresearchpress.com by University of Queensland on 11/19/14 For personal use only.
892
CAN. J. MICROBIOL. VOL. 22, 1976
species mutans, Bratthall serotype c t ) and strain NCTC2 10449 (subspecies sobrinus, Bratthall serotype c) were used for this study. Both were obtained from Dr. I. Shklair, Great Lakes Naval Training Center, Great Lakes, Illinois. The cultures were maintained in fluid thioglycollate medium (Difco) to which 2% calcium carbonate was added. The cultures were grown in a yeast extract - tryptone - glucose medium at 37 "C under an atmosphere of N2/C02 (95:5). Antigen for immunization was prepared by suspending 50 mg of lyophilized stationary phase bacteria in 5 ml of 0.15 M NaCl (saline) and incubating for 30 min at 60 "C to heat-kill the cells. The rabbits were given intravenous injections of the antigen 3 days per week over a 3-week period. Ten days after the last injection blood was withdrawn by cardiac puncture, clarified, and the serum fraction frozen at -20°C. Both the anti-10449 and the antiOMZ176 sera gave bands of precipitation with homologous antigen using Ouchterlony geldiffusion methods. No precipitation bands were observed between preimmune sera and homologous antigen. To prepare the antiserum-coated cells, a 10-ml sample of cells from exponentially growing cultures of each of the two strains was pelleted by centrifugation and resuspended in 0.5 ml of antiserum. Control samples were suspended in an equivalent amount of preimmune serum or 0.1 M phosphate buffer, pH 7.8. The cells were incubated for 30 min at 37 "C, centrifuged, and washed once in 5 volumes of saline. Cells were prepared for sectioning by preliminary fixation for 2 h in 2% glutaraldehyde, overnight fixation in 1% osmium tetroxide in the buffer of Ryter and Kellenberger (13), dehydration through a graded acetone series, and embedment in Spurr's low-viscosity medium (14). Sections were cut using an LKB ultramicrotome with a diamond knife. Sections were poststained with uranyl acetate and lead citrate (12) and examined using either a Zeiss EM9S or a Philips 300 electron microscope. Replicas of freeze-etched ce1Is were prepared with the use of Balzers BA360M freezeetching device by the method of Friedman et al. (3). The replicas were cleaned with 70% HH,SO, and 0.5% NaHOCl and examined using a Philips 300 electron microscope. In thin section of cells of S. mutans OMZ176 ZNCTC,National Collection of Type Cultures.
incubated with antiserum, a thick electron-dense layer can be seen surrounding the cells (Fig. 16). In most cases, the layer appears to be slightly less dense at the junction with the cell wall. Cells incubated in buffer or in preimmune serum did not show this layer (Fig. la). It is difficult to appreciate the three-dimensional aspects of the immune serum : cell complex by examination of thin-sectioned material. In addition. there exists the possibility of introducing fixation artifacts during preparation of thin sections for electron microscopy. Platinum-shadowed carbon replicas of unfixed freeze-fractured and etched s~ecimens were therefore examined. In the control suspensions the cells appeared to be distributed randomly throughout the replica, although, as would be expected, many cells appeared in chains (Fig. 2a). Good fracturing was achieved; in many cells the wall, membrane, and cytoplasm were easily distinguished. The background was uniform in appearance. Macroscopic agglutination was observed in the samples incubated with antiserum. The cells in these preparations were found only in specific areas of the replica. The material in these areas was less granular than the general background and exhibited discrete boundaries (Fig. 26). It thus appears that the cells were embedded within a thick coating of immune serum. In almost all cells either concave or convex surfaces were seen (Fig. 2c). Similar results were seen for S. mutans 10449. Cells of S. mutans strains OMZ176 treated with specific immune serum possess an additional, extensive layer (80-90 nm thick) external to their cell wall and similar to that described by Lai et al. (6, 7, 8). Freeze-fracture followed by etching revealed two additional features of the antibody cell surface interaction. (1) The cleavage plane within the cells appears to be altered in the presence of antiserum. The mechanism of this alteration is unknown. (2) Although the thickness of the layer external to the wall is evident in thin section, the three-dimensional perspective of the agglutinated cells is best observed in extensively etched preparations. In such preparations the immune complex appears more abundant than suggested by thin section. The observed thickness of the antibody - surface antigen layer is considerably greater (in thin section and freeze-etch preparations) than predicted on the basis of the size of the immunoglobulin molecules and measurements of cell wall thickness of S. mutans OMZ176 (2). Although it is possible that other
Can. J. Microbiol. Downloaded from www.nrcresearchpress.com by University of Queensland on 11/19/14 For personal use only.
NOTES
FIG.1. (a) Thin section of untreated S. mutans OMZ176. (b) Thin section of S. mlitans OMZ176 treated with immune serum. Note the thick fibrillar layer external to the cell wall. The bar in this and subsequent figures represents 500 nm.
893
CAN. J. MICROBIOL. VOL. 22, 1976
Can. J. Microbiol. Downloaded from www.nrcresearchpress.com by University of Queensland on 11/19/14 For personal use only.
894
FIG.2. (a) Freeze-etch preparation of control S. mutans OMZ176. Note the cytoplasm (C), cytoplasmic membrane (CM), and cell wall (CW). (b) Freeze-etch preparation of immune serum-treated S. mutans OMZ176. Note the discrete boundaries of the agglutinated cell complex (arrows). (c) Freezeetch preparation of immune serum-treated S. rnutans OMZ176.
Can. J. Microbiol. Downloaded from www.nrcresearchpress.com by University of Queensland on 11/19/14 For personal use only.
serum components may be bound within the complex, it is more likely that the cell surface polymers in their native configuration extend further into the medium surrounding the cells than revealed following fixation and dehydration. In the presence of specific antibody the polymers coated by antiserum are held in the native, extended configuration accounting for the thickness of the observed coating layer. Birdsell et 01. (I) have reported that the darkly staining portion of the cell wall of Bacillus subtilis decreased in thickness by 15% if concanavalin A (Con A) were allowed to interact specifically with the a-glucosylated teichoic acid of the cell wall before fixation and dehydration. An additional "fluffy" layer 25-60 nm in thickness composed of Con A and teichoic acid was external to the darkly staining cell wall. van Driel et al. (15) reported that the membrane teichoic acid (LTA) of Lactobacillus ferrnenti was exposed on the surface of the cell walls. These facts coupled with the recent reports of "secretion" of LTA by a variety of gram-positive organisms (5, 10) suggest that the domain of the bacterial cell surface may be considerably more extensive than indicated by examination of electron micrographs of thinsectioned material. Not only could covalently linked cell wall components extend several nanometres from the cell surface, but also cell membrane components could be in the process of transversing the cell wall for ultimate release into the culture fluid.
2. EMYANITOFF, R. G., and D. C. BIRDSELL.1974. Morphology of hydrated and lypophilized whole cells of Streptococcirs mutans OMZ176. Bacteriol. Proc. 1974: 56. 3. FRIEDMAN, B. S . , P . R. D U G A NR. , M. PFISTER.and C . C. REMSEN.1968. Fine structure and composition of the zoogleal matrix surrounding Zoogloecl ratnigericr. J. Bacteriol. 96: 214d2153. 4. GIBBONS,R. J., and J. V A N HOUTE. 1975. Bacterial adherence in oral microbial ecology. Annu. Rev. Microbiol. 29: 1 9 4 4 . 5. JOSEPH,R., and G. D. SHOCKMAN. 1975. Synthesis and excretion of glycerol teichoic acid during growth of two streptococcal species. Infect. Imrnun. 12: 333-338. 6. L A I , C . , M. A. LISTGARTEN, and B. ROSAN.1973. Serology ofStreptococcrrsscrtrglris: localization ofantigens with unlabeled antisera. Infect. Immun. 8: 475-48 1. 7. L A [ , C . , M. A. LISTGARTEN, and B. ROSAN.1975. Immunoelectron microscopic identification and localization of Strc~ptococc~ts scrtzg~ris with peroxidase-labeled antibody: localization of surface antigens in pure cultures. Infect. Immun. 11: 193-199. 8. LAI, C., M. A. LISTGARTEN, and B. ROSAN. 1975. lmmunoelectron microscopic identification and srrt~giris with localization of Srreprococclrs peroxidase-labeled antibody: localization of Srreptoc~occlrssanglris in intact dental plaque. Infect. Immun. 11: 20C-210. 9. LISTGARTEN, M. A., H . E . MAYO,and R . TREMBLAY. 1975. Development of dental plaque on epoxy resin crowns in man: a light and electron microscopic study. J. Periodontal. Res. 46: 10-26. 10. M A R K H A M J. ,L . , K . W. KNOX,A. J. W I C K E Nand , M. J. HEWETT. 1975. Formation of extracellular lipoteichoic acid by oral streptucocci and lactobacilli. Infect. Immun. 12: 378-386. 11. MERGENHAGEN, S. E., T . R. TEMPEL, and R. SNYDERMAN.1970. Immunologic reactions and periodontal inflammation. J. Dent. Res. 49: 256261. Acknowledgments 12. REYNOLDS, E. S. 1963. The use of lead citrate a t high We thank Dr. T. Hashimoto for his helpful p H a s an electron-opaque stain in electron microscopy. J . Cell Biol. 17: 20g212. discussions of the manuscript, Mr. 0. Payne and 1958. Etude au Mr. D. Balkwill for excellent technical assistance. 13. RYTER,A,, and E . KELLENBERGER. microscope electronique de plasmas contenant d e The work reported here was carried out while I'acide desoxyribonucleique. Z. Naturforsch. 13: one of us (R.G.E.) was supported by an American 597-605. Society for Microbiology Presidents Fellowship 14. SPURR,A. R. 1969. A low-viscosity epoxy resin embedding medium for electron microscopy. J. Ultrain the laboratory of T. E. Rucinsky. struct. Res. 26: 31-43. 15. V A N DRIEL,D. A., A . J. WICKEN, M. R. DICK SON,^^^ I. BIRDSELL, D. C . , R. J. DOYLE,and M. MORGENK . W. KNOX. 1973. Cellular location of the STERN.1975. Organization of the teichoic acid in the lipoteichoic acids of Lactobacill~rsfermenti NCTC cell wall of Bacillus subtilis 168. J. Bacteriol. 121: 6991 and Lactobaci//us casei N C T C 6375. J. Ultra726734. struct. Res. 43: 483-497.
Can. J. Microbiol. Downloaded from www.nrcresearchpress.com by University of Queensland on 11/19/14 For personal use only.
3. RAMSDALE,S. J., and R. E. WILKINSON. 1968. Identification of petroleum sources of beach pollution by gas-liquid chromatography. J. Inst. Petrol. 54: 326332. J. D., and R. R. COLWELL. 1974. Microbial 4. WALKER,
89 1
degradation of model petroleum at low temperatures. Microbial Ecol. 1: 63-95. 5. WESTLAKE,D. W. S., A. JOBSON. R. PHILLIPPE, and F. D. COOK.1974. Biodegradability and crude oil composition. Can. J. Microbial. 20: 9 15-928.
Electron microscopy of antibody-labelled cells of Streptococcus mutans R. G. EMYANITOFF Dc,pcrrtr?7ent q f Microbiology. Loyolti Uni\*c,rsity Stritck Scllool of M ~ d i c i n e Mtryn,ood, , l1linoi.s. U . S . A . 60153
T. E. RUCINSKY Department oJ'Microbiology, Penn.cyh~rrniaState University, University Prrrk, Penn.syIl~ania,U . S . A . 16802
AND
D. C. BIRDSELL Depcrrtment of Btrsic Dentrrl Sri~rrc.es,Urli~.rrsityc(fF1oridrr College. of Dentistry. Gninc,.svill(., Floridrr, U . S . A . 32610 Accepted March 1. 1976
EMYANITOFF, R. G . , T. E. RUCINSKY, and D. C. BIRDSELL. 1976. Electron microscopy of antibody-labelled cells of Streptococcwss mrrttrt1.s. Can. J. Microbiol. 22: 891-895. Examination of immune complexes between cells of Strcprococ~crr.~ r?lr~totr.sand homologous antiserum by the techniques of thin-sectioning and freeze-etching revealed that the cells were embedded within an extensive matrix 8C-90 nm thick with defined boundaries.
EMYANITOFF, R. G., T. E. RUCINSKYet D. C. BIRDSELL. 1976. Electron microscopy of antibody-labelled cells of Streptococclrs mrrtrrrls. Can. J . Microbiol. 22: 891-895. Les immuns complexes form& lors de la reaction entre S t r e p t o c o c c ~ trn~ttans .~ et un antiserum homologue ont ete examines en coupes minces et par decapage i froid. Cet examen montre des cellules enrobees dans une grosse gaine de 80 i 90 nm d'tpaisseur et dont les contours sont dkfinis. [Traduit par le journal]
T h e initial interaction between m a n a n d his coniplex oral flora is based upon specific adherence between surface components o f the organism a n d complimentary components o n the surface t o be colonized (tooth surface, epithelial cell, etc.) (4). Once this initial interaction has been established, most subsequent diversification of species is d u e t o bacterial cell -cell interaction with concomitant nutritional interaction (cross-feeding, etc.). T h e techniques recently developed by Listgarten et al. (9) t o examine dental plaque ill sit11 have underlined the complexity of the flora contained within the plaque matrix. Dental plaque plays m a j o r roles in t w o disease processes: (a) as a source of acids f o r caries production, a n d (6) a s a site f o r release of antigenic conlponents believed t o be involved in the i m m u n e etiology of gingivitis a n d perio'Received June 20, 1975.
dontal disease ( I I). A n understanding o f the specific etiology of either of these disease processes necessitates a n understanding of the interaction a n d possible synergism a m o n g a n d between the niembers of the oral flora. A n imp o r t a n t contribution t o o u r a p p r o a c h t o this understanding results from the work of Lai et al. (6, 7, 8). Using specific antiserum they have been able t o identify c o l u m n a r colonies of Sttvptoc o c c ~ t ssang~tiswithin plaque. Expansion o f this technique t o include other members o f the plaque microbiota would certainly p r o m o t e a better understanding of t h e complexities of dental plaque. T h e study described below was designed t o (a) demonstrate antiserum coating of S. tn~ttans, a n d (b) gain information a b o u t t h e three-dimensional organization o f the antiserum - bacterial cell complex b y using t h e techniques of freeze-etching. Streptococc~ts tnlttans strain O M Z 1 7 6 (sub-
Can. J. Microbiol. Downloaded from www.nrcresearchpress.com by University of Queensland on 11/19/14 For personal use only.
892
CAN. J. MICROBIOL. VOL. 22, 1976
species mutans, Bratthall serotype c t ) and strain NCTC2 10449 (subspecies sobrinus, Bratthall serotype c) were used for this study. Both were obtained from Dr. I. Shklair, Great Lakes Naval Training Center, Great Lakes, Illinois. The cultures were maintained in fluid thioglycollate medium (Difco) to which 2% calcium carbonate was added. The cultures were grown in a yeast extract - tryptone - glucose medium at 37 "C under an atmosphere of N2/C02 (95:5). Antigen for immunization was prepared by suspending 50 mg of lyophilized stationary phase bacteria in 5 ml of 0.15 M NaCl (saline) and incubating for 30 min at 60 "C to heat-kill the cells. The rabbits were given intravenous injections of the antigen 3 days per week over a 3-week period. Ten days after the last injection blood was withdrawn by cardiac puncture, clarified, and the serum fraction frozen at -20°C. Both the anti-10449 and the antiOMZ176 sera gave bands of precipitation with homologous antigen using Ouchterlony geldiffusion methods. No precipitation bands were observed between preimmune sera and homologous antigen. To prepare the antiserum-coated cells, a 10-ml sample of cells from exponentially growing cultures of each of the two strains was pelleted by centrifugation and resuspended in 0.5 ml of antiserum. Control samples were suspended in an equivalent amount of preimmune serum or 0.1 M phosphate buffer, pH 7.8. The cells were incubated for 30 min at 37 "C, centrifuged, and washed once in 5 volumes of saline. Cells were prepared for sectioning by preliminary fixation for 2 h in 2% glutaraldehyde, overnight fixation in 1% osmium tetroxide in the buffer of Ryter and Kellenberger (13), dehydration through a graded acetone series, and embedment in Spurr's low-viscosity medium (14). Sections were cut using an LKB ultramicrotome with a diamond knife. Sections were poststained with uranyl acetate and lead citrate (12) and examined using either a Zeiss EM9S or a Philips 300 electron microscope. Replicas of freeze-etched ce1Is were prepared with the use of Balzers BA360M freezeetching device by the method of Friedman et al. (3). The replicas were cleaned with 70% HH,SO, and 0.5% NaHOCl and examined using a Philips 300 electron microscope. In thin section of cells of S. mutans OMZ176 ZNCTC,National Collection of Type Cultures.
incubated with antiserum, a thick electron-dense layer can be seen surrounding the cells (Fig. 16). In most cases, the layer appears to be slightly less dense at the junction with the cell wall. Cells incubated in buffer or in preimmune serum did not show this layer (Fig. la). It is difficult to appreciate the three-dimensional aspects of the immune serum : cell complex by examination of thin-sectioned material. In addition. there exists the possibility of introducing fixation artifacts during preparation of thin sections for electron microscopy. Platinum-shadowed carbon replicas of unfixed freeze-fractured and etched s~ecimens were therefore examined. In the control suspensions the cells appeared to be distributed randomly throughout the replica, although, as would be expected, many cells appeared in chains (Fig. 2a). Good fracturing was achieved; in many cells the wall, membrane, and cytoplasm were easily distinguished. The background was uniform in appearance. Macroscopic agglutination was observed in the samples incubated with antiserum. The cells in these preparations were found only in specific areas of the replica. The material in these areas was less granular than the general background and exhibited discrete boundaries (Fig. 26). It thus appears that the cells were embedded within a thick coating of immune serum. In almost all cells either concave or convex surfaces were seen (Fig. 2c). Similar results were seen for S. mutans 10449. Cells of S. mutans strains OMZ176 treated with specific immune serum possess an additional, extensive layer (80-90 nm thick) external to their cell wall and similar to that described by Lai et al. (6, 7, 8). Freeze-fracture followed by etching revealed two additional features of the antibody cell surface interaction. (1) The cleavage plane within the cells appears to be altered in the presence of antiserum. The mechanism of this alteration is unknown. (2) Although the thickness of the layer external to the wall is evident in thin section, the three-dimensional perspective of the agglutinated cells is best observed in extensively etched preparations. In such preparations the immune complex appears more abundant than suggested by thin section. The observed thickness of the antibody - surface antigen layer is considerably greater (in thin section and freeze-etch preparations) than predicted on the basis of the size of the immunoglobulin molecules and measurements of cell wall thickness of S. mutans OMZ176 (2). Although it is possible that other
Can. J. Microbiol. Downloaded from www.nrcresearchpress.com by University of Queensland on 11/19/14 For personal use only.
NOTES
FIG.1. (a) Thin section of untreated S. mutans OMZ176. (b) Thin section of S. mlitans OMZ176 treated with immune serum. Note the thick fibrillar layer external to the cell wall. The bar in this and subsequent figures represents 500 nm.
893
CAN. J. MICROBIOL. VOL. 22, 1976
Can. J. Microbiol. Downloaded from www.nrcresearchpress.com by University of Queensland on 11/19/14 For personal use only.
894
FIG.2. (a) Freeze-etch preparation of control S. mutans OMZ176. Note the cytoplasm (C), cytoplasmic membrane (CM), and cell wall (CW). (b) Freeze-etch preparation of immune serum-treated S. mutans OMZ176. Note the discrete boundaries of the agglutinated cell complex (arrows). (c) Freezeetch preparation of immune serum-treated S. rnutans OMZ176.
Can. J. Microbiol. Downloaded from www.nrcresearchpress.com by University of Queensland on 11/19/14 For personal use only.
serum components may be bound within the complex, it is more likely that the cell surface polymers in their native configuration extend further into the medium surrounding the cells than revealed following fixation and dehydration. In the presence of specific antibody the polymers coated by antiserum are held in the native, extended configuration accounting for the thickness of the observed coating layer. Birdsell et 01. (I) have reported that the darkly staining portion of the cell wall of Bacillus subtilis decreased in thickness by 15% if concanavalin A (Con A) were allowed to interact specifically with the a-glucosylated teichoic acid of the cell wall before fixation and dehydration. An additional "fluffy" layer 25-60 nm in thickness composed of Con A and teichoic acid was external to the darkly staining cell wall. van Driel et al. (15) reported that the membrane teichoic acid (LTA) of Lactobacillus ferrnenti was exposed on the surface of the cell walls. These facts coupled with the recent reports of "secretion" of LTA by a variety of gram-positive organisms (5, 10) suggest that the domain of the bacterial cell surface may be considerably more extensive than indicated by examination of electron micrographs of thinsectioned material. Not only could covalently linked cell wall components extend several nanometres from the cell surface, but also cell membrane components could be in the process of transversing the cell wall for ultimate release into the culture fluid.
2. EMYANITOFF, R. G., and D. C. BIRDSELL.1974. Morphology of hydrated and lypophilized whole cells of Streptococcirs mutans OMZ176. Bacteriol. Proc. 1974: 56. 3. FRIEDMAN, B. S . , P . R. D U G A NR. , M. PFISTER.and C . C. REMSEN.1968. Fine structure and composition of the zoogleal matrix surrounding Zoogloecl ratnigericr. J. Bacteriol. 96: 214d2153. 4. GIBBONS,R. J., and J. V A N HOUTE. 1975. Bacterial adherence in oral microbial ecology. Annu. Rev. Microbiol. 29: 1 9 4 4 . 5. JOSEPH,R., and G. D. SHOCKMAN. 1975. Synthesis and excretion of glycerol teichoic acid during growth of two streptococcal species. Infect. Imrnun. 12: 333-338. 6. L A I , C . , M. A. LISTGARTEN, and B. ROSAN.1973. Serology ofStreptococcrrsscrtrglris: localization ofantigens with unlabeled antisera. Infect. Immun. 8: 475-48 1. 7. L A [ , C . , M. A. LISTGARTEN, and B. ROSAN.1975. Immunoelectron microscopic identification and localization of Strc~ptococc~ts scrtzg~ris with peroxidase-labeled antibody: localization of surface antigens in pure cultures. Infect. Immun. 11: 193-199. 8. LAI, C., M. A. LISTGARTEN, and B. ROSAN. 1975. lmmunoelectron microscopic identification and srrt~giris with localization of Srreprococclrs peroxidase-labeled antibody: localization of Srreptoc~occlrssanglris in intact dental plaque. Infect. Immun. 11: 20C-210. 9. LISTGARTEN, M. A., H . E . MAYO,and R . TREMBLAY. 1975. Development of dental plaque on epoxy resin crowns in man: a light and electron microscopic study. J. Periodontal. Res. 46: 10-26. 10. M A R K H A M J. ,L . , K . W. KNOX,A. J. W I C K E Nand , M. J. HEWETT. 1975. Formation of extracellular lipoteichoic acid by oral streptucocci and lactobacilli. Infect. Immun. 12: 378-386. 11. MERGENHAGEN, S. E., T . R. TEMPEL, and R. SNYDERMAN.1970. Immunologic reactions and periodontal inflammation. J. Dent. Res. 49: 256261. Acknowledgments 12. REYNOLDS, E. S. 1963. The use of lead citrate a t high We thank Dr. T. Hashimoto for his helpful p H a s an electron-opaque stain in electron microscopy. J . Cell Biol. 17: 20g212. discussions of the manuscript, Mr. 0. Payne and 1958. Etude au Mr. D. Balkwill for excellent technical assistance. 13. RYTER,A,, and E . KELLENBERGER. microscope electronique de plasmas contenant d e The work reported here was carried out while I'acide desoxyribonucleique. Z. Naturforsch. 13: one of us (R.G.E.) was supported by an American 597-605. Society for Microbiology Presidents Fellowship 14. SPURR,A. R. 1969. A low-viscosity epoxy resin embedding medium for electron microscopy. J. Ultrain the laboratory of T. E. Rucinsky. struct. Res. 26: 31-43. 15. V A N DRIEL,D. A., A . J. WICKEN, M. R. DICK SON,^^^ I. BIRDSELL, D. C . , R. J. DOYLE,and M. MORGENK . W. KNOX. 1973. Cellular location of the STERN.1975. Organization of the teichoic acid in the lipoteichoic acids of Lactobacill~rsfermenti NCTC cell wall of Bacillus subtilis 168. J. Bacteriol. 121: 6991 and Lactobaci//us casei N C T C 6375. J. Ultra726734. struct. Res. 43: 483-497.
