Staining Reaction of Dental Plaque After Various Extraction Procedures T. M. CHRISTIE, R. LACY, and E. NEWBRUN Division of Oral Biology, School of Dentistry, University of California, San Francisco, California 94143, USA After water and dilute alkali extraction, a sample of dental plaque that was stained by the PA-CrA-silver technique showed staining intracellularly on the cell wall and extracellularly. When more prolonged and stronger alkali extraction was used, reacting material was retained on the cell wall and intracellularly. There was a noticeable loss of extracellular reacting material.
Dental plaque is a soft, nonminer.alized bacterial deposit that forms on the exposed surfaces of teeth which are not adequately cleaned.1 The bacteria are embedded in an extracellular matrix. A morphometric analysis demonstrated that the extracellular matrix formed about 30% of the area of dental plaque at the plaque-tooth interface.2 These findings are in reasonable agreement with calculations based on bacterial counts of a plaque smear and the weight of a sample of plaque. Carlsson and Sundstr6m3 showed that the actual area occupied by each component was influenced by the preceding diet. The extracellular matrix of dental plaque consists in part of proteins and glycoproteins derived from saliva4,5 and polysaccharides formed by the bacteria.6,7 Water and various concentrations of alkali (NaOH or KOH) have been used to extract polysaccharides from dental plaque. Chemical analysis of these extracts indicates that levan and some glucans are water-soluble, the levans consisting of about 1 to 2% of the dry weight.8 Glucans and reportedly some hetThis work was supported by Grant DE 02512 from the National Institute of Dental Research, National Institutes of Health, Bethesda, Md. Received for publication June 6, 1974. Accepted for publication October 10, 1974.
eropolysaccharides9 are present in the alkali extract and account for 10 to 15%0 of the dry weight. It is not known, however, if such extraction procedures are selective for only the polysaccharides of the extracellular matrix or if bacterial cell walls or capsular material, intercellular glycogen, and nuclear material are also eluted. Carbohydrate macromolecules that are oxidized by periodic acid may be localized ultrastructurally by the periodic acidchromic acid-silver (PA-CrA-silver) technique.10,11 The purpose of this investigation was to determine if a loss of carbohydrate macromolecules from the sites other than extracellular matrix could be revealed after extraction steps by the PA-CrA-silver technique. Materials and Methods Dental plaque was collected on three separate occasions from all surfaces except the occlusal of the teeth of one volunteer
(29-year-old woman). The donor, following her usual diet, refrained from oral hygiene procedures for 24 hours. On the morning of collection, no breakfast was consumed in order to avoid inclusion of food debris in the sample. Thirty minutes before collection, the volunteer rinsed with a 10% (w/v) sucrose solution for three minutes and then rinsed immediately with distilled water. The sample of plaque was divided into four approximately equal portions. One portion was fixed immediately with a 2.5% (v/v) solution of glutaraldehyde in an 0.1 M sodium cacodylate buffer. The pH of the fixative was 7.2. The other three samples were each placed in 5 ml of the different extraction solutions and dispersed by shaking. They were extracted four times each at room temperature using 5 ml of triple 487
Downloaded from jdr.sagepub.com at East Carolina University on April 24, 2015 For personal use only. No other uses without permission.
J Dent Res May-June 1975
CHRISTIE, LACY, & NEWBRUN
488
glass-distilled water, 0.1 N NaOH, or 1 N NaOH, respectively. The plaque was redispersed and centrifuged (1,240 X g for four minutes) after every change of medium. A second collection of plaque was subjected to longer extraction times with more concentrated alkali. The second sample was divided into three portions and one portion was fixed with glutaraldehyde. The other two portions were extracted three times each at room temperature, one-half hour each time. using I N and 5 N NaOH. The extracted samples were fixed in buffered glutaraldlehyde for at least three hours. All samples were embedded in Vestopal NV polyester.a Thin sections approximately 600 A tlhick were cut with an ultramicrotome' andl collected on stainless steel grids. The sections were stained by tlhe PA-CrAsilver techniqJue.10"1' The stained specimens were examined and photographied witlh an electron microscope.C
Results PLAQUE
FIXED
BUT
NOT
OXIDIZED
WITH
PERIODIC ACID.-Deeply stained small particles a Polysciences Inc., Warringlon, Pa. bSorvall SMT-2, Porter Blum, Norwalk, Conn. c Elmiskop IA, Siemens, Berlin, W Ger.
of irregular size and shape were seen in the extracellular matrix of the plaque (Fig 1). Occasionally, similar but much less heavily stained granules could be observed intracellularly. More often the intracellular material appeared as a lightly stained homogeneous mass. In addition, weakly staining material was found in the cell wall. PLAQUE FIXED AND OXIDIZED WITH PERIODIC
ACID AND CHROMIC ACID.-Samples oxidized with PA-CrA showed a more intense staining of the extracellular material which appeared as fine particles. Bacterial cell walls were deeply stained and intracellular staining was more pronounced (Fig 2). PLAQUE FEXTRACTED WITH WATER AND OXIDIZED WITH PERIODIC ACID AND CHROMIC
ACID.-After extraction with water, reacting material was seen intracellularly, extracellularly, and in the bacterial cell wall. These samples showed a diminished intensity of staining in the cell wall (Fig 3). Control specimens that had not been oxidized resembled in appearance the nonextracted, nonoxidized plaque shown in Figure 1; the intensity of staining, however, was less. PLAQUE EXTRACTED WITH ALKALI AND OXIDIZED WITH PERIODIC ACID AND CHROMIC
ACID.-Samples of plaque extracted with 0.1
FIG 1.-Plaque specimen fixed immediately after collection. Stained with silver methenamine; no
oxidation (mag X52,500). Downloaded from jdr.sagepub.com at East Carolina University on April 24, 2015 For personal use only. No other uses without permission.
Vol1 54 No. 3
N NaOH appeared similar to samples extracted with water. Plaque extracted with I N NaOH consistently retained reacting material in the cell wall. Intracellularly, there was a more intense staining than that noticed in previous samples. Little or no staining was observed extracellularly (Fig 4). The nonoxidized stain control which had been extracted with I N NaOH also showed little or no staining extracellularly but clearly revealed reacting material in the cell wall. Intracellular staining was noticeably less than in the oxidized sample. When stronger alkali (5 N) was used, the extract-
ing procedures resulted in loss of reacting material in the cell wall as well as most of the extracellular material (Fig 5). The intense staining seen intracellularly with 1 N NaOH treated plaque was also seen in these samples. Only minimally reacting material was detectable in the cell wall of nonoxidized control specimens. Discussion The histochemical reaction of plaque before and after extraction by a variety of techniques was studied. Stain controls were used for each treatment. Plaque was sam-
STAINING REACTION OF PLAQUE
489
pled on three separate occasions and the findings were essentially the same. Unextracted plaque contained large amounts of deeply reacting material intracellularly, extracellularly, and in the cell wall. The intracellular material is presumed to be glycogen. The reacting material in the cell wall is probably muramyl glycopeptides and teichoic acids. The extracellular matrix is composed primarily of glycoproteins and water-insoluble glucans. To a lesser extent, water-soluble glucans and levans may be present as evidenced by the small alteration in intensity of staining following water extraction (compare Figs 2 and 3). Some of the extracellular polysaccharides may contribute to the staining of the cell wall while still attached to the enzymes responsible for their production. Since the control specimens for nonspecific staining showed considerable extracellular reacting material in unextracted, unoxidized plaque (Fig 1) , the specificity of the PA-CrA-silver technique may be questioned. Nonspecific staining of ribosomes and nuclei has been observed in mammalian cells.11 When the plaque was extracted with
FIG 2.-Plaque specimen fixed immediately after collection. Oxidized with PA-CrA and stained with silver methenamine (mag x52,500)
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490
CHRISTIE, LACY, r NEWBRUN
J Dent Res May-June 1975
FIG 3.-Plaquie specimen extracte(d for 90 minuLtes with water. Oxidlize( uvith PA-CrA andl staine(l with silver methienamine. Note failure to remove most of extracellullai- icacting material (mag x52,500).
water, the only change noted was some reduction in the staining of the cell wall. This may indicate that the stained extracellular material is made up predominantly of glucan, most of which is insoluble in water. The effect of alkali extraction appears to be dependent on time and concentration. Extraction withi 0.1 N and I N NaOH for 4f) minutites iatl little or no effect on the intensity of the staining reaction in the extracellular matrix and cell wall. Loss of
reacting material from the cell wall after more prolonged (1.5 hours) and more concentrate(d alkali treatment suggests that chemical stutlies of such plaquL e extracts sIhouldl be initerpreted with caution since the extracts undoubtedly contain cell-wall componients as well as extracellular material. Indeed, Hotz, Guiggenheim, and Schmid12 fountcd traces of cell wall components in 1 N KOH extract of pooled plaque and have correctly interpreted such a procedure as affecting the bacterial cell wall.
/zS\
i
FIG 4.-Plaque specimen extractedl for 90 minutes with I N NaOH. Oxiclized with PA-CrA and stained with silver methenamine. Note loss of extracellular reaction material (mag x52,500) .
Downloaded from jdr.sagepub.com at East Carolina University on April 24, 2015 For personal use only. No other uses without permission.
Vol 54 No. 3
STAINING REACTION OF PLAQUE
aSa
r
-I- -'-
491
9
400 nm
|- | .Fin 5.-Plaque sample extracted for 90 minuites witth 5 N NaOH. Oxidlized with PA-CrA and stained with silver methenamine. Note (leci-ease in staining initenisity of cell wvall material compared with Figure 4 (mag x52,500).
eichoic acids can be almost completely extracted from bacterial cell walls with dilulte alkaii (0.5) NaOH) at room temperature.13 Since teiclhoic acids have aminosuLgars or hlexoses attached to the glycerol or ribitol residues, extraction of thiose polymers could accotunt for some of the loss of cell-wall reacting material that was observed. Presumptive evidtence for the presence of glycerol teichioic acidls in Streptococcus mrltans (strains BHT andl FA-I) cell walls has been found.14 These have been extracted with alkali (0.5 N NaOH), purified, and are believed to represent the somatic antigen responsible for reactivity of this group.'5
It should be noted that not only polysaccharides are solubilized at the high pH occurring with alkali extraction procedures. Schachtele, Loken, and Schmittl6 treated the packed sediment of sucrose-grown cultures of oral streptococci with 0.3 N NaOH. Little of the insoluble polysaccharide was dissolved although most of the cellular deoxyribonuicleic acid and protein were solubilized. Fox and Dawesl7,18 reported maximal extraction of proteins from the predominant microorganisms of dental plaque and from hlulman plaque at a pH of 12.7 using 0.5 N NaOH. Mlany of the proteins in plaquie are glycoproteins; some of these slhould be oxidlizedl by periodic acid.19 Thus, the altered staining reactions observed in this study following alkali extractions may
also be attributed to the loss of glyco-
proteins.
Thle observations reportedl here are the Iasis for fturthler investigationi using otlher staining teclhniques and pure cultures of microorganlisms.20
Conclusions (C'hemical investigations of dental plaque bave ulse(d water and alkali extractions to separate the extracellular matrix from the microorganisms of plaque. This study, using the silver methenamine PA-CrA technique, was an attempt to determine the efficacy of such procedlures and to determine if such procedures also remove carbohydrate macromolecules from the bacterial cell wall and from within the cell. Plaque fixed immediately without extraction and without periodate oxidation revealed abundant reacting material particularly in the extracellular matrix. This implies that the silver methenamine PA-CrA teclhniqLue is not absolutely specific for polysaccharides. Parallel samples subjected to periodlate oxidation showed increased staining of cell-wall components and matrix. After water extr-action, reacting material was seen intracellularly, on the cell wall, and, what is most important, a significant amount extracellularly which is water insoluble. Dilute alkali (0.1 N NaOH) also failed to dissolve the extracellular matrix. After more prolonged and stronger alkali
Downloaded from jdr.sagepub.com at East Carolina University on April 24, 2015 For personal use only. No other uses without permission.
492
CHRISTIE, LACY, & NEWBRUN
extraction, reacting material was retained consistently on the cell wall and some intracellularly, but there was a noticeable loss of extracellular reacting material. References 1. LOE, H.: Present Day Status and Direction for Future Research on the Etiology and Prevention of Periodontal Disease, J Periodontol 48: 678-682, 1969. 2. SCHROEDER, H.E., and DE BOEVER, J.: The Structure of Microbial Dental Plaque, in McHUGH, W.H. (ed): Dental Plaque, Edinburgh: E. & S. Livingstone, Ltd., 1970, pp
49-74. 3. CARLSSON, J.; and SUNDSTR6M, B.: Variations in Composition of Early Dental Plaque Following Ingestion of Sucrose and Glucose, Odontol Revy 19: 161-169, 1968. 4. SILVERMAN, G.; and KLEINBERG, I.: Fractionation of Human Dental Plaque and the Characterization of its Cellular and Acellular Components, Arch Oral Biol 12: 1387-1405,
1967. 5. KREMBEL, J.; FRANK, R.M.; and DELUZARCHE, A.: Fractionation of Human Dental Plaque, Arch Oral Biol 14: 563-565, 1969. 6. GUGGENHEIM, B.: Extracellular Polysaccharides and Microbial Plaque, Int Dent J 20:
657-678, 1970. 7. NEWBRUN, E.: Extracellular Homopolysaccharides of Dental Plaque: Synthesis and Degradation, in ROBINOVITCH, M., and SCREEBNY, L. (eds): Dental Plaque and Its Relation to Oral Diseases, Seattle: University of Washington, 1972, pp. 36-58. 8. McDoUGALL, W.A.: Studies on Dental Plaque. IV Levans and the Dental Plaque, Aust Dent J 9: 1-5, 1964. 9. CRITCHLEY, P.: The Formation of Extracellular Polysaccharides from Glucose in Monkey Plaque In Vivo, Caries Res 3: 205-
206, 1969.
J Dent Res May-June 1975 10. HERNANDEZ, W.; RAMBOURG, A.; and LEBLOND, C.P.: Periodic Acid Chromic AcidMethenamine Silver Technique for Glucoprotein Detection in the Electron Microscope, J Histochem Cytochem 16: 507, 1968. 11. RAMBOURG, A.; HERNANDEZ, W.; and LEBLOND, C.P.: Detection of Complex Carbohydrates in the Golgi Apparatus of Rat Cells, J Cell Biol 40: 395-414, 1969. 12. HOTZ, P.; GUGGENHEIM, B.; and SCHMID, R.: Carbohydrates in Pooled Dental Plaque, Caries Res 6: 103-121, 1972. 13. ARCHIBALD, A.R.; COAPES, H.E.; and STAFFORD, G.H.: The Action of Dilute Alkali on Bacterial Cell Walls, Biochem J 113: 899-900, 1969. 14. BLEIWEIS, A.S.; CRAIG, R.A.; ZINNER, D.D.; and JABLON, J.M.: Chemical Composition of Purified Cell Walls of Cariogenic Streptococci, Infect Immun 3: 189-191, 1971. 15. BLEIWEIs, A.S.; CRAIG, R.A.; COLEMAN, S.E.; and VAN DE RIJN, I.: The Streptococcal Cell Wall: Structure, Antigenic Composition, and Reactivity with Lysozyme, J Dent Res 50: 1118-1130, 1971. 16. SCHACHTELE, C.F.; LOKEN, A.E.; and SCHMITT, M.K.: Use of Specifically Labeled Sucrose for Comparison of Extracellular Glucan and Fructan Metabolism by Oral Streptococci, Infect Immun 5: 263-266. 1972. 17. Fox, D.J.; and DAWES, C.: The Effect of Various Factors on the Extraction of Protein from the Predominant Microorganisms in Human Dental Plaque, Arch Oral Biol 15: 1355-1371, 1970. 18. Fox, D.J.; and DAWES, C.: The Extraction of Protein Matrix from Human Dental Plaque, Arch Oral Biol 15: 1069-1077, 1970. 19. FERGUSON, D.B.: The Electrophoresis of Dental Plaque, J Dent Res 43: 956, 1964. 20. SHARMA, M.; DHILLON, A.; and NEWBRUN, E.: Cell-bound Glucosyltransferase Activity of Streptococcus sanguis, Strain 804, Arch Oral Biol 19: 1063-1072, 1974.
Downloaded from jdr.sagepub.com at East Carolina University on April 24, 2015 For personal use only. No other uses without permission.
Dental plaque is a soft, nonminer.alized bacterial deposit that forms on the exposed surfaces of teeth which are not adequately cleaned.1 The bacteria are embedded in an extracellular matrix. A morphometric analysis demonstrated that the extracellular matrix formed about 30% of the area of dental plaque at the plaque-tooth interface.2 These findings are in reasonable agreement with calculations based on bacterial counts of a plaque smear and the weight of a sample of plaque. Carlsson and Sundstr6m3 showed that the actual area occupied by each component was influenced by the preceding diet. The extracellular matrix of dental plaque consists in part of proteins and glycoproteins derived from saliva4,5 and polysaccharides formed by the bacteria.6,7 Water and various concentrations of alkali (NaOH or KOH) have been used to extract polysaccharides from dental plaque. Chemical analysis of these extracts indicates that levan and some glucans are water-soluble, the levans consisting of about 1 to 2% of the dry weight.8 Glucans and reportedly some hetThis work was supported by Grant DE 02512 from the National Institute of Dental Research, National Institutes of Health, Bethesda, Md. Received for publication June 6, 1974. Accepted for publication October 10, 1974.
eropolysaccharides9 are present in the alkali extract and account for 10 to 15%0 of the dry weight. It is not known, however, if such extraction procedures are selective for only the polysaccharides of the extracellular matrix or if bacterial cell walls or capsular material, intercellular glycogen, and nuclear material are also eluted. Carbohydrate macromolecules that are oxidized by periodic acid may be localized ultrastructurally by the periodic acidchromic acid-silver (PA-CrA-silver) technique.10,11 The purpose of this investigation was to determine if a loss of carbohydrate macromolecules from the sites other than extracellular matrix could be revealed after extraction steps by the PA-CrA-silver technique. Materials and Methods Dental plaque was collected on three separate occasions from all surfaces except the occlusal of the teeth of one volunteer
(29-year-old woman). The donor, following her usual diet, refrained from oral hygiene procedures for 24 hours. On the morning of collection, no breakfast was consumed in order to avoid inclusion of food debris in the sample. Thirty minutes before collection, the volunteer rinsed with a 10% (w/v) sucrose solution for three minutes and then rinsed immediately with distilled water. The sample of plaque was divided into four approximately equal portions. One portion was fixed immediately with a 2.5% (v/v) solution of glutaraldehyde in an 0.1 M sodium cacodylate buffer. The pH of the fixative was 7.2. The other three samples were each placed in 5 ml of the different extraction solutions and dispersed by shaking. They were extracted four times each at room temperature using 5 ml of triple 487
Downloaded from jdr.sagepub.com at East Carolina University on April 24, 2015 For personal use only. No other uses without permission.
J Dent Res May-June 1975
CHRISTIE, LACY, & NEWBRUN
488
glass-distilled water, 0.1 N NaOH, or 1 N NaOH, respectively. The plaque was redispersed and centrifuged (1,240 X g for four minutes) after every change of medium. A second collection of plaque was subjected to longer extraction times with more concentrated alkali. The second sample was divided into three portions and one portion was fixed with glutaraldehyde. The other two portions were extracted three times each at room temperature, one-half hour each time. using I N and 5 N NaOH. The extracted samples were fixed in buffered glutaraldlehyde for at least three hours. All samples were embedded in Vestopal NV polyester.a Thin sections approximately 600 A tlhick were cut with an ultramicrotome' andl collected on stainless steel grids. The sections were stained by tlhe PA-CrAsilver techniqJue.10"1' The stained specimens were examined and photographied witlh an electron microscope.C
Results PLAQUE
FIXED
BUT
NOT
OXIDIZED
WITH
PERIODIC ACID.-Deeply stained small particles a Polysciences Inc., Warringlon, Pa. bSorvall SMT-2, Porter Blum, Norwalk, Conn. c Elmiskop IA, Siemens, Berlin, W Ger.
of irregular size and shape were seen in the extracellular matrix of the plaque (Fig 1). Occasionally, similar but much less heavily stained granules could be observed intracellularly. More often the intracellular material appeared as a lightly stained homogeneous mass. In addition, weakly staining material was found in the cell wall. PLAQUE FIXED AND OXIDIZED WITH PERIODIC
ACID AND CHROMIC ACID.-Samples oxidized with PA-CrA showed a more intense staining of the extracellular material which appeared as fine particles. Bacterial cell walls were deeply stained and intracellular staining was more pronounced (Fig 2). PLAQUE FEXTRACTED WITH WATER AND OXIDIZED WITH PERIODIC ACID AND CHROMIC
ACID.-After extraction with water, reacting material was seen intracellularly, extracellularly, and in the bacterial cell wall. These samples showed a diminished intensity of staining in the cell wall (Fig 3). Control specimens that had not been oxidized resembled in appearance the nonextracted, nonoxidized plaque shown in Figure 1; the intensity of staining, however, was less. PLAQUE EXTRACTED WITH ALKALI AND OXIDIZED WITH PERIODIC ACID AND CHROMIC
ACID.-Samples of plaque extracted with 0.1
FIG 1.-Plaque specimen fixed immediately after collection. Stained with silver methenamine; no
oxidation (mag X52,500). Downloaded from jdr.sagepub.com at East Carolina University on April 24, 2015 For personal use only. No other uses without permission.
Vol1 54 No. 3
N NaOH appeared similar to samples extracted with water. Plaque extracted with I N NaOH consistently retained reacting material in the cell wall. Intracellularly, there was a more intense staining than that noticed in previous samples. Little or no staining was observed extracellularly (Fig 4). The nonoxidized stain control which had been extracted with I N NaOH also showed little or no staining extracellularly but clearly revealed reacting material in the cell wall. Intracellular staining was noticeably less than in the oxidized sample. When stronger alkali (5 N) was used, the extract-
ing procedures resulted in loss of reacting material in the cell wall as well as most of the extracellular material (Fig 5). The intense staining seen intracellularly with 1 N NaOH treated plaque was also seen in these samples. Only minimally reacting material was detectable in the cell wall of nonoxidized control specimens. Discussion The histochemical reaction of plaque before and after extraction by a variety of techniques was studied. Stain controls were used for each treatment. Plaque was sam-
STAINING REACTION OF PLAQUE
489
pled on three separate occasions and the findings were essentially the same. Unextracted plaque contained large amounts of deeply reacting material intracellularly, extracellularly, and in the cell wall. The intracellular material is presumed to be glycogen. The reacting material in the cell wall is probably muramyl glycopeptides and teichoic acids. The extracellular matrix is composed primarily of glycoproteins and water-insoluble glucans. To a lesser extent, water-soluble glucans and levans may be present as evidenced by the small alteration in intensity of staining following water extraction (compare Figs 2 and 3). Some of the extracellular polysaccharides may contribute to the staining of the cell wall while still attached to the enzymes responsible for their production. Since the control specimens for nonspecific staining showed considerable extracellular reacting material in unextracted, unoxidized plaque (Fig 1) , the specificity of the PA-CrA-silver technique may be questioned. Nonspecific staining of ribosomes and nuclei has been observed in mammalian cells.11 When the plaque was extracted with
FIG 2.-Plaque specimen fixed immediately after collection. Oxidized with PA-CrA and stained with silver methenamine (mag x52,500)
Downloaded from jdr.sagepub.com at East Carolina University on April 24, 2015 For personal use only. No other uses without permission.
490
CHRISTIE, LACY, r NEWBRUN
J Dent Res May-June 1975
FIG 3.-Plaquie specimen extracte(d for 90 minuLtes with water. Oxidlize( uvith PA-CrA andl staine(l with silver methienamine. Note failure to remove most of extracellullai- icacting material (mag x52,500).
water, the only change noted was some reduction in the staining of the cell wall. This may indicate that the stained extracellular material is made up predominantly of glucan, most of which is insoluble in water. The effect of alkali extraction appears to be dependent on time and concentration. Extraction withi 0.1 N and I N NaOH for 4f) minutites iatl little or no effect on the intensity of the staining reaction in the extracellular matrix and cell wall. Loss of
reacting material from the cell wall after more prolonged (1.5 hours) and more concentrate(d alkali treatment suggests that chemical stutlies of such plaquL e extracts sIhouldl be initerpreted with caution since the extracts undoubtedly contain cell-wall componients as well as extracellular material. Indeed, Hotz, Guiggenheim, and Schmid12 fountcd traces of cell wall components in 1 N KOH extract of pooled plaque and have correctly interpreted such a procedure as affecting the bacterial cell wall.
/zS\
i
FIG 4.-Plaque specimen extractedl for 90 minutes with I N NaOH. Oxiclized with PA-CrA and stained with silver methenamine. Note loss of extracellular reaction material (mag x52,500) .
Downloaded from jdr.sagepub.com at East Carolina University on April 24, 2015 For personal use only. No other uses without permission.
Vol 54 No. 3
STAINING REACTION OF PLAQUE
aSa
r
-I- -'-
491
9
400 nm
|- | .Fin 5.-Plaque sample extracted for 90 minuites witth 5 N NaOH. Oxidlized with PA-CrA and stained with silver methenamine. Note (leci-ease in staining initenisity of cell wvall material compared with Figure 4 (mag x52,500).
eichoic acids can be almost completely extracted from bacterial cell walls with dilulte alkaii (0.5) NaOH) at room temperature.13 Since teiclhoic acids have aminosuLgars or hlexoses attached to the glycerol or ribitol residues, extraction of thiose polymers could accotunt for some of the loss of cell-wall reacting material that was observed. Presumptive evidtence for the presence of glycerol teichioic acidls in Streptococcus mrltans (strains BHT andl FA-I) cell walls has been found.14 These have been extracted with alkali (0.5 N NaOH), purified, and are believed to represent the somatic antigen responsible for reactivity of this group.'5
It should be noted that not only polysaccharides are solubilized at the high pH occurring with alkali extraction procedures. Schachtele, Loken, and Schmittl6 treated the packed sediment of sucrose-grown cultures of oral streptococci with 0.3 N NaOH. Little of the insoluble polysaccharide was dissolved although most of the cellular deoxyribonuicleic acid and protein were solubilized. Fox and Dawesl7,18 reported maximal extraction of proteins from the predominant microorganisms of dental plaque and from hlulman plaque at a pH of 12.7 using 0.5 N NaOH. Mlany of the proteins in plaquie are glycoproteins; some of these slhould be oxidlizedl by periodic acid.19 Thus, the altered staining reactions observed in this study following alkali extractions may
also be attributed to the loss of glyco-
proteins.
Thle observations reportedl here are the Iasis for fturthler investigationi using otlher staining teclhniques and pure cultures of microorganlisms.20
Conclusions (C'hemical investigations of dental plaque bave ulse(d water and alkali extractions to separate the extracellular matrix from the microorganisms of plaque. This study, using the silver methenamine PA-CrA technique, was an attempt to determine the efficacy of such procedlures and to determine if such procedures also remove carbohydrate macromolecules from the bacterial cell wall and from within the cell. Plaque fixed immediately without extraction and without periodate oxidation revealed abundant reacting material particularly in the extracellular matrix. This implies that the silver methenamine PA-CrA teclhniqLue is not absolutely specific for polysaccharides. Parallel samples subjected to periodlate oxidation showed increased staining of cell-wall components and matrix. After water extr-action, reacting material was seen intracellularly, on the cell wall, and, what is most important, a significant amount extracellularly which is water insoluble. Dilute alkali (0.1 N NaOH) also failed to dissolve the extracellular matrix. After more prolonged and stronger alkali
Downloaded from jdr.sagepub.com at East Carolina University on April 24, 2015 For personal use only. No other uses without permission.
492
CHRISTIE, LACY, & NEWBRUN
extraction, reacting material was retained consistently on the cell wall and some intracellularly, but there was a noticeable loss of extracellular reacting material. References 1. LOE, H.: Present Day Status and Direction for Future Research on the Etiology and Prevention of Periodontal Disease, J Periodontol 48: 678-682, 1969. 2. SCHROEDER, H.E., and DE BOEVER, J.: The Structure of Microbial Dental Plaque, in McHUGH, W.H. (ed): Dental Plaque, Edinburgh: E. & S. Livingstone, Ltd., 1970, pp
49-74. 3. CARLSSON, J.; and SUNDSTR6M, B.: Variations in Composition of Early Dental Plaque Following Ingestion of Sucrose and Glucose, Odontol Revy 19: 161-169, 1968. 4. SILVERMAN, G.; and KLEINBERG, I.: Fractionation of Human Dental Plaque and the Characterization of its Cellular and Acellular Components, Arch Oral Biol 12: 1387-1405,
1967. 5. KREMBEL, J.; FRANK, R.M.; and DELUZARCHE, A.: Fractionation of Human Dental Plaque, Arch Oral Biol 14: 563-565, 1969. 6. GUGGENHEIM, B.: Extracellular Polysaccharides and Microbial Plaque, Int Dent J 20:
657-678, 1970. 7. NEWBRUN, E.: Extracellular Homopolysaccharides of Dental Plaque: Synthesis and Degradation, in ROBINOVITCH, M., and SCREEBNY, L. (eds): Dental Plaque and Its Relation to Oral Diseases, Seattle: University of Washington, 1972, pp. 36-58. 8. McDoUGALL, W.A.: Studies on Dental Plaque. IV Levans and the Dental Plaque, Aust Dent J 9: 1-5, 1964. 9. CRITCHLEY, P.: The Formation of Extracellular Polysaccharides from Glucose in Monkey Plaque In Vivo, Caries Res 3: 205-
206, 1969.
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