INFECTION AND IMMUNITY, Apr. 1976, p. 1228-1234Copyright © 1976 American Society for Microbiology

Vol. 13, No. 4Printed in USA.

Dextran-Mediated Interbacterial Aggregation BetweenDextran-Synthesizing Streptococci and Actinomyces viscosus

G. BOURGEAU AND B. C. McBRIDE*

Department of Microbiology, University of British Columbia, Vancouver, British Columbia, V6T 1 W5,Canada

Received for publication 18 November 1975

Streptococcus sanguis and Streptococcus mutans bind to the surface ofActino-myces viscosus, producing large microbial aggregates. Aggregates form rapidlyand are not easily dissociated by vigorous mixing. The binding is mediated bydextran. Glucose-grown streptococci will not aggregate unless they are firstmixed with high-molecular-weight dextran. Aggregation is induced with dex-trans isolated from Leuconostoc, S. sanguis, or S. mutans. Sucrose-grownstreptococci will adhere to A. viscosus without the addition of an exogenoussource of dextran. A. viscosus will bind dextran and then bind glucose-grownstreptococci. Aggregation occurs over a wide pH range and is dependent oncations. The aggregating activity of A. viscosus is both protease and heatsensitive. The aggregating activity of S. sanguis is heat stable but sensitive todextranase.

Human dental plaque is a complex assort-ment of bacteria held together by host-synthe-sized (7) and bacterially synthesized polymers(6). Although the flora is complex, it is at thesame time a specific association of microorga-nisms (9). This specificity is in part a conse-quence of the ability of bacteria to adhere tohost tissue or to other bacteria. Gibbons andNygaard (8) reported that certain oral orga-nisms would aggregate with one another whenpure cultures were mixed together. They sug-gested that this aggregating capability repre-sented a mechanism that would allow bacteriato bind into plaque. Specific interspecies aggre-gation of bacteria has been observed by micro-scopic study of dental plaque. The most strikingexample is the "corncob" association of cocciand rods (12, 13).

In recent publications (14, 14a), we reportedthat high-molecular-weight dextran will in-duce aggregation of the filamentous organismActinomyces viscosus. The reaction is sensi-tive, requiring as few as three molecules ofdextran per bacterial cell, and it is specificfor dextran. The association is strong, as evi-denced by the fact that cells will bind toSephadex beads (dextran) and resist removalby vigorous washing. In the presence of su-crose Streptococcus sanguis and Streptococcusmutans will synthesize dextran that is boundto the cell and dextran that is released intothe media (5, 11, 18). We postulated that theActinomyces could use the cell-free dextranto form aggregates in plaque. In this report we

will provide evidence that cell-bound dextranwill mediate the attachment ofA. viscosus to S.sanguis and S. mutans.

MATERIALS AND METHODSOrganisms. A. viscosus (15987) was obtained from

the American Type Culture Collection. It was grownin brain heart infusion broth (BHI) in static culture,aerobically at 37 C. Growth on a solid medium re-quired incubation in a 10% CO2 atmosphere. S. mu-tans was isolated from human carious lesions asdescribed by Gold et al. (10). S. sanguis was isolatedfrom human dental plaque as described by Carlsson(1). These organisms were grown in Trypticase soybroth (TSB).

Stock cultures were stored at -70 C in growthmedium supplemented with enough sterile glycerolor dimethyl sulfoxide to give a final concentration of10%. It was found that A. viscosus cells that wererepeatedly subcultured gradually lost the ability toaggregate. Because of this, cells were grown fromthe stock cultures once a month.

Dextran. Dextrans of known molecular weightwere purchased from Pharmacia (Montreal, Quebec,Canada). Samples of dextran were isolated fromvarious S. sanguis and S. mutans strains by theprocedure of Sidebotham et al. (7).

Analysis of the isolated dextrans. Total carbohy-drate was measured by the anthrone method asdescribed by Scott and Melvin (16) or by the phenol-sulfuric acid method of Dubois et al. (3), using dex-tran T2000 as the standard. Ketohexose was deter-mined by the cysteine-sulfuric acid method of Discheand Devi (2), using fructose as a standard. Proteinwas measured by the Lowry method, using bovineserum albumin as a standard. The purity of thedextran samples was checked by thin-layer chroma-

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DEXTRAN-MEDIATED INTERBACTERIAL AGGREGATION 1229

tography. Dextran samples were dissolved in dis-tilled water to a final concentration of 2.5 mg/ml.One hundred microliters of the sample was mixedwith 100 M1 of 4 N HCl and sealed in a glass am-

poule. The mixture was hydrolyzed in a boiling wa-

ter bath for 1 h. The hydrolyzed samples were thenfreeze-dried in the presence of NaOH pellets. Theresidue was resuspended in 100 ,l of distilled water,

and 10 A1 was chromatographed on a cellulose thin-layer chromatography sheet (Polygram Cell 300;Macherey-Nagel and Co., Duren, Germany) in n-

butanol-acetic acid-water (3:1:1). The chromato-gram was developed using an aniline diphenyl-amine spray followed by heating at 100 C for 2 min.Dextran T2000, glucose, and fructose were used as

standards.Enzymes. Trypsin (type XI), chymotrypsin (type

III), subtilisin (type VIII), and hyaluronidase (typeIII) were purchased from Sigma. Dextranase, pur-

chased from Sigma, was found to be contaminatedwith protease and numerous glycosidases. The gly-cosidase activities detected are reported in Table 1.Protease was measured using the casein assay de-scribed by Rick (15).

Interbacterial aggregation. Bacterial cells were

harvested by centrifugation, washed three times in0.02 M tris(hydroxymethyl)aminomethane (Tris)buffer, pH 7.2, and resuspended in Tris buffer togive the desired number of cells per milliliter. Toinsure that the streptococcal cells were free of solu-ble dextran, the supernatant from the third washwas assayed to determine whether it could induceaggregation ofA. viscosus. The required numbers ofcells were then mixed together with enough bufferto give a final volume of 1.1 ml. The mixture was

incubated at room temperature for 1 h. At 15, 30,and 45 min the tubes were agitated gently. Theextent of aggregation was measured after 1 h ofincubation by either scoring aggregation on a 0 to+4 basis or by counting the nonaggregated bacteria.In the former procedure the size of the aggregateswas estimated visually, using a stereomicroscope; a

score of zero indicated no visible aggregation and a

score of +4 indicated that the majority of bacteriahad formed a single large clump. Only a limitedamount of information can be obtained in the visualassay. Therefore, a second more precise measure of

activity was made by counting the numbers of orga-nisms remaining in suspension after the aggregateshad settled to the bottom of the tube. Fifteen min-utes after the final shaking, bacterial aggregateshad settled to the bottom of the tube and it was

possible to remove a sample from the top of thereaction mixture. The sample was diluted in expo-

nential steps into sterile buffer and replicate sam-

ples were plated on Mitis-Salivarius agar to deter-mine the number of streptococci and onto BHI agar

to determine total counts. The numbers of A. visco-sus were determined by the difference. The relativerecovery rates of each organism on the two types ofmedia were also determined.

RESULTS

Interbacterial aggregation. When A. visco-sus was mixed with S. sanguis, the cell suspen-

sion rapidly became granular and soon largeclumps of bacteria formed (Fig. 1). Microscopicexamination of these clumps showed that theywere a mixture of the two organisms. The spec-

imen used for photomicrography was preparedby sonicating clumps of aggregated bacteria.This procedure dispersed the organisms wellenough to permit photographs to be taken. Be-fore sonication, the organisms were all presentin large refractile clumps and it was difficult todistinguish rods from cocci. The visibly obviousaggregation could be measured as a decrease inthe numbers of bacteria that remained in sus-

pension in the reaction mixture (Table 2).There was no observable decrease in the num-

bers of cells in suspension in control reactionsin which each organism was incubated alone.The colony-forming units (CFU) ofA. viscosuswere estimated by difference as describedabove. The accuracy of this procedure was veri-fled in an experiment in which each colony on a

BHI plate was identified by microscopy as con-

sisting of either coccal or filamentous bacteria.There was excellent correlation between the

TABLE 1. Contaminating glycosidase activity in commercial dextranase

Buffer (mo- Substrate hydro-

PNP substrate Temp pH lar concn of lyzed (,umol) per Enzyme U per mg(C) sodium cit- min by 0.5 mg of of dextranasen

rate) dextranase

/3-D-GalactopyranoSide 30 4.6 0.05 2.35 x 10-3 2.12 x 102a-D-GalaCtopyranoside 30 4.6 0.05 3.66 x 10-3 1.36 x 102/3-D-Glucoside 25 4.8 0.10 3.30 x 10-3 1.51 x 102a-L-FUCOSide 25 6.0 0.05 7.70 x 10-6 6.50 x 104a-D-GlUCOSide 25 4.8 0.10 1.15 x 10-3 4.35 x 102N-acetyl-,-D-galactosaminide 30 4.6 0.05 3.90 X 10- 1.28 X 102N-acetyl-f3-D-glucosaminide 30 4.6 0.05 8.00 x 10-4 6.25 X 10213-Glucuronide 37 5.0 0.20 9.00 X 10-5 5.60 x 103a-D-MannOSide 25 4.5 0.05 1.70 x 10-4 2.93 X 103

't One unit is the amount of enzyme needed to hydrolyze 1 MLmol of substrate per min under the conditionscited.

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1230 BOURGEAU AND McBRIDE

TABLE 2. Interbacterial aggregation of S. sanguisand A. viscosus

Bacterial mix- Decrease Aggrega-ture Cells/mi in Agetion

S. sanguis 109 0 0A. viscosus 109 0 0S. sanguis + 109 57 +4A. viscosus 109 86S. sanguis + 109 15 +3A. viscosus 108 84S. sanguis + 109 84 +3A. viscosus 107 90S. sanguis + 109 0 0A. viscosus 100,0

difference method and the colony identificationmethod; therefore the less time-consuming dif-ference procedure was used for all experiments.Aggregates could be partially dispersed by vig-orous mixing, but they rapidly reformed uponstanding.S. mutans also co-aggregates with A. visco-

sus, but because S. mutans self-aggregates theassay for co-aggregation is more difficult. Forthis reason S. sanguis was used for the major-ity of experiments reported in this paper.

Co-aggregation is dependent on the presenceof ions. The requirement is not specific and maybe satisfied by either 5 x 10-3 M divalent or 10-2M monovalent cations. Ions tested included Na,K, Mg, Mn, Ca, Ba, and Cs. This is analogousto dextran-induced aggregation of A. viscosus.Co-aggregation was not affected by altering thepH between 5.0 and 8.5.Role of sucrose. Dextran induces aggrega-

tion of A. viscosus, and it seemed reasonablethat dextran on the cell surface of the strepto-cocci might be the factor that mediates co-ag-gregation. To test this hypothesis, S. mutansand S. sanguis were grown in TSB and TSBsupplemented with one of eleven differentsugars to a final concentration of 1%. The cellswere harvested and washed three times in Trisbuffer and then tested for their ability to co-aggregate with A. viscosus. To insure thatthere was no soluble dextran, the streptococciwere washed with buffer until the washings no

longer aggregated A. viscosus. The Actino-myces and one of the streptococci were mixedand incubated, and aggregation was measured.Streptococci grown in TSB supplemented withglucose did not co-aggregate with A. viscosus,

whereas cells grown in the presence of sucrose

aggregated strongly with A. viscosus. No othersugar supplement resulted in aggregation. Theability of A. viscosus to aggregate was not af-fected by the type of sugar in its growth me-

dium.

The importance of sucrose was indicated inanother experiment in which S. sanguis wasgrown to the stationary growth phase in TSB.Sucrose was then added to give a final concen-tration of 5%. The culture was incubated at37 C, and cells were removed at various times,washed, and then assayed for aggregating ac-tivity. After incubation for 4 h in the presenceof sucrose, the cells were able to aggregate(Table 4). Maximum activity was observedafter 12 h of incubation. Control cells that werenot exposed to sucrose did not aggregate at anytime during the 12-h incubation period. In thisexperiment there was a small amount of back-ground aggregation; this was observed sporadi-cally and can probably be attributed to thesmall amount of sucrose that is found as acontaminant in TSB.

Effect of cell concentration. In vitro aggre-gation is dependent on the number of orga-nisms in the assay mixture. When the S. san-guis concentration was kept constant at 10"CFU/ml, the A. viscosus concentration could bedropped to as low as 107 CFU/ml before therewas a large loss in aggregating activity (Table2). In the reverse experiment (Table 3), the A.

TABLE 3. Interbacterial aggregation ofA. viscosusand glucose or sucrose-grown S. sanguis

Decrease in cfu AggregationBacterial mix- Cells/!

ture ml glu- su- glu- su-

cose" crose" cosea crose"

A. viscosus 1010 0 0 0 0S. sanguis 10"' 0 0 0 0A. viscosus + 1010 32 95S. sanguis 1010 35 95 +1 +4A. viscosus + 10'0 0 79S. sanguis 109 0 99 0 +3A. viscosus + 1010 0 74S. sanguis 100 0 99 0 +2A. viscosus + 1010 0 0 0S. sanguis 10' 0 0 0

" S. sanguis grown in TSB supplemented with eitherglucose or sucrose.

TABLE 4. Effect oflength of incubation with sucroseon interbacterial aggregation

Reaction mix- Length of incubation (h)ture SucrosetO 4 8 12

A. viscosus -0 0 0 0S. sanguis + 0 0 0 0A. viscosus + -1 3 3 4S. sanguis +A. viscosus + -S. sanguis - +1 +1 +1 +1

" Sucrose was added and then immediately re-moved by washing.

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DEXTRAN-MEDIATED INTERBACTERIAL AGGREGATION 1231

viscosus concentration was kept constant andthe numbers of S. sanguis were varied. As canbe seen, there was no demonstrable aggrega-tion when the streptococcal population fell be-low 108 CFU/ml. These results indicate that therelative proportions of the organisms are im-portant in the in vitro assay.

Effect of cell age. Cell surfaces can changewith the age of the cells and culture conditions,and such changes may affect the ability of cellsto aggregate. To determine whether this wasoccurring, an experiment was designed inwhich the age of the S. sanguis culture waskept constant at 18 h and the age of the A.viscosus culture was varied from 1 to 4 days.Cells were washed and resuspended to a con-centration of 10'0 cells/ml in buffer. There wasno change in the ability to aggregate. When 4-day-old A. viscosus cells were incubated withS. sanguis cells that varied in age between 8and 48 h, there was no change in the ability toco-aggregate although there appeared to be aslight increase in the tendency to self-aggre-gate on the part of the S. sanguis cells at 48 h.If A. viscosus was grown in TSB rather thanBHI, a different picture emerged. Aggregatingactivity was most pronounced during late logphase and disappeared rapidly during station-ary phase. Thus cultural conditions have a sig-nificant effect on the nature of the Actinomycescell surface.

Effect of exogenously added dextran. In anexperiment designed to prove that dextran me-diates aggregation, glucose-grown S. sanguiswas incubated with dextran (5 mg/ml) for 30

min. Dextran not bound to the cell surface wasremoved by washing the cells in Tris buffer.The dextran-treated S. sanguis cells weremixed with A. viscosus and aggregation wasmeasured (Table 5). Dextran T2000 or dextranisolated from S. sanguis bound to the strepto-coccal cell surface and promoted binding to A.viscosus. Cells that were not treated with dex-tran did not aggregate. Dextran T10, molecularweight 10,000, was not large enough to promoteaggregation. A similar experiment was per-formed with S. mutans (Table 6). The resultswere the same, but there was aggregation ofthe S. mutans in the presence of dextran,which made it necessary to disrupt these aggre-gates by vigorous agitation immediately beforemixing the cells with A. viscosus. When thisprocedure was followed, most of the A. viscosusaggregated with the S. mutans as evidenced bythe loss of CFU from the reaction mixture. Thiswas not caused by release of dextran from thestreptococci since the supernatant from an S.mutans control would not aggregate A. visco-sus.

In the reverse experiment (Table 5) A. visco-sus was incubated with dextran and thenmixed with glucose-grown S. sanguis or S. mu-tans. Interbacterial aggregation was inducedby S. mutans, S. sanguis, or dextran T2000 ordextran isolated from S. sanguis or S. mutans.Dextran T10 had no effect.

Cell modification. Treatment of A. viscosuswith protease severely impaired or eliminatedits ability to aggregate with dextran T2000. Ifdextran is the link between A. viscosus and the

t

* *4

* I-, 4..*.*'*-.'

0

0, * S

'0 F.

I . . Vp .-.

I &

U XI-v Z.b=sx

MEWP!

FIG. 1. Photomicrograph of S. sanguis bound to A. viscosus. Bar represents 10 ,um.

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1232 BOURGEAU AND McBRIDE

streptococci, such treatment should have a sim-ilar effect on co-aggregation. The enzymeslisted in Table 7 were incubated with A. visco-sus cells (1010 cells/ml) for 1 h. The final concen-tration of enzyme was 2 mg/ml. The cells werethen washed three times in Tris buffer andresuspended to the original concentration. A.

TABLE 5. Interbacterial aggregation mediated bydextran bound to S. sanguis or A. viscosus

Decrease in CFU

Exptl' Aggregation _____ Aggre-mixture . gationA. VIS- S. san-cosus guis

1 A. viscosus 0 - 02 S. sanguis - 0 03 S. sanguis + - 0 +1

dextranT2000

4 S. sanguis + - 0 +1S. sanguisdextran

5 A. viscosus + 10 0 +1S. sanguis

6 A. viscosus + 0 0 0S. sanguis+ dextran10

7 A. viscosus + 99 97 +2S. sanguis+ dextranT2000

8 A. viscosus + 99 99 +3S. sanguis+ S. san-guis dex-tran

9 A. viscosus + 80 - +3dextranb

10 A. viscosus + 99 99 +3dextranb +S. sanguis

"S. sanguis (expt. 3-8) orA. viscosus (expt. 9, 10)was incubated with dextran for 60 min and thenwashed to remove unbound dextran.

b Dextan T2000 or S. sanguis dextran.

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viscosus and S. sanguis were mixed in a 1:1ratio. As can be seen in Table 7, protease de-stroyed the ability to aggregate. Heating theA .viscosus to 90 C for 15 min eliminated aggre-gating activity. Dextranase destroyed the abil-ity ofA. viscosus mixed with dextran to bind toglucose-grown S. sanguis.The effect of treating S. sanguis cells with

dextranase (2 mg/ml) and various proteases (2mg/ml) is shown in Table 8. The cells wereincubated with the enzyme for 1 h, washedthree times with Tris buffer, and assayed. Dex-tranase completely eliminated aggregation,suggesting that a dextran molecule is involved.Since the dextranase is badly contaminatedwith other glycosidases, this is not a conclusiveexperiment. To try to get around this problem,the cells were mixed with dextranase in thepresence of low-molecular-weight dextran. Thedextran protected the cells from the enzyme,providing more evidence that the dextranse is

TABLE 6. Interbacterial aggregation mediated bydextran bound to S. mutans

Decrease in CFU (%)Aggregation mix- Aggrega-

ture A. visco- S. mu- tionsus tans

S. mutans 0 +1S. mutans + dex- 0 +1

tran 10S. mutans + dex- 25 +2

tran T2000S. mutans + Mu- 98 +4

tans dextranA. viscosus + S. 33 50 +2mutans

A. viscosus + S. 0 0 +1mutans + dex-tran 10

A. viscosus + S. 99 90 +3mutans + dex-tran T2000

A. viscosus + S. 99 99 +4mutans + mu-tans dextran

TABLE 7. Co-aggregation after modification of the A. viscosus cell surface

AggregationAggregation mixture

Trypsin Hyaluronidase Subtilisin Heat Dextranase None

A. viscosus 0 0 0 0 0 0A. viscosus + dextran 0 +2 0 0 0 +2

T2000A. viscosus + dextran 0 +4 0 +1 0 +4

T2000 + S. sanguis"A. viscosus + S. sanguisb 0 +4 0 +1 +3 +4

S. sanguis was grown in glucose-supplemented TSB broth.S. sanguis was grown in sucrose-supplemented TSB broth.

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TABLE 8. Co-aggregation after modification of the S. sanguis cell surtaceAggregation

Aggregation mixtureTrypsin Hyaluronidase Subtilisin Dextranase Heat None

S. sanguis 0 0 0 0 0 0S. sanguis" + A. viscosus +4 +4 +4 0 +3 0S. sanguisb + A. viscosus +4 +4 +4 +4 +3 0

+ dextran T2000

'" S. sanguis grown in glucose supplemented TSB broth.b A. viscosus grown in sucrose supplemented TSB broth.

the active enzyme. Boiling the streptococcalcells for 20 min or treating them with proteasedid not affect the ability of the cells to aggre-gate.

DISCUSSIONThe experiments described in this paper

prove that the dextran-synthesizing organismsS. sanguis and S. mutans will bind to thesurface ofA. viscosus. In the in vitro assay theconsequence is the formation ofclumps ofbacte-ria; in vivo this would result in interbacterialaggregations within the plaque matrix.Dextran appears to be the molecule that

binds the organisms together. A summary ofthe evidence follows. (i) Only those streptococcithat have been grown in the presence of sucrosewill aggregate. This is to be expected becausesucrose is required for dextran synthesis. (ii)The addition of dextran to streptococci grown inthe absence of sucrose creates a cell that canaggregate. In other words, the streptococci canbind dextran to the cell surface and this dex-tran can then be used to bind to A. viscosus.The reverse is also true-dextran bound to theActinomyces is available for binding to thestreptococci. (iii) An enzyme mixture contain-ing dextranase will destroy the ability of su-crose-grown streptococci to aggregate.The source of dextran does not seem impor-

tant; linear Leuconostoc dextran (dextranT2000) works as well as the soluble and insolu-ble dextrans isolated from S. sanguis and S.mutans. Thus dextrans that would be synthe-sized within the plaque matrix are capable ofmediating interbacterial aggregation.

Low-molecular-weight dextran does not me-diate binding. This is in keeping with the obser-vations that this compound will not induce ag-gregation of S. mutans (6) or A. viscosus (14a).A. viscosus loses the ability to bind to dextranand therefore to bind to the streptococci aftercontinuous subculture on laboratory medium.In some instances the organism will regain itsbinding abilities, but more often the bindingactivity is permanently lost. Therefore, it was

necessary to continually go back to the stockcultures to obtain reactive cells. The lability ofsurface properties is a problem that is oftenencountered in studies of cell recognition sys-tems. It is advisable that those who study mi-crobial adherence use freshly isolated strains intheir experiments. The receptor on the Actino-myces cell wall is a protein or is associated witha protein as it is sensitive to protease and heat-ing. It is unstable when the organism is re-moved from its growth medium. Cells lose theability to aggregate if they are incubated atroom temperature for 6 h, incubated at 4 Covernight, or frozen. The dextran-binding uniton the S. sanguis cell wall is probably not aprotein since it is insensitive to protease orheat. It could be a carbohydrate polymer or themurein in the cell wall.We do not wish to suggest that dextrans are

the only mechanism by which A. viscosus bindsinto a plaque matrix, but the ability of theorganism to bind to dextran makes it a veryplausible mechanism and one that could beused in an appropriate environment. The abil-ity of A. viscosus to adsorb soluble dextranmeans that it could use the dextran in an eco-system removed from the streptococci; alterna-tively it could bind directly to dextran-synthe-sizing organisms.

It is possible that the dextran is a model ofthe in vivo receptor. The natural receptor sys-tem could possibly consist of a number of a-1,6glucose units linked to a host glycoprotein orcell wall of some other bacteria. If this is thecase, then dextran is a model compound. How-ever, it seems likely that given its dextran-binding capability the organism would use it ifthe occasion arose. It is possible that the "corn-cobs" (13) present in plaque are streptococcibound to an Actinomyces by dextran.

ACKNOWLEDGMENTS

The assistance of Mary Gisslow is gratefully acknowl-edged.

This work was supported by the Canadian Medical Re-search Council.

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LITERATURE CITED

1. Carlsson, J. 1965. Zooglea-forming streptococci, resem-

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2. Dische, Z., and A. Devi. 1960. A new colorimetricmethod for the determination of ketohexoses in thepresence of aldoses, ketoheptoses and ketopentoses.Biochim. Biophys. Acta. 39:140-144.

3. Dubois, M., K. A. Giles, and J. K. Hamilton. 1956. Acolorimetric method for determination of sugar andrelated substances. Anal. Chem. 28:350-356.

4. Gibbons, R. J., and S. B. Banghart. 1967. Synthesis ofextracellular dextran by cariogenic bacteria and itspresence in human dental plaque. Arch. Oral Biol.12:11-24.

5. Gibbons, R. J., and M. Nygaard. 1968. Synthesis ofinsoluble dextran and its significance in the forma-tion of gelatinous deposits by plaque-forming strepto-cocci. Arch. Oral Biol. 13:1249-1262.

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7. Gibbons, R. J., and D. M. Spinell. 1969. Salivary in-duced aggregation of plaque bacteria. In W. D. Mc-Hugh (ed.), Symposium on dental plaque, E. and S.Livingstone, Wynnewood, Pa.

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11. Hehre, E. J., and J. M. Neill. 1946. Formation of sero-logically reactive dextrans by streptococci from sub-acute bacterial endocarditis. J. Exp. Med. 83:147-161.

12. Jones, S. J. 1972. A special relationship between spheri-cal and filamentous microorganisms in mature dentalplaque. Arch. Oral Biol. 17:613-616.

13. Listgarten, M. A., H. Mayo, and M. Amsterdam. 1973.Ultrastructure of the attachment device between coc-cal and filamentous organisms in "corncob" forma-tions of dental plaque. Arch. Oral Biol. 18:651-656.

14. McBride, B. C., and G. Bourgeau. 1975. Dextran in-duced aggregation of A. viscosus. J. Dent. Res.54:186.

14a. McBride, B. C., and G. Bourgeau. 1975. Dextran-in-duced aggregation of Actinomyces viscosus. Arch.Oral Biol. 20:837-841.

15. Rick, W. Trypsin. In H. V. Bergmeyer (ed.), Methods ofenzymatic analysis, p. 1018-1021. Academic PressInc., New York.

16. Scott, T. A., and E. H. Melvin. 1953. Determination ofdextran with anthrone. Anal. Chem. 25:1656-1661.

17. Sidebotham, R. L., H. Weigel, and W. H. Brown. 1971.Studies on dextrans and dextranases. Part IX. Dex-trans produced by cariogenic organisms. Carbohydr.Res. 19:151-159.

18. Wood, J. M., and P. Critchley. 1966. The extracellularpolysaccharide produced from sucrose by a cariogenicstreptococcus. Arch. Oral Biol. 11:1039-1042.

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