Simplified technique for sonication and processing of dental plaque samples

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0095-1137/79/05-0579/05$02.00/0

Simplified Technique for Sonication and Processing of Dental

Plaque Samples

MICHAELWEINER,tMICHAEL G. NEWMAN,* ANN HORIKOSHI, ANDVALERIJ GRINENKO School of Dentistry, Section of Periodontics, University of California at Los Angeles, Los Angeles,

California 90024

Received forpublication 8 February 1979

A simplified method for processing dental plaque samples was devised and

comparedtopreviouslyusedmethods. Analysisof 36nonstandardizedsubgingival plaque samples from various states of periodontal health and disease and 12 standardized supragingival plaque samples demonstrated that there was no sig-nificant difference between the recovery offered by the two techniques. Compa-rable recovery, increased convenience, and adaptation to the clinical setting suggeststhat implementation of this simplified technique maybe ofgreatvalue.

One of themajor goalsofperiodontalresearch is the characterization of microorganisms resi-dent in thegingivalsulcus and periodontal lesion (5). Recent technical and conceptual

develop-mentshave greatlyimproved theabilityto iso-late and cultivate bacteria colonizing the tooth surface and the gingival sulcus. For complete characterization,whole plaque samples must be dispersed tobreak up bacterial cell aggregates and diluted to reduce the number of bacteria whichwill be characterized.

Although the dispersive ability of sonic

dis-ruptionwasoriginallydescribed by Williams and

Eikenberg (7), very little information is available regarding itsuseinoral microbiology research. The goal of sonicationas amethodtodisperse

plaquesamplesistoeffectively break up

bacte-rialcellclumpswhileminimizingthe destruction

ofviable cells. Robrishet al. (6) demonstrated

that gram-negative bacteria are moresensitive

to sonic disruption than gram-positive

orga-nisms,suggestingthatsonicdisruptionofplaque

isatechniquefor theenrichment of

gram-posi-tiveorganisms rather thanatechniquefor

gain-ing representative samples. Manganiello et al.

(2) compared anaerobic and aerobic sonic

dis-persion ofsupragingival plaque. Up to 75% of

the supragingival bacteria samples were

re-coveredbyusing anaerobic sonication and pre-reducedanaerobically sterilized (PRAS)

trans-port solutions. Continuous anaerobiosis was

achieved by flushing all PRAS solutions with

oxygen-free gas through sterilized cannulas

in-troducedinto thePRAS vials.

Even though these methods were improve-ments over more conventional techniques not

employing anaerobic techniques of sampling, tPresentaddress: NorthwesternUniversity School of Med-icine, Evanston, IL60201.

dispersion, dilution, plating, and cultivation, they are inconvenient. In general, these tech-niquesare elaborate, expensive, and time

con-suming, requiring severaltechniciansand com-plex equipment.Anew,simplified technique was devised for plaque dispersion and processing whichofferedmanyadvantages over the previ-ously used technique. The purpose of the

pres-entinvestigationwas todetermine whetherthe

simplifiedmethod ofplaque dispersion and

proc-essing offered comparable recovery of microor-ganismstothepreviously used technique.

MATERIALS AND METHODS Plaquesamples. A total of 36 plaque samples were taken fromsubgingivalsites of16patients exhibiting different periodontal conditions, including patients withperiodontalabscesses,aged but healthy patients, patients with pregnancy gingivitis, and those with idiopathicjuvenile periodontitis (periodontosis) (Ta-ble 1). Eighteen samples were processed with the previouslyusedtechniques, whereas the remaining18 samples wereprocessed using thesimplifiedmethods. Twelve standardized supragingivalplaquesamples

werealso taken from the firstmolar inonepatient,so that a more accurate comparison of the sampling techniquescould be made.Sampleswerestandardized in the following manner. Three adjacent teeth, the maxillary second premolarand the first and second molars,weregivenathoroughprophylaxisbyadental hygienist.Subsequently,these teethwerenotbrushed for a period of 72 h, when a supragingival plaque samplewasremoved from1mm2of thebuccal surface of thefirstmolarwithamodifiedgas-flushed syringe

(3). Thearea wasrecleaned,andtheplaquewasagain allowedtodevelopfor72h whenasecondsamplewas

taken from thesamelocation.Thetwosampleswere

processed using either the simplified or previously used techniques.This wasrepeatedsix times inone

individualwhoexhibitedperiodontalhealth (gingival

index - 0), sothat the plaque samples would be as

homogeneousaspossible,facilitatingamoreaccurate 579

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comparisonof thesamplingmethods.

Sample processing.Themethodpreviouslyused for processing plaque samples involved the use of rubber-stoppered 15-ml vials as the transport

con-tainerofthePRASholdingsolution (Fig. 1). Plaque TABLE 1. Source anddistribution ofpatients

No. ofpatients Patient type

New method Old method

Aged,healthy 3 5

Periodontal abscess 2 3

IJpa 1

-Pregnancygingivitis 2

aIJP,Ideopathic

juvenile

periodontitis (peridonto-sis).

sampleswereplacedinto thedispersionsolution which

contained1/4-strengthRingerssolutionsupplemented with 0.0001%resazurin, 0.05%cysteine,and 0.1%

so-diummetaphosphate topreventbacterial

reaggrega-tion after sonicdispersion (2).Tomaintaincontinuous anaerobiosis, sterilized gassing cannulas delivered

ox-ygen-free gasinto the PRAS solutions whenever the rubber stopperswereremoved(2, 3). Plaquedispersal

wasaccomplished byintroducing thesterileendofa

Branson sonic probe into the PRAS solution fora

duration of5satafrequency of 6,. Serial dilutions of

thedispersed plaqueweremadeby transferring 1-ml portionsofdispersionsolution into 9 ml of PRAS full-strength Ringers solution with serological pipettes. Anaerobiosiswasmaintained in the dilution tubesby

usingadditionalgassing cannulas.

The simplified method for plaque processing in-volves theuseof septum-fittedserumvials (M.

Wei-FIG. 1. Comparisonofsimplifiedmethod with previous method forprocessingof dental plaquesamples, showingdispersion(top), dilution (middle), and plating (bottom).

|SIMPLIFIED METHOD |PREVIOUS METHOD

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PROCESSING DENTAL PLAQUE SAMPLES 581

ner,V. Grinenko, B. Kaplan, and M. G. Newman, J. Dent.Res. 57:676) (Fig. 1). After the sample is taken it iswithdrawninto the syringe, when the bluntneedle ofthesampling device is replaced with a sterile sharp needle. Theneedle canpuncture the septum, allowing thesample broach to enter the PRAS solution. The syringe and needle areretracted, leaving the broach in the PRAS solution with the septum forming a gas-tightseal around it. The broach is removed aftersonic disruption (see below) or at the time the serumbottle is disaasembled or discarded. These procedures achieve anaerobiosis as measured by resazurin indi-catorwithout the need for acomplicated gaasing ap-paratus. Plaque dispersal wasthen accomplishedby placingthe vial in a 37°C water bath contained in an ultrasonic dental instrument cleaner (Model T14, L&R) for 30 s. Subsequently, serial dilutions were made in 20-ml septum-fitted test tubes which also contained PRASfull-strength Ringers solution. Por-tions, 0.1 ml each, were then plated onto triplicate Trypticase soy agar plates with 5% sheep's blood (Bal-timore Biological Laboratories) and 0.5% hemin and 0.05% menadione.For the previously used techniques, this was accomplished by using a pipette, whereas tuberculin syringes were used with the simplified methods. Theplates were incubated at 37°C for 5 days inair,airplus 5%C02,and10%C02-10%H2-80% N2 toquantitateorganiasm obligate to those atmospheres. Colony-fonning units were counted for plates contain-ingbetween 30 and 300 colonies. Viable counts were calculatedbymultiplying the colony-forming units by theappropriate dilution factors.

All colonies on one quadrant of the plate (described above) werepicked as representative of each particu-larsample. Theseisolates were then subcultured and reincubated in the same atmosphere as orginally iso-lated.All bacteria isolated from the anaerobic atmos-phere and those obligate to the air or air plusC02 environmentswereidentifiedon thebasis of colonial andcellular morphology,Gram stain, and atmospheric tolerances.

RESULTS

The mean viable count for the subgingival plaque samples obtained from all periodontal conditionsforthesimplified techniquewas1.34 x 106 organiama, as compared to 1.54 X 106 organisms for the previous methods (Table 2).

TheStudent'st testdemonstratedthat the dif-ference between themeanrecovery countswas

notsignificant (statisticalsignificanceatthe 5%

level of confidence) (Table 3). The anaerobic

and gram-negative nature of the subgingival

plaque samples was analyzed to determine

whether a full range of organisms, typically

found with thepreviously usedmethods, could

beisolatedbyusing thenewertechniques (Table

3). No significant differences were found be-tweenthemeanrecovery of anaerobesor

gram-negative organi8msfor the two sampling

tech-niques. Analysis of variance (1) also

demon-strated no significant differences between the

TABLE 2. Recovery ofbacteriafrom subgingival plaque

Bacteria

Method Sample Anaer- Gram

source Viable obic nega-counts M tive

Aged, healthy 1.36 x10r patient 1.20 x104 2.36 x10W

1.00x105 2.12 x10W 1.64 x105 2.03 x106 1.04 x106 1.04xlo6 1.20x 106 2.00x103 3.00 x106 2.56x10' Periodontal 3.20 x105 abscess 1.28 x106 1.92 x105 1.32x106 1.20 x107 Avg 1.54 x10' Aged, healthy 1.12 x106 patient 1.92 x 105 1.08 x106 8.80x103 1.04 x105 1.20 x106 2.00 x105 1.12 x105 Periodontal 9.60x103 abscess 4.00x10' 2.50x105 IJPF 4.80x103 1.00xî07 Pregnancy 1.50x106 gingivitis 2.00x106 3.00x106

1.00x106

2.00x106

Avg 1.34 x10'

12.5 17.5 17.5 80.6 98.0 20.0 74.3 65.8 44.0 52.5 45.7 5.0 77.5 39.5 90.5 74.9 62.5 88.5 34.4 54.7 81.3 77.7 12.0 8.0 20.0 6.7 10.0 16.0 75.0 42.3 47.4 20.8 96.3 45.5 42.3 31.1 51.2 48.2 40.7 17.5 71.0 70.2 45.0 74.3 63.6 60.0 42.5 47.8 0.0 72.5 55.8 48.1 44.3 82.5 92.3 53.1 53.2 96.9 77.7 12.0 8.0 20.0 6.7 10.0 33.3 82.1 47.4 31.6 70.8 61.5 36.4 50.0 44.8 42.1 51.3 43.5 aIJP,Ideopathicjuvenileperiodontitis(periodontosis).

twomethodsfor the anaerobicorgram-negative

distribution.

Tomorepreciselydetermine theabilityof the

twotechniquestogive

comparable

results,

1,080

bacterial isolateswerecharacterized,and shown inTable4.Gram-positive

cocci,

which forin the

most difficult to disperse cell aggregates,

ac-counted for 30.5% of the samples processed by

thepreviouslyusedtechniques,and 43.1%of the

samplesprocessedwith the

simplified

methods. Student's t test and analysis of variance dem-onstrated no significant difference (at the 5% level ofconfidence)between themeanrecoveries

of gram-positive cocci or the distributions.

Gram-negative rods, shown to be most

sonic-sensitivetodisruption,wererecoveredas46.5% of the plaque samples for the

previously

used

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TABLE 3. Student'st testandanalysis of variance between the recoveryofmicroorganisms bytwosampling techniques

Degrees Observed Critical Observed Critical Plaque sample Recoveryterms of free- Students t F score

Fo.os

dom tscore

Standardized, supragingival Viabilitycounts 10 0.117 1.81 1.03 5.05

%anaerobic 10 0.1 34 1.81 1.72 5.05

%gramnegative 10 0.264 1.81 1.30 5.05

Nonstandardized,subgingival Viabilitycounts 34 0.235 1.69 1.41 3.24

%anaerobic 34 1.47 1.69 1.07 3.24

% gramnegative 34 1.15 1.69 1.27 3.24

aNote thatsignificanceisdeterminedbythe observedt scoreorFscoreexceeding theto.o5or

Fo0o5

critical values (1). No dataweresignificantatthe 5% level ofconfidence.

TABLE 4. Comparisonof thedifferential recovery betweentwosamplingtechniques: nonstandardized

plaque

Gram- Gram- Gram- Gram-method positive positive

negative negative

cocci rods cocci rods Previous 30.5% 16.5% 6.7% 46.4% Simplified 43.1% 13.4% 4.2% 39.3%

tScore 1.36 1.12 1.52 1.06

F Score 1.15 1.62 1.23 1.47

a

to.os

critical value= 1.69;

Fo0o5

critical value=3.24.

Notethatsignificanceisdeterminedby theobserved t score orFscore exceeding the to.o5 critical valueor theFo.o5critical value.Nodataweresignificantatthe 5% level of confidence.

methods and as 30.3% for the simplified

tech-niques.Nosignificant differenceswerefound for

the recovery of gram-negative rods using the

Student'sttestandanalysisof variance. The recovery oforganismsfrom the standard-ized plaque samples, using the simplified

method, averaged3.4 x 107organisms,whereas the recovery forthepreviously used technique

was 3.6 x 107 organisms (Table 5). Anaerobic recoveryaveraged48.8%for thesimplified tech-nique and 47.5% for the previous methods. Re-covery ofgram-negativebacteria was46.2% for thesimplifiedmethods and48.9%for the previ-ouslyusedtechniques. Student's ttest and anal-ysis of variancewerecalculatedontheresults of viable counts and anaerobic and gram-negative recovery,but no significantdifferencesbetween sampling techniques could befound (at the5%

level ofconfidence).

DISCUSSION

The results indicate that the mean recovery rates and distribution of microorganisms as measured byviabilitycounts and percentage of anaerobic and gram-negative organisms were

comparablefor thesimplified technique and pre-viously used techniques of plaque processing.

TABLE 5. Recoveryof bacteriafromstandardized supragingivalplaque

Bacteria

Sam-Method ple

Viable

Anaer- Gram

no. _counts obic nea

obi tive

Previous 1 7.7 x 106 50.1 57.6

2 9.1x 106 62.1 59.1 3 7.4x 10' 45.4 26.3 4 6.4 x 107 64.1 71.6 5 7.1 x 106 27.1 36.7 6 5.4x 107 36.4 41.1 Avg 3.6x 107 47.5 48.9 Simplified 1 9.1x 106 64.4 51.7 2 1.5 x 107 51.2 42.1 3 5.1x 107 30.1 28.1 4 3.5x 107 76.4 79.1 5 9.7 x 106 28.2 31.2 6 8.4x 107 42.7 38.7 Avg 3.4x 107 48.8 46.2

This was true for standardized as wellas non-standardizedplaquesamples.Comparabilitywas

shown bythefactthat the recovery of

microor-ganisms by the simplified techniques was not

significantlydifferentfrom the recovery offered

bythepreviouslyusedmethods. The additional characterization of bacterial isolates showed that even the most sonic-sensitive organisms, such as gram-negative rods, can bereadily iso-latedby the simplified method of plaque

proc-essing.

In analyzing these results, it is important to point out theinaccuracies of comparing unstan-dardizedplaquesamples, as well as the problems associated with methods of plaque standardiza-tion. The unstandardized plaque samples were taken from patients with various periodontal

conditions, including those with idiopathic ju-venile periodontitis (periodontosis), those with

periodontal abscess, aged but healthy patients, andthose with pregnancygingivitis.Since recent

WEINER ET AL. J. CLIN.

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studies have shown that qualitative and quanti-tative differences exist between the bacterial

floraassociated with these different periodontal

conditions, direct comparison of sampling tech-niques cannot be accurately assessed by using samples from different clinical entities. How-ever, the results from the two methods were similar to each other, indicating the ability to accurately recover the residentmicroorganisms associated with the particular state of periodon-talhealth or disease.

Although exact quantification of the supragin-gival plaque samples was not achieved (4), the standardization procedure employed in this study gave homogeneous samples. Comparable recovery ofmicroorganisms from these samples

using bothmethods ofplaque processing

dem-onstrates thesimilarityof the two methods. Theresults suggested that thesimplified tech-nique ofplaque dispersion and processing affords comparable recovery to the previously used methods. The primary reason for employing this technique is the increased convenience of this method ofplaqueprocessing. Since the previous methodrequiredtheremoval of the rubber stop-persfor all dispersionand dilution procedures,

it may be moresusceptibletocontamination (M. G. Newman, unpublished data). With the vast

expansion ofknowledgeinoralmicrobiology, a

convenient and accurate technique for plaque

sampling, readily adaptable to the clinical

set-ting,will be of greatvalue.

ACKNOWLEDGMENT

Thisstudy wassupported byPublic Health Service grant NIH-NIDR DE 04441-02 from the National Institutes of Health.

LITERATURE CITED

1. Comrey, A. L. 1975. Elementarystatistics: a problem solving approach. Dorsey Press,Homewood, Ill. 2. Mangamiello,A. D.,S. S. Socransky, C. Smith,D.

Propas, V.Orham,and I. LDogon.1977.Attempts toincreaseviablecountrecovery of human supragingi-val dentalplaque.J.Periodontal Res. 12:107-119. 3. Newman,M.G.,and S. S.Socransky.1977.

Predomi-nantcultivable microbiota inperiodontosis.J. Perio-dontal Res. 12:120-128.

4.Poole, A. E., and M. N. Gilmour. The variability of unstandardizedplaquesobtained fromsingleor multi-plesubjects.Arch. Oral Biol.16:681-687,1971.

5. Proceedings of the International Symposium on

PeriodontalResearch.1977.Chicago, IEl.

6. Robrish, S. A.,S. B. Grove,R.S. Bernstein, P.T. Marucha, S. S. Socransky, and B. Amdur. 1976.

Effectofsonictreatment onpureculturesand aggre-gatesof bacteria. J. Clin. Microbiol. 3:474-479.

7. Williams,N.B.,and C. F.Eikenberg.1952.Effectsof sonic vibration on the numbers ofmicroorganisms cul-tivated fromhuman saliva. J. Dent. Res. 31:428439.

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