Orcd Microbioi Itntnutioi 1992: 7: 187-191 W. C. Rubright\ W. Johnson^ J. D. Spivey^ J. Jakobsen"
A comparison of three methods for estimating dental plaque removal from individual teeth
'Department of Periodontics, College of Dentistry, ^Department of Microbiology, College of Medicine, 'Department of Periodontics, ''Department of Preventive and Community Dentistry, College of Dentistry, University of Iowa, Iowa City, USA
Rubright WC, John.son W, Spivey JD, Jakobsen J. A eotnparison of three methods for estittiatitig dental plaque retnoval from individual teeth. Oral Micfobiol Itntnutioi 1992: 7: 187-191. The purpose of this study was to detemiine the efftcietiey of detital plaque removal, //; vitro, by physical force. Of the 3 tnethods used, turbidity was the most sensitive for tneasuring tnicrogratn atnoutits of plaque. Air-dried weight atid dental plaque imprint assays (DPIA) cotnpletnented turbidity tneasuretnents by providing a weight standard and a new method for visualizing plaque bacterial strueture before the stained itnprint was transfortned into tiutnerical data. The atnount of dental plaque retnoved frotn a tooth by physical force can be tneasured and described as a pereentage of the total retnovable plaque on that tooth.
A tnajor probletn exists in trying to determine the efftciency of various physical methods of retnoving dental plaque because there are no standards for measuring total plaque on the tooth. What is needed is a tneasuretnent of plaque retnoved as a percentage of total plaque, Sotne previous studies used visual obset vations or photographs to disclose (7) stained detital plaque or show plaque retnovai by tooth-cleaning devices (5). Utifortunately, tnost plaque stains, including erytlirosin and crystal violet, do not petietrate into the deeper layers of plaque bacteria; thus, these methods do tiot accurately represent the total atnount of plaque present oti the tooth before atid after cleanitig. Sitice dental plaque is the tnajor etiological agent in human periodontal disease (3), it is necessary to tetnove as tnuch plaque as possible to retnove specific baeteria or gtoups of bacteria associated with periodontal disease. Unfortunately, individual pathogenic sttains of bacteria cannot be singled out for temoval; therefore, it is tiecessary to retnove as much of the total plaque as possible to retnove bacteria assoeiated with dental diseases. There is little doubt that physical force can remove sotne plaque from teeth, but there are little quantitative data (9, 14) to indicate how tnuch plaque cati be tetnoved by various types or tnagnitudes of foree. Thete are 2 tnajor shortcotnings in designing physical force studies: a
method is needed to eoUect and concentrate microgratn quantities of plaque retnoved frotn a single tooth after its dispersal by force, and an end point is needed to tneasure the plaque retnaining on the tooth after the initial applicatioti of foree. The purpose of this study was to develop tnethods to overcome these 2 study design litnitations and to investigate the relationship between physical force and dental plaque retnoval. During the course of this study a tiew assay was developed and cotnpared to two traditional plaque assays. The new assay, known as the dental plaque imprint assay (DPIA), provided excellent eellular detail and a sensitive colorimetrie measurement of plaque mass.
Key words: dental plaque: plaque assay William C, Rubright, Department of Periodontics, College of Dentistry, University of Iowa, Iowa City, lA 52242, USA Accepted for publication August 21, 1991
reading of the water frotn the third cleanitig was less than 0.35% nephelotneter turbidity units (% NTU) on the 10 scale of the turbiditneter. Each brush (//403 bristle brush, Dretnel, Racitie, WI), held in a flexible handpiece, was used to displace plaque. The resulting plaque suspension was passed through a 25 gauge needle to disperse large particles. Volumes of 0.5, 1.0, 2.0, 4.0, 6.0, 8.0 atid 10.0 tnl of the initial plaque suspension were diluted with distilled water to a final volutne of 25 ml. The control fluid consisted of 25.0 tnl of distilled water. Turbidity tneasurements were made on each 25-tnl satnple and were expressed as % NTU (Sargent Welch Turbiditneter, Skokie, IL).
Air-dried weight. Following each turbidity tneasurement, 25 ml of the diluted Total plaque mass initial plaque suspension or distilled Turbidity. Three freshly extracted hu- water (control satnple) was filtered tnan tnolar teeth were tutnble-washed through a preweighed 47-tnm-diameter, for 5 min in running tap water (2000 0.45-/(tn pore, polycarbonate filter (Nutnl/tnin at 24-30°C) to remove blood eleopore, Pleasanton, CA), shiny side and debris. The root of each tooth was up. The prefiltration mean weight for held with a hemostat so that the ana- thefilterswas 14.717 tng (SEM = 0.106). totnical crown was held under the sur- A vacuutn (150-200 tiimHg) for filfaee of 200 tnl distilled water contained tration was obtained with a hand putnp in a 240-ml sterile plastic beaker. Before (Nalgetie, Cucatnonga, CA), Filtration tiylon brushes were used to remove pressure was stopped when the moisture plaque from teeth, each brush was ring inside the glass funnel approached cleaned 3 titnes by running the brush at the outer edge of the filter. The filter 1725 rptn for 1 tniti under water against was retnoved frotn the holditig deviee the inner wall of the vessel. The brush and then plaeed cell-side-up on the surwas considered clean if the turbidity faee of a clean 5 x 7.6 ctn glass slide. Material and methods
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The bottom of the slide was fiamed over the outer cone of a Bunsen burner flatne (4 passes, 2-3 s each) to heat-fix the plaque to the filter, to arrest bacterial growth or degradation and to protnpt drying of the filter. Thirty seconds later, the filter was tnoved to a clean, plastic tissue culture dish with cover where the plaque-containing filter was allowed to air-dry overnight at room temperature. Filters were weighed on a Cahn 25 automatic electrobalance with a Staticmaster bar (Cahn, Cerritos, CA) loeated near the weight pan. The difference between the original filter weight and the filter weight after filtration was designated plaque dry weight. Filtration of a similar amount of distilled water served as a eontrol for dry weight measurements.
imprint area was removed with cotton applicators moistened with the extraction fluid. The stained plaque itnpritits were examined with the light microscope. The crystal violet dye was extracted frotn the dental plaque itnprints and control slides with a crystal violet extraction fluid eonsisting of a solution of 20%i alkylbenzyldetnethy-ammoniutn ehloride (50'% aqueous solution) and 10% glacial acetic acid. A 5.0 ml volume of extraction fluid was pipetted over the surface of the stained plaque itnprint. The fiuid was collected in a Petri dish and then rcpipetted over the itnprint 10 titnes. The absorbance of the extract was tneasured in a Speetronie-21 spectrophotometer (Bausch & Lotnb, Rochester, NY) at a wave length of 587 nm. If the optical density readings exceeded 1.0, the sample was diluted 1:1 with the extraction fiuid. Dentat ptac/ue itnprint assay (DPIA). Additional 25-ml aliquots of the diluted plaque suspension were first atialyzed for turbidity and then filtered as pre- Total plaque measurement viously described. Filtration pressure A series of experitnents established a was stopped when the tnoisture ring in- profile of plaque removal frotn a sitigle side the glass funnel apptoached the tooth after 8 consecutive brushings. outer edge of the filter. Sotne residual Freshly extracted single tnolars were tnembrane moisture was needed to en- washed for 5 tnin, A diamond disc rotathance plaque adhesion to the glass slide ing at 3450 rptn was used to tnake a surface. The outer edge of the filter, cell shallow ditch at a level near the ceside down, was touched to the surface of mentoenamel junction (CEJ). The tooth a clean 5 x 7.6 cm glass slide to establish was returned to the running water bath position and the rest of the filter was for I additional min to remove powallowed to fall to place, A latex-gloved dered tooth debris. An orthodontic ligafitiger tip was used to stnooth out any ture wire was then plaeed withiti the bubbles or wrinkles on the outer surface ditch and eircumferentially wound of the filter metnbrane. The slide and around the base of the anatotnieal filter were passed over the outer eone of crown as a means of isolating the crowti a Bunsen burner fiame 4 titnes to heat- from the tooth root, A hetnostat was fix the dental plaque to the .surface of used to hold the tooth by its roots under the glass slide. After cooling for 30 s, the surface of 100 tnl distilled water in the filter membrane was peeled from the a sterile 120-ml volutne plastic beaker, glass surfaee, leaving behind the dental A ^403 nylon bristle brush (Dretnel) rotplaque imprint. The filter was placed ating at 1725 rptn was held against all into a sterile plastic tissue culture dish exposed surfaces of the tooth crown for for subsequent dry weight measure- 2 tnin. Seven additional 2-min periods ments. Permoutit histological mounting of brushing of the exposed tooth surface mediutn (Fisher Scientific, Fair Lawn, using a separate, clean brush and water NJ) was applied with a cotton applica- vessel were eonducted. Turbidity and tor to all areas of the glass slide surface air-dried weight measurements were tiot occupied by the plaque or control then perfortned, in triplicate, on 25-ml imprint to repel dye from the nonitn- aliquots of the plaque suspension obprinted surface. Plaque itnprints and tained from each brushing. Another 25control slides were air-dried overnight ml aliquot of the suspension was used and stained 15 min with 5.0 ml of a for the DPIA. Total plaque consisted of fresh, filtered 0.1% crystal violet stain. the sum of all 8 tneasuretnents tninus Exeessive stain was removed by dipping the eontrol value. By this method, the each slide 10 titnes in 3 separate vessels amount of plaque retnoved by any of containing 300 ml of distilled water. the 8 brushings can be expressed as a Prior to the extraetion process, any stain percentage of the total plaque on that appearing outside the itnprint or cotitrol tooth.
Practical application
Removal of sotne of the plaque frotn one surface of the tooth's crown was used to test the ability of the previously described plaque tneasuretnent tnethods to demonstrate the relationship of plaque removal by physical force (partial brushing) to residual plaque on the tooth crown's total surface area. Freshly extracted and washed single tnolars were circutnferentially disked tiear the CEJ and an orthodontic ligature wire was tightly wound into the groove as previously described. Several strands of the wire were positioned above the surface of the tooth; those strands served as a physical barrier to isolate the exposed surfaces of the crown frotn the roots. A hetnostat was used to hold the tooth crown under the surface of 100 tnl of water in a sterile plastic beaker. A nylon bristle brush rotating at 1725 rptn was used to displace plaque from the mesial surface of the tooth into the water. The resulting plaque suspension was designated as the amount of dental plaque removed by brushing. After the retnoval of plaque on one side of the tooth's crown, 5 two-min brushings with nylon brushes rotating at 1725 rptn were used to dislodge the retnainder of the dental plaque into 112tnl vessels each containing 100 tnl of distilled water. The 5 resulting plaque suspensions were designated residual plaque. The plaque tnass in each suspension was detertnined by triplicate tneasuretnents of 25-tnl aliquots for turbidity and dry weight plus a sitigle tneasurement for DPIA on the remaining 25 tnl portion of each plaque suspension. Total removable plaque is defitied as the atnount of plaque retnoved by the initial physical force (partial brushing) plus the sum of the subsequent 5 brushings. Statistical analysis
A eorrelation coefficient (12) was used to indicate the degree of correlation between litie point plots of turbidity, airdried weight and DPIA detertninations. The analysis of variatice and Duncan's tnultiple range test (13) were used to compare groups of tnean values to detertnine if there were any signifieant differetices. Results
The histogram in Fig. 1 shows turbidity, dry weight and DPIA measuretnents of
Dental plaque assays
NTU %
DPIA
Weight
Turbidity
OD587nm
U8 500
Milliliters of Original Plaque Suspension Fig. 1. Measurements of diluted plaque suspension showing the relationship between turbidity, air-dried weight and DPIA
increasing volumes of the original dental plaque suspensions. Three freshly extracted tnolars were used for this experitiient. All of the values represented in Fig. 1 are monotonic; from control (left) up through the highest concentration of plaque suspension, 10 ml (right). The turbidity and DPIA measurements were slightly more sensitive than the air-dried weight measurement values when the lowest plaque volume was compared with the controls. At the lowest volutne of the plaque suspension, I ml, the airdried weight tniethod was not able to distinguish a statistically significant difference in measuretnietit between that amount of plaque suspension atid the control fluid. The bar graphs show a near litiear pattern of iticreasing tnean values as increasing volurnes of the diluted initial plaque suspension are exatnined. When the data in Fig. 1 were represented as linear regression slopes, there was a high correlation coefficient betweeti turbidity and air-dried weight (;- = 0.997), between turbidity and DPIA (r = 0.999) and air-dried weight atid DPIA (;• = 0.997). Comparative tneasuretnents of the turbidity of the original plaque suspension and filtrate showed that the 0.45-/nn pore filter tnembrane removed plaque particles frotn the suspension (data not shown). Although this figure shows resulting data for one experitnent, repeated measurements on other teeth showed that these values were reproducible. Although there was a direct relationship between the volutne of plaque suspension filtered and the amount of stain extracted from the dental plaque itnprints, additional information was needed about the efficiency of the trans-
fer of plaque frotn filter to glass surface. The graph (Fig. 2) cotnpares the airdried weight of increasing volumes of the initial plaque suspension concentrated on polycarbonate filters without transfer to the glass surface to the airdried weights of plaque retnainitig on the filters after the DPIA transfer process. The difference in weights before and after transfer represents the atnount of plaque transferred to the glass slide (arrows). The data show ati increase iti air-dried weight (/"= 203.99, P> 0.0001) that corresponds to increased atnounts of the filtered plaque suspension at each volutne. However, the bars representing plaque tnass temaining on the filter tnetnbranes after transfer remain at a
189
similar height at each change of plaque volume. Results of previous turbidity, airdried weight and DPIA tneasuretnents of a diluted plaque suspension showed that all 3 methods could be used to detennine dental plaque tnass. The next step was to detertnine the nutnber of brushings required to establish an endpoint for retnoval of plaque frotn the one tooth. Most of the plaque was retnoved by the first brushing (Fig. 3). The first brushing retnoved 55.10% by turbidity, 65.22% by air-dried weight and 90.00% by DPIA. By contrast, the eighth brushing removed 6.11% (turbidity), 4.08% (air-dried weight) and 1.02% DPIA of the plaque. The control tneasuretnents were lower than any of the brushing values by each of the 3 tneasuretnents: 0.200 % NTU, 32.20 //g and 0.022 absorbance. Because of the low percentage of plaque retnoval after the fifth brushing, the endpoint for plaque retnoval was established as the sum of the plaque retnoved iti the first 5 brushings. Using that endpoint, another experitnetit was conducted to detertnine if turbidity, air-dried weight and DPIA tneasuretnents would give sitnilar values when the percentage of partial plaque retnoval is being estitnated. Single teeth were brushed for 15 s and the resulting suspensions were assayed for plaque retnoval by all 3 tnethods. The atnount of plaque detected by each method, as a percent of total plaque retnoval, as tur-
SOOn
B
Plaque weight Plaque weight remaining on filter after DPIA
as
300-
'5 200-
10
mis of Original Plaque Suspension Fig. 2. Efl'ieiency of plaque transfer. The vertical liites and arrows represent the percentage of plaque transferred from filter to glass surface
190
Rubright et al. 1 Turbidity P Weight
[]DPIA
j
X
>
§ O)
Wei
=)
1000
r.
;
•
mn M\ ^ hn ^n 4
5
6
7
BRUSHINGS /g. i. Effect of consecutive brushings on plaque removal bidity (25%), air-dried weight (31%) and DPIA (33%). All 3 methods detected a similar amount of plaque retnoved by partial brushing. Although the data shown in Fig. 3 demonstrate plaque removal from a single tooth, repeated measurements of plaque removal from other teeth showed sitnilar values. Discussion Traditional methods of measuring plaque, turbidity and air-dried weight were able to accurately measure microgram amounts of plaque removed from a single tooth; however, there was no way to visualize what was being measured. The DPIA was able to accurately measure plaque and it also allowed visualization of the plaque morphology. The results in Fig. I show that each of the 3 test methods (turbidity, air-dried weight and DPIA) were sensitive enogh to measure microgram amounts of soft plaque removed from teeth. Turbidity measuretnents were the most sensitive of the 3 methods used to determine plaque mass in this study. These measurements were able to distinguish between the least concentration (I ml, 47 /^g) of the plaque suspension and the cotitrol fluid. In other experiments conducted but not presented in this article, the plaque suspension was further diluted and its turbidity was still statistically different from the control value. In addition, turbidity measurements were simple to perform and nondestructive; therefore sub-
sequent tests could be made by other methods from the same sample. Other investigators reported that plaque turbidity (6) and dry weight (8) were accurate and reproducible methods of measuritig dental plaque. Turbidity cannot be related to end point growth in serial dilutions or colony counts because plaque contains both viable and dead cells. Turbidity tneasunnents cannot stand by themselves without sotne fixed standard. In this experitnent, air-dried weights were used to indicate the amount of plaque mass in each satnple. Filtration through membranes was found to be a useful tnethod of concentrating bacteria from suspension (2, 11). The results of this study suggest that large volumes of fluid containing microgratn atnounts of plaque can be quantified by concentration on filter surfaces for air-dried weight measurements. Measurements of air-dried weight reached their lower limit of sensitivity at about the 2 tnl level, 110 /(g. The absorbance of color retnoved frotn the DPIA was sensitive to the 1.0 tnl level (47 /^g). Sitrtilarly, plaque can be imprinted onto glass slides for microscopic observation after filtration. Dental plaque's natural ability to adhere to smooth surfaces (4, 15) appears to be enhanced in this study by the use of heat as a method of attaching plaque to the surface of a glass slide. The major advantage of the DPIA over the other methods is that the investigator can visualize the dental plaque before the dye is extracted for spectrophotometric
tneasuretnent. For example, if ati inexpensive anitnal hair bristle brush were used to retnove plaque from extracted teeth there tnay have been enough particulate debris from the bristles to account for considerable turbidity or airdried weight. This tnay lead to inaccurate measuretnents of plaque. Similarly, during the consecutive brushings of the tooth, plaque was still being removed frotn the tooth after the eighth brushing. The ctystal violet-stained dental plaque itnprint permitted microscopic examination to verify the presence of plaque bacteria. The DPIA tnethod is accurate when the lowest plaque satnple is in the range of 40-50 jx%. This lack of sensitivity with stnall amounts of plaque may be the reason that an accurate comparison of plaque removed by the first brushing could not be made to subsequent brushings (Fig. 3). However, the DPIA appeared to be accurate in its ability to quantify larger atnounts of plaque when the physical force tneasuretnent (partial removal) was related to the first 5 brushings. Sotne caution is required in using the DPIA tnethod. If the filter should slip on the glass surface duritig the transfer, the result may be a stnudged imprint. Likewise, if the plaque suspension was filtered to absolute dryness, the transfer to glass would not work. If the dull side of the filter is up duritig the llltratioti process the itnprint will contain bacteria that are matted together. This results in poor microscopic resolution of the plaque. The membrane filter must be positioned shiny side up in the holding device during filtration so that the bacteria are clearly defined. Sotne investigators have tnade some progress iti filter methods to itnprove cell resolution by use of itntnersion oil to clear the filter (11) after collecting bacteria on its surface; others have used chetnical agents to dissolve away the filter (1), thereby leaving the bacteria on the surface of glass slides. Recently, a pressure transfer technique was described (10) for tnoving matnmalian cells frotn filter surface to glass slides, where the cells could be seen and coutited, thereby eliminating the filter image. The atnount of dental plaque removed from a tooth by physical force can be measured and described as a percentage of the total plaque on that tooth. In addition, all 3 methods gave sitnilar values so all 3 appear applicable to the measurement of dental plaque. Turbidity is the simplest and most accu-
Dental plaque assays rate tnethod of the 3. It is a notidestructive tnethod. Its apparent shortcotnitig is the inability of the investigator to visualize what is being tneasured. Airdried weight tneasurements are accurate and dependable, but those tneasurements are slow and tedious and require use of an expensive electro-balance. The DPIA appears to be an efficient tnethod of quantifying plaque retnoval and, in addition, offers the investigator a chance to visualize plaque bacterial morphology. The DPIA is tiot intended to be the sole assay, but its main use is as an adjutictive tnethod to turbidity and or air-dried weight tneasuretnents.
References 1. Bales CE, Durfee GR. Cytologic technique. In: Koss LG, ed. Diagnostic cytology. Philadelphia: 1187-1266. JB Lippincot, 1979: 2. Bitton G, Dutton RJ, Foran JA. New
rapid technique for counting microorganisms directly on membrane filters. Stain Technol 1983: 58: 343-346. 3. Caton J. Periodontal diagnosis and diagnostic aids. In: Nevins M, Becker W, Kornman K, ed. Ptoceeditigs of the World Workshop in Clinical Periodontics. Princeton: 1990, 1-8. 4. Christensen GO, Simpson WA, Beachey EN. Adhesion of bacteria to animal tissue complex tnechanistii. In: Savage DC, Fletcher M, ed. Bacterial adhesion. New York: Plenutn Press, 1985. 5. Eaton KA, Kieser JB, Baker R. Assesstnent of plaque by image analysis. J Clin Periodontics 1985: 12: 135-140. 6. Gawronski TH, Staat RH, Folke LEA. Quantitation of human plaque by turbidimetry. J Dent Res 1973: 52: 633. 7. Lang NP, Ostergard E, Loe H. A fluorescent plaque disclosing agent. J Periodont Res 1972: 7: 59-67. 8. McDermid AS, Marsh PD, Keevil CW, EUwood DC. Additive inhibitory effects of combinations of fluoride and chlorhexidine on acid production by Streptococcus nmtatts and Streptococcus sanguis. Caries Res 1985: 19: 64-71.
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9. Mehrotra KK, Kapoor KK, Ptadhan BP. Bhushan A. Assessment of plaque tenacity on enamel surface. J Periodont Res 1983: 18: 386-392. 10. Oud PS. Zahniser DJ, Garcia GL, et al. Pressure-fixation tnethod of transferring cells from polycarbonate filters to glass slides. Atial Quantitative Cytol 1984: 6: 131-137. 11. Romero S. Schell RF. Pennell DR. Rapid method for the differentiation of gram-positive and gram-negative bacteria on membrane filters. J Clin Microbiol 1988: 26: 1378-1382. 12. SAS user's guide. Basics, version 5 edition. Cary, NC: SAS Institute, 1985: 862-874. 13. SAS user's guide. Statistics, version 5 edition. Cary, NC: SAS Institute. 1985: 434-506. 14. TuUberg A. An experitnental study of the adhesion of bacterial layers to some restorative dental materials. Scand J Detit Res 1986: 94: 164-173. 15. van Houte J. Bacterial adherence and dental plaque formation. Infection 1982: 10: 252-266.
A comparison of three methods for estimating dental plaque removal from individual teeth
'Department of Periodontics, College of Dentistry, ^Department of Microbiology, College of Medicine, 'Department of Periodontics, ''Department of Preventive and Community Dentistry, College of Dentistry, University of Iowa, Iowa City, USA
Rubright WC, John.son W, Spivey JD, Jakobsen J. A eotnparison of three methods for estittiatitig dental plaque retnoval from individual teeth. Oral Micfobiol Itntnutioi 1992: 7: 187-191. The purpose of this study was to detemiine the efftcietiey of detital plaque removal, //; vitro, by physical force. Of the 3 tnethods used, turbidity was the most sensitive for tneasuring tnicrogratn atnoutits of plaque. Air-dried weight atid dental plaque imprint assays (DPIA) cotnpletnented turbidity tneasuretnents by providing a weight standard and a new method for visualizing plaque bacterial strueture before the stained itnprint was transfortned into tiutnerical data. The atnount of dental plaque retnoved frotn a tooth by physical force can be tneasured and described as a pereentage of the total retnovable plaque on that tooth.
A tnajor probletn exists in trying to determine the efftciency of various physical methods of retnoving dental plaque because there are no standards for measuring total plaque on the tooth. What is needed is a tneasuretnent of plaque retnoved as a percentage of total plaque, Sotne previous studies used visual obset vations or photographs to disclose (7) stained detital plaque or show plaque retnovai by tooth-cleaning devices (5). Utifortunately, tnost plaque stains, including erytlirosin and crystal violet, do not petietrate into the deeper layers of plaque bacteria; thus, these methods do tiot accurately represent the total atnount of plaque present oti the tooth before atid after cleanitig. Sitice dental plaque is the tnajor etiological agent in human periodontal disease (3), it is necessary to tetnove as tnuch plaque as possible to retnove specific baeteria or gtoups of bacteria associated with periodontal disease. Unfortunately, individual pathogenic sttains of bacteria cannot be singled out for temoval; therefore, it is tiecessary to retnove as much of the total plaque as possible to retnove bacteria assoeiated with dental diseases. There is little doubt that physical force can remove sotne plaque from teeth, but there are little quantitative data (9, 14) to indicate how tnuch plaque cati be tetnoved by various types or tnagnitudes of foree. Thete are 2 tnajor shortcotnings in designing physical force studies: a
method is needed to eoUect and concentrate microgratn quantities of plaque retnoved frotn a single tooth after its dispersal by force, and an end point is needed to tneasure the plaque retnaining on the tooth after the initial applicatioti of foree. The purpose of this study was to develop tnethods to overcome these 2 study design litnitations and to investigate the relationship between physical force and dental plaque retnoval. During the course of this study a tiew assay was developed and cotnpared to two traditional plaque assays. The new assay, known as the dental plaque imprint assay (DPIA), provided excellent eellular detail and a sensitive colorimetrie measurement of plaque mass.
Key words: dental plaque: plaque assay William C, Rubright, Department of Periodontics, College of Dentistry, University of Iowa, Iowa City, lA 52242, USA Accepted for publication August 21, 1991
reading of the water frotn the third cleanitig was less than 0.35% nephelotneter turbidity units (% NTU) on the 10 scale of the turbiditneter. Each brush (//403 bristle brush, Dretnel, Racitie, WI), held in a flexible handpiece, was used to displace plaque. The resulting plaque suspension was passed through a 25 gauge needle to disperse large particles. Volumes of 0.5, 1.0, 2.0, 4.0, 6.0, 8.0 atid 10.0 tnl of the initial plaque suspension were diluted with distilled water to a final volutne of 25 ml. The control fluid consisted of 25.0 tnl of distilled water. Turbidity tneasurements were made on each 25-tnl satnple and were expressed as % NTU (Sargent Welch Turbiditneter, Skokie, IL).
Air-dried weight. Following each turbidity tneasurement, 25 ml of the diluted Total plaque mass initial plaque suspension or distilled Turbidity. Three freshly extracted hu- water (control satnple) was filtered tnan tnolar teeth were tutnble-washed through a preweighed 47-tnm-diameter, for 5 min in running tap water (2000 0.45-/(tn pore, polycarbonate filter (Nutnl/tnin at 24-30°C) to remove blood eleopore, Pleasanton, CA), shiny side and debris. The root of each tooth was up. The prefiltration mean weight for held with a hemostat so that the ana- thefilterswas 14.717 tng (SEM = 0.106). totnical crown was held under the sur- A vacuutn (150-200 tiimHg) for filfaee of 200 tnl distilled water contained tration was obtained with a hand putnp in a 240-ml sterile plastic beaker. Before (Nalgetie, Cucatnonga, CA), Filtration tiylon brushes were used to remove pressure was stopped when the moisture plaque from teeth, each brush was ring inside the glass funnel approached cleaned 3 titnes by running the brush at the outer edge of the filter. The filter 1725 rptn for 1 tniti under water against was retnoved frotn the holditig deviee the inner wall of the vessel. The brush and then plaeed cell-side-up on the surwas considered clean if the turbidity faee of a clean 5 x 7.6 ctn glass slide. Material and methods
188
Rubright et al.
The bottom of the slide was fiamed over the outer cone of a Bunsen burner flatne (4 passes, 2-3 s each) to heat-fix the plaque to the filter, to arrest bacterial growth or degradation and to protnpt drying of the filter. Thirty seconds later, the filter was tnoved to a clean, plastic tissue culture dish with cover where the plaque-containing filter was allowed to air-dry overnight at room temperature. Filters were weighed on a Cahn 25 automatic electrobalance with a Staticmaster bar (Cahn, Cerritos, CA) loeated near the weight pan. The difference between the original filter weight and the filter weight after filtration was designated plaque dry weight. Filtration of a similar amount of distilled water served as a eontrol for dry weight measurements.
imprint area was removed with cotton applicators moistened with the extraction fluid. The stained plaque itnpritits were examined with the light microscope. The crystal violet dye was extracted frotn the dental plaque itnprints and control slides with a crystal violet extraction fluid eonsisting of a solution of 20%i alkylbenzyldetnethy-ammoniutn ehloride (50'% aqueous solution) and 10% glacial acetic acid. A 5.0 ml volume of extraction fluid was pipetted over the surface of the stained plaque itnprint. The fiuid was collected in a Petri dish and then rcpipetted over the itnprint 10 titnes. The absorbance of the extract was tneasured in a Speetronie-21 spectrophotometer (Bausch & Lotnb, Rochester, NY) at a wave length of 587 nm. If the optical density readings exceeded 1.0, the sample was diluted 1:1 with the extraction fiuid. Dentat ptac/ue itnprint assay (DPIA). Additional 25-ml aliquots of the diluted plaque suspension were first atialyzed for turbidity and then filtered as pre- Total plaque measurement viously described. Filtration pressure A series of experitnents established a was stopped when the tnoisture ring in- profile of plaque removal frotn a sitigle side the glass funnel apptoached the tooth after 8 consecutive brushings. outer edge of the filter. Sotne residual Freshly extracted single tnolars were tnembrane moisture was needed to en- washed for 5 tnin, A diamond disc rotathance plaque adhesion to the glass slide ing at 3450 rptn was used to tnake a surface. The outer edge of the filter, cell shallow ditch at a level near the ceside down, was touched to the surface of mentoenamel junction (CEJ). The tooth a clean 5 x 7.6 cm glass slide to establish was returned to the running water bath position and the rest of the filter was for I additional min to remove powallowed to fall to place, A latex-gloved dered tooth debris. An orthodontic ligafitiger tip was used to stnooth out any ture wire was then plaeed withiti the bubbles or wrinkles on the outer surface ditch and eircumferentially wound of the filter metnbrane. The slide and around the base of the anatotnieal filter were passed over the outer eone of crown as a means of isolating the crowti a Bunsen burner fiame 4 titnes to heat- from the tooth root, A hetnostat was fix the dental plaque to the .surface of used to hold the tooth by its roots under the glass slide. After cooling for 30 s, the surface of 100 tnl distilled water in the filter membrane was peeled from the a sterile 120-ml volutne plastic beaker, glass surfaee, leaving behind the dental A ^403 nylon bristle brush (Dretnel) rotplaque imprint. The filter was placed ating at 1725 rptn was held against all into a sterile plastic tissue culture dish exposed surfaces of the tooth crown for for subsequent dry weight measure- 2 tnin. Seven additional 2-min periods ments. Permoutit histological mounting of brushing of the exposed tooth surface mediutn (Fisher Scientific, Fair Lawn, using a separate, clean brush and water NJ) was applied with a cotton applica- vessel were eonducted. Turbidity and tor to all areas of the glass slide surface air-dried weight measurements were tiot occupied by the plaque or control then perfortned, in triplicate, on 25-ml imprint to repel dye from the nonitn- aliquots of the plaque suspension obprinted surface. Plaque itnprints and tained from each brushing. Another 25control slides were air-dried overnight ml aliquot of the suspension was used and stained 15 min with 5.0 ml of a for the DPIA. Total plaque consisted of fresh, filtered 0.1% crystal violet stain. the sum of all 8 tneasuretnents tninus Exeessive stain was removed by dipping the eontrol value. By this method, the each slide 10 titnes in 3 separate vessels amount of plaque retnoved by any of containing 300 ml of distilled water. the 8 brushings can be expressed as a Prior to the extraetion process, any stain percentage of the total plaque on that appearing outside the itnprint or cotitrol tooth.
Practical application
Removal of sotne of the plaque frotn one surface of the tooth's crown was used to test the ability of the previously described plaque tneasuretnent tnethods to demonstrate the relationship of plaque removal by physical force (partial brushing) to residual plaque on the tooth crown's total surface area. Freshly extracted and washed single tnolars were circutnferentially disked tiear the CEJ and an orthodontic ligature wire was tightly wound into the groove as previously described. Several strands of the wire were positioned above the surface of the tooth; those strands served as a physical barrier to isolate the exposed surfaces of the crown frotn the roots. A hetnostat was used to hold the tooth crown under the surface of 100 tnl of water in a sterile plastic beaker. A nylon bristle brush rotating at 1725 rptn was used to displace plaque from the mesial surface of the tooth into the water. The resulting plaque suspension was designated as the amount of dental plaque removed by brushing. After the retnoval of plaque on one side of the tooth's crown, 5 two-min brushings with nylon brushes rotating at 1725 rptn were used to dislodge the retnainder of the dental plaque into 112tnl vessels each containing 100 tnl of distilled water. The 5 resulting plaque suspensions were designated residual plaque. The plaque tnass in each suspension was detertnined by triplicate tneasuretnents of 25-tnl aliquots for turbidity and dry weight plus a sitigle tneasurement for DPIA on the remaining 25 tnl portion of each plaque suspension. Total removable plaque is defitied as the atnount of plaque retnoved by the initial physical force (partial brushing) plus the sum of the subsequent 5 brushings. Statistical analysis
A eorrelation coefficient (12) was used to indicate the degree of correlation between litie point plots of turbidity, airdried weight and DPIA detertninations. The analysis of variatice and Duncan's tnultiple range test (13) were used to compare groups of tnean values to detertnine if there were any signifieant differetices. Results
The histogram in Fig. 1 shows turbidity, dry weight and DPIA measuretnents of
Dental plaque assays
NTU %
DPIA
Weight
Turbidity
OD587nm
U8 500
Milliliters of Original Plaque Suspension Fig. 1. Measurements of diluted plaque suspension showing the relationship between turbidity, air-dried weight and DPIA
increasing volumes of the original dental plaque suspensions. Three freshly extracted tnolars were used for this experitiient. All of the values represented in Fig. 1 are monotonic; from control (left) up through the highest concentration of plaque suspension, 10 ml (right). The turbidity and DPIA measurements were slightly more sensitive than the air-dried weight measurement values when the lowest plaque volume was compared with the controls. At the lowest volutne of the plaque suspension, I ml, the airdried weight tniethod was not able to distinguish a statistically significant difference in measuretnietit between that amount of plaque suspension atid the control fluid. The bar graphs show a near litiear pattern of iticreasing tnean values as increasing volurnes of the diluted initial plaque suspension are exatnined. When the data in Fig. 1 were represented as linear regression slopes, there was a high correlation coefficient betweeti turbidity and air-dried weight (;- = 0.997), between turbidity and DPIA (r = 0.999) and air-dried weight atid DPIA (;• = 0.997). Comparative tneasuretnents of the turbidity of the original plaque suspension and filtrate showed that the 0.45-/nn pore filter tnembrane removed plaque particles frotn the suspension (data not shown). Although this figure shows resulting data for one experitnent, repeated measurements on other teeth showed that these values were reproducible. Although there was a direct relationship between the volutne of plaque suspension filtered and the amount of stain extracted from the dental plaque itnprints, additional information was needed about the efficiency of the trans-
fer of plaque frotn filter to glass surface. The graph (Fig. 2) cotnpares the airdried weight of increasing volumes of the initial plaque suspension concentrated on polycarbonate filters without transfer to the glass surface to the airdried weights of plaque retnainitig on the filters after the DPIA transfer process. The difference in weights before and after transfer represents the atnount of plaque transferred to the glass slide (arrows). The data show ati increase iti air-dried weight (/"= 203.99, P> 0.0001) that corresponds to increased atnounts of the filtered plaque suspension at each volutne. However, the bars representing plaque tnass temaining on the filter tnetnbranes after transfer remain at a
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similar height at each change of plaque volume. Results of previous turbidity, airdried weight and DPIA tneasuretnents of a diluted plaque suspension showed that all 3 methods could be used to detennine dental plaque tnass. The next step was to detertnine the nutnber of brushings required to establish an endpoint for retnoval of plaque frotn the one tooth. Most of the plaque was retnoved by the first brushing (Fig. 3). The first brushing retnoved 55.10% by turbidity, 65.22% by air-dried weight and 90.00% by DPIA. By contrast, the eighth brushing removed 6.11% (turbidity), 4.08% (air-dried weight) and 1.02% DPIA of the plaque. The control tneasuretnents were lower than any of the brushing values by each of the 3 tneasuretnents: 0.200 % NTU, 32.20 //g and 0.022 absorbance. Because of the low percentage of plaque retnoval after the fifth brushing, the endpoint for plaque retnoval was established as the sum of the plaque retnoved iti the first 5 brushings. Using that endpoint, another experitnetit was conducted to detertnine if turbidity, air-dried weight and DPIA tneasuretnents would give sitnilar values when the percentage of partial plaque retnoval is being estitnated. Single teeth were brushed for 15 s and the resulting suspensions were assayed for plaque retnoval by all 3 tnethods. The atnount of plaque detected by each method, as a percent of total plaque retnoval, as tur-
SOOn
B
Plaque weight Plaque weight remaining on filter after DPIA
as
300-
'5 200-
10
mis of Original Plaque Suspension Fig. 2. Efl'ieiency of plaque transfer. The vertical liites and arrows represent the percentage of plaque transferred from filter to glass surface
190
Rubright et al. 1 Turbidity P Weight
[]DPIA
j
X
>
§ O)
Wei
=)
1000
r.
;
•
mn M\ ^ hn ^n 4
5
6
7
BRUSHINGS /g. i. Effect of consecutive brushings on plaque removal bidity (25%), air-dried weight (31%) and DPIA (33%). All 3 methods detected a similar amount of plaque retnoved by partial brushing. Although the data shown in Fig. 3 demonstrate plaque removal from a single tooth, repeated measurements of plaque removal from other teeth showed sitnilar values. Discussion Traditional methods of measuring plaque, turbidity and air-dried weight were able to accurately measure microgram amounts of plaque removed from a single tooth; however, there was no way to visualize what was being measured. The DPIA was able to accurately measure plaque and it also allowed visualization of the plaque morphology. The results in Fig. I show that each of the 3 test methods (turbidity, air-dried weight and DPIA) were sensitive enogh to measure microgram amounts of soft plaque removed from teeth. Turbidity measuretnents were the most sensitive of the 3 methods used to determine plaque mass in this study. These measurements were able to distinguish between the least concentration (I ml, 47 /^g) of the plaque suspension and the cotitrol fluid. In other experiments conducted but not presented in this article, the plaque suspension was further diluted and its turbidity was still statistically different from the control value. In addition, turbidity measurements were simple to perform and nondestructive; therefore sub-
sequent tests could be made by other methods from the same sample. Other investigators reported that plaque turbidity (6) and dry weight (8) were accurate and reproducible methods of measuritig dental plaque. Turbidity cannot be related to end point growth in serial dilutions or colony counts because plaque contains both viable and dead cells. Turbidity tneasunnents cannot stand by themselves without sotne fixed standard. In this experitnent, air-dried weights were used to indicate the amount of plaque mass in each satnple. Filtration through membranes was found to be a useful tnethod of concentrating bacteria from suspension (2, 11). The results of this study suggest that large volumes of fluid containing microgratn atnounts of plaque can be quantified by concentration on filter surfaces for air-dried weight measurements. Measurements of air-dried weight reached their lower limit of sensitivity at about the 2 tnl level, 110 /(g. The absorbance of color retnoved frotn the DPIA was sensitive to the 1.0 tnl level (47 /^g). Sitrtilarly, plaque can be imprinted onto glass slides for microscopic observation after filtration. Dental plaque's natural ability to adhere to smooth surfaces (4, 15) appears to be enhanced in this study by the use of heat as a method of attaching plaque to the surface of a glass slide. The major advantage of the DPIA over the other methods is that the investigator can visualize the dental plaque before the dye is extracted for spectrophotometric
tneasuretnent. For example, if ati inexpensive anitnal hair bristle brush were used to retnove plaque from extracted teeth there tnay have been enough particulate debris from the bristles to account for considerable turbidity or airdried weight. This tnay lead to inaccurate measuretnents of plaque. Similarly, during the consecutive brushings of the tooth, plaque was still being removed frotn the tooth after the eighth brushing. The ctystal violet-stained dental plaque itnprint permitted microscopic examination to verify the presence of plaque bacteria. The DPIA tnethod is accurate when the lowest plaque satnple is in the range of 40-50 jx%. This lack of sensitivity with stnall amounts of plaque may be the reason that an accurate comparison of plaque removed by the first brushing could not be made to subsequent brushings (Fig. 3). However, the DPIA appeared to be accurate in its ability to quantify larger atnounts of plaque when the physical force tneasuretnent (partial removal) was related to the first 5 brushings. Sotne caution is required in using the DPIA tnethod. If the filter should slip on the glass surface duritig the transfer, the result may be a stnudged imprint. Likewise, if the plaque suspension was filtered to absolute dryness, the transfer to glass would not work. If the dull side of the filter is up duritig the llltratioti process the itnprint will contain bacteria that are matted together. This results in poor microscopic resolution of the plaque. The membrane filter must be positioned shiny side up in the holding device during filtration so that the bacteria are clearly defined. Sotne investigators have tnade some progress iti filter methods to itnprove cell resolution by use of itntnersion oil to clear the filter (11) after collecting bacteria on its surface; others have used chetnical agents to dissolve away the filter (1), thereby leaving the bacteria on the surface of glass slides. Recently, a pressure transfer technique was described (10) for tnoving matnmalian cells frotn filter surface to glass slides, where the cells could be seen and coutited, thereby eliminating the filter image. The atnount of dental plaque removed from a tooth by physical force can be measured and described as a percentage of the total plaque on that tooth. In addition, all 3 methods gave sitnilar values so all 3 appear applicable to the measurement of dental plaque. Turbidity is the simplest and most accu-
Dental plaque assays rate tnethod of the 3. It is a notidestructive tnethod. Its apparent shortcotnitig is the inability of the investigator to visualize what is being tneasured. Airdried weight tneasurements are accurate and dependable, but those tneasurements are slow and tedious and require use of an expensive electro-balance. The DPIA appears to be an efficient tnethod of quantifying plaque retnoval and, in addition, offers the investigator a chance to visualize plaque bacterial morphology. The DPIA is tiot intended to be the sole assay, but its main use is as an adjutictive tnethod to turbidity and or air-dried weight tneasuretnents.
References 1. Bales CE, Durfee GR. Cytologic technique. In: Koss LG, ed. Diagnostic cytology. Philadelphia: 1187-1266. JB Lippincot, 1979: 2. Bitton G, Dutton RJ, Foran JA. New
rapid technique for counting microorganisms directly on membrane filters. Stain Technol 1983: 58: 343-346. 3. Caton J. Periodontal diagnosis and diagnostic aids. In: Nevins M, Becker W, Kornman K, ed. Ptoceeditigs of the World Workshop in Clinical Periodontics. Princeton: 1990, 1-8. 4. Christensen GO, Simpson WA, Beachey EN. Adhesion of bacteria to animal tissue complex tnechanistii. In: Savage DC, Fletcher M, ed. Bacterial adhesion. New York: Plenutn Press, 1985. 5. Eaton KA, Kieser JB, Baker R. Assesstnent of plaque by image analysis. J Clin Periodontics 1985: 12: 135-140. 6. Gawronski TH, Staat RH, Folke LEA. Quantitation of human plaque by turbidimetry. J Dent Res 1973: 52: 633. 7. Lang NP, Ostergard E, Loe H. A fluorescent plaque disclosing agent. J Periodont Res 1972: 7: 59-67. 8. McDermid AS, Marsh PD, Keevil CW, EUwood DC. Additive inhibitory effects of combinations of fluoride and chlorhexidine on acid production by Streptococcus nmtatts and Streptococcus sanguis. Caries Res 1985: 19: 64-71.
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9. Mehrotra KK, Kapoor KK, Ptadhan BP. Bhushan A. Assessment of plaque tenacity on enamel surface. J Periodont Res 1983: 18: 386-392. 10. Oud PS. Zahniser DJ, Garcia GL, et al. Pressure-fixation tnethod of transferring cells from polycarbonate filters to glass slides. Atial Quantitative Cytol 1984: 6: 131-137. 11. Romero S. Schell RF. Pennell DR. Rapid method for the differentiation of gram-positive and gram-negative bacteria on membrane filters. J Clin Microbiol 1988: 26: 1378-1382. 12. SAS user's guide. Basics, version 5 edition. Cary, NC: SAS Institute, 1985: 862-874. 13. SAS user's guide. Statistics, version 5 edition. Cary, NC: SAS Institute. 1985: 434-506. 14. TuUberg A. An experitnental study of the adhesion of bacterial layers to some restorative dental materials. Scand J Detit Res 1986: 94: 164-173. 15. van Houte J. Bacterial adherence and dental plaque formation. Infection 1982: 10: 252-266.
