Svanberg² investigated toothbrushes used by people who were severely infected with Streptococcus mutans. Toothbrushes still were contaminated with S. mutans concentrations of 10⁴ colony-forming units per milliliter of solution even after being stored in dry air for 24 hours after use. Dayoub and colleagues³ detected microorganisms on toothbrushes that had been kept dry for five days after use. Glass and colleagues^4,5 demonstrated the ability of the herpes simplex virus to remain viable on a dried toothbrush for at least 48 hours and in a moist environment for more than seven days. Noga and colleagues⁶ discovered retention of Candida albicans on toothbrushes.

Reinfection of the oral cavity is possible owing to injuries of the gingiva that can occur during toothbrushing.¹ The area of the toothbrush in which the tufts are anchored is especially prone to heavy contamination.⁷ Fluids and food debris can be drawn into the spaces between tufts by capillary action, and this may lead to bacterial growth.⁸ The conventional technique of fastening bristles with a metal anchor in the center creates small cavities between the tufts. There also is a great predrilled hole in the center of each bundle of filaments.⁹ In the last few years, the relatively new technique of in-mold tufting was believed to be microbiologically sound, since for the most part it prevents empty spaces in the toothbrush head.^8,10 But this hypothesis needs to be proven. A further innovative technique of anchoring bristles recently has been developed. The filaments are placed in the mold individually, which means that gaps and spaces are eliminated entirely. The main difference from the other two techniques exists in the fact that each filament is not collected into a bundle but is attached to the head separately. This leads to greater distances between each filament. Therefore, rinsing of the toothbrush after use is more effective.

The area of the toothbrush in which the tufts are anchored is especially prone to heavy contamination. Fluids and food debris can be drawn into the spaces between tufts by capillary action, and this may lead to bacterial growth.

The aim of our study was to compare the adherence of microorganisms to toothbrushes made with three different techniques of anchoring bristles: staple-set tufting, in-mold tufting and individual in-mold placement of filaments. The following hypotheses had to be considered:

1. The in-mold tufting has an advantage over staple-set tufting in that it does not leave any gaps or holes.
   
2. The individual in-mold placement of filaments is considered to be superior to both in-mold tufting and staple-set tufting because the distances between the filaments are extended and the spaces and gaps associated with staple-set tufting can be avoided.
   
3. Regarding the hygiene of these three types of anchoring, the biological results of the microbial retention of brushes made with the individual in-mold placement of filaments technique are generally superior to those for brushes made with in-mold tufting, which in turn is better than the conventional method using metal anchors.
   

	MATERIALS, SUBJECTS AND METHODS

TOP ABSTRACT MATERIALS, SUBJECTS AND METHODS RESULTS DISCUSSION CONCLUSION REFERENCES

 
In our study, we examined toothbrushes made with different anchoring techniques to determine their retention of cariogenic microorganisms, such as S. mutans, lactobacilli and Candida species.

Materials. We tested 120 toothbrushes, 40 toothbrushes for each anchoring technique.

– Staple-set tufting, represented by toothbrush A, was patented in 1877 by J.V. Gane in France.¹¹ With this principle, the filaments are collected into bundles, bent in half with a metal anchor in the center and driven into predrilled holes in the toothbrush. The German Institute for Standardization norm determines a tensile strength of 25 newtons for each bundle, which is guaranteed by the metal anchor. This type of anchoring inevitably creates gaps, which are connected with the outer surface of the toothbrush. This can cause problems with oral hygiene.¹²

– In-mold tufting, represented by toothbrush B, was patented by Krause and colleagues¹² in 1922 at the German Patent Office in Berlin. With this principle, the bristles are cut at the base, collected into bundles, welded together and attached to the head. Synthetic material is injected into the cavities (molds) surrounding the bundles.

– The individual in-mold placement of filaments, represented by toothbrush C, was patented at the International Patent Office of the World Intellectual Property Organization in Geneva in 1996.¹³ The filaments are placed in the molds individually and are fastened into the molds with synthetic material.

Specific brands were not of any importance in this study. Toothbrush C was a prototype that has not yet been put on the market. The prototype was tested in this study for the first time. All toothbrushes, including the prototype, were manufactured by Coronet-Werke GmbH (Wald-Michelbach, Germany).

Photographic documentation and analysis using a scanning electron microscope. Before their use in the study, we documented the toothbrushes photographically and analyzed them under a light microscope and a scanning electron microscope.

– For the light-microscopic photographs, we selected five heads of each toothbrush type and cut the heads into several vertical and horizontal layers. We used an Orthoplan light microscope (Leica, Wetzlar, Germany) for photographic documentation. The photographs were taken with an Orthomat (Leica) camera and Kodak Ektachrome 320T film (Kodak, Harrow, England).

– For the scanning electron microscopic photographs, we cleaned the 15 toothbrush heads thoroughly with Leit-C (Chemicals Neubauer, Münster, Germany) and fixed them on plates. Before scanning them, we sputter-coated the heads in a coating unit (SCD-040, Balzers AG, Balzers, Liechtenstein) and covered them with a 100- to 150-nanometer–thick film. We took the photographs using a scanning electron microscope (PSEM 500, Philips, Eindhoven, Netherlands) according to the method described by Althaus and colleagues,⁸ Mueller and colleagues¹⁴ and Wetzel and colleagues.¹⁵ The working voltage was 25 kilovolts. We used Kodak T-Max 100 film (Kodak, Rochester, N.Y.).

Each child used two toothbrushes, cleaning the maxillary and mandibular teeth on one side with one toothbrush and those on the opposite side with a second toothbrush.

Subjects and methods. Our subjects were 45 children aged 6 to 13 years with carious lesions who had consulted the Department of Paediatric Dentistry at Justus-Liebig-University of Giessen, Dental School, Giessen, Germany. We instructed all subjects as to how to brush their teeth correctly. Each child used two toothbrushes, cleaning the maxillary and mandibular teeth on one side with one toothbrush and those on the opposite side with a second toothbrush. We randomized the children into three groups:

– group 1 (15 patients, average age: 8.7 years), using toothbrushes A and B;

– group 2 (15 patients, average age: 8.6 years), using toothbrushes A and C;

– group 3 (15 patients, average age: 8.7 years), using toothbrushes B and C.

We showed the children how to use the Bass toothbrushing method.¹⁶ Induction took three minutes. Each child brushed using a small pea-sized mass of toothpaste containing fluoride. Afterward, we rinsed the toothbrushes in 50 mL of tap water under standardized conditions. We rotated every toothbrush 10 times in a water-filled vessel. We carried out the microbiological examinations either immediately after use or after the toothbrushes were air-dried for two hours or eight hours. For drying, we laid the toothbrushes separately with the heads pointing upward on absorbent tissue paper in a sterile box at room temperature. The distance between each toothbrush was about 5 centimeters. The box was not actively ventilated so as to avoid transmission of germs. We examined 10 toothbrushes of each type at every time interval.

Dental examination. We performed diagnosis and evaluation of caries according to the general principles for an international standardization of caries laid down by Baume.¹⁷

Microbiological screening. To isolate the microorganisms from the toothbrushes, we immersed the heads in 15 mL of Sputasol (Oxoid, Basingstoke, Hampshire, England) solution (consisting of 0.1 gram dithithreitol, 0.78 g sodium chloride, 0.02 g potassium chloride, 0.112 g disodium hydrogen phosphate and 0.02 g potassium dihydrogen phosphate) and placed them in an ultrasonic device (Bandelin Sonorex, Bandelin Electronic GmbH, Berlin, Germany). We centrifuged 1 mL bacterial suspension at 20,000 rotations per minute for four minutes, and we rejected 800 microliters of the supernatant. We resuspended the pellet in the remaining 200 µL, and we plated 20 µL on each side for carrying out a bacteria test (CRT bacteria test, Ivoclar Vivadent, Schaan, Liechtenstein). This test consists of two different agar slides: rogosa agar, specific for lactobacilli, and mitis salivarius bacitracin agar, specific for S. mutans. We decided to follow the manufacturer’s instructions and not count the colonies, and, therefore, we did not use reference tables for determining bacterial frequency. Instead, we counted the number of microorganisms contained in 20 µL of the suspension as an absolute figure and calculated them according to the extracted volume of 1 mL suspension. For the detection of Candida species, we also cultured samples on sabouraud agar plates (Merck KgaA, Darmstadt, Germany). Candida species were identified according to their ability to produce germ tubes.¹⁸ In cases of uncertainty, some colonies were cultured additionally on CHROMagar Candida (CHROMagar, Paris). All culture media were incubated at 37 C for 48 hours.

Statistical analysis. We, in conjunction with the Department of Medical Informatics of Justus-Liebig-University of Giessen, evaluated the differences in contamination between direct examination of the used toothbrushes and examination after intervals of drying using the univariate two-factor analysis of variance.¹⁹ We fixed the levels of significance at P .05 (significant), P .01 (very significant) and P .001 (most significant). We carried out statistical analysis of the data using the software package SPSS for Windows Version 8.0 (SPSS, Chicago).²⁰

	RESULTS

TOP ABSTRACT MATERIALS, SUBJECTS AND METHODS RESULTS DISCUSSION CONCLUSION REFERENCES

 
Characteristics of the toothbrush heads. Figure 1 shows the heads of the three toothbrush types. The head of toothbrush A has three edges, with the top and the apex of the head being the smallest parts. The tufting area is 31 x 12 millimeters. Thirty-seven tufts are arranged in 12 vertical rows. The head of toothbrush B is formed like a narrow triangle, is 32 x 12 mm and has a vertical arrangement of 11 rows containing 38 tufts. The head of the toothbrush C is oval-shaped and has three rounded edges with the filaments placed in the mold individually. It has a concave surface to which the filaments are affixed and measures 24 x 10 mm. There are 978 individual filaments in toothbrush C.

View larger version (40K): [in this window] [in a new window]   Figure 1. Overview of the toothbrushes A, B and C. A. Staple-set tufting. B. In-mold tufting. C. Individual in-mold placement of filaments.

Staple-set tufting. Figure 2A shows a longitudinal section of the toothbrush head. The bundles are not fixed tightly enough to the synthetic material. This creates gaps and holes, which form part of the outer surface. The gaps are made visible by the scanning electron microscope (Figure 2B).

View larger version (77K): [in this window] [in a new window]   Figure 2. Principle of staple-set tufting. A. Light-microscopic photograph of toothbrush A. B. Scanning electron microscopic photograph of toothbrush A.

View larger version (41K): [in this window] [in a new window]   Figure 3. Principle of in-mold tufting. A. Light-microscopic photograph of toothbrush B. B. Scanning electron microscopic photograph of toothbrush B.

View larger version (28K): [in this window] [in a new window]   Figure 4. Principle of individual in-mold placement of filaments. A. Light-microscopic photograph of toothbrush C. B. Scanning electron microscopic photograph of toothbrush C.

Statistical analysis shows that the anchoring systems and the drying intervals both had a highly significant effect on the microbial contamination of the brushes.

Microbiological results. Figure 5 shows the microbial contamination of toothbrushes A, B and C after different intervals of air-drying. The table shows the maximum and the minimum values of the germs retrieved from the toothbrushes at different time points. The mean values and the standard deviation also are given. The results produced a great difference between the minimum and the maximum values; in one case, they ranged from zero to 20,000 CFU/mL. S. mutans was most in evidence, followed by lacto-bacilli. In all cases in which we detected Candida species, there were colonies of only C. albicans. But this microorganism was not actually found in every case.

View larger version (33K): [in this window] [in a new window]   Figure 5. Quantities (mean values) and comparative statistical significance of Streptococcus mutans, lactobacilli and Candida albicans isolated from toothbrushes A, B and C. CFU/mL: Colony-forming units per milliliter.

The results show that the differences in the microbial contamination of toothbrushes A and B are not significant.

An obviously smaller amount of S. mutans and lactobacilli was retrieved from product C. The difference in results between toothbrushes A and C concerning the retention of S. mutans and lactobacilli was most significant (P .001). The same applied to the retention of S. mutans and lactobacilli (P .001) on toothbrushes B and C. Because only a few toothbrushes in all three groups contained C. albicans, there was no sound basis for a statistical analysis in this respect.

	DISCUSSION

TOP ABSTRACT MATERIALS, SUBJECTS AND METHODS RESULTS DISCUSSION CONCLUSION REFERENCES

 
The aim of our study was to investigate the different principles of toothbrush filament anchoring with regard to their microbial contamination. The study confirms and elaborates on the results of previous investigations.^{4,9,10,21–25}

Althaus and colleagues⁸ compared the staple-set tufting and in-mold tufting principles in toothbrushes used by children. They found that all cavities of the in-mold tufted toothbrush were sealed thoroughly, thus eliminating any spaces for food debris and microorganisms. It was hoped that in-mold tufting would become an important innovation in toothbrush hygiene. However, the investigations of Bienengraeber and colleagues⁷ revealed that the new type of in-mold tufting was beneficial insofar as the liquid synthetic anchoring material fills the hollow spaces between filaments completely and smoothly.

Hypothesis 1. Our study refutes the hypothesis that in-mold tufting is innovative for oral hygiene. The levels of microbial contamination of the toothbrushes found in our study do not reveal noteworthy differences between contamination of the conventionally anchored filaments and that of in-mold–tufted filaments. Our first hypothesis, therefore, could not be confirmed.

Hypothesis 2. In our study, we compared the individual in-mold placement of filaments, a technique never before tested, with the two other principles mentioned above. The results showed a noticeable reduction in bacterial contamination with this new technique compared with the other two principles of anchoring. Thus, our second hypothesis was upheld.

Hypothesis 3. The third hypothesis—that it is possible to grade the three types of anchoring according to their microbial contamination—also was disproved. The improvement in hygiene associated only with the new principle of anchoring obviously is owed to the fact that the toothbrushes can be rinsed more easily, because the filaments are anchored individually. As a result, there is less danger of microorganisms’ becoming entrapped. Because the filaments are anchored individually, there is no need for tufting. Presumably, not only the tight hold of the synthetic material at the base of the bundles, but also the distance between the filaments themselves are of immense importance. Forming the filaments into bundles tends to have certain disadvantages with regard to hygiene because moisture can escape only slowly and, thus, an adequate breeding ground for microorganisms is created. As early as 1946, McCauley²⁶ noticed that interrow spacing of the tufts facilitated cleansing (with disinfectants) and drying.

	CONCLUSION

TOP ABSTRACT MATERIALS, SUBJECTS AND METHODS RESULTS DISCUSSION CONCLUSION REFERENCES

 
We came to the conclusion that the bundling of toothbrush filaments increases the risk of bacterial adherence regardless of which anchoring technique is used. The new method of anchoring, which combines compact and individual attachment of the filaments to the head of the toothbrush, leads to sound hygienic conditions. This innovative principle, which was investigated for the first time in our study, may be of a great innovative importance not only to dental but also to medical brushes. It also perhaps could improve the hygiene standards of brushes used in private households.