|Year : 2015 | Volume
| Issue : 1 | Page : 26-32
The minimum inhibitory concentration of different candidal disinfecting agents
Aiman A Ali1, Fahad A Al Harbi2
1 Department of Biomedical Dental Sciences, College of Dentistry, University of Dammam, Saudi Arabia
2 Department of Substitutive Dental Sciences, College of Dentistry, University of Dammam, Saudi Arabia
|Date of Web Publication||20-Jan-2015|
Aiman A Ali
Department of Biomedical Dental Sciences. College of Dentistry, University of Dammam, P.O. Box 60710, Dammam 31555
Purpose: Despite the availability of a large number of commercial denture cleansing products, only a few are used by wearers of dentures. This may be due to negligence on the part of the wearers of dentures and or the high cost of the products. We, therefore, felt inspired to study the antifungal effect of different materials usually available in the kitchen.
Materials and Methods: One hundred and sixty resin acrylic samples were prepared and divided into three groups of 50 samples each and immersed into variable concentrations of: Sodium chloride (Group I), Sodium bicarbonate (Group II), and vinegar (Group III). In addition, 10 samples were immersed into water as a control group (Group IV). Minimum inhibitory concentration and the minimum immersion time needed were studied for each group.
Results: The solutions were only found to be effective against Candida albicans in concentrations higher than 50 ml/200 ml, 10 g/200 ml and 5 g/200 ml of vinegar, sodium bicarbonate and sodium chloride respectively. The same concentrations were more effective when acrylic resin plates were immersed for 8 hours rather than 1 hour (P < 0.05).
Conclusion: Sodium chloride, sodium bicarbonate and vinegar are not strong enough as denture cleansing agents in low concentrations. However, high concentrations used for 8 hours might be helpful for the user of dentures.
نادرًا ما يستخدم مرضى التعويضات الفموية غسول أطقم الأسنان على الرغم من توفر العديد منها، وذلك إما لغلاء أسعارها أو لجهلهم بها. هذا ما دفعنا لدراسة فعالية بعض المواد المتوفرة في كل بيت كمضادات فطرية.
تم إعداد 061 عينة من الراتنج حيث وزعت على ثلاث مجموعات كل منها تحتوي على 05 عينة إضافة لعشر عينات في المجموعة الضابطة. غمرت عينات المجموعة الأولى في تركيزات مختلفة من محلول كلور الصوديوم (ملح الطعام)، و غمرت عينات المجموعة الثانية في تركيزات مختلفة من محلول بيكربونات الصوديوم, بينما غمرت عينات المجموعة الثالثة في تركيزات مختلفة من الخل. أما عينات المجموعة الضابطة فقد غمرت بالماء. بعد ذلك تم دراسة الزمن والتركيز الأدنى اللازم لتطهير العينات.
وجد بأن هذه المحاليل فعالة في القضاء على فطريات المبيضات فقط بتركيز أعلى مــن 002 / 05 مل، 002 / 01مل و002 / 5 مل من الخل، بيكربونات الصوديوم وكلور الصوديوم على التوالي. كما وجد بان فعالية نفس التركيزات تزداد بزيادة زمن الغمر من 1إلى 8 ساعات وقد أظهر تحليل النتائج أنها ذات قيمة إحصائية.
ومن ذلك نستنتج أن الخل وملح الطعام وبيكربونات الصوديوم غير فعالة في تطهير أطقم الأسنان عندما تستخدم بتركيزات منخفضة. ولكن استخدام هذه المواد بتركيزات عالية ولمدة 8 ساعات قد يكون فعالا.
Keywords: Denture cleansing agents, sodium bicarbonate, sodium chloride, vinegar
|How to cite this article:|
Ali AA, Al Harbi FA. The minimum inhibitory concentration of different candidal disinfecting agents. Saudi J Med Med Sci 2015;3:26-32
|How to cite this URL:|
Ali AA, Al Harbi FA. The minimum inhibitory concentration of different candidal disinfecting agents. Saudi J Med Med Sci [serial online] 2015 [cited 2019 Sep 21];3:26-32. Available from: http://www.sjmms.net/text.asp?2015/3/1/26/149668
| Introduction|| |
Candida albicans is an opportunistic pathogen that commonly affects individuals with a compromised immune system. In the oral cavity, candidiasis is often related to the use of dentures and leads to the development of a condition referred to as denture-induced stomatitis. Denture-induced stomatitis is an inflammatory reaction of the denture-bearing mucosa, affecting approximately 65% of the complete upper dentures. 
Different treatment modalities for oral candidiasis have been proposed. ,, Topical application of creams or mouthwashes is effective only for a short period, and very often the purpose is defeated by the constant flushing by saliva. Systemic administration of drugs is not very effective in controlling this type of Candida infection because the organism, usually limits its activity to the oral mucosa. Moreover, they have a variety of adverse effects. Koray et al. suggested a more conservative intervention for the management of oral candidiasis with oral mouth rinses, rather than risk the adverse effects and complications with the use of systemic drugs.  A drug with favorable pharmacologic profile has been incorporated into the commonly employed polymer systems such as heat and chemically activated polymethyl methacrylate, tissue conditioners and soft liners which could function as efficient drug-releasing agents. 
The chemical methods involve denture immersion in chemical products such as sodium perborate, sodium hypochlorite, coconut soap, hypochloride, phosphoric and benzoic acids, chlorhexidine digluconate and enzymes such as proteases and mutanases.  Tablets of sodium perborate-based denture cleanser are commonly used for prosthesis cleaning and to help mechanical hygiene. Gornitsky et al. verified the antimicrobial activity of these solutions on microorganisms that adhered to prostheses, but suggested that the use of the prosthesis cleaning agents might be controlled.  Sodium hypochlorite is inexpensive, presents a broad spectrum of activity and requires a short period of disinfection. , An in vitro study by Montagner et al. suggested that sodium hypochlorite-based substances and hydrogen peroxide are more efficient disinfectants against C. albicans than 2% chlorhexidine solution.  Despite its efficiency as a disinfectant, sodium hypochlorite has some disadvantages including its corrosive activity on metal surfaces, irritant effect on the skin and other cells with destruction of cloth including cotton. 
Gluteraldehyde-based solutions are often indicated in dentistry.  High antimicrobial activity of gluteraldehyde has been described in the literature and its effectiveness is related to the period of exposure. Angelillo et al. and Silva et al. demonstrated the effectiveness of this solution against C. albicans. , However, there is concern about its toxicity, a characteristic that is considered a limitation for its use. 
Chlorhexidine is more effective than Nystatin or Amphotericin B in killing adherent C. albicans cells. , The reduction in biofilm growth and adhesion supports the role of chlorhexidine as a potential therapeutic agent in the prevention and treatment of denture stomatitis. However, the sensitivity of other strains of C. albicans to such a treatment requires further evaluation.  Although chlorhexidine presents excellent antimicrobial properties such as low-concentration efficiency, substantivity, minimal perception by the gastrointestinal tract, the capacity to reduce biofilm formation and disorganize preformed biofilm; it also presents some side-effects, including mild discomfort or burning sensation when it comes into contact with the oral mucosa. In addition, there is epithelium exfoliation, teeth staining, enhancement of supra-gingival calculus and color alteration of artificial teeth and denture basis. 
Sodium bicarbonate has been used for the disinfection of dentures and orthodontic appliances. Many studies have shown that sodium bicarbonate at high concentrations has an antimicrobial effect over several microorganisms isolated from the oral cavity, including C. albicans.  In a study by Sousa, 5% sodium bicarbonate was shown to be a viable alternative, at least with regards to its anti-Candida adherence effect, since it significantly reduced the adherence of C. albicans to the surface of the thermally activated specimens of acrylic resins. Under the conditions of their study, both white vinegar and corega tablets were found to be inadequate in reducing the number of C. albicans colonies adhering to the specimens. 
The use of vinegar as a disinfectant is not often discussed in dentistry. Acetic acid is one component of vinegar. Vinegar is as effective as the most-often employed disinfectants against C. albicans.  Considering that C. albicans is the main etiologic agent of oral candidosis, the possible use of this solution in the disinfection of prostheses might be very promising as therapeutic and preventive, especially because of its low toxicity.  Studies on the possible effects on other properties of the resin seem to be necessary.
In vitro experiments have already shown that low fungicidal doses of acetic acid induce programmed cell death of C. albicans.  Other researchers recently studied the effectiveness of a 10% vinegar solution in the control of Candida species in the oral cavity of wearers of complete upper dentures.  They noted a reduction in the amount (CFU/ml) of candida species in the saliva and the presence of denture stomatitis in the studied patients, after immersing the complete dentures in a 10% vinegar solution for 8 h in the night for 45 days.
The variation of the results of published papers motivated us to conduct this investigation to study the minimum inhibitory concentrations (MIC) and the minimum immersion time needed (MITN) for the use of common materials available in every kitchen against C. albicans.
| Materials and methods|| |
One hundred sixty resin acrylic samples were prepared and divided into 5 groups as follows:
Samples, with dimensions of 10 mm × 10 mm × 2 mm in heat cure acrylic were fabricated at the Prosthodontic Laboratory (Trevalon/Universal Clear-Dentsply. Germany). All samples of all the groups were immersed in a container filled with artificial saliva (Moi.stir-Indiana-USA) with Candidal species (ATCC 10231) and incubated in 37°C for 1 week. They were all washed with tap water and inoculated on Sabouraud's dextrose agar plate (Gibco, Paisley, Scotland) and incubated aerobically at 37°C for 48 hours.
- Group I: 50 samples to be immersed in different concentrations of sodium chloride.
- Group II: 50 samples to be immersed in different concentrations of sodium bicarbonate.
- Group III: 50 samples to be immersed in different concentrations of vinegar.
- Group IV: (group control): 10 samples to be immersed in water.
To determine the MITN and MIC on Candidal colonization, infected samples of acrylic plates were kept in containers with different concentrations of:
Half of the plates (5 samples each) from each solution were taken out after 1 hour and the other half (5 samples each) after 8 hours. All samples were washed with tap water and inoculated on Sabouraud's dextrose agar plate (Gibco, Paisley, Scotland) and incubated aerobically at 37°C for 48 hours. After incubation, C. albicans colonies were separated from the acrylic resin using a hard vortex mix for 10 minutes. This was later centrifuged at 4500 rpm for 5 minutes. After centrifuging, the acrylic resin plates were removed from the tubes and the concentrated pellets collected from the tube. Effectiveness of each concentration of the disinfecting agents for a specific time were evaluated using 2 different methodologies (Platelet count and microscopic evaluation) as follows: Culture test and slide count.
- Sodium chloride (1 g/200 ml, 2 g/200 ml, 5 g/200 ml, 10 g/200 ml, 20 g/200 ml) (10 samples in each concentration).
- Sodium bicarbonate solutions (1 g/200 ml, 2 g/200 ml, 5 g/200 ml, 10 g/200 ml, 20 g/200 ml) (10 samples in each concentration).
- Vinegar solutions (25 ml/200 ml, 50 ml/200 ml, 75 ml/200 ml, 100 mg/200 ml, 125 ml/200 ml) (10 samples in each concentration).
- Water (10 samples).
One hundred microliters (100 μl) of the centrifuged pellet were taken and cultured in plates filled with SD agar and incubated for 24 hours at 37°C. After incubation, colonies of C. albicans were counted in the plates using a marker pen counter (colony counter "Scienceware - Bel-Art Products"). Colonies that covered the entire surface of the plate were considered to be overgrowth [Figure 1].
|Figure 1: Culture of samples immersed in different concentrations of Sodium Chloride showing the decrease of number of Candidal colonies when the concentration increased (Plates count).|
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A volume of 7.5 μl was taken from the concentrated pellet and added to 2.5 μl of trypan blue 0.4% solution in phosphate (MP-Biomedicals). The new 10 μl solution was placed on a slide count (Neubauer Slide Counter "Chambers-Marienfeld") for microscopic evaluation. A slide count contained 4 main squares, each divided into 16 smaller squares. Candida were counted on the 2 main squares and multiplied by 2 to obtain the total number of Candida on each slide.
Trypan blue stain enabled the counting of live and dead Candida numbers under a light microscope at ×10 magnification. Live Candida appear as transparent circles with blue borders while dead Candida appear as blue circles [Figure 2].
|Figure 2: Microscopic picture showing the difference between dead and alive Candida albicans.|
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Statistical software SPSS version 19.0 was used for data analysis. The results of fungal growth on various evaluations and chemicals are presented in terms of mean ± standard deviation. Two-way (repeated measure) analysis of variance was applied for the comparison of mean fungal growth of three chemical groups and on various concentrations. Post-hoc Scheffe's test was applied for pair-wise comparison of chemical groups and intra group comparison of mean fungal growth on various concentrations. P ≤ 0.05 was considered a result of statistical significance.
| Results|| |
All plates immersed in (25 ml/200 ml, 50 ml/200 ml) concentrations of vinegar showed overgrowth of Candidal colonies whether the plates were immersed for 1 hour or 8 hours. However, the number of colonies decreased in high concentrations, and when samples were immersed for a long time (8 h). Similarly, all plates immersed in different concentrations of Sodium bicarbonate (1 g/200 ml, 2 g/200 ml, 5 g/200 ml, 10 g/200 ml) showed an overgrowth of Candidal colonies for plates immersed for either 1 or 8 hours. The number of colonies decreased only in a 20 g/200 ml concentration. All plates immersed in different concentrations of Sodium chloride (1 g/200 ml, 2 g/200 ml, 5 g/200 ml) showed an overgrowth of Candidal colonies for plates immersed for either 1 or 8 hours. The number of colonies decreased when the concentration increased to 20 g/200 ml and 10 g/200 ml, and when immersed for 8 hours [Table 1], [Table 2] and [Figure 3]. All plates immersed in water for 1 or 8 hours showed an overgrowth of Candida colonies.
|Figure 3: Effect of different disinfectants in different concentrations for 1 and 8 h (Plates count).|
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|Table 1: Comparison of fungal growth of Candida albicans on various levels of concentration and chemicals with normal and microscopic evaluation.|
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|Table 2: Comparison of fungal growth of Candida on various levels of concentration and chemicals with normal and microscopic evaluation according to time intervals.|
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In the slide count of samples immersed in different concentrations of vinegar, the number of live Candida decreased to 0.00 in the increased concentration of 125 ml/200 ml when samples were immersed for 1 hour, and in 100 ml and 125 ml/200 ml concentrations when samples were immersed for 8 hours.
Similarly, when the concentration of Sodium bicarbonate increased, the number of live C. albicans decreased to 0.00 in 20 g/200 ml concentration when samples were immersed for 8 hours only.In group I, when the concentration of Sodium chloride increased, the number of live C. albicans decreased to 0.00 in 20 g/200 ml concentration when samples were immersed for 1 hour, and in 10 grams and 20 g/200 ml concentrations when samples were immersed for 8 hours. All plates immersed in water for 1 or 8 hours showed overgrowth [Figure 4].
|Figure 4: Effect of different disinfectants in different concentrations for 1 and 8 hours (microscopic evaluation)|
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| Discussion|| |
Geriatric patients are those most frequently affected by denture stomatitis. Despite the large number of commercial denture cleansing products currently available, only a few wearers use any, possibly from ignorance and/or their cost. Others might be aware of denture cleansing products, but may not have them at home. Therefore, this paper intends to discuss the MIC and MITN of the three most common materials found in every house on C. albicans. These materials are Salt (Sodium chloride), vinegar and Sodium bicarbonate which are available in every kitchen.
Samples were immersed in the disinfecting agents for 8 hours, the length of time the wearer of dentures normally has them off at night. Other samples were immersed for only 1 hour to see if the same agents were effective for a short duration.
The most common item is salt or Sodium chloride. All geriatric patients have access to salt which is available in every kitchen in the world. Very little information is available on the PubMed about the effect of Sodium chloride on C. albicans. The results of this study showed that Sodium chloride affects C. albicans only in concentrations higher than 10 g/200 ml when samples are immersed for at least 8 hours. Another study showed that the use of 0.66 per cent of NaCl was significantly effective in reducing viable microorganisms compared to tap water. 
Vinegar is the second most common material in kitchens. The use of vinegar as a disinfectant has not often been discussed in dentistry although da Silva  stated in his study that vinegar was as effective as the most-often employed disinfectants against C. albicans. However, results of this study showed that vinegar was effective in the highest concentration only (125 ml/200 ml) when samples were immersed for 1 hour. It was effective in 75 ml/200 ml concentration or higher when samples were immersed for 8 hours. In another study, white vinegar was found to be inefficient in reduction of the number of C. albicans colonies that adhered to specimens.  However, other studies , have reported that white vinegar was the most effective disinfecting agent for tested microorganisms and acrylic resins. The differences in concentrations used in the various studies and the various commercial types of vinegar should be taken into account.
The results of this study showed that Sodium bicarbonate decreased the number of C. albicans only when it was used in very high concentrations (20 g/200 ml), while in another study Sodium bicarbonate significantly reduced the adherence of C. albicans to the surface of the thermally activated acrylic resin specimens even when used in 5% concentration only. 
In this study, it was found that the duration of immersion of acrylic resin plates was very important. The effect of the disinfecting agents increased with an increase in the duration of immersion of the dentures. Therefore, the same concentration of Sodium chloride (10 g/200 ml) was not as effective when samples were immersed for 1 hour. The number of live C. albicans decreased significantly when the dentures were immersed in the same concentration for a longer period of 8 hours. Similarly, the same concentrations of vinegar (75 ml/200 ml, 100 ml/200 ml, and 125 ml/200 ml) were not as effective for 1 hour as they were when the dentures were immersed for 8 hours.
Immersion of samples once (either for 1 hour or 8 hours) is one of the main limitations of this study. Other studies repeated the test for 45 days to study the effects of the disinfecting agent over a long period.  The results of this study may be improved if the immersion is repeated over a longer period (weeks or months). However, some studies have reported that increasing the time of emersion may cause an alteration of the physical surface of the acrylic resin. ,
It should be noted that materials studied in this paper have no toxic effect on oral tissues and are always available for geriatric patients.
| Conclusion|| |
Sodium chloride, sodium bicarbonate and vinegar are not strong enough as denture cleansing agents in low concentrations. However, the use of high concentrations for 8 hours might be helpful for wearers of dentures.
| Acknowledgment|| |
The authors would like to thank the Deanship of Scientific Research, University of Dammam, Kingdom of Saudi Arabia for funding this research project (Project No. 201022). The authors would like also to thank Mr. Intisar Siddiqui, Biostatistician and Lecturer, College of Dentistry, University of Dammam, for his assistance with statistical analysis.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4]
[Table 1], [Table 2]